Methods of reducing large granular lymphocyte and natural killer cell levels

Abstract

To provide methods of reducing large granular lymphocyte and natural killer cell levels.SOLUTION: The present disclosure relates to methods for treating diseases or disorders associated with LGL and / or NK cells, methods for reducing or depleting LGL and / or NK cells, and methods for inducing ADCC activity using antibodies that bind to a cell surface protein on LGL and / or NK cells and comprise enhanced ADCC activity. The present invention also relates to a method for depleting or reducing the number of large granular lymphocytes and natural killer cells in a human subject upon administration of a CD94, CD57, or NKG2A binding molecule that consists of a part that specifically binds to CD94, CD57, or NKG2A receptors and an immunoglobulin Fc part. In a specific embodiment, a method of the present invention depletes or reduces the number of large granular lymphocytes and natural killer cells in the spleen, blood, bone marrow, joints, or other tissues.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 826,660, filed Mar. 29, 2019, and U.S. Provisional Patent Application No. 62 / 982,578, filed Feb. 27, 2020, the disclosures of each of which are hereby incorporated by reference in their entireties.

[0002] Submission of Sequence Listing in ASCII Text File The following submission in ASCII text file is hereby incorporated by reference in its entirety: Computer - Readable Form (CRF) of the Sequence Listing (filename: 186542000140SEQLIST.TXT, date of record: Mar. 25, 2020, size: 17 KB).

[0003] The present disclosure relates to methods for reducing levels of large granular lymphocytes and natural killer cells in humans.

Background Art

[0004] Lymphocytes are a subset of white blood cells that specifically recognize and respond to foreign antigens. There are three major classes of lymphocytes: T lymphocytes (T cells), B lymphocytes (B cells), and natural killer (NK) cells. Large granular lymphocytes (LGL) account for 8-15% of peripheral blood lymphocytes (200-400 / μL) and are characterized by abundant cytoplasm with azurophilic granules (Loughran TP Jr. Blood. 1993;82(1):1-14). Azurophilic granules contain cytolytic components such as perforin and granzymes. LGL are divided into two major categories: cytotoxic T cells and NK cells. LGL T cells usually express CD3, CD8, and CD57 and show TCR gene rearrangement, while NK cells express CD56, are negative for surface CD3, may express CD8, and do not show TCR gene rearrangement (Alekshun et al., Cancer Control 2007, Vol. 14, No. 2, p141-150). NK cell LGL (CD3-) belong to the innate immune system and have the ability of non-major histocompatibility complex-restricted cytotoxicity (Alekshun et al., Cancer Control 2007, Vol. 14, No. 2, p141-150).

[0005] There are three distinct diseases involving LGLs: T-cell large granular lymphocyte (T-LGL) leukemia, chronic lymphoproliferative disorder of NK cells (CLPD-NK, previously NK-LGL), and aggressive NK-cell leukemia, such as aggressive natural killer leukemia (ANKL) and extranodal NK / T-cell lymphoma, nasal type (ENKL). T-cell LGL leukemia is the most frequent LGL disorder in Western countries, accounting for 85% of all cases. The median age at diagnosis is 60 years, and there is no gender predilection. The etiology of the disease is governed by the clonal expansion of LGLs that are resistant to activation-induced cell death due to constitutive survival signaling (Lamy et al., Blood, 2017, Vol. 129, No. 9, 1082-1094). Approximately one-third of patients with T- and NK LGL leukemia are asymptomatic at the time of diagnosis. Initial presentations are mainly related to neutropenia and include recurrent oral aphthous ulcers and fevers secondary to bacterial infections. These infections usually involve the skin, oropharynx, and perirectal areas, but severe sepsis can occur. However, some patients may have profound, persistent neutropenia but no infections over extremely long periods. The frequency of recurrent infections varies from 15% to 39% in different series. Fatigue and B symptoms are observed in 20% to 30% of cases. Splenomegaly is reported with a frequency that varies from 20% to 50%, and lymphadenopathy is rare. Half of the patients show lymphocyte counts between 4×10 9 / L and 10×10 9 / L, and the LGL count is usually in the range of 1 to 6×10 9 / L. In 7% to 36% of cases, lower LGL counts (0.5 to 1×10 9 / L) may be observed. Severe neutropenia and moderate neutropenia are observed in 16% to 48% and 48% to 80% of cases, respectively. Anemia is frequent, and transfusion-dependent patients are observed in 10% to 30% of cases (Lamy 2017).

[0006] Most patients with LGL leukemia will eventually require treatment at some point during the progression of the disease. Disease-related death is mainly due to severe infections that occur in approximately 10% of the patient population. Overall survival at 10 years is 70% (Lamy 2017). Primary therapy relies on the use of single immunosuppressive oral agents such as methotrexate (10 mg / m2 per week), cyclophosphamide (100 mg per day) or cyclosporine (3 mg / kg per day). Based on retrospective studies, the median overall response rate (ORR) is 50%, with similar responses to each of the three drugs. The complete response (CR) rate is relatively low: 21% for methotrexate, 33% for cyclophosphamide, and 5% for cyclosporine. The duration of response is 21 months for methotrexate, and the relapse rate is high, i.e., 67%.

[0007] During chronic methotrexate treatment, liver (hepatitis) and lung dysfunction (hypersensitivity pneumonitis) may occur. Due to its mutagenic potential, it is recommended to discontinue cyclophosphamide administration after 8 - 12 months. During cyclosporine treatment, renal function and blood pressure must be carefully monitored.

[0008] In addition to NK or T LGL leukemia, NK or LGL cells play important roles in rheumatoid arthritis (RA), Felty syndrome, aggressive NK leukemia, inclusion body myositis (IBM), inflammatory bowel disease (IBD) and other diseases. Felty syndrome (FS) is characterized by the triad of destructive joint complications, splenomegaly and neutropenia in seropositive rheumatoid arthritis (RA). The complete triad is not an absolute requirement, but an absolute neutrophil count (ANC) of generally less than 1500 / mm 3 persistent neutropenia is required to establish the diagnosis. Approximately 30 - 40% of FS patients have peripheral blood proliferation of LGL. Clonal T-LGL populations are very similar in FS and T-LGLL, with expression of CD3+, CD28-, CD57+ as well as inhibitory and activating NK receptors on LGL. The symptoms and management of LGLL and SF are similar.

[0009] Unlike the case in synovial fluid (SF) obtained from normal human subjects without LGL or NK cells, SF obtained from RA patients has LGL and NK cells that express high levels of CD94 or CD57 or NKG2A. CD94 has been found to be an important regulator of synovial fluid NK cell cytokine synthesis.

[0010] IBM is the most common inflammatory muscle disease in the elderly. This disease is characterized by slowly progressive weakness and wasting of both distal and proximal muscles, most pronounced in the finger flexors and knee extensors. Inflammation is evident from infiltration of muscle fibers by immune cells. Granular lymphocyte proliferation is present in both blood and muscle, providing additional biomarkers for IBM and suggesting a mechanistic relationship with the neoplastic disease T-cell large granular lymphocytic leukemia. Most (58%) patients with IBM have an abnormal population of large granular lymphocytes in their blood, meeting the standard diagnostic criteria for T-cell LGLL. Muscle immunohistochemical analysis demonstrated infiltration of muscle by large granular lymphocytes in all 15 of the IBM patients, but in only 1 of 28 patients with dermatomyositis or polymyositis.

[0011] Current therapies for diseases involving large granular lymphocytes (LGL) do not selectively reduce or deplete the levels of LGL or NK cells. Therefore, it would be beneficial to develop more effective and safe therapies for treating diseases mediated by LGL and NK cells.

Prior Art Documents

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0013] The present invention relates to a method for depleting or reducing the number of large granular lymphocytes and natural killer cells in a human subject upon administration of a CD94 or CD57 or NKG2A binding molecule comprising a portion that specifically binds to the CD94 or CD57 or NKG2A receptor and an immunoglobulin Fc portion.

[0014] The present invention provides a treatment method for treating LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, IBM or IBD in a subject, comprising administering to the subject an effective amount of an antibody that specifically binds to human CD94, human CD57 or human NKG2A, the antibody comprising a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region.

[0015] The present invention provides a method for reducing or depleting the number of peripheral blood LGL or NK cells in a human subject, the method comprising administering to the subject an antibody comprising an immunoglobulin Fc region that is specific for any of CD94 or CD57 or NKG2A or an additional cell surface protein that is specific for LGL cells, between about 0.01 and about 25 mg / kg, that does not contain fucose or contains an Fc mutation that enhances its binding to CD16, wherein the administration of the antibody reduces the number of peripheral blood LGL or NK cells below the limit of detection and the level remains below detectable for at least about 1 week after dosing of the antibody. In some embodiments, the reduction of LGL or NK cells occurs within the first 24 hours after administration. In some embodiments, the reduction of LGL or NK cells is reversible. In some embodiments, the reduction of LGL or NK cells leads to a reduction in LGL leukemia symptoms. In some embodiments, the reduction of LGL or NK cells leads to a reduction in Felty syndrome symptoms. In some embodiments, the reduction of LGL or NK cells leads to a reduction in IBM symptoms. In some embodiments, the reduction of LGL or NK cells leads to a reduction in aggressive NK leukemia symptoms.

[0016] In one aspect, provided herein is a method of treating LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, IBM or IBD in a subject, the method comprising administering to the subject an effective amount of an antibody that specifically binds to human CD94, human CD57 or human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity as compared to the wild-type IgG1 Fc region. In some embodiments, administration of the antibody reduces the number of peripheral blood LGLs or NK cells below the limit of detection and / or the levels remain below detectable for at least about one week after dosing of the antibody. In some embodiments, the reduction of LGLs or NK cells occurs within the first 24 hours after administration. In some embodiments, the reduction of LGLs or NK cells is reversible. In some embodiments, the reduction of LGLs or NK cells leads to a reduction of LGL leukemia symptoms. In some embodiments, the reduction of LGLs or NK cells leads to a reduction of Felty's syndrome symptoms. In some embodiments, the reduction of LGLs or NK cells leads to a reduction of IBM symptoms. In some embodiments, the reduction of LGLs or NK cells leads to a reduction of aggressive NK leukemia symptoms. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the Fc region of the antibody comprises non-fucosylated human IgG1 Fc.

[0017] In another aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an antibody that specifically binds to a cell surface protein selected from human CD94, human CD57 or human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity as compared to the wild-type IgG1 Fc region, and wherein the disease or disorder is selected from chronic lymphoproliferative disorders of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis or inflammatory bowel disease. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood LGLs or NK cells in the subject.

[0018] In another aspect, a method of reducing the number of peripheral blood LGL and / or NK cells in a subject, the method comprising administering to the subject an effective amount of an antibody that specifically binds to a cell surface protein selected from human CD94, human CD57, or human NKG2A, the antibody comprising a human immunoglobulin Fc region that comprises enhanced ADCC activity as compared to a wild-type IgG1 Fc region, wherein the subject has a disease or disorder selected from LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, or inflammatory bowel disease, is provided herein.

[0019] In another aspect, a method of inducing ADCC activity in a subject, the method comprising administering to the subject an effective amount of an antibody that specifically binds to a cell surface protein selected from human CD94, human CD57, or human NKG2A, the antibody comprising a human immunoglobulin Fc region that comprises enhanced ADCC activity as compared to a wild-type IgG1 Fc region, wherein the subject has a disease or disorder selected from chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, or inflammatory bowel disease, and wherein administration of the antibody to the subject results in a reduction in the number of peripheral blood LGL and / or NK cells in the subject, is provided herein.

[0020] In some embodiments that can be combined with any of the above embodiments, at least about 1,000 receptors per cell, at least about 2,000 receptors per cell, at least about 3,000 receptors per cell, at least about 4,000 receptors per cell, at least about 5,000 receptors per cell, or at least about 7,000 receptors per cell of the cell surface protein are expressed on the surface of peripheral blood LGL and / or NK cells in the subject.

[0021] In some embodiments that can be combined with any of the foregoing embodiments, the reduction in the number of peripheral blood LGL or NK cells in a subject comprises at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or from about 10% to about 80% reduction as compared to the number of peripheral blood NK cells in a human subject prior to administration of the antibody. In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject occurs within the first 24 hours after administration of the antibody to the subject. In some embodiments, the number of peripheral blood LGL and / or NK cells in a subject is reduced below the limits of clinical diagnosis of the disease or disorder. In some embodiments, the number of peripheral blood LGL and / or NK cells in a subject is reduced to equal to or less than 2×10 9 cells / L (e.g., in a peripheral blood sample obtained from the subject). In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject below the limits of clinical diagnosis of the disease or disorder persists in the subject for at least about 1 week after administration of the antibody to the subject. In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject to equal to or less than 2×10 9 cells / L (e.g., in a peripheral blood sample obtained from the subject) persists in the subject for at least about 1 week after administration of the antibody to the subject. In some embodiments, the number of peripheral blood LGL and / or NK cells in a subject is reduced below the limit of detection of peripheral blood LGL and / or NK cells in the subject. In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject below the limit of detection of peripheral blood LGL and / or NK cells persists in the subject for at least about 1 week after administration of the antibody to the subject. In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject is reversible.

[0022] In some embodiments that can be combined with any of the foregoing embodiments, administration of the antibody to a subject results in a reduction in the number of peripheral blood NK cells in the subject. In some embodiments, the NK cells in the subject are CD3 negative and CD56 positive, CD3 negative and CD16 positive, CD3 negative and CD57 positive, CD3 negative and CD94 positive, or CD3 negative and NKG2A positive. In some embodiments, the antibody has an EC50 between about 3 ng / ml and about 40 ng / ml.

[0023] In some embodiments that can be combined with any of the foregoing embodiments, administration of the antibody to a subject does not result in a reduction in T cells in the subject. In some embodiments, the T cells in the subject are CD3 positive and CD4 positive or CD3 positive and CD16 negative.

[0024] In some embodiments that can be combined with any of the foregoing embodiments, the subject is human.

[0025] In some embodiments that can be combined with any of the foregoing embodiments, administration of the antibody to a subject does not result in tumor lysis syndrome in the subject.

[0026] In some embodiments that can be combined with any of the foregoing embodiments, the antibody comprises a non-fucosylated human IgG1 Fc region.

[0027] In some embodiments that can be combined with any of the foregoing embodiments, the antibody binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, the human cell Fc gamma receptor IIIA comprises a valine residue or a phenylalanine residue at amino acid residue position 158. In some embodiments, the human cell Fc gamma receptor IIIA comprises the sequence of SEQ ID NO: 8 or 9.

