CD3 binding antibodies

Abstract

To provide anti- CD3 antibodies that more closely mimic TCR / MHC interactions and are desirable for minimizing the release of toxic cytokines while maintaining effective tumor cytolysis.SOLUTION: Novel human CD3 antigen binding polypeptides and their preparation and their use in the treatment and / or diagnosis of various diseases, as well as bispecific antibody molecules capable of activating immune effector cells and their use in the diagnosis and / or treatment of various diseases. In some embodiments, the anti-CD3 antibody is characterized or selected to have a reduced tendency to induce cytokine release upon binding to a competent T cell, e.g., due to the release of IL-2 and IFN γ.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross Reference 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 applications are incorporated herein by reference in their entireties. [Background technology]

[0002] background The body's immune system plays a role in 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 and neutralizes products recognized by the body as foreign. In contrast, the cellular system includes cells such as T cells and macrophages that remove and neutralize foreign invaders.

[0003] T cell activation is critical for stimulating immune responses. T cells exhibit immunological specificity and direct most cell-mediated immune responses. T cells do not secrete antibodies but are required for antibody secretion by B lymphocytes. T cell activation requires the engagement of the T cell receptor complex and multiple cell surface molecules, such as CD4 or CD8 molecules. Antigen-specific T cell receptors (TcRs) consist of disulfide-linked heterodimers, membrane glycoproteins with chains, alpha and beta (α and β), or gamma and delta (γ and δ). TcRs are noncovalently associated with a complex of invariant proteins called CD3.

[0004] T cells are known to exert potent antitumor effects in numerous experimental settings. Antibodies that can effectively recruit T cells to tumor cells have been available, for example, bispecific antibodies directed against tumor-associated antigens (TAAs) and agonistic T cell membrane proteins such as the TCR / CD3 complex and CD28. These bispecific antibodies can activate T cells regardless of their TCR specificity, resulting in the specific lysis of cells bearing the respective TAA.

[0005] However, although anti-CD3 bispecific antibodies can direct T cell-mediated lysis to malignant cells, clinical trials with CD3-based bSAbs have shown high toxicity in patients. Nonspecific T cell activation from bSAbs can occur antigen-independently due to Fc / Fc receptor (FcR) interactions, or antigen-dependently when the antigen is expressed on both normal and tumor cells. Both mechanisms may have been responsible for the toxicity observed in previous clinical trials. See, for example, Link et al. (1998) Int. J. Cancer 77(2). :251-6;Durben et al. Molecular Therapy(2 015);23 4, 648-655. The resulting cytokines Release syndrome has been an important block in the development of these antibodies for therapeutic purposes.

[0006] The interaction of the T cell receptor (TCR) with its peptide-MHC ligand determines the activity of the T cell. The binding properties of this interaction have been studied in great detail and have been shown to regulate T cell function. The strength and nature of the TCR-peptide / MHC interaction determine whether the T cell exerts effector function or is inactivated and deleted. Antibodies against CD3 activate T cells by altering the conformation of the CD3 epsilon chain and, depending on the epitope, can have either an agonistic or antagonistic effect on the T cell (Yoon et al., 1994 Immunity 1:5 63-569). Given the significant side effects of many T cell agonists, it may be preferable to maintain potent antitumor efficacy while reducing the release of proinflammatory cytokines. However, partial agonist anti-CD3 antibodies suboptimally alter the CD3ε chain, resulting in ineffective signaling, and most anti-CD3 antibodies are full agonists for both pathways. Whether these effector functions can be uncoupled is unclear. Many existing anti-CD3 antibodies (e.g., SP-34, UCHT1, OKT3) have affinities in the 1–50 nM KD range, which may not be optimal for therapeutic use.

[0007] CD3-specific antibodies and bispecific antibodies derived therefrom are provided by the present invention.

[0008] public CD3 antibodies are disclosed, for example, in U.S. Patent Nos. 5,585,097, 5,929,212, 5,968,509, 6,706,265, 6,750,325, 7,381,803, and 7,728,114. Bispecific antibodies with CD3 binding specificity are disclosed, for example, in U.S. Patent Nos. 7,262,276, 7,635,472, 7,862,813, and 8,236,308, each of which is specifically incorporated by reference herein. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 5,585,097 [Patent Document 2] U.S. Patent No. 5,929,212 [Patent Document 3] U.S. Patent No. 5,968,509 [Patent Document 4] U.S. Patent No. 6,706,265 [Patent Document 5] U.S. Patent No. 6,750,325 [Patent Document 6] U.S. Patent No. 7,381,803 [Patent Document 7] U.S. Patent No. 7,728,114 [Patent Document 8] U.S. Patent No. 7,262,276 [Patent Document 9] U.S. Patent No. 7,635,472 [Patent Document 10] U.S. Patent No. 7,862,813 [Patent Document 11] U.S. Patent No. 8,236,308 [Non-patent literature]

[0010] [Non-Patent Document 1] Link et al. (1998) Int. J. Cancer 77(2):251-6 [Non-patent document 2] Durben et al. Molecular Therapy(2015);23 4, 648-655 [Non-patent document 3] Yoon et al.,1994 Immunity 1:563-569 Summary of the Invention [Means for solving the problem]

[0011] overview The compositions and methods of use thereof bind to CD3 and activate signaling through CD3 (e.g., CD3 + The present invention provides for a family of closely related antibodies that activate T cells. The antibody family comprises a set of CDR sequences as defined herein. Antibody families offer many benefits that contribute to their usefulness as clinical therapeutic agent(s). Antibodies within a family include members with a range of binding affinities, allowing for the selection of specific sequences with desired affinities. The ability to fine-tune affinity allows for the selection of specific sequences with desired affinities depending on the individual being treated. It is particularly important to control the level of CD3 activation in the body, thereby reducing toxicity.

[0012] In some embodiments, the anti-CD3 antibody is about 10 -6 ~about 10 -11 The antibodies may have an affinity (KD) for CD3 in the range of 0.1 to 1.0.

[0013] Anti-CD3 antibodies with affinities (KD) of 50 nM or greater, 100 nM or greater, 500 nM or greater, or 1 μM or greater may be desirable to more closely mimic TCR / MHC interactions and minimize toxic cytokine release while maintaining effective tumor cell lysis. In some embodiments, anti-CD3 antibodies are characterized or selected for a reduced propensity to induce cytokine release upon binding to competent T cells, e.g., for the release of IL-2 and IFNγ. Antibodies may be selected for therapeutic use to optimize tumor cell killing and reduced cytokine release, e.g., within a family of antibody sequences described herein, as antibodies that induce cytokine release that is less than about half the maximum observed for family members in comparative assays, and may also be, e.g., less than about 25% or less of the maximum observed for family members in comparative assays. In some embodiments, bispecific or multispecific antibodies are provided that comprise at least a heavy chain variable region from an antibody family, and may include heavy and light chain variable regions provided herein. Bispecific antibodies comprise at least the heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise heavy and light chain variable regions. In some such embodiments, the second antibody specifically binds to a target antigen such as a tumor-associated antigen, e.g., an integrin, a pathogen antigen, a checkpoint protein, etc. Various bispecific antibody forms are within the scope of the present invention, including, but not limited to, single-chain polypeptides, two-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and multiples thereof.

[0014] Each of the CD3-specific antibodies comprises a VH domain comprising CDR1, CDR2, and CDR3 sequences in a human VH framework. Family 2 CDR sequences may be located, by way of example, in the region of approximately amino acid residues 26-33, 51-58, and 97-112 for CDR1, CDR2, and CDR3, respectively, of the exemplary variable region sequences provided in SEQ ID NOS: 1-18. It will be understood by those skilled in the art 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.

[0015] The CDR sequences of Family 2 antibodies may have the following sequence formula: X represents a variable amino acid and may be a specific amino acid as indicated below. [ka] where: X5 can be any amino acid, in some embodiments X5 is D, A or H, and in some embodiments X5 is D. X6 can be any amino acid, in some embodiments, X6 is D or N, and in some embodiments, D6 is D. In some embodiments, the CDR1 sequence of a Family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 26-33. [ka] In some embodiments, the CDR2 sequence of a Family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 51-58. [ka] where: X 9’’ may be any amino acid, and in some embodiments, X 9’’is D or S, and in some embodiments, X 9’’ is D, X 11’’ may be any amino acid, and in some embodiments, X 11’’ is R or S, X 12’’ may be any amino acid, and in some embodiments, X 12’’ is L or R.

