Anti-NKG2a antibodies and uses thereof

Abstract

To provide isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to human natural killer cell inhibitory receptor group 2A (NKG2A) proteins with high affinity and that exhibit therapeutically desirable functional properties, e.g., for treatment of cancer.SOLUTION: The invention provides an isolated monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds human natural killer cell inhibitory receptor group 2A (NKG2A) proteins and exhibits at least one of: reduced binding and / or interaction of HLA-E to / with human NKG2A protein; reversed NKG2A-mediated inhibitory signaling; enhanced natural killer cell response; enhanced functional activity of T cells; reduced binding to human Fc gamma receptor (FcγR); and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 768,471, filed November 16, 2018, and U.S. Provisional Application No. 62 / 927,211, filed October 29, 2019, both of which are incorporated by reference in their entireties.

[0002] The present invention relates to anti-NKG2A (natural killer cell inhibitory receptor group 2A) antibodies and pharmaceutical compositions thereof. The present invention also relates to methods for using such antibodies, including methods for treating diseases such as cancer by administering anti-NKG2A antibodies and pharmaceutical compositions thereof.

[0003] References to sequence listings submitted electronically via EFS-WEB This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 4, 2019, is named 13119-WO-PCT_SL.txt and is 273,467 bytes in size. [Background technology]

[0004] Cancer is a global epidemic. According to the Global Health Data Exchange, it is one of the leading causes of disease and the second leading cause of death, causing approximately 17% of deaths worldwide. (Hannah Ritchie and Max Roser, "Causes of Death - Share of deaths by cause, World, 2017," OurWorldInData.org, 2018, available at https: / / ourworldindata.org / grapher / share-of-deaths-by-cause-2016). According to the World Health Organization, even in 2010, the economic impact of cancer was $1.16 trillion, and in 2018, cancer accounted for an estimated 9.6 million deaths worldwide. ("Cancer." World Health Organization. World Health Organization, 2018, available at https: / / www.who.int / news-room / fact-sheets / detail / cancer). According to National Cancer Institute estimates, in 2019, more than 1.7 million new cases of cancer will be diagnosed and more than 600,000 patients will die from cancer in the United States. ("Cancer Stat Facts: Cancer of Any Site." SEER Training Modules, US National Cancer Institute, Institutes of Health, National Cancer Institute, 2019<https: / / seer.cancer.gov / statfacts / html / all.html> ).

[0005] Traditional cancer treatments include surgery, radiation therapy, and chemotherapy, among other therapies. In recent years, immuno-oncology, or immunotherapy, has emerged as a new option for treating cancer using the body's immune system. Immuno-oncology differs from traditional cancer treatments, which, for example, have attempted to directly target tumors and / or destroy tumor blood supplies. Instead, immuno-oncology is designed to harness the patient's own immune system to help restore or enhance the patient's anti-tumor immune response. Without an immuno-oncology approach, the patient's own immune response often fails to hinder tumor growth for a variety of reasons. For example, many tumors have developed specialized mechanisms to evade the patient's immune response. Tumor cells may also have lost expression of antigens that can be recognized by the patient's immune system. In other cases, the rapid growth of a tumor may even outstrip the immune system's ability to effectively control the tumor. (Abbas et al., "Chap. 18: Immunity to Tumors," in Cellular and Molecular Immunology, 9 th ed. Elsevier, Inc., (2018)). Understanding how the immune system influences cancer development and how it can be used to treat cancer presents a challenging, multifaceted problem. For example, many patients do not respond to existing immuno-oncology treatments, and some develop resistance mechanisms such as T-cell exhaustion, in which a specific type of white blood cell, T-cells, no longer function properly. (Dempke et al., Eur. J. of Cancer, 74: 55-72 (2017)). Summary of the Invention [Problem to be solved by the invention]

[0006] Patients need improved treatments for diseases such as cancer that improve upon conventional treatments, including currently available cancer immunotherapies. There is a great need for novel immuno-oncology agents, used either alone or in combination with existing agents, to improve patient response rates and overcome drug resistance. [Means for solving the problem]