[0028] In some embodiments that can be combined with any of the foregoing embodiments, the antibody (a) specifically binds to human CD94 and does not bind to the epitope on human CD94 that is the same as that bound by anti-CD94 antibody clones HP-3D9, DX22, 131412 or 12K45, (b) specifically binds to human CD57 and does not bind to the epitope on human CD57 that is the same as that bound by anti-CD57 antibody clone NK-1, or (c) specifically binds to human NKG2A and does not bind to the epitope on human NKG2A that is the same as that bound by anti-NKG2A antibody clone Z199.

[0029] In some embodiments that can be combined with any of the foregoing embodiments, the antibody (a) specifically binds to human CD94 and binds to human CD94 with an affinity greater than that of anti-CD94 antibody clones HP-3D9, DX22, 131412 and 12K45, (b) specifically binds to human CD57 and binds to human CD57 with an affinity greater than that of anti-CD57 antibody clone NK-1, or (c) specifically binds to human NKG2A and binds to human NKG2A with an affinity greater than that of anti-NKG2A antibody clone Z199.

[0030] In some embodiments, the disease or disorder is Felty's syndrome, and administration of the antibody to a subject results in reduction of one or more Felty's syndrome symptoms in the subject.

[0031] In some embodiments, the disease or disorder is inclusion body myositis, and administration of the antibody to a subject results in reduction of one or more inclusion body myositis symptoms in the subject.

[0032] In some embodiments, the disease or disorder is aggressive NK leukemia, and administration of the antibody to a subject results in reduction of one or more aggressive NK leukemia symptoms in the subject.

[0033] In some embodiments, the disease or disorder is rheumatoid arthritis, and administration of the antibody to a subject results in reduction of one or more rheumatoid arthritis symptoms in the subject.

[0034] In some embodiments, the disease or disorder is LGL leukemia, and administration of the antibody to the subject results in reduction of one or more LGL leukemia symptoms in the subject.

[0035] In some embodiments, the disease or disorder is CLPD-NK, and administration of the antibody to the subject results in reduction of one or more CLPD-NK symptoms in the subject.

[0036] In another aspect, provided herein is a method of treating CLPD-NK in a human subject in need thereof, comprising administering to the human subject an effective amount of an antibody, wherein the antibody specifically binds to human NKG2A and the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity as compared to a wild-type IgG1 Fc region. In some embodiments, the antibody does not bind to an epitope on human NKG2A that is identical to the anti-NKG2A antibody clone Z199. In some embodiments, the antibody binds to human NKG2A with a greater affinity than the anti-NKG2A antibody clone Z199.

[0037] In another aspect, provided herein is a method of treating CLPD-NK in a human subject in need thereof, comprising administering to the human subject an effective amount of an antibody, wherein the antibody specifically binds to human CD94 and the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity as compared to a wild-type IgG1 Fc region. In some embodiments, the antibody does not bind to an epitope on human CD94 that is identical to the anti-CD94 antibody clones HP-3D9, DX22, 131412 or 12K45. In some embodiments, the antibody binds to human CD94 with a greater affinity than the anti-CD94 antibody clones HP-3D9, DX22, 131412 and 12K45.

[0038] In some embodiments that can be combined with any of the foregoing embodiments, administration of the antibody to a human subject results in a reduction in the number of peripheral blood LGL or NK cells in at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 10% - about 90% of human subjects, compared to the number of peripheral blood NK cells in the human subject prior to administration of the antibody. In some embodiments, NK cells in a human subject are CD3 negative and CD56 positive, CD3 negative and CD16 positive, CD3 negative and CD57 positive, CD3 negative and CD94 positive or CD3 negative and NKG2A positive. In some embodiments, administration of the antibody to a human subject does not result in a reduction in T cells in humans. In some embodiments, T cells in a human subject are CD3 positive and CD4 positive or CD3 positive and CD16 negative. In some embodiments, administration of the antibody to a human subject does not result in tumor lysis syndrome in humans. In some embodiments, the antibody comprises a non-fucosylated human IgG1 Fc region. In some embodiments, the antibody binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, human cell Fc gamma receptor IIIA comprises a valine residue or a phenylalanine residue at amino acid residue position 158. In some embodiments, human cell Fc gamma receptor IIIA comprises the sequence of SEQ ID NO: 8 or 9. In some embodiments, administration of the antibody to a human subject results in improvement of one or more CLPD-NK conditions in humans.

[0039] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0040] The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that illustrates exemplary embodiments in which the principles of the invention are utilized and the accompanying drawings.

Brief Description of the Drawings

[0041]

Figure 1A

Figure 1B

[0042]

Figure 2A-1

Figure 2A-2

Figure 2B-1

Figure 2B-2

[0043]

Figure 3

[0044]

Figure 4A

Figure 4B

[0045]

Figure 5A

Figure 5B

[0046]

Figure 6A

Figure 6B

[0047]

Figure 7A

Figure 7B

[0048]

Figure 8A

Figure 8B

[0049]

Figure 9

[0050]

Figure 10

Mode for Carrying Out the Invention

[0051] Some embodiments are described below by way of example with reference to the application of examples. It should be understood that numerous specific details, relationships, and methods are shown in order to provide a complete understanding of the features described herein. However, those skilled in the relevant art will readily recognize that the features described herein can be implemented without one or more of the specific details or using other methods. The features described herein are not limited by the exemplified order of acts or events, as some acts can occur in a different order and / or concurrently with other acts or events. Further, not all exemplified acts or events are necessary to carry out the methodology in accordance with the features described herein.

[0052] As used herein, the singular forms "a", "an", and "the" also include the plural forms unless specifically stated otherwise. Further, the terms "including", "includes", "having", "has", "with" or variations thereof, as used in any of the detailed description and / or claims, are to be construed as inclusive in the same manner as the term "comprising". The term "comprising", as used herein, is synonymous with "including" or "containing" and is inclusive or unrestricted.

[0053] Any reference to "or" herein, unless otherwise specified, includes "and / or". As used herein, the term "about" in relation to a number refers to that number plus or minus 10% of that number. The term "about" in relation to a range refers to the range extending from minus 10% from the lowest value and plus 10% from the highest value.

[0054] I. Use and Method of Treatment As discussed above, LGL and NK cells have been implicated in the etiology of a number of diseases and disorders. Many of these disorders or diseases are characterized by the accumulation of clonal or non-clonal LGL and NK cells.

[0055] In some embodiments, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of an antibody that specifically binds to a cell surface protein selected from human CD94, human CD57, or human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region, and wherein the disease or disorder is selected from chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, or inflammatory bowel disease.

[0056] In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood LGL and / or NK cells in the subject. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood LGL cells in the subject. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood NK cells in the subject.

[0057] Also provided herein is a method of reducing the number of peripheral blood LGL and / or NK cells in a subject, comprising administering to the subject an effective amount of an antibody that specifically binds to a cell surface protein selected from human CD94, human CD57, or human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region, and wherein the subject has a disease or disorder selected from LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, or inflammatory bowel disease.

[0058] Also provided herein is a method of inducing ADCC activity in a subject, comprising administering to the subject an antibody that specifically binds to a cell surface protein selected from human CD94, human CD57, or human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region that comprises enhanced ADCC activity compared to the wild-type IgG1 Fc region, the subject has a disease or disorder selected from chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, or inflammatory bowel disease, and administration of the antibody to the subject results in a reduction in the number of peripheral blood LGL and / or NK cells in the subject.

[0059] In some embodiments, the antibody specifically binds to human CD94 or human NKG2A. In some embodiments, the antibody specifically binds to human CD94. In some embodiments, the antibody specifically binds to human NKG2A. In some embodiments, the disease or disorder is CLPD-NK. In some embodiments, the disease or disorder is LGL leukemia. In some embodiments, the disease or disorder is Felty syndrome. In some embodiments, the disease or disorder is rheumatoid arthritis. In some embodiments, the disease or disorder is aggressive NK leukemia. In some embodiments, the disease or disorder is myosistis. In some embodiments, the disease or disorder is inflammatory bowel disease. In some embodiments, the disease or disorder is T-large granular lymphocyte leukemia (T-LGLL). In some embodiments, the disease or disorder is natural killer-large granular lymphocyte leukemia (NK-LGLL). In some embodiments, the disease or disorder is CLPD-NK and the antibody specifically binds to human CD94. In some embodiments, the disease or disorder is CLPD-NK and the antibody specifically binds to human NKG2A. In some embodiments, the disease or disorder is T-LGLL and the antibody specifically binds to human CD94. In some embodiments, the disease or disorder is T-LGLL and the antibody specifically binds to human NKG2A. In some embodiments, the disease or disorder is NK-LGLL and the antibody specifically binds to human CD94. In some embodiments, the disease or disorder is NK-LGLL and the antibody specifically binds to human NKG2A.

[0060] Also provided herein is a method of treating CLPD-NK in a human subject in need of treatment for CLPD-NK, the method comprising administering to the human subject an effective amount of an antibody, wherein the antibody specifically binds to human NKG2A and the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region. In some embodiments, administration of the antibody to the human subject results in improvement of CLPD-NK symptoms in the human.

[0061] Also provided herein is a method of treating CLPD-NK in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an antibody, wherein the antibody specifically binds to human CD94 and comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region. In some embodiments, administration of the antibody to the human subject results in amelioration of CLPD-NK symptoms in the human.

[0062] In some embodiments, the terms treating, treatment, ameliorate, ameliorating, reducing, reduction of a symptom, reduction of one or more symptoms, and other grammatical equivalents thereof, when used in reference to treating one or more symptoms, include alleviating, lessening, or ameliorating one or more symptoms of a disease or disorder, preventing additional symptoms, ameliorating or preventing the underlying cause of the symptoms, inhibiting the disease or disorder, e.g., suppressing the development of the disease or disorder, alleviating the disease or disorder, causing regression of the disease or disorder, alleviating the condition caused by the disease or disorder, or arresting the symptoms of the disease or disorder, and include prevention. In some embodiments, these terms further include achieving a therapeutic benefit and / or a prophylactic benefit. In some embodiments, a therapeutic benefit refers to eradication or amelioration of the underlying disease or disorder being treated. A therapeutic benefit is also achieved by eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease or disorder, such that in some embodiments, improvement is observed in a patient, even though the patient may still be suffering from the underlying disease or disorder. For prophylactic benefit, a pharmaceutical composition is administered to a patient at risk of developing a particular disease or disorder or to a patient reporting one or more of the physiological symptoms of a disease or disorder, even though a diagnosis of the disease or disorder has not been made.

[0063] In some embodiments, an effective amount, a therapeutically effective amount, or a pharmaceutically effective amount can be a sufficient amount of at least one pharmaceutical composition or compound (e.g., an antibody of the present disclosure) being administered to alleviate to some extent one or more of the symptoms of the disease or condition being treated.

[0064] In some embodiments, at least about 2,000 receptors per cell (e.g., at least about 1,000 receptors per cell, at least about 2,000 receptors per cell, at least about 3,000 receptors per cell, at least about 4,000 receptors per cell, at least about 5,000 receptors per cell, at least about 7,000 receptors per cell, at least about 10,000 receptors per cell, at least about 20,000 receptors per cell, at least about 30,000 receptors per cell, at least about 40,000 receptors per cell, at least about 50,000 receptors per cell, at least about 60,000 receptors per cell, at least about 70,000 receptors per cell, at least about 80,000 receptors per cell, at least about 90,000 receptors per cell, at least about 100,000 receptors per cell, at least about 200,000 receptors per cell, at least about 300,000 receptors per cell, at least about 400,000 receptors per cell, at least about 500,000 receptors per cell, at least about 600,000 receptors per cell, at least about 700,000 receptors per cell, at least about 800,000 receptors per cell, at least about 900,000 receptors per cell, at least about 1,000,000 receptors per cell or more) of a cell surface protein (e.g., human CD94, human CD57 or human NKG2A) are expressed on the surface of peripheral blood LGL and / or NK cells in a subject. In some embodiments, the number of receptors of a cell surface protein (e.g., human CD94, human CD57 or human NKG2A) on the surface of peripheral blood LGL and / or NK cells is compared between a sample (e.g., a biological specimen) obtained from a healthy (e.g., normal) subject and a sample obtained from a subject having a disease or disorder (e.g., NK cell (CLPD-NK), LGL leukemia, Felty syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis or inflammatory bowel disease). In some embodiments, the expression of the cell surface protein (e.g., human CD94, human CD57 or human NKG2A) is specific to the surface of LGL and / or NK cells.In some embodiments, the expression of a cell surface protein (e.g., human CD94, human CD57 or human NKG2A) is specific to the surface of LGL and / or NK cells in a sample obtained from a subject having a disease or disorder (e.g., human CD94, human CD57 or human NKG2A). The number of cell surface proteins (e.g., receptors) expressed on the surface of peripheral blood LGL and / or NK cells in a subject can be measured using any method known in the art, such as flow cytometry as described in Examples 1-3. In some embodiments, by using the same biological specimen, the inventors show the expression of CD94 or CD57 or NKG2A and additional cell surface proteins specific to LGL cells.

[0065] In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject occurs within the first 24 hours after administration of an antibody to the subject, e.g., within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours or within about 24 hours.

[0066] In some embodiments, the number of peripheral blood LGL and / or NK cells in a subject (e.g., in a peripheral blood sample obtained from the subject) is reduced below the limit of clinical diagnosis of a disease or disorder. In some embodiments, the number of peripheral blood LGL and / or NK cells in a subject is 2×10 9Reduced to equal or less than 1 cell / L (e.g., in a peripheral blood sample obtained from a subject). See, for example, Lamy, T. et al. (2017) Blood 129:1082-1094. In some embodiments, the reduction of the number of peripheral blood LGL and / or NK cells in a subject to below the limit of clinical diagnosis of a disease or disorder is present in the subject for at least about 1 week, e.g., at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months or longer after administration of the antibody to the subject. In some embodiments, the reduction of the number of peripheral blood LGL and / or NK cells in a subject to equal or less than 2×10 9 cells / L (e.g., in a peripheral blood sample obtained from a subject) occurs for at least about 1 week, e.g., at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months or longer after administration of the antibody to the subject.

[0067] In some embodiments, the number of peripheral blood LGL and / or NK cells in a subject is reduced below the detection limit of peripheral blood LGL and / or NK cells in the subject. In some embodiments, the reduction of the number of peripheral blood LGL and / or NK cells in a subject below the detection limit of peripheral blood LGL and / or NK cells is present in the subject for at least about 1 week, such as at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months or longer after administration of the antibody to the subject. In some embodiments, peripheral blood LGL and / or NK cells are detected by flow cytometry (such as performed on a peripheral blood sample obtained from the subject) using the following markers: CD3−CD8−CD16+CD56+CD57+ (for the CLPD-NK immunophenotype) or CD3+CD8+CD16+CD56−CD57+ (for the T-LGLL immunophenotype).