[0016] In some embodiments, the CD3 sequence of the Family 2 anti-CD3 antibody is of the formula [ka] wherein X 11” and X 12” is as defined above. In some embodiments, the CDR3 sequence of a Family 2 anti-CD3 antibody comprises a sequence set forth in any of SEQ ID NOs: 1-18, residues 97-112. In some embodiments, the CD3-binding VH domain of a Family 2 antibody is paired with a light chain variable region domain. In some such embodiments, the light chain is a fixed light chain.

[0017] 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, the CDR1 sequence comprises amino acid residues 27-32, 50-52, and 89-97 for CDR1, CDR2, and CDR3, respectively.

[0018] In some embodiments, the CDR sequences of the antibodies of the present invention are sequences that have at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the CDR sequence or set of CDR sequences set forth in SEQ ID NOs: 1 to 18. In some embodiments, the CDR sequences of the present invention are sequences that have at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to any of SEQ ID NOs: 1 to 18. The CDRs may contain one, two, three or more amino acid substitutions relative to any one CDR sequence or set of CDR sequences. In some embodiments, the amino acid substitution(s) are at one or more of positions 5 or 10 in CDR1, 2, 6 or 7 in CDR2, and 1, 8, 9 or 10 in CDR3 relative to the Family 2 formula above.

[0019] In some embodiments, bispecific antibodies of the invention comprise a CD3-binding variable region described herein paired with a light chain. In some embodiments, the light chain comprises the variable region sequence set forth in SEQ ID NO: 19, or a variable region comprising the set of CDR sequences and framework sequences of SEQ ID NO: 19. Various Fc sequences are used, including, but not limited to, human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. In some embodiments, the second arm of the bispecific antibody 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, for example, as shown in FIG. 2B. In some embodiments, the anti-BCMA arm comprises the variable region sequence set forth in SEQ ID NO: 20, or the tandem variable region sequence set forth in SEQ ID NO: 21. The Fc sequence of the anti-BCMA arm may be, but is not limited to, human IgG1, IgG2a, IgG2b, IgG3, IgG4, etc. The CDR sequences may be those contained in SEQ ID NO: 20. In some embodiments, the CDR sequences comprise amino acid residues 26-33, 51-58, and 97-108 for CDR1, CDR2, and CDR3, respectively.

[0020] In another embodiment, there is provided a pharmaceutical composition comprising at least a CD3-binding VH domain of the invention, a monospecific, bispecific, etc. antibody or antibody-like protein comprising at least a CD3-binding VH domain of the invention, and a pharmaceutically acceptable excipient. The composition may be lyophilized, suspended in solution, etc., or provided in a unit dose formulation.

[0021] In some embodiments, methods for the treatment of cancer are provided, the methods comprising administering to an individual in need thereof an effective amount of a monospecific, bispecific, etc., antibody of the invention. Where the antibody is bispecific, the second antigen-binding site may 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 tract, brain tumor, 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 an infectious pathogen.

[0022] In some embodiments, methods for treating infectious diseases are provided, comprising administering to an individual in need thereof an effective amount of a monospecific, bispecific, etc. antibody of the invention. When the antibody is bispecific, the second antigen-binding site may specifically bind to a pathogen antigen, e.g., a bacterium, virus, or parasite.

[0023] In other embodiments, methods are provided for producing bispecific antibodies of the invention, comprising expressing antibody sequences, e.g., one or more light chain coding sequences, one or more heavy chain coding sequences, in a single host cell, which in various embodiments may be a prokaryotic cell or a eukaryotic cell, such as a mammalian cell. [Brief explanation of the drawings]

[0024] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. According to common practice, various features of the drawings may be It is emphasized that the drawings are not to scale. Conversely, dimensions of various features are arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0025] [Figure 1] 1A-1C. Figure 1A shows an alignment of the CDR1, 2, and 3 regions of antibody family 2 members of SEQ ID NOs: 1-18, which specifically bind to human CD3, corresponding to residues 26-33, 51-58, and 97-112. Figure 1B shows the CDR1, 2, and 3 regions of the fixed light chain (SEQ ID NO: 19), as well as exemplary anti-BCMA sequences (SEQ ID NOs: 20 and 21). Figure 1C provides the CDR sequences of a reference anti-CD3 antibody (SEQ ID NO: 22), ID304704. [Figure 2-1] 2A-2E. Schematic diagrams of bispecific human antibodies. 2A is an anti-CD3:anti-tumor antigen bispecific antibody with a common light chain (all three unique chains). 2B is an anti-CD3:anti-tumor antigen bispecific antibody with two unique light chains (all four unique chains). 2C is an anti-CD3:anti-tumor antigen bispecific antibody with only the tumor antigen-binding domain chain of the heavy chain (three unique chains). 2D is an anti-CD3:anti-tumor antigen bispecific antibody with an ScFv tumor antigen-binding domain (all three unique chains). 2E is an anti-CD3:anti-tumor antigen bispecific antibody with an ScFv anti-CD3 binding domain (all three unique chains). [Figure 2-2] 2A-2E. Schematic diagrams of bispecific human antibodies. 2A is an anti-CD3:anti-tumor antigen bispecific antibody with a common light chain (all three unique chains). 2B is an anti-CD3:anti-tumor antigen bispecific antibody with two unique light chains (all four unique chains). 2C is an anti-CD3:anti-tumor antigen bispecific antibody with only the tumor antigen-binding domain chain of the heavy chain (three unique chains). 2D is an anti-CD3:anti-tumor antigen bispecific antibody with an ScFv tumor antigen-binding domain (all three unique chains). 2E is an anti-CD3:anti-tumor antigen bispecific antibody with an ScFv anti-CD3 binding domain (all three unique chains). [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 SEQ ID NOs of the anti-CD3 VH sequences. Column 2 shows the MFI values ​​for Jurkat cell binding of the parent monospecific anti-CD3. Column 3 shows the MFI values ​​for cynomolgus monkey T cell binding of the parent 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 with the bispecific antibody that binds to the BCMA protein coated on plastic at the indicated dose. Column 6 shows the picograms of IL-6 released by pan T cells stimulated with the bispecific antibody that binds to the BCMA protein coated on plastic at the indicated dose. Column 7 shows the picograms of IL-10 released by pan T cells stimulated with the bispecific antibody that binds to the BCMA protein coated on plastic at the indicated dose. Column 8 shows picograms of IFN-γ released by pan T cells stimulated with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 9 shows picograms of TNFα released by pan T cells stimulated with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 10 shows the EC50 of bispecific antibody-mediated U266 tumor cell lysis in the presence of human pan T cells. Column 11 shows the percent lysis of U266 tumor cells at a dose of 333 ng / mL of bispecific antibody in the presence of bispecific antibody and human pan T cells. Column 12 shows the protein binding affinity of the anti-CD3 arm of the bispecific antibody measured by octet. Column 13 shows the MFI values ​​for Jurkat cell binding of the bispecific antibody. [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 via redirection of 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 with the addition of bispecific antibodies. The X-axis indicates the concentration of antibody used, and the Y-axis indicates the % lysis of tumor cells 6 hours after antibody addition. [Figure 5] The killing activity of bispecific U266 correlated with the release of IL-2. A comparison of bispecific antibody-mediated tumor cell lysis activity and IL-2 cytokine release is shown in a scatter plot. The correlation between IL-2 production and U266 tumor cell lysis is R2=0.37. [Figure 6] The killing activity of bispecific U266 correlated with the release of IFN-γ. A comparison of bispecific antibody-mediated tumor cell lysis activity and IFN-γ cytokine release is shown in a scatter plot. The correlation between IFN-γ production and U266 tumor cell lysis is R2=0.53. [Figure 7] The killing activity of bispecific U266 correlated with anti-CD3 binding affinity. A comparison of bispecific antibody-mediated U266 tumor cell lysis activity and anti-CD3 binding affinity is shown in a scatter plot. The correlation between U266 killing EC50 and protein binding affinity is R2=0.93. [Figure 8] 8A-8D. Bispecific antibody-mediated tumor cell lysis. The αCD3_F1F:aBCMA bispecific antibody was assayed for its ability to kill three different BCMA+ tumor cells and one BCMA-negative cell line by redirection of activated primary T cells. In this experiment, tumor cells were mixed with activated pan-T cells at an E:T ratio of 10:1 with the addition of the bispecific antibody. 8A shows the killing of RPMI-8226 cells, 8B shows the killing of NCI-H929 cells, 8C shows the killing of U-266 cells, and 8D shows the killing of negative control K562 cells. The X-axis indicates the concentration of antibody used, and the Y-axis indicates the % lysis of tumor cells 6 hours after antibody addition. [Figure 9]9A-9D. Bispecific antibody-mediated IL-2 release. The level of IL-2 cytokine release was measured after culturing resting human T cells with various tumor cell lines and increasing amounts of the αCD3_F1F:aBCMA bispecific antibody. 9A shows IL-2 release stimulated by RPMI-8226 cells, 9B shows IL-2 release stimulated by NCI-H929 cells, 9C shows IL-2 release stimulated by U266 cells, and 9D shows IL-2 release stimulated by negative control K562 cells. [Figure 10] 10A-10D. Bispecific antibody-mediated IFNγ release. The level of IFNγ cytokine release was measured after culturing resting human T cells with various tumor cell lines and increasing amounts of the αCD3_F1F:aBCMA bispecific antibody. 10A shows IFNγ release stimulated by RPMI-8226 cells, 10B shows IFNγ release stimulated by NCI-H929 cells, 10C shows IFNγ release stimulated by U-266 cells, and 10D shows IFNγ release stimulated by negative control K562 cells. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description To facilitate the understanding of this invention, several terms are defined below.