[0007] In some aspects, the present invention provides isolated monoclonal antibodies (e.g., humanized and human monoclonal antibodies) that bind to the human NKG2A protein (SEQ ID NO: 1), i.e., anti-NKG2A antibodies, e.g., anti-hNKG2A antibodies, that exhibit desirable functional properties. In one aspect, the desirable functional properties of the anti-NKG2A antibodies disclosed herein include stimulating an immune response, e.g., treating cancer. In another aspect, the desirable functional properties of the anti-NKG2A antibodies disclosed herein include treating subjects infected with viruses, including human patients. In some embodiments, the anti-NKG2A antibodies disclosed herein treat infectious diseases. In another aspect, the anti-NKG2A antibodies disclosed herein treat autoimmune conditions. In other embodiments, the anti-NKG2A antibodies of the present invention are used as antagonist anti-NKG2A antibodies to stimulate and / or enhance an immune response in a subject, e.g., to stimulate and / or enhance an anti-tumor response of the immune system, including natural killer cells and / or T cells. In other embodiments, the anti-NKG2A antibodies of the present invention are used in combination with other antibodies to treat a variety of conditions, including cancer, infectious diseases, including viral infections, and autoimmune diseases. Thus, in some embodiments, the anti-NKG2A antibodies disclosed herein are used, either alone or in combination with other treatments, e.g., other immuno-oncology treatments and / or chemotherapy and / or surgery, to treat a variety of conditions or diseases, including cancer and viral infections. In other embodiments, the anti-NKG2A antibodies disclosed herein are used in methods for detecting NKG2A protein in a sample.

[0008] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A and has the following characteristics: (a) reducing (e.g., blocking) the binding and / or interaction of an NKG2A ligand (e.g., HLA-E in humans) with the human NKG2A protein; (b) reversing NKG2A-mediated inhibitory signaling; (c) does not bind to human NKG2C protein or binds with low affinity; (d) binding to human and / or cynomolgus monkey NKG2A; (e) enhancing natural killer cell responses; (f) enhancing the functional activity of T cells; (g) reduced binding to human Fc gamma receptors (FcγR); (h) inducing and / or enhancing anti-tumor immune responses; (i) inducing and / or enhancing an antiviral immune response; and / or (j) having low immunogenicity in subjects, including human subjects; The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, which exhibits at least one of the following:

[0009] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof has the following characteristics: (a) an EC of about 0.6 nM or less for binding to human NKG2A protein as measured by a cell binding assay 50 having value, (b) an EC of about 9.0 nM or greater for binding to human NKG2C protein as measured by a cell binding assay. 50 having value, (c) a second EC for binding to human NKG2C protein with respect to binding to human NKG2A protein; 50 EC values ​​approximately 15 times lower than 50 having value, (d) an IC of about 1.0 nM or less with respect to reducing the binding and / or interaction of HLA-E with human NKG2A protein as measured by a cell-blocking assay 50 having value, (e) a K of about 0.4 nM or less as measured by Scatchard analysis D and binds to the human NKG2A protein. (f) a K of about 61 nM or less as measured by surface plasmon resonance D and bind to human NKG2A. (g) a K of about 1.0 nM or less as measured by Scatchard analysis D It binds to cynomolgus monkey NKG2A, (h) be internalized upon binding to NKG2A-expressing cells; (i) increasing interferon-gamma (IFNγ) production; (j) an EC of about 0.5 nM or less 50 Indicating internationalization in values, and / or (k) The half-life of the anti-NKG2A antibody:NKG2A complex is about 40 seconds or longer. In some embodiments, the half-life of the anti-NKG2A antibody:NKG2A complex is measured using surface plasmon resonance analysis.

[0010] In one embodiment, the anti-NKG2A antibodies or antigen-binding fragments thereof disclosed herein reduce (eg, block) the interaction between human NKG2A protein and human NKG2A ligand (ie, HLA-E).

[0011] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein, comprising: (a) a heavy chain variable domain comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15; (b) a heavy chain variable domain comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 154, 14, and 15; or (c) a heavy chain variable domain comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 155, 14, and 15. The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

[0012] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein and comprises a heavy chain and a light chain variable region, each, (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:167; and / or (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:9, SEQ ID NO:164, or SEQ ID NO:169; An isolated monoclonal antibody or antigen-binding fragment thereof is provided.

[0013] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[0014] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:164.

[0015] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 167 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 169.

[0016] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein, wherein the heavy and light chains comprise: (a) the amino acid sequences of SEQ ID NOs: 7 and 5, respectively; (b) the amino acid sequences of SEQ ID NOs: 7 and 19, respectively; or (c) the amino acid sequences of SEQ ID NOs: 35 and 36, respectively. The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, consisting essentially of:

[0017] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof competes with or binds to the same epitope as an anti-NKG2A antibody disclosed herein for binding to an NKG2A protein.