[0068] In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject is reversible. In some embodiments, the reduction in the number of peripheral blood LGL and / or NK cells in a subject is reversible within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months or longer after administration of the antibody to the subject.

[0069] In some embodiments, administration of the antibody to a subject results in a reduction in the number of peripheral blood LGLs and / or NK cells in the subject. In some embodiments, NK cells in the subject are CD3 negative and CD56 positive, CD3 negative and CD16 positive, CD3 negative and CD57 positive, CD3 negative and CD94 positive, or CD3 negative and NKG2A positive. In some embodiments, NK cells in the subject are CD3 negative and CD56 positive. In some embodiments, NK cells in the subject are CD3 negative and CD16 positive. Biomarkers expressed by NK cells (e.g., CD3, CD16, CD56) can be measured using any method known in the art, such as flow cytometry as described in Examples 1-3.

[0070] In some embodiments, the description that a cell or population of cells is positive (+) for, or expresses, a particular marker (e.g., CD3, CD4, CD8, CD16, CD56, CD57, CD94, NKG2A, etc.) refers to the detectable presence of the particular marker on or in the cell. In some embodiments, the description that a cell or population of cells is positive, +, for, or expresses, a surface marker (e.g., cell surface protein) is detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, and the staining is at a level substantially above that detected by performing the same procedure using an isotype-matched control and / or a fluorescence minus one (FMO) gating control under otherwise identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker, such that the presence of cell surface expression of the particular marker is detectable by flow cytometry.

[0071] In some embodiments, a description that a cell or population of cells is negative (-) for or does not express a particular marker (e.g., CD3, CD4, CD8, CD16, CD56, CD57, CD94, NKG2A, etc.) refers to the absence of a detectable presence of the particular marker on or in the cell. In some embodiments, a description that a cell or population of cells is negative, - for or does not express a surface marker (e.g., a cell surface protein) is detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, and the staining is at a level substantially the same as or less than the staining detected by performing the same procedure using an isotype-matched control and / or a fluorescence minus one (FMO) gating control under otherwise identical conditions, and / or at a level lower than that of cells known to be positive for the marker, and / or at a level substantially the same as or less than that of cells known to be negative for the marker, such that the absence of cell surface expression of the particular marker is detectable by flow cytometry.

[0072] In some embodiments, the antibody has an EC50 for reducing peripheral blood LGL and / or NK cells in a subject of between about 1 ng / ml and about 100 ng / ml, e.g., any of about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml or about 100 ng / ml. In some embodiments, the antibody has an EC50 between about 3 ng / ml and about 40 ng / ml. In some embodiments, the antibody has an EC50 of about 3 ng / ml. In some embodiments, the antibody has an EC50 of about 40 ng / ml. The EC50 can be measured using any method known in the art, e.g., as described in the Examples.

[0073] In some embodiments, administration of the antibody to a subject does not result in a reduction of T cells in the subject. In some embodiments, the T cells in the subject are CD3 positive and CD4 positive or CD3 positive and CD16 negative. Biomarkers expressed by T cells (e.g., CD3, CD16, CD4) can be measured using any method known in the art, such as flow cytometry as described in the Examples.

[0074] In some embodiments, the subject is a human, primate, non-human primate (e.g., African green monkey, rhesus monkey, etc.), domestic mammalian, game mammalian or captive mammalian. In some embodiments, the subject is a human. In some embodiments, the human subject is an infant, child, juvenile, young adult, adult or elderly. In some embodiments, the subject has a disease involving LGL and / or NK cells, such as CLPD-NK, LGL leukemia, Felty syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis or inflammatory bowel disease.

[0075] In some embodiments, administration of the antibody to a subject does not result in tumor lysis syndrome in the subject. Tumor lysis syndrome can be measured or diagnosed according to any method known in the art, such as the Cairo-Bishop classification system for tumor lysis syndrome (see, e.g., Cairo and Bishop (2004) Br J Haematol, 127(1):3-11).

[0076] In some embodiments, the antibodies of the present disclosure bind to CD94 or CD57 or NKG2A. In some embodiments, the antibodies of the present disclosure deplete and / or reduce the levels of LGL and / or NK cells. In some embodiments, the antibodies of the present disclosure have a demonstrable benefit for patients (e.g., human patients) having a disease or disorder, such as CLPD-NK, LGL leukemia, rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD and other diseases associated with LGL and / or NK cells. In some embodiments, the antibodies of the present disclosure have better tolerability and fewer side effects than first and second line therapies for a disease or disorder (e.g., CLPD-NK, LGL leukemia, Felty's syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis or inflammatory bowel disease), such as chemotherapy, alemtuzumab and splenectomy. In some embodiments, the antibodies of the present disclosure demonstrate more selective depletion of disease-inducing cells (e.g., peripheral blood LGL and / or NK cells) compared to current non-selective therapies, such as chemotherapy, alemtuzumab and splenectomy. Thus, in some embodiments, the present disclosure provides a method of reducing or depleting the number of LGL and / or NK cells in a human subject upon administration of a molecule (e.g., an antibody of the present disclosure) that binds to a cell surface protein on LGL and / or NK cells, such as CD94 or CD57 or NKG2A or an additional cell surface protein specific for LGL and / or NK cells and that comprises (a) a region that specifically binds to the target and (b) an immunoglobulin Fc region.

[0077] A. Administration and dosing regimen (i) Route of administration In some embodiments, administering, administration, etc. refer to methods used to enable delivery of a therapeutic or pharmaceutical composition to a desired site of biological action. In some embodiments, the antibodies (and any additional therapeutic agents) of the disclosure for use in any of the methods provided herein can be administered to a subject (e.g., a human) by any suitable means including parenteral, intralung, intranasal, and intralesional administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies of the disclosure are administered by intravenous injection. The dosing of the antibodies of the disclosure can be by any suitable route, e.g., by injection such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.

[0078] (ii) Dosing regimen The antibodies of the disclosure for use in any of the methods provided herein can be administered to a subject using various dosing schedules or regimens including, but not limited to, single or multiple administrations over various time points, bolus dosing, and pulse infusion. The specific dosage of the antibodies of the disclosure to be administered will vary according to specific target specificity, type of disease or disorder, subject, and nature and severity of the disease, the health status of the subject, the therapeutic regimen (e.g., whether combination therapeutic agents are used), and the selected route of administration. In some embodiments, the dosage of the antibodies of the disclosure can be from about 0.0001 mg per kg of subject body weight to 100 mg per kg of subject body weight. Exemplary dosing regimens of the antibodies of the disclosure require multiple administrations of the antibody over an extended period of time, e.g., at least 6 months.

[0079] B. Diseases There are three distinct diseases involving LGL: T-cell large granular lymphocyte (T-LGL) leukemia, chronic lymphoproliferative disorder of NK cells (CLPD-NK, formerly NK-LGL), and aggressive NK-cell leukemia, e.g., aggressive natural killer leukemia (ANKL) and extranodal NK / T-cell lymphoma, nasal type (ENKL).

[0080] In addition to NK or T LGL leukemia, NK or LGL cells play important roles in rheumatoid arthritis (RA), Felty syndrome, aggressive NK leukemia, inclusion body myositis (IBM), inflammatory bowel disease (IBD) and other diseases. Non-limiting examples of diseases and disorders in which LGL and NK cells play a role include LGL leukemia, rheumatoid arthritis, Felty syndrome, aggressive NK leukemia, IBM and IBD. Advantageously, using the methods described herein, essentially, using diseased cells to eliminate each other via mechanisms such as ADCC using NK cells, for example, the number of abnormal or diseased NK cells (e.g., CLPD-NK, ANKL or ENKL cells) can be reduced. For exemplary descriptions of the symptoms of these diseases, see, for example, Lamy, et al, Blood, 2017 x Vol. 129, No. 9; Loughran Blood, VOI 82, NO 1 (July I), 1993: pp 1-14; Semenzato G, et al, Blood. 1997;89(1):256-260; and Bourgault-Rouxel, et al, Leuk Res.2008;32(1):45-48.

[0081] (i)CLPD-NK Chronic lymphoproliferative disorder of NK cells (CLPD-NK) is also referred to as NK-LGL leukemia, chronic NK cell lymphocytosis, chronic NK-LGL lymphoproliferative disorder (LPD), granular lymphocytic proliferative disorder of NK cell lineage, NK cell LGL lymphocytosis or low-grade granular NK cell LPD, and is generally characterized by a persistent (e.g., 6 months or longer) increase (e.g., ≧2×10 9 / L) of peripheral blood NK cells.

[0082] Symptoms of CLPD-NK include variable cytopenia, such as neutropenia and anemia, fatigue, fever, night sweats, recurrent infections, rheumatoid arthritis, lymphadenopathy, hepatosplenomegaly, skin lesions, hematological neoplasms, vasculitis, neuropathy and autoimmune disorders.

[0083] In some embodiments of the methods provided herein, the disease or disorder is CLPD-NK, and administration of the antibody results in a reduction of one or more CLPD-NK symptoms in the subject. In some embodiments, a reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in a reduction of one or more CLPD-NK symptoms in the subject.

[0084] Symptoms of CLPD-NK can be measured by any method known in the art, for example, anemia, neutropenia, clinical tests for measuring complete blood count and / or magnetic resonance imaging (MRI), CT scan, palpation or ultrasound (e.g., for determining hepatosplenomegaly), bone marrow examination and flow cytometry. Methods for measuring symptoms of CLPD-NK are described, for example, in Swerdlow, S.H. et al. (2016) Blood 127:2375-2390.

[0085] (ii) LGL leukemia Large granular lymphocytic (LGL) leukemia is a chronic lymphoproliferative disorder characterized by a chronic increase in large granular lymphocytes (LGL) in the peripheral blood and is called T-cell LGL leukemia.

[0086] Symptoms of LGL leukemia include splenomegaly, B symptoms (e.g., systemic symptoms such as fever, night sweats and weight loss), anemia, neutropenia and recurrent infections. Rheumatoid arthritis is common in people with T-cell LGL leukemia.

[0087] In some embodiments of the methods provided herein, the disease or disorder is LGL leukemia, and administration of the antibody results in a reduction of one or more LGL leukemia symptoms in the subject. In some embodiments, a reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in a reduction of one or more LGL leukemia symptoms in the subject.

[0088] The symptoms of LGL leukemia can be measured by any method known in the art, for example, anemia, neutropenia and other cytopenias, clinical tests for measuring complete blood count, magnetic resonance imaging (MRI), CT scan, palpation or ultrasound (e.g., for determining splenomegaly), bone marrow examination and flow cytometry. Methods for measuring the symptoms of LGL leukemia are described, for example, in Swerdlow, S.H. et al. (2016) Blood 127:2375-2390.

[0089] (iii) Felty syndrome Felty syndrome is an autoimmune disease characterized by rheumatoid arthritis, splenomegaly (e.g., inflammatory splenomegaly) and reduction in the number of neutrophils in the blood. Symptoms of Felty syndrome include painful, stiff and / or swollen joints, physical findings associated with rheumatoid arthritis, splenomegaly, neutropenia, infections, dry keratoconjunctivitis, fever, weight loss, fatigue, skin discoloration, ulcers (e.g., sores), hepatomegaly, anemia, thrombocytopenia, abnormal liver function, enlarged lymph nodes and vasculitis.

[0090] In some embodiments of the methods provided herein, the disease or disorder is Felty syndrome and administration of the antibody results in reduction of one or more Felty syndrome symptoms in the subject. In some embodiments, reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in reduction of one or more Felty syndrome symptoms in the subject. Symptoms of Felty syndrome include, but are not limited to, joint inflammation, joint pain and splenomegaly.

[0091] The symptoms of Felty syndrome can be measured using any method known in the art, such as anemia, neutropenia, thrombocytopenia and other cytopenias, clinical tests for measuring complete blood count, magnetic resonance imaging (MRI), CT scan or ultrasound (e.g., for determining splenomegaly and / or hepatomegaly), clinical tests for abnormal liver function, palpation for determining splenomegaly and / or hepatomegaly, flow cytometry, Disease Activity Score-28 (DAS-28, such as that used to monitor rheumatoid arthritis symptoms) and DAS-28 with erythrocyte sedimentation rate (ESR).

[0092] (iv) Rheumatoid arthritis Rheumatoid arthritis mainly affects joints, but may also affect other organs, and is an autoimmune disorder that may be associated with cardiovascular disease, osteoporosis, interstitial lung disease, infections, cancer, fatigue and depression. Symptoms of rheumatoid arthritis include swollen, tender, warm joints, joint inflammation, joint pain, joint stiffness, splenomegaly, rheumatoid nodules (e.g., in the skin), necrotizing granulomas, vasculitis, pyoderma gangrenosum, Sweet syndrome, drug reactions, erythema nodsum, lobe pannicultis, atrophy of finger skin, palmar erythema, skin fragility, diffuse alopecia areata, pulmonary fibrosis, Caplan syndrome, exudative pleural effusion, atherosclerosis, myocardial infarction, stroke, pericarditis, endocarditis, left ventricular failure, valvulitis, fibrosis of the heart and / or blood vessels, anemia, increased platelet count, low white blood cell count, renal amyloidosis, episcleritis, scleritis, keratoconjunctivitis sicca, keratitis, vision loss, liver problems, peripheral neuropathy, multiple mononeuritis, carpal tunnel syndrome, myelopathy, atlantoaxial subluxation, vertebrae slipping, fatigue, low-grade fever, malaise, morning stiffness, loss of appetite, weight loss, osteoporosis, cancer (e.g., lymphoma, skin cancer) and periodontitis may be associated.

[0093] In some embodiments of the methods provided herein, the disease or disorder is rheumatoid arthritis, and administration of the antibody results in reduction of one or more rheumatoid arthritis symptoms in a subject. In some embodiments, reduction in the number of peripheral blood LGL and / or NK cells in a subject after administration of the antibody results in reduction of one or more rheumatoid arthritis symptoms in the subject.