[0027] Before the present active agents and methods are described, it is to be understood that this invention is not limited to the particular methodology, products, devices, and agents described, as such methods, devices, and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0028] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "drug candidates" refers to one or a mixture of such candidates, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the devices, formulations, and methodologies described herein that may be used in connection with the inventions described herein.

[0030] Where a range of values ​​is provided, each intervening value is understood to be one-tenth of the unit of the lower limit between the upper and lower limit of that range and any other stated or intervening value in that stated range, unless the context clearly dictates otherwise, that is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either of those included limits are also included in the invention.

[0031] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0032] Generally, conventional methods of protein synthesis, recombinant cell culture and protein isolation, and recombinant DNA technology, which are within the skill of those in the art, are employed in the present invention. Such techniques are explained fully in the literature, see, for example, Maniatis, Fritsch & Sambr. ook, Molecular Cloning:A Laboratory Manu al(1982);Sambrook, Russell and Sambrook,Molecular Cloning: A Laboratory Manual(2001);Harlow, Lane and Harlow, Using Antibod ies:A Laboratory Manual:Portable Protocol No.I, Cold Spring Harbor Laboratory(1998);and Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory tory;(1988).

[0033] definition "Comprising" means that the listed 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.

[0034] "Consisting essentially of" refers to a limitation of the scope of the described composition or method to particular materials or steps that do not materially affect the basic and novel characteristic(s) of the invention.

[0035] "Consisting of" refers to the exclusion from a composition, method, or kit of any element, step, or ingredient not specified in the claim.

[0036] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or may be therapeutic, in that a disease is partially or completely cured and / or adverse effects resulting from the disease are cured. As used herein, "treatment" encompasses any treatment of a disease in a mammal, hereinafter including (a) treatment to prevent the onset of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it. These include (a) preventing the onset of a disease, (b) inhibiting the disease, i.e., arresting its onset, or (c) alleviating or regressing the disease. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Of particular interest is the treatment of ongoing disease, where treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably administered prior to complete loss of function in the affected tissue. The subject therapies may also be administered during, and in some cases after, the symptomatic stage of the disease.

[0037] By "therapeutically effective amount" is intended the amount of active agent necessary to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that induces, ameliorates, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or improves resistance to a disorder.

[0038] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, and the like. Subjects may be humans, but also include other mammals, particularly mammals useful as experimental models for human disease, e.g., mice, rats, and the like.

[0039] The terms "cancer," "neoplasm," and "tumor" are used interchangeably herein to refer to cells that exhibit autonomous, unregulated growth, i.e., exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells that may be detected, analyzed, or treated herein include precancerous (e.g., benign), malignant, premetastatic, metastatic, and nonmetastatic cells. Cancers of virtually any tissue are known. The phrase "cancer burden" refers to the number of cancer cells or cancer volume in a subject. Thus, reducing cancer burden refers to reducing the number of cancer cells or cancer volume in a subject. As used herein, the term "cancer cell" refers to any cell that is or is derived from a cancer cell, e.g., a clone of a cancer cell. Many types of cancer are known to those skilled in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, and circulating cancers such as leukemias, including B-cell leukemia, T-cell leukemia, and others, among others. 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, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

[0040] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors, such as natural killer cells, neutrophils, and macrophages, recognize bound antibody on target cells and cause lysis of the target cells. 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.

[0041] An "effector cell" is a leukocyte that expresses one or more constant region receptors and performs an effector function.

[0042] A "cytokine" is a protein released by one cell and acts on another cell as an intercellular mediator. Cytokines of interest include, but are not limited to, cytokines released from activated T cells, such as IL-2, IFNγ, etc.

[0043] "Non-immunogenic" refers to a substance that does not initiate, induce, or enhance an immune response, including an adaptive immune response and / or an innate immune response.

[0044] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). 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 a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.

[0045] "Pharmaceutically acceptable excipient" generally means an excipient that is safe, non-toxic, and useful in preparing the desired pharmaceutical composition, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0046] "Pharmaceutically acceptable salts and esters" refers to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the compound can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. 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 arenesulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include those formed with carboxy, sulfonyloxy, and phosphonoxy groups present in the compound, e.g., C 1-6 This includes esters formed from alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a mono-acid mono-salt or ester or a di-salt or ester; similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The compounds named in this 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 both the parent (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may exist in two or more stereoisomeric forms, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (racemic or not) of such stereoisomers.

[0047] The terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably to indicate that the substance can be administered to or on a human without producing undesired physiological effects to an extent that would interfere with administration of the composition.

[0048] The "homology" between two sequences is determined by sequence identity. When the two sequences being compared are of different lengths, sequence identity preferably refers to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. Sequence identity can be conventionally determined using a computer program such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the method of Smith and Waterman, Advances in Genetics, to find the segment with the highest sequence identity between two sequences. n Applied Mathematics 2 (1981), 482-489 is utilized. When using Bestfit or other sequence alignment programs to determine whether a particular sequence has, for example, 95% identity with the reference sequence of the present invention, the parameters are preferably adjusted so that the percentage of identity is calculated over the entire length of the reference sequence and so that homology gaps of up to 5% of the total number of nucleotides in the reference sequence are allowed. When using Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. Deviations that appear in the comparison of a given sequence with the above sequences of the present invention can be caused by, for example, additions, deletions, substitutions, insertions, or recombinations. Such sequence comparisons are preferably performed using the program "fasta20u66" (version 2.0u66, September 1998, William R. Pearson and the University of ersity of Virginia;WRPearson(1990), Met hods in Enzymology 183, 63-98, see attached examples, and htt See also http: / / workbench.sdsc.edu / .) For this purpose, the "default" parameter settings may be used.

[0049] "Variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will have at least about 80% sequence identity, more preferably at least about 90% sequence homology. Amino acid sequence variants may have substitutions, deletions, and / or insertions at specific positions within the reference amino acid sequence.

[0050] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

[0051] As used herein, the term "host cell" (or "recombinant host cell") is intended to refer to a cell that has been genetically modified, or can be genetically modified 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 particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in some generations due to mutation 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.

[0052] "Binding affinity" generally refers to the strength of the sum total 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 the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. A low affinity antibody binds weakly to an antigen (or receptor). High affinity antibodies bind antigens (or receptors) more tightly and remain bound longer, whereas high affinity antibodies tend to dissociate more easily.

[0053] Unless otherwise specified, the term "conjugate" as described and claimed herein is defined as a heterologous molecule formed by the covalent attachment of one or more antibody fragment(s) to one or more polymer molecule(s), wherein the heterologous molecule is water-soluble, i.e., soluble in physiological fluids such as blood, and the heterologous molecule does not include any structured aggregates. A conjugate of interest is PEG. In the context of the above definition, the term "structured aggregate" refers to (1) any aggregate of molecules in aqueous solution having a spheroid or spheroidal shell structure, such that the heterologous molecule is not in a micelle or other emulsion structure, and is not immobilized in a lipid bilayer, vesicle, or liposome, and (2) an aggregate of molecules in a solid or insolubilized form that does not release the heterologous molecule into solution upon contact with an aqueous phase, such as a chromatography bead matrix. Thus, the term "conjugate" as defined herein encompasses the heterologous molecule in a precipitate, sediment, biodegradable matrix, or other solid that can release the heterologous molecule into aqueous solution upon hydration of the solid.

[0054] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody. The label may be detectable in itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0055] "Solid phase" refers to a non-aqueous matrix to which an antibody of the invention can adhere. Examples of solid phases encompassed herein include those formed in part or in whole of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the context, the solid phase may constitute the well of an assay plate; in others, it is a purification column (e.g., an affinity chromatography column). The term also includes discontinuous solid phases of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

[0056] Antibodies, also called immunoglobulins, typically comprise at least one heavy chain and one light chain, and the amino-terminal domains of the heavy and light chains are variable in sequence and are therefore commonly referred to as variable region domains, or variable heavy (VH) domains or variable light (VH) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be achieved with variable sequence in the heavy chain alone, and a variety of non-natural configurations of antibodies are known and used in the art.