[0018] In another aspect, the present invention provides a method for treating NKG2A by isolating the following amino acid residues when bound to human NKG2A protein, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS): (a) LSIDNEEMKF (SEQ ID NO: 156); (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157); (c) LAFKHEIKDSDN (SEQ ID NO: 158); and (d) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to

[0019] In some embodiments, the monoclonal antibody blocks the binding of an NKG2A ligand (eg, HLA-E in humans) to the human NKG2A protein.

[0020] In another aspect, the present invention provides a method for the production of NKG2A-binding proteins that, when bound to human NKG2A, bind to the following amino acid residues as determined by HDX-MS and / or rapid photochemical oxidation (FPOP) epitope mapping of the protein: (a) LSIDNEEMKF (SEQ ID NO: 156) (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157) (c) LAFKHEIKDSDN (SEQ ID NO: 158) (d) L; and (e) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to

[0021] In some embodiments, the antibody blocks binding of an NKG2A ligand (eg, HLA-E in humans) to the human NKG2A protein.

[0022] In some embodiments, the isolated monoclonal antibody is a full-length antibody. In other embodiments, the isolated monoclonal antibody is a full-length IgG1 antibody. In some other embodiments, the isolated monoclonal antibody is an antibody fragment. In other embodiments, the antibody fragment is a Fab, Fab', (Fab')2, Fv, or scFv fragment. In other embodiments, the isolated monoclonal antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0023] In one aspect, the present invention provides an isolated full-length monoclonal antibody that specifically binds to human NKG2A protein, comprising: (a) the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and / or (b) the light chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:5; Isolated full-length monoclonal antibodies are provided.

[0024] In some embodiments, the heavy chain of the isolated full length monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:5.

[0025] In another aspect, the isolated full-length monoclonal antibody specifically binds to human NKG2A protein, wherein the heavy chain consists essentially of the amino acid sequence set forth in SEQ ID NO:7 and the light chain consists essentially of the amino acid sequence set forth in SEQ ID NO:5.

[0026] In another aspect, the invention provides isolated nucleic acid molecules encoding the heavy chain variable region and / or the light chain variable region of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the nucleic acid molecule is complementary DNA (cDNA).

[0027] In another aspect, the present invention provides an expression vector comprising a nucleic acid molecule described herein. In another aspect, the present invention provides a host cell transformed with an expression vector described herein.

[0028] In another aspect, the invention provides an immunoconjugate comprising an antibody described herein linked to an agent.

[0029] In another aspect, the invention provides methods of producing an antibody, comprising culturing a host cell described herein. In some embodiments, the method further comprises recovering the antibody from the host cell.

[0030] In another aspect, the present invention provides bispecific molecules comprising an anti-NKG2A antibody described herein linked to a second functional moiety.

[0031] In another aspect, the present invention provides a composition comprising an anti-NKG2A antibody described herein or a bispecific molecule described herein and a pharmaceutically acceptable carrier and / or a soluble neutral-active hyaluronidase glycoprotein. In some embodiments, the composition further comprises an additional therapeutic agent. In other embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In other embodiments, the anti-PD-1 antibody is nivolumab and the anti-CTLA antibody is ipilimumab. In other embodiments, the antibodies described herein are for use as a medicament for treating cancer.

[0032] In some embodiments, the cancer treated using the anti-NKG2A antibodies, immunoconjugates, bispecific molecules, and compositions described herein is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, renal cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, gastric cancer, melanoma, renal cancer, urothelial cancer, glioblastoma multiforme, or virus-associated cancer. In other embodiments, the cancer is cervical cancer, squamous cell carcinoma of the head and neck (HNSCC), pancreatic cancer, non-small cell lung carcinoma - adenocarcinoma type (NSCLC-AD), non-small cell lung carcinoma - squamous cell type (NSCLC-SQC), gastric cancer, melanoma, colorectal cancer (CRC), endometrial cancer, ovarian cancer, renal cell carcinoma (RCC), urothelial carcinoma (UCC), breast cancer, small cell lung carcinoma, glioblastoma multiforme, prostate cancer (also known as adenocarcinoma of the prostate or PRC), or non-Hodgkin's lymphoma.