[0094] In some embodiments, symptoms and disease state / progression of rheumatoid arthritis are measured according to the 2010 ACR / EULAR classification criteria for rheumatoid arthritis (see, e.g., Aletaha et al., (2010) Annals of Rheumatic Diseases, 69(9):1580-8). Rheumatoid arthritis symptoms can also be measured by any method known in the art, e.g., erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, anti-citrullinated protein antibody, anemia and other cytopenias, increased platelet count, low white blood cell count, complete blood count, clinical tests for measuring renal amyloidosis, X-ray, MRI, CT scan, medical imaging such as ultrasound (e.g., ultrasound examination using a high-frequency transducer; Doppler ultrasound), flow cytometry, Disease Activity Score-28 (DAS-28), and DAS-28 with erythrocyte sedimentation rate (ESR).

[0095] (v) Aggressive NK leukemia Aggressive NK cell leukemia is an aggressive disease with systemic proliferation of NK cells and a rapidly deteriorating clinical course. Aggressive NK leukemia may also be referred to as aggressive NK cell lymphoma. Symptoms of aggressive NK cell leukemia include constitutional symptoms (e.g., malaise, weight loss, fatigue), hepatosplenomegaly, lymphadenopathy, coagulation disorders, hemophagocytic syndrome, multiple organ failure, infections such as Epstein-Barr virus, necrosis, and allergic reactions (e.g., to insect bites, e.g., mosquito bites) that can result in systemic symptoms such as fever, swollen lymph nodes, abdominal pain, diarrhea, and anaphylaxis.

[0096] In some embodiments of the methods provided herein, the disease or disorder is aggressive NK cell leukemia, and administration of the antibody results in reduction of one or more aggressive NK cell leukemia symptoms in the subject. In some embodiments, reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in reduction of one or more aggressive NK cell leukemia symptoms in the subject.

[0097] Symptoms of aggressive NK leukemia can be measured by any method known in the art, for example, anemia, neutropenia and other cytopenias, clinical tests for measuring complete blood count and / or magnetic resonance imaging (MRI), CT scan, palpation or ultrasound (e.g., for determining splenomegaly), bone marrow examination and flow cytometry. Methods for measuring symptoms of aggressive NK leukemia are described, for example, in Swerdlow, S.H. et al. (2016) Blood 127:2375-2390.

[0098] (vi) Inclusion body myositis Inclusion body myositis (IBM) is an inflammatory muscle disease characterized by an autoimmune and degenerative process that results in progressive weakness and wasting of distal and / or proximal muscles, also known as sporadic inclusion body myositis. Generally, IBM is characterized by infiltration of immune cells into muscle tissue. In some cases, patients with IBM have elevated creatine kinase levels in the blood. Symptoms of IBM include progressive muscle weakness, muscle wasting / atrophy, frequent tripping and falling, difficulty manipulating fingers, foot drop, limited mobility, balance disorders, muscle pain, dysphagia and fatigue.

[0099] In some embodiments of the methods provided herein, the disease or disorder is IBM, and administration of the antibody results in reduction of one or more IBM symptoms in the subject. In some embodiments, reduction in the number of peripheral blood LGL and / or NK cells in the subject after administration of the antibody results in reduction of one or more IBM symptoms in the subject.

[0100] The symptoms of IBM can be measured by any method known in the art, such as muscle biopsy, blood tests (e.g., for measuring creatine kinase), electromyogram (EMG) studies, blood tests for measuring antibodies against NT5C1A, flow cytometry, and, without limitation, myositis disease activity assessment tools including the Myositis Intention to Treat Activity Index (MITAX) and the Myositis Disease Activity Assessment Visual Analogue Scales (MYOACT).

[0101] (vii) Inflammatory bowel disease Inflammatory bowel disease (IBD) refers to a class of inflammatory conditions of the colon and small intestine. Types of IBD include ulcerative colitis and Crohn's disease. Symptoms of IBD include diarrhea, fever, fatigue, abdominal pain, abdominal cramps, bloody stools, reduced appetite, and weight loss.

[0102] In some embodiments of the methods provided herein, the disease or disorder is IBD and administration of the antibody results in reduction of one or more IBD symptoms in a subject. In some embodiments, reduction in the number of peripheral blood LGL and / or NK cells in a subject after administration of the antibody results in reduction of one or more IBD symptoms in the subject.

[0103] The symptoms of IBD can be measured by any method known in the art, such as clinical blood tests for anemia, other cytopenias or infections, fecal occult blood tests, colonoscopy, flexible sigmoidoscopy, upper gastrointestinal endoscopy, capsule endoscopy, balloon-assisted enteroscopy, x-rays, CT scans, MRI scans, ultrasound, and flow cytometry.

[0104] II. Antibodies In some embodiments, molecules (e.g., antibodies) that bind to CD94, CD57, NKG2A, or other cell surface proteins expressed on LGL and / or NK cells are provided herein. Also provided herein are molecules (e.g., antibodies) having an immunoglobulin Fc portion with modifications that bind to CD94 or CD57 or NKG2A and include mutations that reduce fucosylation, non-fucosylate, or enhance ADCC activity and / or improve the affinity of the Fc region for Fc receptors such as CD16.

[0105] In some embodiments, the antibodies provided herein bind to human CD94, human CD57, or human NKG2A. In some embodiments, the antibodies provided herein bind to CD94, CD57, or NKG2A.

[0106] In some embodiments, the term antibody is used in the broadest sense and includes, but is not limited to, various antibody structures including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibodies of the present disclosure are isolated antibodies. An “isolated” antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the antibody's research, diagnostic, and / or therapeutic use and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of the antibody, in some embodiments greater than 99% by weight, as determined, for example, by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, for example, by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using, for example, Coomassie blue or silver staining. An isolated antibody can include an antibody in vivo in situ within a recombinant cell since at least one component of the antibody's natural environment will not be present. However, typically, an isolated antibody is prepared by at least one purification step.

[0107] In some embodiments, a monoclonal antibody is an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies making up the population are generally identical, excluding potential variant antibodies that contain, for example, variants that are naturally occurring or that arise during the production of the monoclonal antibody preparation and are present in trace amounts, and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, in some embodiments, a monoclonal antibody is obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies can be produced using any method known in the art. For example, monoclonal antibodies to be used in accordance with the present disclosure can be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals that contain all or part of the human immunoglobulin locus.

[0108] A. Enhancement of ADCC activity In some embodiments, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-directed cytotoxicity, or ADCC refers to a cell-mediated reaction in which non-specific cytotoxic cells that produce Fc receptors, such as natural killer cells (NK cells), neutrophils, and macrophages, recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. The major mediator cells are natural killer (NK) cells. NK cells express FcγRIII (Ravetch et al. (1991) Annu. Rev. Immunol., 9:457-92). In some embodiments, ADCC activity refers to the ability of an antibody or Fc fusion protein to induce an ADCC response.

[0109] In some embodiments, the antibodies provided herein have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the enhancement of ADCC activity refers to an antibody or an Fc region of an antibody that mediates or induces ADCC more efficiently and / or effectively than a natural or wild-type antibody and / or a natural or wild-type Fc region of the antibody in the presence of effector cells in vitro or in vivo, which can be determined using, for example, an ADCC assay as described herein or as well known in the art. In some embodiments, the effector cells are leukocytes that produce one or more Fc receptors and perform effector functions. In some embodiments, such cells produce at least FcγRIII and exhibit ADCC effector function. Examples of ADCC-mediated human leukocytes include peripheral blood mononuclear cells (PBMCs), natural killer cells (NKs), monocytes, cytotoxic T cells, and neutrophils.

[0110] In some embodiments, the ADCC activity is determined using, for example, peripheral blood mononuclear cells (PBMCs) and / or NK effector cells 51Using an in vitro assay as described in the examples using a Cr release assay, for example, see Shields et al. (2001) J. Biol. Chem., 276:6591-6604, or it can be directly evaluated using another suitable method. ADCC activity can be expressed as the number of remaining cells after an ADCC assay (e.g., see Example 2) or the concentration (e.g., EC50) of an antibody or Fc fusion protein at which lysis of target cells is half-maximal. In some embodiments, ADCC activity is determined using an ex vivo assay using PBMC and / or NK cells as described in the examples, and the ADCC activity of the antibodies of the present disclosure is described as the percentage of target cells remaining after an ADCC assay and / or the EC50 of the antibody (i.e., the concentration of the antibody of the present disclosure at which half-maximal depletion or lysis of target cells is achieved). The EC50 of an antibody can be determined using any method known in the art, for example, using a dose-response curve and GraphPad Prism as described in the examples. In some embodiments, the antibodies provided herein induce ADCC activity, and the EC50 measured using an ex vivo assay as described in the examples is from about 1 ng / ml to about 100 ng / ml (e.g., any of about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml or about 100 ng / ml).In some embodiments, the antibodies of the present disclosure exhibit an EC50 that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% lower than the EC50 of a control antibody (e.g., a wild-type control antibody or an antibody known or commercially available in the art against the same target).

[0111] In some embodiments, EC50 refers to the concentration of a compound (e.g., an antibody) that induces an intermediate response between baseline and maximum after a specified exposure time. For example, EC50 can be used to measure the potency of an antibody that mediates and / or induces effector functions, such as ADCC activity. In some embodiments, the EC50 of a dose-response curve represents the concentration of a compound (e.g., an antibody) at which 50% of the maximum effect is observed.

[0112] In some embodiments, the antibodies of the present disclosure have higher maximum target cell lysis compared to a control antibody (e.g., a wild-type control antibody or an antibody known or commercially available in the art against the same target). For example, the antibodies of the present disclosure can exhibit maximum target cell lysis that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% higher than that of a control antibody (e.g., a wild-type control antibody or an antibody known or commercially available in the art against the same target).

[0113] (i) Enhanced binding to Fc receptors In some embodiments, the antibodies provided herein include a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type Fc region. In some embodiments, the antibodies provided herein bind to human cell Fc receptors to a greater extent than antibodies comprising a wild-type Fc region. In some embodiments, the Fc receptor (FcR) is a receptor capable of binding to the Fc region of an antibody. Certain Fc receptors can bind to IgG (i.e., gamma-receptors), such receptors including subclasses of FcyRI, FcyRII, and FcyRIII, as well as their allelic variants and alternative splicing events. For an overview of Fc receptors, see Ravetch and Kinet: Annu. Rev. Immunol. 9, 457 (1991); Capel et al. Immunomethods, 4, 25 (1994); and de Haas et al., J. Lab. Clin. Med. 126, 330 (1995).

[0114] In some embodiments, the antibodies provided herein bind to human cell Fc gamma receptor IIIA to a greater extent than antibodies comprising a wild-type Fc region. In some embodiments, human cell Fc gamma receptor IIIA comprises a valine or phenylalanine residue at amino acid residue position 158. See, for example, UniProt accession P08637 or VAR_003960. In some embodiments, human cell Fc gamma receptor IIIA comprises the sequence of SEQ ID NO: 8 or 9. Human cell Fc gamma receptor IIIA 158F

Chemical formula

Chemical formula

[0115] In some embodiments, the antibodies provided herein are of the IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgA (IgA1 or IgA2), IgD, IgM or IgE isotype. In some embodiments, the antibodies provided herein are of the IgG isotype. In some embodiments, the antibodies provided herein are of the IgG1 isotype. In some embodiments, the antibodies provided herein bind to human cell Fc gamma receptor IIIA (FcγRIIIA) to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. In some embodiments, the human cell Fc gamma receptor IIIA comprises a valine or phenylalanine residue at amino acid residue position 158. Exemplary assays for determining binding to human cell Fc gamma receptor IIIA are known in the art; see, e.g., Lazar, G.A. et al. (2006) Proc. Natl. Acad. Sci. 103:4005-1010; and Ferrara, C. et al. (2011) Proc. Natl. Acad. Sci. 108:12669-12674.

[0116] In some embodiments, the Fc region is the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In some embodiments, the Fc region includes a native Fc region or a variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the carboxyl terminus. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In some embodiments, the numbering of amino acid residues in the Fc region or the constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. In some embodiments, the wild-type Fc region or native Fc region is an Fc region that contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. In some embodiments, the variant Fc region is an Fc region that contains an amino acid sequence that differs from the native or wild-type sequence of the Fc region by at least one amino acid. In some embodiments, the variant Fc region has at least one amino acid substitution, for example, approximately 1 to 10 or 1 to 5 amino acid substitutions. In some embodiments, the Fc region variant is at least approximately 80% (e.g., at least about 90% or at least about 95%) homologous to the native or wild-type sequence Fc region and / or the Fc region of the original polypeptide. In some embodiments, at least one amino acid substitution in the variant Fc region enhances the effector function of the variant Fc region compared to the native or wild-type Fc region. In some embodiments, the effector function is a biological activity resulting from the Fc region of an antibody that varies depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0117] The binding affinity of an antibody for an Fc receptor can be evaluated using any method known in the art, for example, using surface plasmon resonance and / or ELISA as described in Shields et al. (2001) J. Biol. Chem., 276:6591-6604. In some embodiments, the affinity of the antibodies of the present disclosure for FcγRIIIA is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold or higher than that of the wild-type control.

[0118] In some embodiments, affinity refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or target). For example, the affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein.

[0119] In some embodiments, a description that a molecule (e.g., an antibody and / or Fc region) binds to a greater extent than another molecule (e.g., an antibody and / or Fc region), or a description that a molecule (e.g., an antibody and / or Fc region) binds with greater affinity than another molecule (e.g., an antibody and / or Fc region), or other grammatical equivalents, refers to a molecule (e.g., an antibody and / or Fc region) that binds more tightly (e.g., has a lower dissociation constant) to a target (e.g., an Fc receptor, cell surface protein) than another molecule (e.g., an antibody and / or Fc region) in a binding assay (e.g., as described herein and / or as commonly known in the art) under substantially the same conditions. For example, a description that antibody "X" binds to an Fc receptor to a greater extent than antibody "Y" indicates that in a binding assay (e.g., as described herein and / or as commonly known in the art) under substantially the same conditions, antibody "X" binds more tightly (e.g., has a lower dissociation constant) to the Fc receptor than antibody "Y". In another example, a description that antibody "X" binds to a target (e.g., a cell surface protein) with greater affinity than antibody "Y" indicates that in a binding assay (e.g., as described herein and / or as commonly known in the art) under substantially the same conditions, antibody "X" binds more tightly (e.g., has a lower dissociation constant) to the target (e.g., a cell surface protein) than antibody "Y".

[0120] (ii) Reduction of fucosylation In some embodiments, the antibodies of the present disclosure are not fucosylated or lack fucose, e.g., the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, and are glycosylated antibody variants comprising an Fc region. In some embodiments, for antibodies with reduced fucose or lacking fucose, the ADCC function is improved. Antibodies that are not fucosylated or lack fucose have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. In some embodiments, the non-fucosylated or fucose-lacking antibody compositions of the present disclosure are compositions in which less than about 50% of the N-linked glycans attached to the Fc region of the antibodies in the composition contain fucose.