[0057] A "functional" or "biologically active" antibody or antigen-binding molecule (herein including heavy-chain-only antibodies and bispecific three-chain antibody-like molecules (TCAs)) is one that is capable of exerting one or more of its native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, e.g., a TCA, may have the ability to specifically bind to an antigen, and the binding may trigger or modify a cellular or molecular event, such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a TCA, may also block ligand activation of a receptor or act as an agonist or antagonist. The ability of an antibody or other binding molecule, e.g., a TCA, to exert one or more of its native activities depends on several factors, including proper folding and assembly of the polypeptide chain.

[0058] The term "antibody" as used herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomeric, dimeric, multimeric, and multispecific antibodies (e.g., bispecific antibodies). Antibodies include heavy chain-only antibodies, triple-chain antibodies, single-chain Fvs, nanobodies, and the like, as well as antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0059] The term antibody can refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule, including engineered subclasses with modified Fc portions that provide reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species. In one embodiment, the immunoglobulins are predominantly human.

[0060] The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy and light chain variable domains each contain four FRs that primarily adopt a beta-sheet structure, connected by three hypervariable regions that form loops connecting, and in some cases, part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, (See Md.) The constant domains are not directly involved in binding the antibody to an antigen. However, they exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

[0061] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. A hypervariable region may comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or those residues from the "hypervariable loops". "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0062] A variable region of interest comprises at least one CDR sequence, usually at least two CDR sequences, and more usually three CDR sequences, from a Family 2 variable region as provided herein. While exemplary CDR designations are provided herein, those of skill in the art will understand that many definitions of CDRs are commonly used, including the Kabat definition ("Zhao et al. A germline knowledge-based computational approach"). nal approach for determining antibody complementarity determining.” Mol Immunol. 2010;47:694-700), which is The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformational ons of immunoglobulin hypervariable regi ons." Nature. 1989;342:877-883). Definitions of CDRs for other purposes include, but are not limited to, 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: implica tions for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139. and others, each of which is specifically incorporated herein by reference.

[0063] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include 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 advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0064] Antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567, and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Mera antibodies include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0065] As used herein, an "intact antibody chain" comprises a full-length variable region and a full-length constant region (Fc). An intact, "traditional" antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, hinge, CH2, and CH3 for secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (a native-sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and cytotoxicity. These include downregulation of surface receptors, and constant region mutations that alter effector profiles, Fc receptor binding, etc.

[0066] Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be provided as different "classes." There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; 2004 / 02 29310). 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 sequences of their constant domains.

[0067] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with a receptor, e.g., FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using various assays, e.g., as disclosed in the definitions herein. A "dead" Fc is one that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high-affinity Fc receptors.

[0068] A "native-sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. 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, as well as naturally occurring Fc regions. These variants are included.

[0069] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0070] The variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at positions 234, 235, and 237 of the EU index (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at positions 330 and 331 of the EU index reduce complement binding (Tao et al., J. Exp. Med. 1999, 10:143-144). 78:661 (1993), and Canfield and Morrison, J. Exp. Med. 173:1483 (1991). The IgG2 residues at positions 233-236 and the IgG4 residues at positions 327, 330, and 331 are related to human IgG1. The substitution significantly reduces ADCC and CDC (see, e.g., Armour KL. et al. al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001. J Biol Chem. 27 6(9):6591-604). Other Fc variants are possible, including, but not limited to, those in which regions capable of forming disulfide bonds are deleted, or in which specific amino acid residues are removed or methionine residues are added at the N-terminus of the native Fc form. Thus, in one embodiment of the invention, one or more Fc portions of an ScFc molecule may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, the molecule may comprise an Fc variant.

[0071] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting, deleting, or adding amino acid residues for complement binding or Fc receptor binding. For example, deletions can be made in complement binding sites, such as, but not limited to, the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0072] Fc may be in a form having native glycosylation, increased glycosylation compared to the native form, or decreased glycosylation compared to the native form, or may be in a non-glycosylated or deglycosylated form. Increased, decreased, removed, or other modifications of glycosylation can be achieved by methods common in the art, such as chemical methods, enzymatic methods, or by expressing it in a genetically engineered production cell line. Such cell lines may include microorganisms (e.g., Pichia Pastoris) and mammalian cell lines (e.g., CHO cells) that naturally express glycosylation enzymes. Furthermore, microorganisms or cells may be engineered to express glycosylation enzymes or may be unable to express glycosylation enzymes (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-Ni (See, for example, Shiya, et al., BMC Biotechnology 7:84 (2007); and WO07 / 055916.) As an example of a cell engineered to have altered sialylation activity, the alpha-2,6-sialyltransferase 1 gene is , Chinese hamster ovary cells, and sf9 cells have been engineered. Thus, antibodies expressed by these engineered cells are sialylated by exogenous gene products. Additional methods for obtaining Fc molecules with modified amounts of sugar residues compared to native molecules include separating the molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography (see, e.g., WO 07 / 117505). The presence of certain glycosylated moieties has been shown to alter immunoglobulin function. For example, removal of carbohydrate chains from Fc molecules results in a drastic 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 preventing unwanted immune responses in vivo. Further important modifications include sialylation and fucosylation; the presence of sialic acid in IgG correlates with anti-inflammatory activity (see, e.g., Kaneko, et al., Science 313:760 (2006)), whereas removal of fucose from IgG leads to enhanced ADCC activity (see, e.g., Shoj-Hosaka, et al., J. Biochem., 140:777 (2006).

[0073] In alternative embodiments, antibodies of the present invention may have Fc sequences with enhanced effector function, e.g., by increasing binding affinity to FcγRIIIA and enhancing 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 removal of fucose by glycoengineering can increase binding to FcγRIIIA, which translates to >50-fold higher ADCC activity compared to wild-type IgG1 controls. Protein engineering has generated multiple mutants that increase the affinity of Fc binding to FcγRIIIA through amino acid mutations in the Fc portion of IgG1. In particular, the triple alanine mutant S298A / E333A / K334A exhibits a 2-fold increased binding to FcγRIIIA and ADCC function. The S239D / I332E (2x) and S239D / I332E / A330L (3x) mutants have significantly increased binding affinity to FcγRIIIA and enhanced ADCC capacity in vitro and in vivo. Other Fc mutants identified by yeast display also showed improved binding to FcγRIIIA and enhanced tumor cell killing in mouse xenograft models. See, e.g., Liu et al. (2014) JBC 289(6):3571-90, specifically incorporated herein by reference.

[0074] The term "antibody comprising an Fc region" refers to an antibody that comprises 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 the nucleic acid encoding the antibody. Thus, antibodies with an Fc region according to the present invention can include antibodies with or without K447.

[0075] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. The CD3-binding antibodies of the present invention comprise a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association, but may also contain additional antibodies, e.g., VH in the absence of VL sequences, for use in multispecific configurations. Even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity may be lower than that of a two-domain binding site.

[0076] 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 few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear 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.

[0077] "Humanized" forms of non-human (e.g., 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; Chothi a et al(1989)Nature 342:877;Riechmann et al. al(1992)J. Mol. Biol. 224, 487-499;Foote and Winter,(1992)J. Mol. Biol. 224:487-499;Presta et al(1993)J. Immunol. 151, 2623- 2632; Werther et al (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001) Thromb. Haemost. 85:379-389. For further details, see U.S. Patent Nos. 5,225,539, 6,548,640, 6,982,322, and 6,982,323. 1, 5,585,089, 5,693,761, 6,407,213, Jones et al (1986) Nature, 321:522-525; and Ri See Echmann et al (1988) Nature 332:323-329.

[0078] As used herein, the term "single-chain antibody" refers to a single polypeptide chain comprising one or more antigen-binding domains that bind to an epitope of an antigen, such domains being derived from or sharing sequence identity with the variable regions of antibody heavy or light chains. H or V L Gene segments, D and J H gene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L The V-, D-, and J-gene segments can be encoded by gene segments from a variety of animals, including humans, birds, fish, sharks, mammals, rodents, non-human primates, camels, llamas, rabbits, etc.

[0079] The CD3 binding antibodies of the present invention find particular utility in multispecific configurations, including but not limited to, bispecific antibodies, trispecific antibodies, etc. A wide variety of methods and protein configurations are known and have been used in bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0080] First generation BsMAbs consisted of two heavy chains and two light chains, one each from two different antibodies. The two Fab regions were directed against two antigens. The Fc region, composed of the two heavy chains, forms a third binding site with Fc receptors on immune cells (see, e.g., Lindhofer et al., The Journal of Immunology, Vol. 155, p219-225, 1995). ). The antibodies may be from the same species or different species. For example, cell lines expressing rat and mouse antibodies will secrete functional bispecific Abs due to preferentially species-restricted pairing of heavy and light chains. In other embodiments, the Fc regions are engineered to only fit together in a specific way.