[0121] In some embodiments, fucosylated or fucosylation refers to fucose residues within the oligosaccharides attached to the peptide backbone of the antibodies of the present disclosure. Specifically, a fucosylated antibody contains an α(1,6)-linked fucose on the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the antibody Fc region, e.g., at position Asn297 of the human IgG1 Fc domain (EU numbering of Fc region residues). Asn297 may also be located approximately +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in immunoglobulins.

[0122] In some embodiments, the degree of fucosylation is, for example, the percentage of fucosylated oligosaccharides relative to all oligosaccharides as identified by methods known in the art, such as in an N-glycosidase F-treated antibody composition evaluated by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a composition of fully fucosylated antibodies, at least 90% or essentially all of the oligosaccharides contain a fucose residue, i.e., are fucosylated. Thus, individual antibodies in such a composition typically contain a fucose residue in each of the two N-linked oligosaccharides in the Fc region. In some embodiments, in a composition of fully non-fucosylated antibodies, less than about 10% of the oligosaccharides are fucosylated or are essentially free of fucosylation, and individual antibodies in such a composition do not contain a fucose residue in either of the two N-linked oligosaccharides in the Fc region. In a composition of partially fucosylated antibodies, only some of the oligosaccharides contain fucose. Subject to the condition that the composition does not contain essentially all individual antibodies lacking a fucose residue in the N-linked oligosaccharides in the Fc region and does not contain essentially all individual antibodies containing a fucose residue in both of the N-linked oligosaccharides in the Fc region, individual antibodies in such a composition may or may not contain a fucose residue in one or both of the N-linked oligosaccharides in the Fc region. In one embodiment, a composition of partially fucosylated antibodies has a degree of fucosylation of from about 10% to about 80% (e.g., from about 50% to about 80%, from about 60% to about 80% or from about 70% to about 80%).

[0123] In some embodiments, the glycosylated antibody variant comprises an Fc region, and the carbohydrate structure attached to the Fc region lacks fucose. In such variants, the ADCC function is improved. Examples of defucosylated or fucose-deficient antibodies are described in US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0124] Antibodies with reduced fucosylation or non-fucosylated antibodies can be generated using any method known in the art. In some embodiments of the antibodies of the present disclosure, at least one or two of the heavy chains of the antibody may be non-fucosylated. For example, the antibodies of the present disclosure with reduced fucosylation or non-fucosylated antibodies of the present disclosure have an alpha1,6-fucosyltransferase (Fut8) knockout and / or overexpress beta1,4-N-acetylglycosaminyltransferase III (GnT-III) and / or Golgi mu-mannosidase II (ManII) and can be produced in cell lines. Antibodies with reduced fucosylation or non-fucosylated antibodies can also be produced using cell lines that lack "FUT8", an alpha-1,6 fucosyltransferase that catalyzes the transfer of fucose, for example, using Chinese hamster ovary (CHO) cells lacking FUT8 (Yamane-Ohnuki et al., 2004), or using small interfering RNA (siRNA) to block the expression of the FUT8 gene (Mori et al., 2004). Other cell lines that can be used to produce non-fucosylated or defucosylated antibodies or antibodies with reduced fucosylation are known in the art, for example, Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., especially in Example 11), and knockout cell lines, for example, alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)) as well as cells overexpressing beta1,4-N-acetylglycosaminyltransferase III (GnT-III) and Golgi mu-mannosidase II (ManII).

[0125] In some embodiments, the antibodies of the present disclosure have reduced fucose relative to the amount of fucose on the same antibody produced in wild-type CHO cells. For example, the antibody may have less fucose than it would otherwise have if produced by natural CHO cells (e.g., CHO cells that produce a native glycosylation pattern, e.g., CHO cells that contain the native FUT8 gene). In some embodiments, the antibodies provided herein have less than about 50%, 40%, 30%, 20%, 10%, 5% or 1% of the N-linked glycans containing fucose. In certain embodiments, the antibodies provided herein have no N-linked glycans containing fucose, i.e., the antibody is completely fucose-free, or has no fucose, or is not fucosylated, or is afucosylated. The amount of fucose can be determined by one of ordinary skill in the art, for example, by calculating the average amount of fucose in the sugar chain at Asn297 relative to the total of all glycostructures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at position approximately 297 in the Fc region (Eu numbering of Fc region residues), although Asn297 may also be located between approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. In some embodiments, at least one or both of the heavy chains of the antibody are not fucosylated.

[0126] Antibodies lacking 1,6-fucose in their heavy chain glycosylation may have enhanced binding affinity for the FcγRIII receptor and increased ADCC activity (see, for example, Shields et al., 2002; Shinkawa et al, 2002; Okazaki, 2004; Dall'Ozzo, 2004). In some embodiments, the antibodies provided herein have a modified Fc region that includes mutations that reduce fucosylation, are non-fucosylated and / or enhance ADCC activity and / or improve the affinity of the Fc region for Fc receptors, such as FcγRIII and CD16. In some embodiments, a molecule (e.g., an antibody provided herein) may induce cytotoxicity (ADCC) directed by the antibody to a greater extent than a fucosylated or wild-type antibody, and may deplete or reduce the number of LGL and NK cells.

[0127] In some embodiments, the antibodies of the disclosure are engineered to improve ADCC activity by reducing fucosylation. In some embodiments, the molecules provided herein (e.g., an antibody provided herein) may induce cytotoxicity (ADCC) directed by the antibody to a greater extent than a fucosylated or wild-type antibody, and may deplete or reduce the number of LGL and / or NK cells. In some embodiments, at least one or two of the heavy chains of the antibodies of the disclosure are not fucosylated. In some embodiments, the antibodies of the disclosure are modified such that the carbohydrates of the antibody are not fucosylated. In some embodiments, the antibodies of the disclosure are modified such that less than about 90% of the carbohydrates of the antibody, such as any of less than about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5% or about 1% contain fucose. In some embodiments, the antibodies of the disclosure are modified such that less than about 40% of the carbohydrates of the antibody contain fucose. In some embodiments, the antibodies provided herein are not fucosylated.

[0128] In some embodiments, the molecules (e.g., antibodies) provided herein are fucosylated or induce higher levels of antibody-directed cytotoxicity (ADCC) than wild-type antibodies and may deplete or reduce the number of LGL and NK cells.

[0129] (iii) Mutations that enhance ADCC activity The antibodies of the disclosure may include a variant Fc region. In some embodiments, the variant Fc region includes at least one amino acid substitution in the Fc region that improves ADCC activity. For example, the antibodies of the disclosure may have a variant IgG1 Fc region that includes one or more of the Fc mutations selected from S239D, A330L, I332E, F243L, and G236A. In another example, the antibodies of the disclosure may have a human IgG1 Fc variant region that includes one or more of the Fc mutations selected from S239D, A330L, I332E, F243L, and G236A. Other amino acid substitutions known to enhance ADCC activity, such as those described in Lazar et al., PNAS 103, 4005-4010 (2006); Shields et al., J. Biol. Chem. 276, 6591-6604 (2001); Stewart et al., Protein Engineering, Design and Selection 24, 671-678 (2011), and Richards et al., Mol Cancer Ther 7, 2517-2527 (2008), may also be used.

[0130] (iv) Reduced internalization In some embodiments, the antibodies of the present disclosure have a low degree of receptor-induced internalization, for example, compared to a wild-type control antibody against the same target or an antibody known in the art or commercially available. Antibodies with less internalization have higher receptor occupancy on the cell surface and higher levels of receptor-antibody complexes on the cell surface, which can enhance ADCC activity. The antibodies of the present disclosure can be tested in vitro for their ability to internalize their target (e.g., any of CD94, CD57, or NKG2A). Antibody candidates that do not have internalization activity or have low internalization activity can be further tested for binding to cynomolgus monkey and / or human-derived targets (e.g., any of cynomolgus monkey and / or human CD94, cynomolgus monkey and / or human CD57, or cynomolgus monkey and / or human NKG2A). Antibodies that bind to cynomolgus monkey and / or human targets can be used for cell killing assays (e.g., ADCC assays) in vitro and in vivo. The cell killing activity (e.g., ADCC activity) of the selected antibodies can be compared to commercially available antibodies or antibodies known in the art.

[0131] B. Preparation of Antibodies The antibodies of the present disclosure can be produced using any technique and / or method known in the art. Techniques for preparing antibodies, for example, monoclonal antibodies (mAbs) against substantially any target antigen, are well known in the art. See, for example, Koehler and Milstein, Nature 256: 495 (1975) and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtained by injecting a composition containing an antigen (e.g., any one of CD94, CD57, or NKG2A, or a portion thereof) into a mouse, harvesting the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to generate hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against the antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma cultures. One of ordinary skill in the art will understand that when the antibody is administered to a human subject, the antibody will bind to a human antigen (e.g., any one of human CD94, human CD57, or human NKG2A, or a portion thereof).

[0132] MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography using Protein A or Protein G Sepharose, size exclusion chromatography, and ion exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and 2.9.1-2.9.3. See also Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

[0133] After first generating an antibody against an immunogen (e.g., any one of CD94, CD57, or NKG2A, or a portion thereof), the antibody was sequenced and then prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art as discussed below.

[0134] In an exemplary method of making the antibodies of the present disclosure, a recombinant target (e.g., any one of CD94, CD57, or NKG2A) can be utilized for immunization of mice. For example, an antibody made after immunization of a mouse as described above can be analyzed by ELISA and flow cytometry for specific or selective binding to its target (e.g., any one of CD94, CD57, or NKG2A). The antibody can be selected based on its ability to bind to the target (e.g., any one of CD94, CD57, or NKG2A).

[0135] In some embodiments, non-human primate antibodies can be made. General techniques for producing therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., WO91 / 11465 (1991), and in Losman et al., Int. J. Cancer 46: 310 (1990).

[0136] In some embodiments, the antibody can be a human antibody. In some embodiments, the antibody can be a monoclonal human antibody. In some embodiments, a human antibody has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or a sequence encoding other human antibodies. Such antibodies can be obtained from transgenic mice engineered to produce specific human antibodies in response to an antigen challenge, such as any of CD94, CD57, or NKG2A, or a portion thereof. Methods for generating fully human antibodies using either a combinatorial approach or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol. 3:544-50). In certain embodiments, the claimed methods and procedures can utilize human antibodies produced by such techniques.As other methods for generating fully human antibodies, for example, phage display as described in Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40, for example, in normal humans or from humans showing a specific disease state as described in Dantas-Barbosa et al., 2005, or for example, transgenic animals (e.g., mice) that have been genetically engineered to produce human antibodies using the standard immunization protocols discussed above as described in Green et al., 1999, J. Immunol. Methods 231:11-23, Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994) can be mentioned.

[0137] (i) in vitro cell killing assay The production of the antibodies of the present disclosure may include testing the in vitro ADCC activity of the antibodies. The improvement of the cell killing or ADCC activity of the antibodies of the present disclosure (e.g., antibodies that are not fucosylated and / or have mutations or amino acid substitutions that enhance ADCC activity, such as antibodies that include a variant or mutated Fc region and / or have a low level of internalization) can be tested for depletion of LGL and / or NK cells. Depletion of LGL and / or NK cells can be tested using an exemplary in vitro model that recapitulates the activity in humans (Tomasevic, et al, Growth Factors, 2014; 32(6): 223-235; Huang, et al, JCI insight, 2016;1(7):e86689). Peripheral blood lymphocytes (PBLs) isolated from the blood of normal (i.e., healthy) donors are incubated with antibodies having human Fc regions with and without fucose and / or with and without Fc region mutations. The killing levels of LGL or NK cells in the PBLs (e.g., in the PBL sample) are measured using any method known in the art, such as flow cytometry (e.g., as described in the examples). The cell killing activity of the antibodies (e.g., ADCC activity) can be tested, as described above, using various biological specimens obtained from patients having diseases such as chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty's syndrome, IBM and RA with LGL and / or aggressive NK leukemia, such as whole cell homogenates of blood, synovial fluid, bone marrow and spleen, using the assays described above.

[0138] In addition to the above-described cell killing assay, in vitro ADCC and antibody-dependent cellular phagocytosis (ADCP) assays can be performed using the antibodies of the present disclosure, e.g., selected candidate antibodies of the present disclosure, purified target cells (e.g., LGL or NK cells) and / or effector cells, e.g., NK cells or monocytes / macrophages, to assay cell killing, ADCC and / or ADCP activity of the antibodies of the present disclosure. For example, cell killing, ADCC and / or ADCP assays and other assay methods known in the art can be used as described in Kolbeck et al., J Allergy Clin Immunol. 2010;125(6):1344-1353.e2; Gomez-Roman et al., J. Immunol. Methods, 2006, 308, pp. 53-67; and Ackerman et al., J. Immunol. Methods, 2011, 366, pp. 8-19. The in vitro activity of the antibodies of the present disclosure can be compared to commercially available antibodies or antibodies known in the art against the same target.

[0139] (ii) in vivo cell killing assay The production of the antibodies of the present disclosure can include testing the in vivo ADCC activity of the antibodies to show, for example, the activity of an antibody candidate selected in vivo for depletion or reduction in levels of LGL or NK cells. The in vivo cell killing activity (e.g., ADCC and / or ADCP activity) of the antibodies of the present disclosure can be determined using any method known in the art. For example, the ability of the antibodies of the present disclosure to deplete or reduce LGL or NK cells in vivo can be tested using methods known in the art in cynomolgus monkeys. For example, in an exemplary method of testing the in vivo cell killing activity (e.g., ADCC and / or ADCP activity) of the antibodies of the present disclosure, a cohort of cynomolgus monkeys is bled one day prior to administration of a single dose of the antibody of the present disclosure, e.g., antibody treatment, to identify pre-administration levels of LGL and NK cells by flow cytometry. After administration of the antibody of the present disclosure, e.g., during treatment with the antibody of the present disclosure, the monkeys are bled at the following time points: 1 hour, 1 day, 7 days, 14 days, and 30 days. The levels of LGL and NK cells in blood and other biological specimens, e.g., synovial fluid, bone marrow, and spleen, are determined by flow cytometry at each time point. The in vivo activity of the antibodies of the present disclosure can be compared to commercially available antibodies or antibodies known in the art against the same target. For example, an anti-CD94 antibody of the present disclosure, e.g., an anti-CD94 mAb candidate, can be compared to the anti-CD94 antibody DX22, a commercially available anti-CD94 mAb, which has been reported to cross-react with cynomolgus CD94. One of ordinary skill in the art will readily appreciate that the ADCC activity of the antibodies of the present disclosure (e.g., transgenic animals such as transgenic mice) can be assayed in vivo using other methods known in the art for testing ADCC activity in vivo.