[0081] Another type of bispecific antibody involves chemically linked Fab fragments consisting of only the Fab region. Two chemically linked Fab or Fab2 fragments form an artificial antibody that binds to two different antigens, making it a type of bispecific antibody. The antigen-binding fragments (Fab or Fab2) of two different monoclonal antibodies are produced and linked by chemical means such as thioethers (Glennie, MJ et al., Journal of f immunology 139, p 2367-75, 1987;Peter Borchmann et al., Blood, Vol.100, No.9, p3101 -3107, 2002).

[0082] Various other methods for producing multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two antibodies. Single-chain variable fragments (scFvs) are fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains, linked by a short linker peptide of 10 to approximately 25 amino acids. The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Bispecific single-chain variable fragments (di-scFvs, bi-scFvs) can be engineered by linking two scFvs with different specificities. A single peptide chain containing two VH and two VL regions is produced, resulting in a bivalent scFv.

[0083] Bispecific tandem scFvs are also known as bispecific T cell engag- ers (BiTEs). Bispecific scFvs have two variable regions that are too short to fold together (approximately ScFvs can be created using a linker peptide (5 amino acids long) that forces dimerization of the scFv. This type is known as a diabody (Adams et al., British Journal of Cancer 77, pp. 1405-1405, 1998). Dual-Affinity Re-Targeting (DART) platform technology (Macrogenics, Rockville, Md.) This fusion protein technology uses two single-chain variable fragments (scFvs) of different antibodies on a single peptide chain of approximately 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 at the C-terminus and the second binding domain is at the N-terminus of the effector domain.

[0084] Tetravalent and bispecific antibody-like proteins also include DVD-Ig, which is engineered from two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, p1290-1297, 2007). To achieve this, the V domains of two mAbs are fused in tandem with the N-terminal variable domain of the light (VL) chain of a first antibody via a short linker (TVAAP), followed by the VL and Ck of another antibody, to form the light chain of a DVD-Ig protein. Similarly, the variable regions of the heavy (VH) chains of two mAbs are fused in tandem with the N-terminal first antibody via a short linker (ASTKGP), followed by the constant domains of the heavy chain of another antibody to form the heavy chain of a DVD-Ig protein (VH1 / VL1). In the DVD-Ig design, all light and heavy chain constant domains are conserved because they are important for the formation of disulfide-bonded, intact IgG-like molecules. Cotransfection of mammalian cells with expression vectors encoding the light and heavy chains of DVD-Ig results in the secretion of a single species of IgG-like molecule with a molecular weight of approximately 200 kDa. This molecule has four binding sites, two from each mAb.

[0085] 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 / 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 comprising an Fc portion containing CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen, such binding domain being derived from or sharing sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions may be VH and / or V L Gene segments, D and J H gene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment.

[0086] As used herein, "TCA proteins utilize heavy chain-only antibodies" or "heavy chain antibodies" or "heavy chain polypeptides" refers to single chain antibodies comprising heavy chain constant region CH2 and / or CH3 and / or CH4 domains, but not CH1 domains. In one embodiment, a heavy chain antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH2 and CH3 domains. In another embodiment, a heavy chain antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In a further embodiment, a 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 have been truncated are also included herein. In a further embodiment, a heavy chain comprises an antigen-binding domain. Heavy chain-only antibodies consist of a heavy chain, a nucleotide sequence, and at least one CH (CH1, CH2, CH3, or CH4) domain, but do not include the hinge region. Heavy chain-only antibodies may be in the form of a dimer in which the two heavy chains are disulfide-linked or otherwise covalently or non-covalently linked to each other. Heavy chain antibodies may belong to the IgG subclass, although antibodies belonging to other subclasses, such as IgM, IgA, IgD, and IgE subclasses, are also included herein. In certain embodiments, heavy chain antibodies are of the IgG1, IgG2, IgG3, or IgG4 subclass. The subtype is IgG1, in particular.

[0087] Heavy chain antibodies constitute approximately one-quarter of the IgG antibodies produced by camelids, e.g., camels and llamas (Hamers-Casterman C., et al. Na ture. 363, 446-448(1993)). These antibodies are formed by two heavy chains but lack light chains. As a result, the variable antigen-binding portion is called a VHH domain, which represents the smallest naturally occurring intact antigen-binding site, 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 can be generated against various antigens by immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46(1999)), and VHH portions can be easily cloned and expressed in yeast. (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression level, solubility and stability can be compared with classical significantly higher than the levels of F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526(1997)). Sharks , and antibodies called VNARs have also been shown to have a single VH-like domain (Nuttall et al. Eur. J. Biochem. 270, 3543-3 554(2003);Nuttall et al. Function and Bi oinformatics 55, 187-197(2004);Dooley et al. al., Molecular Immunology 40, 25-33(2003 )).

[0088] Antibodies or antigen-binding molecules, including heavy-chain-only antibodies and bispecific three-chain antibody-like molecules (TCAs), herein "bind" to an antigen of interest with sufficient affinity that such antibodies or binding molecules are useful as diagnostic and / or therapeutic agents in targeting the antigen and do not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or other binding molecule to non-target antigens is 10% or less, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0089] protein The present invention relates to a method for detecting and determining whether a CD3-mediated signal transduction is mediated by a CD3-mediated signal transduction pathway (e.g., CD3 + A family of closely related antibodies is provided that activates T cells. Each antibody within the family comprises a set of CDR sequences defined herein and is exemplified by the provided VH sequences of SEQ ID NOS: 1-18. A family of antibodies offers many benefits that contribute to its usefulness as a clinical therapeutic agent(s). The antibodies within the family include members with a range of binding affinities, allowing for the selection of specific sequences with desired affinities. The ability to fine-tune affinity is particularly important for managing the level of CD3 activation in treated individuals, thereby reducing toxicity. For example, when small, abundant tumor antigens (less than 10,000 molecules per cell) are targeted, high-affinity CD3 binders (<30 nM) would be preferred. When highly abundant tumor antigens (greater than 50,000 molecules per cell) are targeted, CD3 binders with low affinity (>50 nM) would be preferred. Apart from affinity, another factor assessed is the ability of the antibody to induce cytokine release, e.g., cytokine production, upon binding to T cells. Where a reduction in cytokine release is desired, it may be the tendency of antibodies to induce the release of IL-2, IFNγ, etc.

[0090] Suitable antibodies can be selected from those provided herein for development and use, including but not limited to, for use as bispecific antibodies. Determination of affinity for candidate proteins can be performed using methods known in the art, such as Biacore assays. Members of an antibody family can be selected from approximately 10 -6 ~about 10 -11 The antibody may have an affinity for CD3 with a Kd of about 10 -6 ~about 10 -10 , about 10 -6 ~about 10 -9 , about 10 -6 ~about 10 -8 , about 10 -8 ~about 10 -11 , about 10 -8 ~about 10 -10 , about 10 -8 ~about 10 -9 , about 10 -9 ~about 10 -11 , about 10 -9 ~about 10 -10 , or any value within these ranges. Affinity selection may be confirmed using biological evaluation, for example, for T cell activation in in vitro or preclinical models, and evaluation of potential toxicity. Cytokine release determination may be assessed using any convenient method, including, but not limited to, the assays described in the Examples.

[0091] Engagement of the T cell receptor (TCR) by binding an MH-peptide complex or an anti-TCR / CD3 antibody initiates T cell activation. Examples of anti-TCR / CD3 antibodies that activate T cells are OKT3 and UCHT1. These anti-CD3 antibodies cross-compete 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 their binding affinity to CD3 and the epitope on CD3, anti-CD3 antibodies activated T cells with different functional outcomes. In vitro incubation of human T cells with low-affinity anti-CD3 antibodies resulted in incomplete activation of T cells and reduced production of IL-2 and IL-10. In contrast, high-affinity CD3 binders activated T cells to produce significantly more IL-2 and other cytokines. Low-affinity anti-CD3 antibodies are considered partial agonists, selectively inducing some effector functions, such as potent tumor killing and CD69 upregulation, while not inducing others, such as IL-2 and IL-10 production. High-affinity binders of the invention are full agonists, activating many immune effector functions of T cells. The strength of the interaction between CD3 and the recognized epitope resulted in qualitatively different activation of T cells. The maximal cytokine production of T cells activated by low-affinity anti-CD3 antibodies was lower than that of T cells activated by high-affinity anti-CD3 antibodies. In some embodiments, antibodies of the invention, when combined with T cells in an activation assay, result in lower release of one or both of IL-2 and IL-10 when compared to a reference anti-CD3 antibody in the same assay; the reference antibody can be ID304703 (SEQ ID NO: 22), or an antibody of comparable affinity. The maximum release of IL-2 and / or IL-10 may be less than about 75% of the release by the reference antibody, may be less than about 50% of the release by the reference antibody, may be less than about 25% of the release by the reference antibody, or may be less than about 10% of the release by the reference antibody.