[0140] Other known antibodies against a target (e.g., any of CD94, CD57, or NKG2A) can also be used in the methods provided herein. For example, the anti-CD94 mAbs of the present disclosure can be tested together with the following anti-CD94 antibodies: HP-3D9 (LSBio catalog number LS-C134679-100, Abnova catalog number: MAB6947), 2I2, 131412 (R&D Systems catalog number: MAB1058), 13B146 (US Biological catalog number: 030068), 13B147 (US Biological catalog number: 030069), 1H1 (Abnova catalog number: MAB10543), 3G2 (Biorbyt catalog number: orb69389), DX22 (Biolegend catalog number 305502), REA113 (Miltenyi Biotec catalog number: 130-098-967), KP43; EPR21003, AT13E3 (ATGen catalog: ATGA0487) and B-D49 (e.g., for in vitro or in vivo ADCC activity, or for any other feature described herein).

[0141] (iii) Humanized The antibodies of the present disclosure can be humanized according to any method known in the art. In some embodiments, a humanized antibody is a chimeric antibody that includes amino acid residues derived from non-human hypervariable regions (HVRs) and amino acid residues derived from human framework regions (FRs). In certain embodiments, a humanized antibody includes substantially all of at least one, and usually two, variable domains, and all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody can optionally include at least a portion of an antibody constant region derived from a human antibody. In some embodiments, an antibody, e.g., a humanized form of a non-human antibody, refers to an antibody that has been humanized.

[0142] For example, monoclonal antibodies can be humanized by transferring mouse CDRs derived from the heavy and light variable chains of murine immunoglobulins to the corresponding variable domains of a human antibody. The mouse framework regions (FRs) in chimeric monoclonal antibodies can also be replaced with human FR sequences. To maintain the stability and antigen specificity of the humanized monoclonal antibody, one or more human FR residues can be replaced with murine counterpart residues. Humanized monoclonal antibodies can be used for the therapeutic treatment of a subject. Techniques for generating humanized monoclonal antibodies are well known in the art, such as those described in Jones et al., 1986, Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest et al., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993, 150:2844.

[0143] (iv) Selection The antibodies of the present disclosure can be selected based on the above parameters, such as enhanced in vitro and / or in vivo cell killing activity (e.g., ADCC and / or ADCP activity), enhanced binding to one or more Fc receptors, level of fucosylation (e.g., reduced fucosylation or defucosylation) and / or affinity for its target protein (e.g., any of CD94, CD57 or NKG2A).

[0144] In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their binding characteristics (e.g., affinity) to human and / or cynomolgus CD94, CD57 or NKG2A. In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their internalization ability as described above, for example. In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their cell killing, ADCC and / or ADCP activities in vivo and / or in vitro as described above, for example.

[0145] In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their solubility. In some embodiments, the antibodies of the present disclosure are selected when they are soluble at a concentration higher than about 10 mg / mL. In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on the level of soluble aggregates formed in the antibody solution. For example, the antibodies of the present disclosure are selected when they have a low level of soluble aggregates (e.g., less than 5%, less than 4%, less than 3%, less than 2% or less than 1% soluble aggregates). In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their ability to maintain binding to their target (e.g., any of CD94, CD57 or NKG2A) during storage at any of about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, for example, for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months or longer. In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their stability (e.g., no degradation products as measured by SDS-PAGE, for example) during storage at any of about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C or about 8°C, for example, for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months or longer.

[0146] In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their toxicology. Toxicological analysis of the antibodies of the present disclosure can be performed using any method known in the art. In an exemplary toxicological analysis, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, are tested for toxicity in cynomolgus monkeys at a dose that is even higher than 5-fold (e.g., about 5-fold higher, about 10-fold higher, about 15-fold higher, about 20-fold higher, about 25-fold higher, about 30-fold higher, about 35-fold higher, about 40-fold higher, about 45-fold higher, about 50-fold higher, about 55-fold higher, about 60-fold higher, about 65-fold higher, about 70-fold higher, about 75-fold higher, about 80-fold higher, about 85-fold higher, about 90-fold higher, about 95-fold higher, about 100-fold higher, or higher) than the dose predicted to be used in human subjects.

[0147] In some embodiments, the antibodies of the present disclosure, such as the humanized antibodies of the present disclosure, can be selected based on their ability to deplete and / or reduce the level of LGL and / or NK cells in vitro and / or in vivo. Depletion or reduction of the level of LGL and / or NK cells can be measured using any method known in the art. For example, depletion of LGL and / or NK cells can be measured using, for example, cell killing, ADCC, and / or ADCP assays as described above and / or as described in the Examples. In some embodiments, the final mAb candidates, if humanized, can be characterized for binding to human and cynomolgus monkey CD94 or CD57 or NKG2a, internalization, ADCC ability, and in vivo activity.

[0148] Furthermore, in some embodiments, the final candidate should be soluble at a concentration higher than 10 mg / mL, have low levels of soluble aggregates (<5%), maintain its binding to the target as measured by ELISA (>90% potency), and have no degradation products as measured by SDS PAGE when incubated at 2 - 8°C for 3 months. In some embodiments, the toxicological analysis of the final humanized candidate can be performed in cynomolgus monkeys at a dose more than 5-fold higher than the dose predicted to be used in human subjects.

[0149] In some embodiments, an antibody that binds to CD94 or CD57 or NKG2A can deplete or reduce the levels of LGL or NK cells and can have a clear benefit for patients (e.g., human patients) such as those with LGL leukemia, rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, etc. Further, antibody treatment can have better tolerance and fewer side effects than first- and second-line therapies including chemotherapy (chemo), chemotherapy, alemtuzumab, and splenectomy. Antibody treatment can demonstrate more selective depletion of disease-inducing cells compared to current non-selective therapies. Non-limiting examples of diseases and disorders in which LGL and NK cells play a role include LGL leukemia, rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, etc. Accordingly, the present invention provides a method for reducing or depleting the number of LGL or NK cells in a human subject upon administration of a molecule that binds to a cell surface protein on LGL or NK cells, such as CD94 or CD57 or NKG2A or an additional cell surface protein specific for LGL cells, and that comprises (a) a region that specifically binds to the target and (b) an immunoglobulin Fc region.

[0150] C. Antibody Target The antibodies of the present disclosure can specifically bind to CD94, CD57 or NKG2A. Also included by the present disclosure are antibodies that bind to cell surface proteins expressed on LGL and / or NK cells. Techniques for preparing antibodies against substantially any target antigen, such as monoclonal antibodies (mAbs), are well known in the art, for example, as described above. Thus, antibodies that bind to cell surface proteins expressed on LGL and / or NK cells can be used in any of the methods, compositions, articles of manufacture or kits disclosed herein.

[0151] In some embodiments, the terms binding, specifically binding, or specific refer to a measurable, reproducible interaction, for example, the binding between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which can be an epitope) binds to this target with greater affinity, avidity, more readily, and / or for a longer period than it binds to other targets. In one embodiment, the degree of binding of the antibody to an irrelevant target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (K D ) of <1 μM, <100 nM, <10 nM, <1 nM or <0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from various species. In another embodiment, specific binding can include, but is not required to include, exclusive binding.

[0152] In some embodiments, the antibodies of the present disclosure bind to human CD94, human CD57 or human NKG2A. In some embodiments, the antibodies of the present disclosure bind to cynomolgus CD94, cynomolgus CD57 or cynomolgus NKG2A. In some embodiments, the antibodies of the present disclosure bind to human and cynomolgus CD94, human and cynomolgus CD57 or human and cynomolgus NKG2A.

[0153] In some embodiments, the antibodies provided herein bind to human CD94 (Natural Killer Cell Antigen CD94; CD94 Entrez Gene ID: 3824; KLRD1 (HGNC Symbol); UniProtKB Identifier: Q13241; HGNC: 6378; Ensembl: ENSG00000134539 OMIM: 602894; KP43), human CD57 (CD57; B3GAT1 (beta-1,3-glucuronyltransferase 1); LEU7; GLCUATP 3; GlcAT-P 4; HNK1, NK-1, NK1; HGNC: 921; Entrez Gene: 27087; Ensembl: ENSG00000109956 OMIM: 151290; UniProtKB: Q9P2W7) or human NKG2A (NKG2-A / NKG2-B type II membrane-anchored protein; gene: KLRC1, UniProtKB: P26715 (NKG2A_HUMAN); CD159 antigen-like family member A; HGNC: 6374).

[0154] In some embodiments, the antibodies of the disclosure bind to human CD94 protein or a portion thereof, or a protein having at least 80% (e.g., any of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) homology to human CD94 protein or a portion thereof. The amino acid sequences of exemplary human CD94 proteins are provided in the sequences of SEQ ID NOs: 1-3:

Chemical formula

[0155] In some embodiments, the antibodies of the present disclosure bind to a human NKG2A protein or a portion thereof or a protein having at least 80% (e.g., any of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) homology to the human NKG2A protein or a portion thereof. The amino acid sequences of exemplary human NKG2A proteins are provided in the sequences of SEQ ID NOs: 4 and 5:

Chemical formula

Chemical formula

[0156] In some embodiments, the antibodies of the present disclosure bind to a human CD57 protein or a portion thereof or a protein having at least 80% (e.g., any of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) homology to the human CD57 protein or a portion thereof. The amino acid sequences of exemplary human CD57 proteins are provided in the sequences of SEQ ID NOs: 6 and 7:

Chemical formula

[0157] In some embodiments, the antibodies of the present disclosure bind to their target (e.g., any of CD94, CD57, or NKG2A) at an epitope that is the same as or different from an epitope of an antibody known in the art for the same target. In some embodiments, the antibodies of the present disclosure bind to an epitope different from an epitope of an antibody known in the art. In some embodiments, the antibodies of the present disclosure specifically bind to human CD94 and do not bind to an epitope on human CD94 that is the same as that of anti-CD94 antibody clones HP-3D9, DX22, 131412, or 12K45. In some embodiments, the antibodies of the present disclosure specifically bind to human CD57 and do not bind to an epitope on human CD57 that is the same as that of anti-CD57 antibody clone NK-1. In some embodiments, the antibodies of the present disclosure specifically bind to human NKG2A and do not bind to an epitope on human NKG2A that is the same as that of anti-NKG2A antibody clone Z199.

[0158] In some embodiments, if the antibody of the present disclosure does not bind to its target (e.g., any of CD94, CD57, or NKG2A) at an epitope that is the same as that of another antibody to the same target, such as a commercially available antibody or an antibody known in the art for the same target, the antibody of the present disclosure does not block the binding of the other antibody to the target, for example, by 50% or more, in a competition assay. In some embodiments, if the antibody of the present disclosure does not bind to its target (e.g., any of CD94, CD57, or NKG2A) at an epitope that is the same as that of another antibody to the same target, such as a commercially available antibody or an antibody known in the art for the same target, the other antibody does not block the binding of the antibody of the present disclosure to the target, for example, by 50% or more, in a competition assay.

[0159] In some embodiments, the antibodies of the present disclosure bind to their target (e.g., any of CD94, CD57, or NKG2A) with a greater affinity than an antibody known in the art for the same target. In certain embodiments, the affinity of the antibody for its target (e.g., any of CD94, CD57, or NKG2A) is the dissociation constant (K D) can be represented by. Affinity can be measured by common methods known in the art, such as flow cytometry or Western blotting. In some embodiments, K D is measured using a radiolabeled antigen-binding assay (RIA) performed using the Fab version of the antibody of the present disclosure and its target (e.g., any of CD94, CD57, or NKG2A). In some embodiments, K D is measured using a surface plasmon resonance assay. Exemplary assays are described, for example, in Drake, A.W. and Klakamp, S.L. (2007) J. Immunol. Methods 318:147-152.

[0160] In some embodiments, the antibody of the present disclosure specifically binds to human CD94 and binds to human CD94 with a greater affinity than the anti-CD94 antibody clones HP-3D9, DX22, 131412, and 12K45. In some embodiments, the antibody of the present disclosure specifically binds to human CD57 and binds to human CD57 with a greater affinity than the anti-CD57 antibody clone NK-1. In some embodiments, the antibody of the present disclosure specifically binds to human NKG2A and binds to human NKG2A with a greater affinity than the anti-NKG2A antibody clone Z199. In some embodiments, the antibody of the present disclosure binds to its target (e.g., any of CD94, CD57, or NKG2A) with an affinity that is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, or greater than any of these, compared to another antibody known in the art to the same target.

[0161] In certain embodiments, the antibody of the present disclosure has a K Dhas. In certain embodiments, the antibodies of the present disclosure have a K for binding to their target (e.g., any of CD94, CD57, or NKG2A) of less than about 1 μM D has. In certain embodiments, the antibodies of the present disclosure have a K for binding to their target (e.g., any of CD94, CD57, or NKG2A) of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM D has. In some embodiments, the antibodies of the present disclosure have a K for binding to their target (e.g., any of CD94, CD57, or NKG2A) of less than about 100 pM, less than about 75 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, less than about 1 pM, less than about 0.5 pM, or less than about 0.1 pM D has.

[0162] Other known antibodies against a target (e.g., any of CD94, CD57, or NKG2A) can also be used in the methods provided herein. For example, the following anti-CD94 antibodies can be used: HP-3D9 (LSBio catalog number LS-C134679-100, Abnova catalog number MAB6947), 2I2, 131412 (R&D Systems catalog number MAB1058), 13B146 (US Biological catalog number 030068), 13B147 (US Biological catalog number 030069), 1H1 (Abnova catalog number MAB10543), 3G2 (Biorbyt catalog number orb69389), DX22 (Biolegend catalog number 305502), REA113 (Miltenyi Biotec catalog number 130-098-967), KP43, EPR21003, AT13E3 (ATGen catalog: ATGA0487), and B-D49.

[0163] III. Pharmaceutical Formulations In some embodiments, a pharmaceutical composition, composition, or pharmaceutical formulation refers to a biologically active compound (e.g., an antibody of the present disclosure) mixed, optionally, with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickening agent, excipient, etc.

[0164] A pharmaceutical composition, pharmaceutical formulation, and / or composition of any of the antibodies of the present disclosure for use in any of the methods described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing such an antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0165] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers, such as phosphoric, citric and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGP and methods of use including rHuPH20 are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0166] The formulations herein may also contain two or more active ingredients, preferably those having complementary activities that do not adversely affect each other, as required for the particular indication being treated (e.g., a disease or disorder).