[0092] In some embodiments of the present invention, bispecific or multispecific antibodies are provided, which may have any of the configurations discussed herein, including, but not limited to, three-chain bispecifics. Bispecific antibodies comprise at least the heavy chain variable region of an antibody specific for a protein other than CD3, and may comprise heavy and light chain variable regions. In some such embodiments, the second antibody specificity binds to a target antigen such as a tumor-associated antigen, e.g., an integrin, a pathogen antigen, a checkpoint protein, or the like. Various forms of bispecific antibodies are within the scope of the present invention, including, but not limited to, single-chain polypeptides, two-chain polypeptides, three-chain polypeptides, four-chain polypeptides, and multiples thereof.

[0093] The family of CD3-specific antibodies comprises a VH domain comprising CDR1, CDR2, and CDR3 sequences in a human VH framework. The CDR sequences may be located, by way of example, in the region of approximately amino acid residues 26-33, 51-58, and 97-112 for CDR1, CDR2, and CDR3, respectively, of the exemplary variable region sequences provided in SEQ ID NOS: 1-18. It will be understood by those skilled in the art 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.

[0094] The CDR sequences of Family 2 antibodies may have the following sequence formula: X represents a variable amino acid and may be a specific amino acid as indicated below. [ka] wherein X5 can be any amino acid, and in some embodiments, X5 is D, A, or H, and in some embodiments, X5 is D. X6 can be any amino acid, and in some embodiments, X6 is D or N, and in some embodiments, D6 is D. In some embodiments, the CDR1 sequence of a Family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 26-33.

[0095] [ka] In some embodiments, the CDR2 sequence of a Family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 51-58.

[0096] [ka] where: X 9’ may be any amino acid, and in some embodiments, X 9’ is D or S, and in some embodiments, X 9’ is D, X 11’ may be any amino acid, and in some embodiments, X 11’ is R or S, X12' can be any amino acid, and in some embodiments, X 12’ is L or R.

[0097] In some embodiments, the CDR3 sequence of a Family 2 anti-CD3 antibody has the formula: [ka] X 11’ and X 12’ is defined above. In some embodiments, the CDR3 sequence of a Family 2 anti-CD3 antibody comprises the sequence set forth in any of SEQ ID NOs: 1-18, residues 97-112.

[0098] 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 contained in SEQ ID NO: 19. In some embodiments, the CDR1 sequence comprises amino acid residues 27-32, 50-52, and 89-97 for CDR1, CDR2, and CDR3, respectively.

[0099] In some embodiments, the CDR sequences of Family 2 antibodies have a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to the CDR sequence or set of CDR sequences set forth in any one of SEQ ID NOs: 1 to 18. In some embodiments, the CDR sequences of the invention comprise one, two, three, or more amino acid substitutions relative to the CDR sequence or set of CDR sequences of any one of SEQ ID NOs: 1 to 18. In some embodiments, the amino acid substitution(s) are at one or more of positions 5 or 10 of CDR1, 2, 6, or 7 of CDR2, and 1, 8, 9, or 10 of CDR3 relative to the above formula.

[0100] When the protein of the present invention is a bispecific antibody, one binding moiety, i.e., the VH / VL combination or only the VH, may be specific for human CD3, while the other arm may be specific for target cells including cancer cells such as ovarian, breast, gastrointestinal, brain, head and neck, prostate, colon, and lung cancer cells, as well as hematological tumors such as leukemia, lymphoma, sarcoma, carcinoma, neural cell tumor, squamous cell carcinoma, germ cell tumor, metastasis, undifferentiated tumor, seminoma, melanoma, myeloma, neuroblastoma, mixed cell tumor, and B-cell tumors, including neoplasia due to infectious agents and other malignancies, pathogen-infected cells, or autoreactive cells causing inflammation and / or autoimmunity. The non-CD3 portion may also be specific for an immunomodulatory protein, as described herein.

[0101] Tumor-associated antigens (TAAs) are relatively restricted to tumor cells, while tumor-specific antigens (TSAs) are unique to tumor cells. TSAs and TAAs are typically parts of intracellular molecules expressed on the cell surface as part of the major histocompatibility complex.

[0102] Tissue-specific differentiation antigens are molecules present on tumor cells and their normal cell counterparts. Tumor-associated antigens known to be recognized by therapeutic mAbs fall into several different categories. Hematopoietic differentiation antigens are usually glycoproteins associated with the cluster of differentiation (CD) and include CD20, CD30, CD33, and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates found on the surface of both normal and tumor cells. Antigens involved in proliferation and differentiation signaling are often growth factors and growth factor receptors. Growth factors that are targets of antibodies in cancer patients include CEA, epidermal growth factor receptor (EGFR, also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1, also known as TNFRSF10A), and TRAILR2 (also known as TNFRSF10B). Antigens involved in angiogenesis are usually 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. The tumor stroma and extracellular matrix are essential support structures for tumors. Stromal and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) and tenascin.

[0103] Examples of therapeutic antibodies useful in bispecific configurations include, but are not limited to, rituximab, ibritumomab, tiuxetan, tositumomab, brentuximab, vedotin, gemtuzumab, ozogamicin, alemtuzumab, IGN101, adecatumumab, labetuzumab, huA33, pemtumomab, oregovomab, CC49 (minletumomab), cG250, J591, MOv18, MORAb-00 3 (faretuzumab), 3F8, ch14.18, KW-2871, hu3S193, IgN311, bevacizumab, IM-2C6, CDP791, etaracizumab, volociximab, cetuximab, panitumumab, nimotuzumab, 806, trastuzumab, pertuzumab, MM-121, AMG102, METMAB, SCH900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, IIIA4, mapatumumab (HGS-ETR1), HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, and 81C6.

[0104] The most actively investigated immune checkpoint receptors in the context of clinical cancer immunotherapy, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4, also known as CD152) and programmed cell death protein 1 (PD1, also known as CD279), are both inhibitory receptors. The clinical activity of antibodies that block either of these receptors means that antitumor immunity can be enhanced at multiple levels, and combinatorial strategies can be designed guided by mechanistic considerations and preclinical models.

[0105] The two ligands of PD1 are PD1 ligand 1 (PDL1, also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed on cancer cells and inhibits T cell activation / function by binding to its receptor PD1 on T cells.

[0106] 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 family of killer inhibitory receptors have each been associated with inhibition of lymphocyte activity and, in some cases, induction of lymphocyte anergy. Antibody targeting of these receptors can be used in the methods of the invention.

[0107] Agents that agonize immune costimulatory molecules are also useful in the methods of the present invention. Such agents include agonists or CD40 and OX40. CD40 is a costimulatory protein found on antigen-presenting cells (APCs) and is required for their activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The major activation signaling molecules for CD40 are IFNγ and CD40 ligand (CD40L). Stimulation by CD40 activates macrophages.

[0108] The targeted anti-CCR4 (CD194) antibody has potential anti-inflammatory and anti-tumor activities. This invention includes a humanized monoclonal antibody against CC chemokine receptor 4 (CCR4). CCR2 is expressed on inflammatory macrophages, which can be found in various inflammatory conditions, such as rheumatoid arthritis, and has also been identified as being expressed on tumor-promoting macrophages. CCR2 is also expressed on regulatory T cells, and the CCR2 ligand, CCL2, mediates the recruitment of regulatory T cells to tumors. Regulatory T cells suppress the response of anti-tumor T cells, and therefore their inhibition or depletion is desirable.

[0109] Production of the Protein of the Invention Antibodies can be prepared by chemical synthesis but are typically produced by recombinant DNA technology methods, such as co-expression of all chains comprising a protein in a single recombinant host cell, or co-expression of a heavy chain polypeptide and an antibody (e.g., a human antibody). Additionally, antibody heavy and light chains can be expressed using a single polycistronic expression vector. Purification of individual polypeptides is achieved using standard protein purification techniques, such as affinity (protein A) chromatography, size exclusion chromatography, and / or hydrophobic interaction chromatography. Bispecifics differ sufficiently in size and hydrophobicity that purification can be performed using standard procedures.

[0110] The amount of antibody and heavy chain polypeptides produced in a single host cell can be minimized through engineering of the antibody and heavy chain constant regions so that homodimerization is favored over heterodimerization, for example by introducing self-complementary interactions (see, e.g., WO 98 / 50431 for the possibility of "protruding into the cavity" strategies, etc. (see WO 96 / 27011)). Thus, another aspect of the invention provides a method of producing a bispecific agent in a recombinant host cell, the method comprising expressing in the recombinant host cell nucleic acid sequences encoding at least two heavy chain polypeptides, the heavy chain polypeptides differing in their constant regions sufficiently to reduce or prevent homodimer formation but increase bispecific formation.

[0111] Where proteins comprise three chains (e.g., FlicAb), they can be produced by co-expression of the three chains (two heavy chains and one light chain) that make up the molecule in a single recombinant host cell.

[0112] For recombinant production of the proteins 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 expression. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0113] In a preferred embodiment, the host cell according to the method of the present invention is capable of expressing high levels of human immunoglobulins, i.e., at least 1 pg / cell / day, preferably at least 10 pg / cell / day, more preferably at least 20 pg / cell / day or more, without the need for amplification of a single-stranded encoding nucleic acid molecule in said host cell.

[0114] Pharmaceutical Compositions Another aspect of the present invention is to provide pharmaceutical compositions comprising one or more proteins of the present invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, pharmaceutically acceptable carriers include, but are not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to carry therapeutic ingredients, or combinations thereof.

[0115] Therapeutic formulations of proteins used in accordance with the present invention are prepared for storage by mixing the protein having the desired purity, for example, in the form of a lyophilized formulation or aqueous solution, with any pharmaceutically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, 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, e.g., serum albumin, gelatin, and the like; The composition may comprise a protein such as a protein or immunoglobulin, a hydrophilic polymer such as polyvinylpyrrolidone, an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine, a monosaccharide, disaccharide, or other carbohydrate including glucose, mannose, or dextrin, a chelating agent such as EDTA, a sugar such as sucrose, mannitol, trehalose, or sorbitol, a salt-forming counterion such as sodium, a metal complex (e.g., Zn-protein complex), and / or a non-ionic surfactant such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0116] Anti-CD3 antibody formulations are disclosed, for example, in U.S. Patent Publication No. 20070065437, the entire disclosure of which is specifically incorporated herein by reference. Similar formulations can be used with the proteins of the invention. The main components of such formulations are a pH buffer effective in the range of 3.0 to 6.2, salt, surfactant, and an effective amount of a bispecific agent having anti-CD3 specificity.

[0117] How to use In particular, when the antigen-binding composition is a multispecific antibody appropriate for the condition being treated, e.g., where one binding moiety specifically binds to a tumor-associated antigen for treatment of associated cancer cells (e.g., a binding moiety specific for a pathogen of interest for treatment of an associated infectious disease), methods are provided for treating or ameliorating diseases, including, but not limited to, infectious diseases, autoimmune diseases, primary or metastatic cancers, etc., in a regimen comprising contacting target cells with the antigen-binding composition of the invention. Such methods comprise administering a therapeutically effective amount or an effective amount of an agent of the invention to a subject in need of treatment, including, but not limited to, a combination of the agent with a chemotherapeutic agent, radiation therapy, or surgery.

[0118] The effective amount of the compositions of the present invention for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or an animal, whether other drugs are being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals (e.g., companion animals such as dogs, cats, and horses, and laboratory mammals such as rabbits, mice, and rats) can also be treated. The treatment dose can be titrated to optimize safety and efficacy.

[0119] Dosage levels can be easily determined by a clinician with ordinary skill, and can be changed as needed, for example, to modify the subject's response to treatment. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host being treated and the specific method of administration. Dosage unit forms generally contain about 1 mg to about 500 mg of active ingredient.

[0120] In some embodiments, the therapeutic dose of the agent is about 0.0001 to 100% of the host's body weight. The dosage may be in the range of 1 mg / kg, more commonly 0.01 to 5 mg / kg. For example, dosages may be 1 mg / kg body weight or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The therapeutic agents of the present invention are typically administered on multiple occasions. The interval between single doses may be weekly, monthly, or yearly. Intervals may also be irregular, as indicated by measuring the patient's blood concentration of the therapeutic agent. Alternatively, the therapeutic agents of the present invention may be administered as a sustained-release formulation, in which case fewer administrations may be required. Dosage and frequency will vary depending on the half-life of the polypeptide in the patient.

[0121] In prophylactic applications, relatively low doses may be administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In other therapeutic applications, relatively high doses may be required at relatively short intervals until the progression of the disease is reduced or halted, and preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

[0122] In yet other embodiments, the methods of the invention include treating, reducing, or preventing tumor growth, tumor metastasis, or tumor invasion of cancers such as carcinoma, blood cancers such as leukemia and lymphoma, melanoma, sarcoma, glioma, etc. For prophylactic uses, a 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 of, lessen the severity of, or delay the onset of, the disease, including biochemical, histological, and / or behavioral symptoms of the disease (and its complications and intermediate pathological phenotypes manifested during the progression of the disease).

[0123] Compositions for the treatment of disease may be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular means. Typical routes of administration are intravenous or intratumoral, although other routes may be equally effective.

[0124] Typically, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid vehicles prior to injection can also be prepared. The preparations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolide, or copolymers, for enhanced adjuvant effect, as discussed above (Langer, Science 249:1527, 1990, and Hanes, Advanced Drug Delivery Reviews 28:9). 7-119, 1997). The drug of the present invention can provide sustained or pulsed release of the active ingredient. The pharmaceutical compositions may be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to permit administration of the pharmaceutical compositions. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0125] The toxicity of the proteins described herein can be measured, for example, by LD 50 (the dose lethal to 50% of the population) or LD 100 The therapeutic index can be determined by standard pharmaceutical procedures in cell cultures or experimental animals by determining the therapeutic effect (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used in formulating a non-toxic dosage range for use in humans. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0126] Pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and the like. It is recognized that the compositions of the present invention, when administered orally, should be protected from digestion. This is typically achieved by complexing molecules with the compositions to make them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in an appropriately resistant carrier, such as a liposome or protective barrier. Means for protecting drugs from digestion are well known in the art.

[0127] Compositions for administration typically contain the antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, weight, and the like, in accordance with the particular mode of administration selected and the needs of the patient (see, e.g., Remington's Pharmaceutical Sciences (15th ed., 1980), and Goodman & Gillman, The Pharmacological Basis of Pharmaceutical Sciences, 1990). is of Therapeutics (Hardman et al., eds., 1996)).

[0128] Also within the scope of the present invention are kits containing the active agents of the present invention and formulations thereof, as well as instructions for use. The kits may further include at least one additional reagent, such as a chemotherapeutic agent. 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 supplied on or with the kit, or otherwise accompanying the kit.

[0129] The composition can be administered for therapeutic treatment. The composition is administered to a patient in an amount sufficient to substantially ablate target cells, as described above. An amount sufficient to achieve this is defined as a "therapeutically effective dose" that can improve overall survival. Single or multiple administrations of the composition can be administered depending on the dosage and frequency required and tolerated by the patient. The specific dose required for treatment depends on the mammal's condition and medical history, as well as other factors such as age, weight, sex, administration route, and efficacy.

[0130] Having fully described the present invention, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the spirit or scope of the invention. [Example]

[0131] Example 1 Genetically engineered rats expressing heavy chain-only antibodies The human IgH locus is H 6-DJ H The human V region was constructed and assembled in several parts, including the modification and ligation of the rat C region gene linked downstream of the V region. H Two BACs carrying separate clusters of genes were assembled (human V H 6-DJ H -rat C) fragment was co-injected with a BAC encoding the fragment.

[0132] Transgenic rats were generated that carry an artificial heavy chain immunoglobulin locus in an unrearranged state. The included constant region genes encode IgM, IgD, IgG2b, IgE, IgA, and a 3' enhancer. RT-PCR and serum analysis (ELISA) of the transgenic rats revealed productive rearrangement of the transgenic immunoglobulin locus and expression of heavy chain-only antibodies of various isotypes in the serum. The transgenic rats were generated as described in U.S. Patent Publication No. 2009 / 0098134 A1. These transgenic rats were mated with rats carrying previously described mutated endogenous heavy and light chain loci. Analysis of these animals demonstrated inactivation of rat immunoglobulin heavy and light chain expression and high levels of heavy chain antibody expression with variable regions encoded by human V, D, and J genes. Immunization of the transgenic rats resulted in the production of high-titer serum responses of antigen-specific heavy chain antibodies. These transgenic rats expressing heavy chain antibodies with human V, D, and J regions were designated UniRats.