[0167] The active ingredients can be entrapped, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization, such as colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, in hydroxy methylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0168] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an antibody or immunoconjugate, and the matrix is in the form of a shaped article, e.g., a film or a microcapsule.

[0169] The formulations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0170] IV. Kits and Manufactured Articles In another aspect of the disclosure, provided are kits or articles of manufacture containing materials useful for the methods provided herein, e.g., treating the above-described diseases or disorders, reducing the number of peripheral blood LGL and / or NK cells in a subject or inducing ADCC activity in a subject. The kit or article of manufacture can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials, such as glass or plastic. The container can hold a composition that is either the container itself or a composition combined with another composition effective for the methods provided herein, e.g., treating the above-described diseases or disorders, reducing the number of peripheral blood LGL and / or NK cells in a subject or inducing ADCC activity in a subject, and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the disclosure. The label or package insert indicates that the composition is used for the methods provided herein, e.g., treating the above-described diseases or disorders, reducing the number of peripheral blood LGL and / or NK cells in a subject or inducing ADCC activity in a subject. Further, the kit or article of manufacture can include (a) a first container containing a composition comprising an antibody of the disclosure and (b) a second container containing a composition comprising a further therapeutic agent. The kit or article of manufacture in this embodiment of the invention can further include a package insert indicating that the composition can be used, e.g., to treat a specific disease or disorder as described herein to reduce the number of peripheral blood LGL and / or NK cells in a subject or to induce ADCC activity in a subject. Alternatively, or additionally, the kit or article of manufacture can further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution or dextrose solution.It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0171] The following description is presented to enable those skilled in the art to make and use various embodiments. The descriptions of specific devices, techniques, and applications are provided by way of example only. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but should be accorded a scope consistent with the claims.

Examples

[0172] (Example 1: Analysis of CD94 expression in immune cells obtained from healthy donors and patients with chronic lymphoproliferative disorders (CLPD-NK) of T large granular lymphocyte leukemia (T-LGLL) and NK cells.) This example describes the results of experiments to determine the levels of CD94 receptor expression in immune cells obtained from healthy donors and patients with T-LGLL and NK-LGLL.

[0173] Materials and Methods Healthy Donors and Patient Samples Buffy coats from fresh, healthy donors were obtained from the Stanford Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Paque (GE Healthcare, Chicago, IL) separation and cryopreserved in Bambanker cell freezing medium (Bulldog-Bio, Portsmouth, NH). Briefly, buffy coats were diluted 1:1 with phosphate-buffered saline (PBS), followed by layering the diluted buffy coats over Ficoll and centrifuging at 760 g. The PBMC layer was isolated, washed with PBS, and then downstream analysis was performed. Peripheral blood leukocytes (PBLs) were isolated by erythrocyte lysis. Cryopreserved patient LGLL PBMCs were obtained. Tissue samples were provided by the Cooperative Human Tissue Network. Tissue dissociation was performed using the Miltenyi Biotec tumor dissociation kit according to the manufacturer's instructions.

[0174] Flow cytometry analysis Approximately 1 × 10 5 cells to 5 × 10 5 cells were plated into non-tissue culture-treated, 96-well V-bottom plates and incubated for 10 minutes at room temperature in human FcγR blocking antibody (Biolegend, San Diego, CA). Cells were then stained with a 1:1000 dilution of eFluor506 viability dye (ThermoFisher, Waltham, MA) for 30 minutes on ice, followed by a wash step with FACS buffer (PBS with 2% fetal bovine serum). Antibody cocktails were added to the cells and incubated for 30 minutes on ice, followed by additional wash steps in FACS buffer. Ultracomp beads (ThermoFisher, Waltham, MA) were used for antibody compensation. The antibodies used in this study are provided in Table 1.

[0175] All data acquisition and fluorescence compensation were performed using a CytoFlex (Beckman Coulter, Atlanta, GA). Data analysis was performed using FlowJo software. Single cells were gated using forward scatter area and forward scatter height, followed by gating of viable cells using eFluor506 and forward scatter area. Monocytes were gated using a CD14+ strategy, T cells were gated using a CD3+ / CD4+ / CD8+ strategy, B cells were gated using a CD3-CD19+ strategy, NK cells were gated using a CD3- / CD56+ / CD57+ / CD16+ strategy, T-LGL leukemia cells were gated using a CD3+CD16+ strategy, NK-LGL leukemia cells were gated using a CD3-CD16+ strategy, and epithelial cells were identified using a CD45- strategy. Granulocytes were individually gated by forward and side scatter.

[0176] CD94, CD56, and NKG2A expression was determined in individual immune cell populations using the markers described above.

[0177] Receptor quantification The number of CD94, CD56, and NKG2A receptors was quantified by staining PBMCs with APC-conjugated anti-target antibodies and gating based on appropriate immune cell populations (e.g., as described above). Quantum APC soluble fluorochrome molecule equivalents (MESF) calibration standard beads (Bangs Laboratories, Inc. Fishers, IN) were analyzed simultaneously to enable conversion of median fluorescence intensity (MFI) measurements to MESF units according to the manufacturer's protocol. Background fluorescence was removed by subtracting FMO (fluorescence minus one) and isotype control MESF values. Subsequently, the MESF values were divided by the fluorophore-to-protein ratio (provided by the manufacturer) to convert to antibody binding capacity or receptor number.

[0178] Antibodies Table 1 provides the antibodies used in the experiments described in Examples 1 to 3.

Table 1-1

Table 1-2

[0179] Results Healthy donors PBMC samples obtained from 6 healthy donors were used to screen for CD94 expression. Additional 3 PBMC samples and 2 peripheral blood leukocyte (PBL) samples obtained from healthy donors were used to screen for CD57 and NKG2A expression. Target expression in granulocytes was analyzed in 2 PBL samples. As shown in Table 2, CD94 was highly expressed in NK cells (>50%) as shown by staining using all 4 anti-CD94 commercially available antibody clones. CD94 expression was low or not detected in monocytes, CD3+CD4+ T cells and B cells. A small subset of CD3+CD8+ T cells expressed CD94 (approximately 10 - 30%). NK cells (CD3-CD56+ and CD3-CD16+) expressed CD57 and NKG2A in the ranges of 60 - 70% and 40 - 45%, respectively. 15% of CD3+CD4+ T cells and 45% of CD3+CD8+ T cells also expressed CD57. Granulocytes did not express any of the targets. All target expression results reproduced the results reported in the literature (see, for example, Loughran TP J. (1993) Blood, 82(1):1-14; Zambello R (2014) Tansl Med UniSa, 8:4-11). Overall, these results showed that CD94 is selectively expressed in NK cells and a subset of CD3+CD8+ T cells.

Table 2

[0180] Blood samples obtained from healthy donors were also analyzed to determine the number of CD94 receptors on CD14+ monocytes, CD3+CD4+ and CD3+CD8+ T cells, CD3-CD19+ B cells, granulocytes (based on FSC / SSC), and CD3-CD57+, CD3-CD16+ and CD3- / CD56+ NK cells.

[0181] The results of flow cytometric analysis of CD94 in PBL samples from representative healthy donors are provided in Figure 1A. CD94 was highly expressed on NK cells, and the number of CD94 receptors was in the range of approximately 40,000 to approximately 50,000 per cell. CD94 was not detected on CD14+ monocytes, granulocytes (FSC / SSC), CD3+CD4+ T cells, and CD3-CD19+ B cells. CD3+CD8+ T cells expressed CD94 in the range of 15 - 40%. Overall, these results showed that CD94 was selectively expressed in all NK cells and a subset of CD8+ T cells and was not detected in other cells in PBL samples from representative healthy donors.

[0182] Figure 1B shows the analysis of cell surface CD94 receptor density to quantify CD94 expression in immune cells in samples obtained from PBMC and PBL samples of six healthy donors. Specifically, anti-CD94 mAb clone HP-3D9 was used in CD14+, CD3+CD4+, CD3-CD19+, CD3+CD8+CD94-, CD3+CD8+CD94+, CD3-CD56+, CD3-CD57+ and CD3-CD16+ cells to evaluate CD94 expression in PBMC samples of six healthy donors. Furthermore, PBL samples obtained from two healthy donors were used to evaluate CD94 expression in granulocytes. CD94 receptor expression was abundant in NK cells and ranged from approximately 70,000 to approximately 120,000 receptors / cell (mean = 117,200). In monocytes (CD14+), granulocytes, T cells (CD3+CD4+, CD3+CD8+) and B cells (CD3-CD19+), CD94 expression was less than 4,000 receptors / cell. Most (60 - 85%) of the CD3+CD8+ T cells were CD94 negative. Overall, these results showed that CD94 receptor density was high in all NK cells, low in a subset of CD8+ cells, and not detected in other cell populations in PBMC and PBL samples from healthy donors.

[0183] T-LGLL patients Blood samples obtained from T-LGLL patients were analyzed to determine the number of CD94 receptors on CD14+ monocytes, CD3+CD4+ T cells, CD3-CD19+ B cells, CD3+CD16- T lymphocytes, CD3+CD16+ leukemic cells and CD3-CD16+ NK cells. CD3+CD16+ leukemic cells corresponded to >55% of lymphocytes in PBMC samples from these patients compared to <10% in PBMC samples obtained from healthy individuals. Figure 2A shows CD94 bright provides an analysis of CD94 expression and receptor quantification in cells obtained from samples of T-LGLL patients, and Figure 2B shows CD94 dim expression and receptor quantification in cells obtained from samples of T-LGLL patients.

[0184] As shown in FIGS. 2A-2B, CD94 was expressed in CD3+CD16+ leukemic cells as well as in subsets of CD3+CD16- T lymphocytes and CD3-CD16+ NK cells. Expression of CD94 in leukemic cells was variable between CD94 bright and CD94 dim patient samples. As shown in FIG. 2A, in the CD94 bright sample, CD3+CD16+ leukemic cells showed >170,000 CD94 receptors and a MFI of 10,000. The high expression of CD94 in CD3+CD16+ leukemic cells in the CD94 bright sample suggested that these cells would be completely depleted by ADCC if they received binding by an anti-CD94 antibody. As shown in FIG. 2B, in the CD94 dim sample, CD3+CD16+ leukemic cells showed approximately 12,000 CD94 receptors and a MFI of 1,000. Others had previously reported that a subset of T-LGLL patients, including patients with an immune cell marker profile similar to the CD94 dim patient sample in FIG. 2B, were negative for CD94 expression (see, e.g., Barila (2019) Leukemia), so this finding was surprising. C94 expression in T-LGLL patient monocytes (CD14+), CD3+CD4+ T cells and CD3-CD19+ B cells was not detected in either the CD94 dim patient sample or the CD94 bright patient sample. Overall, these results showed that CD94 was expressed in leukemic cells, NK cells, subsets of CD3+CD16- T cells and not detected in other cells in PBMCs obtained from T-LGLL patients. This analysis represents the first determination of the number of CD94 receptors on T-LGLL cells. The finding of CD94 expression in CD3+CD16+ leukemic cells in the CD94 dim patient sample suggested that 12,000 receptors expressed on leukemic cells were sufficient to completely deplete these cells by ADCC if they received binding by an anti-CD94 antibody.

[0185] CLPD-NK patients Flow cytometry analysis of CD94 expression and receptor quantification was also performed on blood samples obtained from patients with chronic lymphoproliferative disorder of NK cells (CLPD-NK). CD3-CD16+ leukemia cells corresponded to 70% of lymphocytes in the PBMC samples of this patient, as compared to 5-10% in PBMC samples obtained from healthy individuals.

[0186] As shown in Figure 3, CD94 was expressed in CD3-CD16+ NK leukemia cells, and the receptor density was 500,000 per cell. CD94 expression was not detected in CLPD-NK patient monocytes (CD14+), CD4+ T cells (CD3+CD4+), and B cells (CD3-CD19+). CD94 was expressed in a small percentage (18%) of CD3+CD8+ T cells. Overall, these results showed that CD94 expression was high in leukemia CLPD-NK cells, low in CD3+CD8+ T cells, and not detected in other PBMCs obtained from CLPD-NK patients. This analysis is considered to correspond to the first determination of the number of CD94 receptors on CLPD-NK cells. The extremely high expression of CD94 in leukemia cells suggested that these cells would be completely depleted by ADCC when bound by anti-CD94 antibodies.

[0187] (Example 2: Analysis of the effect of anti-NKG2A antibody on NKG2A expression and ADCC activity in immune cells obtained from healthy donors and patients with chronic lymphoproliferative disorder of NK cells (CLPD-NK).) This example describes the results of experiments to determine the levels of NKG2A receptor expression in immune cells obtained from healthy donors and patients with CLPD-NK. This example also shows the results of experiments measuring the effect of anti-NKG2A antibody on antibody-dependent cell-mediated cytotoxicity (ADCC).

[0188] Materials and Methods Antibody-Dependent Cell-Mediated Cytotoxicity Assay Approximately 1×10 5 ~2×10 5Individual fresh or frozen PBMCs were plated in 96-well U-bottom plates that had been tissue culture-treated in RPMI with 10% low IgG FBS. The cells were incubated overnight in 10-fold dilutions of human IgG1 isotype control antibody, NKG2A Z199 fucosylated antibody or NKG2A Z199 non-fucosylated antibody, and the antibody concentration was 10 1 ~10 -6 μg / ml. The cells were stained with CD3, CD56 and CD16 to identify remaining NK cells (e.g., as described in Example 1). At least 10,000 events were collected by flow cytometry in the lymphocyte population. The percentage of remaining NK / leukemia cells was calculated by normalizing the absolute number by the number of cells in the isotype-treated condition. The EC50 was determined by GraphPad Prism.

[0189] Results Healthy donors Blood obtained from healthy donors was analyzed to determine the number of NKG2A receptors on CD56 bright NK cells. As shown in Figure 4A, the number of NKG2A receptors on CD3-CD56+ NK cells was 800,000.

[0190] To determine whether NK cells can mediate ADCC against NK cells, an ADCC assay was performed on freshly isolated PBMCs from healthy donors using Z199 fucosylated and non-fucosylated anti-NKG2A antibodies. As shown in Figure 4B, NK cells were depleted in a dose-dependent manner, with EC50s of 40 ng / ml and 3 ng / ml for fucosylated and non-fucosylated Z199 antibodies, respectively. Overall, these results showed that the Z199 NKG2A antibody selectively reduced healthy donor NK cells in a dose-dependent manner, and the non-fucosylated antibody was approximately 13-fold more potent than the fucosylated antibody.