[0133] Example 2 Genetically engineered rats expressing fixed light chain antibodies Transgenic human antibody repertoires are composed of diverse (V) H -DJ H ) n A rearranged human Vk-Jk1-Ck light chain was generated from the rearranged heavy chain. To this end, a rearranged human light chain, Vk-Jk1-Ck, was integrated into the rat germline by DNA microinjection, and the resulting transgenic animals were bred with a previously described rat strain that naturally expresses the human heavy chain repertoire (Osborn et al., 2013). This new rat strain was named OmniFlic.

[0134] Immunization of OmniFlic rats with many different antigens resulted in the production of high levels of antigen-specific IgG, similar to other transgenic rats harboring the same IgH locus. Repertoire analysis by RT-PCR revealed highly variable V expression levels at both the transcript and protein levels. H Gene rearrangements were identified, and only one light chain product was identified that was expressed at high levels.

[0135] Antigen-specific binders derived from OmniFlic were obtained by next-generation sequencing and selection from cDNA libraries (yeast, E. coli, and phage), and diverse heavy chain transcripts were identified upon sequencing. For expression in mammalian cells, the hypermutated heavy chain constructs were transfected in combination with the original transgenic Igk sequence. This rearranged Vk-Jk1-Ck showed no mutational changes, and the same light chain was consistently expressed alongside the various heavy chain products to generate monoclonal human IgG.

[0136] Example 3 Generation of antigen-specific antibodies in transgenic rats For the production of antigen-specific heavy chain antibodies in rats, genetically engineered expression rats were immunized in two ways.

[0137] Immunization with recombinant PD-L1 and BCMA extracellular domains. The recombinant PD-L1 and BCMA extracellular domains were purchased from R&D Systems, diluted in sterile saline, and combined with adjuvants. The immunogens were combined with either complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), or Titermax and Ribi adjuvants. The first immunization (priming) with the immunogen in CFA or Titermax was administered to the left and right legs. After the first immunization with the immunogen in CFA, two or more immunizations (boosts) with IFA, or four or more immunizations with Ribi and one more immunization with Titermax were administered to each leg. This series of immunizations resulted in the development of B cells producing high-affinity antibodies. The immunogen concentration was 10 micrograms per leg. Serum was collected from rats at the time of the final blood draw to determine serum titers.

[0138] For the generation of anti-human CD3δε antibodies, genetically engineered rats were immunized using a DNA-based immunization protocol.

[0139] OmniFlic rats were cultured using GENOVAC antibody technology from Aldevron, I Human and cynomolgus monkey CD3-epsilon / delta constructs were immunized at the University of Illinois (Fargo, ND). Draining lymph nodes were harvested after a final boost and RNA isolation. After cDNA synthesis, the IgH heavy chain antibody repertoire was characterized by next-generation sequencing and proprietary in-house software. Candidate antigen-specific VH sequences that showed evidence of antigen-specific positive selection were selected. Hundreds of VH sequences encoding FlicAbS were selected for recombination and cloned into expression vectors. Fully human FlicAb IgG1 antibodies were subsequently expressed in HEK cells for analysis by flow cytometry and ELISA. Human FlicAb was tested for binding to primary human T cells and Jurkat cells by flow cytometry. Human FlicAb was also tested in ELISA using recombinant CD3δε protein. All FlicAb sequences with positive binding to human T cells are listed in Figures 1 and 2. Selected sequences were further characterized in T cell activation assays.

[0140] Example 4. Antibody characteristics The data table in Figure 3 summarizes the behavior of Family 2 anti-CD3 antibodies in monospecific and bispecific formats. Column 1 shows the sequence IDs of the anti-CD3 VH sequences. Column 2 shows the MFI values ​​for Jurkat cell binding of the parent monospecific anti-CD3. Column 3 shows the MFI values ​​for cynomolgus monkey T cell binding of the parent 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 with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 6 shows the picograms of IL-6 released by pan T cells stimulated with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 7 shows the picograms of IL-10 released by pan T cells stimulated with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 8 shows picograms of IFNγ released by pan T cells stimulated with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 9 shows picograms of TNFα released by pan T cells stimulated with bispecific antibodies that bind to BCMA protein coated on plastic at the indicated doses. Column 10 shows the EC of bispecific antibody-mediated U266 tumor cell lysis in the presence of human pan T cells. 50 Column 11 shows the percent lysis of U266 tumor cells at a dose of 333 ng / mL of bispecific antibody in the presence of bispecific antibody and human pan T cells. Column 12 shows the protein binding affinity of the anti-CD3 arm of the bispecific antibody, as measured by Octet. Column 13 shows the MFI values ​​of Jurkat cell binding of the bispecific antibody.

[0141] Example 5 Characterization of Bispecific Antibodies 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 through the redirection of 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 along with the addition of bispecific antibodies. As shown in Figure 4, the X-axis indicates the concentration of antibody used, and the Y-axis indicates the % lysis of tumor cells 6 hours after antibody addition.

[0142] A comparison of bispecific antibody-mediated tumor cell lytic activity and IL-2 cytokine release is shown in the scatter plots in Figure 5. The correlation between IL-2 production and lysis of U266 tumor cells was observed in R 2 = 0.37. A comparison of bispecific antibody-mediated tumor cell lysis activity and IFN-γ cytokine release is shown in the scatter plot in Figure 6. The correlation between IFN-γ production and U266 tumor cell lysis was found to be R 2 =0.53. A comparison of bispecific antibody-mediated U266 tumor cell lytic activity and anti-CD3 binding affinity is shown in the scatter plots in Figure 7. EC 50 The correlation between the affinity and protein binding affinity is R 2 =0.93.

[0143] Example 6 Tumor Cell Lysis The αCD3_F1F:αBCMA bispecific antibody was assayed for its ability to kill three different BCMA+ tumor cells and one BCMA-negative cell line via redirection of activated primary T cells. In this experiment, tumor cells were mixed with activated pan-T cells at an E:T ratio of 10:1 with the addition of the bispecific antibody. The results are shown in Figures 8A-6D. Panel A shows the killing of RPMI-8226 cells, panel B shows the killing of NCI-H929 cells, panel C shows the killing of U-266 cells, and panel D shows the killing of negative control K562 cells. The X-axis indicates the concentration of antibody used, and the Y-axis indicates the % lysis of tumor cells 6 hours after antibody addition.

[0144] Figure 9 shows the level of IL-2 cytokine release measured after culturing resting human T cells with various tumor cell lines and increasing amounts of αCD3_F1F:αBCMA bispecific antibody. Figure 9A shows the release of IL-2 stimulated by RPMI-8226 cells, Figure 9B shows the release of IL-2 stimulated by NCI-H929 cells, Figure 9C shows the release of IL-2 stimulated by U-266 cells, and Figure 9D shows the release of IL-2 stimulated by negative control K562 cells.

[0145] Figure 10 shows the level of IFNγ cytokine release measured after culturing resting human T cells with various tumor cell lines and increasing amounts of the αCD3_F1F:aBCMA bispecific antibody. Figure 10A shows the release of IFNγ stimulated by RPMI-8226 cells, Figure 10B shows the release of IFNγ stimulated by NCI-H929 cells, Figure 10C shows the release of IFNγ stimulated by U266 cells, and Figure 10D shows the release of IFNγ stimulated by negative control K562 cells.

[0146] The examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0147] While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications and equivalent substitutions may be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A pharmaceutical product containing a multispecific antibody for treating a disease or symptom, Multiple specific antibodies, (1) A first binding moiety having binding specificity to human CD3 deltype silon, (i) (A) A CDR1 sequence containing GFTFDDYA (SEQ ID NO: 29), a CDR2 sequence containing ISWNSGSI (SEQ ID NO: 24), and a CDR3 sequence containing AKDSRGYGDYRLGGAY (SEQ ID NO: 41); or (B) CDR1 sequence containing GFTFHNYA (SEQ ID NO: 34), CDR2 sequence containing ISWNSGSI (SEQ ID NO: 24), and CDR3 sequence containing AKDSRGYGDYSLGGAY (SEQ ID NO: 43) Heavy chain variable region including; and (C) CDR1 sequence containing QSVSSN (Sequence ID: 35), CDR2 sequence containing GAS, and CDR3 sequence containing QQYNNWPWT (Sequence ID: 45) Light chain variable region including A first joint including; and (2) A second binding site having binding specificity to tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) other than CD3, wherein the TAA or TSA is not a B-cell maturation antigen (BCMA). including, Pharmaceuticals.

2. The pharmaceutical product according to claim 1, wherein the disease is an autoimmune disease.

3. The pharmaceutical product according to claim 1, wherein the disease is cancer.

4. The pharmaceutical product according to claim 3, wherein a chemotherapeutic agent is further administered.

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