[0191] T cell NKG2A expression from healthy donor PBMC samples was also analyzed. As shown in Figure 5A, 20% of CD3+CD8+ T cells expressed NKG2A and had 285,000 receptors. To examine whether NKG2A-negative cells were resistant to anti-NKG2A Z199 antibody-mediated ADCC killing, an ADCC assay was performed using fresh PBMC obtained from healthy donors. Cells were incubated overnight with fucosylated and non-fucosylated IgG1 isotype controls and anti-NKG22A Z199 antibody. As shown in Figure 5B, the majority of NKG2A-negative CD3+CD8+ T cells were not depleted at all when using the Z199 antibody at the tested concentrations. Overall, these results showed that the Z199 NKG2A antibody did not deplete NKG2A-negative T cells obtained from healthy donors.

[0192] CLPD-NK patients Blood obtained from CLPD-NK patients was analyzed to determine the number of NKG2A receptors on CD3-CD16+ NK leukemia cells. As shown in Figure 6A, 100% of CD3-CD16+ NK leukemia cells expressed NKG2A and had 500,000 NKG2A receptors.

[0193] To determine whether NK leukemia cells could mediate ADCC against NK leukemia cells, an ADCC assay was performed using cells from previously frozen CLPD-NK patient samples with non-fucosylated IgG1 isotype control and anti-NKG2A Z199 antibody. As shown in Figure 6B, NK cells were depleted in a dose-dependent manner and the EC50 was 3 ng / ml. Since NK leukemia cells were the only cells with cytotoxic activity in this patient sample (as demonstrated by CD16 expression), the observed depletion of leukemia cells suggested that NK leukemia cells mediated ADCC against the same cell type. Overall, these results showed that non-fucosylated anti-NKG2A Z199 antibody effectively depleted NK leukemia cells (CD3-CD16+).

[0194] Blood obtained from CLPD-NK patients was also analyzed to determine the number of NKG2A receptors on CD3+CD16- T cells. As shown in Figure 7A, CD3+CD16- T cells were negative for NKG2A expression. To determine whether NKG2A-negative cells were resistant to anti-NKG2A Z199 antibody-mediated ADCC killing, an ADCC assay was performed using previously frozen CLPD-NK patient samples together with non-fucosylated IgG1 isotype control and anti-NKG2A Z199 antibody. As shown in Figure 7B, NKG2A-negative CD3+CD16- T cells were not depleted at all when using the tested concentrations of anti-NKG2A Z199 antibody. These results indicated that non-fucosylated anti-NKG2A Z199 antibody did not deplete NKG2A-negative T cells obtained from CLPD-NK patients.

[0195] (Example 3: Analysis of NKG2A and CD94 expression in liver-derived cells.) This example describes the results of experiments to determine the levels of CD94 and NKG2A receptor expression in liver-derived immune cells obtained from healthy donors.

[0196] Materials and Methods Single and viable liver-derived cells (CD45-) and lymphocyte populations (CD45 / CD4 / CD8 / CD19 / CD56+) were analyzed by flow cytometry as described in Example 1.

[0197] Results Single and viable liver-derived cells (CD45-) and lymphocyte populations (CD45 / CD4 / CD8 / CD19 / CD56+) were examined and screened for CD94 and NKG2A expression. As shown in Figure 8A, CD94 was highly expressed in NK cells of normal liver samples and had approximately 200,000 CD94 receptors per cell. CD94 expression was also present in subsets of T cells (CD45+CD3+CD4+ / CD8+). CD94 expression was not detected in epithelial cells (CD45-) and B cells (CD45+CD3-CD19+). As shown in Figure 8B, NKG2A was detected only in NK cells and had 200,000 receptor numbers. Overall, these results showed that CD94 and NKG2A were expressed in NK cells in normal liver samples. CD94 and NKG2A expression was also detected at low levels in T cells in normal liver samples.

[0198] (Example 4: Analysis of antibody-dependent cellular cytotoxicity (ADCC) mediated by anti-NKG2A antibody) To determine whether T leukemia cells can mediate ADCC against T leukemia cells, an ADCC assay was performed using cells obtained from previously frozen T-LGLL patient samples (PBMC) together with a non-fucosylated IgG1 isotype control and the Z199 antibody.

[0199] Cells were treated overnight with the isotype and non-fucosylated Z199 antibody using five concentrations in the range of 0 - 1 μg / ml. The Y-axis is shown as the number of remaining leukemia cells (CD3+CD16+) under conditions treated with Z199 and human IgG1 isotype.

[0200] As shown in FIGS. 9A and 9B, T-LGLL cells were depleted in a dose-dependent manner by the Z199 antibody, but not by the isotype control. Since T-LGL leukemia cells were the only cells with cytotoxic activity in this patient sample (as demonstrated by the expression of CD16), the depletion of leukemia cells suggests that the leukemia cells mediated ADCC against the same cell type.

[0201] These results demonstrate that the non-fucosylated anti-NKG2A antibody Z199 efficiently depleted T leukemia cells.

[0202] (Example 5: Effect of IL-2 on CD94 expression) CD94 expression was measured over time in normal NK cells cultured with IL-2.

[0203] NK cells purified from PBMC of healthy donors were cultured in IL-2 (50 ng / ml) from day 0 to day 4. CD94 expression, shown as the median fluorescence intensity by flow cytometry, was determined by comparison with fluorescence minus one (FMO) and isotype controls.

[0204] As shown in FIG. 10, CD94 expression increased over time during culture with IL-2 treatment. These results demonstrate that CD94 expression on NK cells was upregulated in the presence of IL-2.

[0205] The present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, but the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety. In certain embodiments, for example, the following items are provided. (Item 1) A method for treating a disease or disorder in a subject, comprising administering to the subject an antibody that specifically binds to a cell surface protein selected from the group consisting of human CD94, human CD57, and human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region, and wherein the disease or disorder is selected from the group consisting of chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, and inflammatory bowel disease. (Item 2) The method according to item 1, wherein administration of the antibody results in a reduction in the number of peripheral blood LGL or NK cells in the subject. (Item 3) A method for reducing the number of peripheral blood LGL and / or NK cells in a subject, comprising administering to the subject an antibody that specifically binds to a cell surface protein selected from the group consisting of human CD94, human CD57, and human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region, and wherein the subject has a disease or disorder selected from the group consisting of LGL leukemia, Felty syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, and inflammatory bowel disease. (Item 4) A method for inducing ADCC activity in a subject, comprising administering to the subject an antibody that specifically binds to a cell surface protein selected from the group consisting of human CD94, human CD57, and human NKG2A, wherein the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region, and wherein the subject has a disease or disorder selected from the group consisting of chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, and inflammatory bowel disease, and wherein administration of the antibody to the subject results in a reduction in the number of peripheral blood LGL and / or NK cells in the subject. (Item 5) The method according to any one of items 2 to 4, wherein at least about 2,000 receptors per cell of the cell surface protein are expressed on the surface of the peripheral blood LGL and / or NK cells in the subject. (Item 6) The method according to any one of items 2 to 5, wherein the reduction in the number of peripheral blood LGL or NK cells in the subject comprises a reduction of at least about 25% compared to the number of peripheral blood NK cells in the human subject before administration of the antibody. (Item 7) The method according to any one of items 2 to 6, wherein the reduction in the number of peripheral blood LGL and / or NK cells in the subject occurs within the first 24 hours after administration of the antibody to the subject. (Item 8) The method according to any one of items 2 to 7, wherein the number of peripheral blood LGL and / or NK cells in the subject is reduced below the limit of clinical diagnosis of the disease or disorder. (Item 9) The method according to item 8, wherein the reduction in the number of peripheral blood LGL and / or NK cells in the subject below the limit of clinical diagnosis of the disease or disorder is present in the subject for at least about 1 week after administration of the antibody to the subject. (Item 10) The method according to any one of items 2 to 9, wherein the number of peripheral blood LGL and / or NK cells in the subject is reduced below the limit of detection of the peripheral blood LGL and / or NK cells in the subject. (Item 11) The method according to item 10, wherein the reduction in the number of peripheral blood LGL and / or NK cells in the subject below the limit of detection of the peripheral blood LGL and / or NK cells in the subject is present in the subject for at least about 1 week after administration of the antibody to the subject. (Item 12) The method according to any one of items 2 to 11, wherein the reduction in the number of peripheral blood LGL and / or NK cells in the subject is reversible. (Item 13) The method according to any one of items 1 to 12, wherein administration of the antibody to the subject results in a reduction in the number of peripheral blood NK cells in the subject. (Item 14) The method according to item 13, wherein the NK cells in the subject are CD3-negative and CD56-positive, CD3-negative and CD16-positive, CD3-negative and CD57-positive, CD3-negative and CD94-positive, or CD3-negative and NKG2A-positive. (Item 15) The method according to item 13 or item 14, wherein the antibody has an EC50 between about 3 ng / ml and about 40 ng / ml. (Item 16) The method according to any one of items 1 to 15, wherein administration of the antibody to the subject does not result in a reduction in T cells in the subject. (Item 17) The method according to item 16, wherein the T cells in the subject are CD3-positive and CD4-positive or CD3-positive and CD16-negative. (Item 18) The method according to any one of items 1 to 17, wherein the subject is human. (Item 19) The method according to any one of items 1 to 18, wherein administration of the antibody to the subject does not result in tumor lysis syndrome in the subject. (Item 20) The method according to any one of items 1 to 19, wherein the antibody comprises a non-fucosylated human IgG1 Fc region. (Item 21) The method according to any one of items 1 to 20, wherein the antibody binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. (Item 22) The method according to item 21, wherein the human cell Fc gamma receptor IIIA comprises the sequence of SEQ ID NO: 8 or 9. (Item 23) The antibody is (a) Specifically binds to human CD94 and does not bind to the epitope on human CD94 that is the same as that bound by anti-CD94 antibody clones HP-3D9, DX22, 131412 or 12K45, (b) Specifically binds to human CD57 and does not bind to the epitope on human CD57 that is the same as that bound by anti-CD57 antibody clone NK-1, or (c) Specifically binds to human NKG2A and does not bind to the epitope on human NKG2A that is the same as that bound by anti-NKG2A antibody clone Z199, The method according to any one of items 1 to 22. (Item 24) The antibody is (a) Specifically binds to human CD94 and binds to human CD94 with an affinity greater than that of anti-CD94 antibody clones HP-3D9, DX22, 131412 and 12K45, (b) Specifically binds to human CD57 and binds to human CD57 with an affinity greater than that of anti-CD57 antibody clone NK-1, or (c) Specifically binds to human NKG2A and binds to human NKG2A with an affinity greater than that of anti-NKG2A antibody clone Z199, The method according to any one of items 1 to 22. (Item 25) The disease or disorder is Felty syndrome, and administration of the antibody to the subject results in reduction of one or more Felty syndrome symptoms in the subject, The method according to any one of items 1 to 24. (Item 26) The disease or disorder is inclusion body myositis, and administration of the antibody to the subject results in reduction of one or more inclusion body myositis symptoms in the subject, The method according to any one of items 1 to 24. (Item 27) The disease or disorder is aggressive NK leukemia, and administration of the antibody to the subject results in reduction of one or more aggressive NK leukemia symptoms in the subject, The method according to any one of items 1 to 24. (Item 28) The method according to any one of items 1 to 24, wherein the disease or disorder is rheumatoid arthritis and the administration of the antibody to the subject results in a reduction of one or more rheumatoid arthritis symptoms in the subject. (Item 29) The method according to any one of items 1 to 24, wherein the disease or disorder is LGL leukemia and the administration of the antibody to the subject results in a reduction of one or more LGL leukemia symptoms in the subject. (Item 30) The method according to any one of items 1 to 24, wherein the disease or disorder is CLPD-NK and the administration of the antibody to the subject results in a reduction of one or more CLPD-NK symptoms in the subject. (Item 31) A method for treating CLPD-NK in a human subject in need of treatment for CLPD-NK, comprising administering to the human subject an effective amount of an antibody, wherein the antibody specifically binds to human NKG2A and the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region. (Item 32) The method according to item 31, wherein the antibody does not bind to the same epitope on human NKG2A as the anti-NKG2A antibody clone Z199. (Item 33) The method according to item 31 or item 32, wherein the antibody binds to human NKG2A with a greater affinity than the anti-NKG2A antibody clone Z199. (Item 34) A method for treating CLPD-NK in a human subject in need of treatment for CLPD-NK, comprising administering to the human subject an effective amount of an antibody, wherein the antibody specifically binds to human CD94 and the antibody comprises a human immunoglobulin Fc region having enhanced ADCC activity compared to the wild-type IgG1 Fc region. (Item 35) The method according to item 34, wherein the antibody does not bind to the same epitope on human CD94 as the anti-CD94 antibody clones HP-3D9, DX22, 131412 or 12K45. (Item 36) The method according to item 34 or item 35, wherein the antibody binds to human CD94 with a greater affinity than anti-CD94 antibody clones HP-3D9, DX22, 131412, and 12K45. (Item 37) The method according to any one of items 31 to 36, wherein the administration of the antibody to the human subject results in a reduction in the number of peripheral blood LGL or NK cells in at least about 25% of the human subjects compared to the number of peripheral blood NK cells in the human subject before the administration of the antibody. (Item 38) The method according to any one of items 31 to 37, wherein the NK cells in the human subject are CD3-negative and CD56-positive, CD3-negative and CD16-positive, CD3-negative and CD57-positive, CD3-negative and CD94-positive, or CD3-negative and NKG2A-positive. (Item 39) The method according to any one of items 31 to 38, wherein the administration of the antibody to the human subject does not result in a reduction in T cells in the human. (Item 40) The method according to item 39, wherein the T cells in the human subject are CD3-positive and CD4-positive or CD3-positive and CD16-negative. (Item 41) The method according to any one of items 31 to 40, wherein the administration of the antibody to the human subject does not result in tumor lysis syndrome in the human. (Item 42) The method according to any one of items 31 to 41, wherein the antibody comprises a non-fucosylated human IgG1 Fc region. (Item 43) The method according to any one of items 31 to 42, wherein the antibody binds to human cell Fc gamma receptor IIIA to a greater extent than an antibody comprising a wild-type human IgG1 Fc region. (Item 44) The method according to item 43, wherein the human cell Fc gamma receptor IIIA comprises the sequence of SEQ ID NO: 8 or 9. (Item 45) The method according to any one of items 31 to 44, wherein administration of the antibody to the human subject results in improvement of one or more CLPD-NK symptoms in the human.

Claims

[Claim 1] The invention described in the specification.

Images