[0033] In some embodiments, the anti-NKG2A antibody is for use in enhancing an immune response. In other embodiments, the invention provides for the use of an anti-NKG2A antibody described herein in the manufacture of a medicament for the treatment of cancer.

[0034] In one aspect, the invention provides a method for treating or delaying the progression of cancer in a human subject, comprising administering to the human subject an effective amount of an anti-NKG2A antibody, immunoconjugate, bispecific molecule, or composition described herein. In some embodiments, the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, renal cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, gastric cancer, melanoma, renal cancer, urothelial cancer, glioblastoma multiforme, or a virus-associated cancer. In other embodiments, the cancer is cervical cancer, squamous cell carcinoma of the head and neck (HNSCC), pancreatic cancer, non-small cell lung carcinoma - adenocarcinoma type (NSCLC-AD), non-small cell lung carcinoma - squamous cell type (NSCLC-SQC), gastric cancer, melanoma, colorectal cancer (CRC), endometrial cancer, ovarian cancer, renal cell carcinoma (RCC), urothelial carcinoma (UCC), breast cancer, small cell lung carcinoma, glioblastoma multiforme, prostate cancer (also known as adenocarcinoma of the prostate or PRC), or non-Hodgkin's lymphoma.

[0035] In other embodiments, the method further comprises administering to the human subject one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are chemotherapeutic agents, radiotherapeutic agents, and / or immunotherapeutic agents. In other embodiments, the one or more additional therapeutic agents are an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab and the anti-CTLA-4 antibody is ipilimumab.

[0036] In another aspect, the invention provides a method of stimulating an immune response in a human subject, comprising administering to the human subject an effective amount of an anti-NKG2A antibody, bispecific molecule, or composition described herein. In some embodiments, the human subject has a tumor, and an anti-tumor immune response is stimulated. In other embodiments, the human subject has a chronic viral infection, and an anti-viral immune response is stimulated.

[0037] In another aspect, the present invention provides a method for detecting the presence of NKG2A protein in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and the NKG2A protein, and detecting the formation of the complex. In some embodiments of the method, the antibody or antigen-binding fragment thereof forms a complex with the NKG2A protein 15 times more rapidly than with the NKG2C protein.

[0038] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, GenBank and other sequence entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0039] [Figure 1A] Figures 1A-C illustrate three methods used to discover and test the anti-NKG2A antibodies disclosed herein. Figure 1A illustrates, at a high level, the hybridoma method and anti-NKG2A antibody discovery and development process. Figure 1B illustrates the antibody library generation method used for antibody discovery. Figure 1C illustrates the single B cell cloning (SBCC) method. [Figure 1B]Figures 1A-C illustrate three methods used to discover and test the anti-NKG2A antibodies disclosed herein. Figure 1A illustrates, at a high level, the hybridoma method and anti-NKG2A antibody discovery and development process. Figure 1B illustrates the antibody library generation method used for antibody discovery. Figure 1C illustrates the single B cell cloning (SBCC) method. [Figure 1C] Figures 1A-C illustrate three methods used to discover and test the anti-NKG2A antibodies disclosed herein. Figure 1A illustrates, at a high level, the hybridoma method and anti-NKG2A antibody discovery and development process. Figure 1B illustrates the antibody library generation method used for antibody discovery. Figure 1C illustrates the single B cell cloning (SBCC) method. [Figure 2] Figure 2 illustrates the mutation scanning analysis used to optimize the discovered anti-NKG2A antibodies. Specifically, Figure 2 shows the steps for generating variants of the 13F3.A4 I107T antibody and characterizing their binding to the NKG2A protein. This analysis enabled the inventors to generate variants of the 13F3.A4 I107T antibody with improved properties and provided a rich set of information regarding the effect of single amino acid substitutions on the binding of the 13F3.A4 I107T antibody to the NKG2A protein. Figure 2 discloses SEQ ID NOS: 183-190, respectively, in order of appearance from top to bottom. [Figure 3] Figure 3 is an exemplary heatmap generated using mutation scanning analysis that allows interpretation of the sequence-activity relationship of single amino acid substitutions. Figure 3 discloses SEQ ID NO: 191 (germline) and SEQ ID NO: 192 (parent). [Figure 4] Figure 4 shows the CDR positions analyzed for the 13F3.A4 I107T anti-NKG2A antibody using mutation scanning analysis. Figure 4 discloses SEQ ID NOs: 30, 15, 192, 11, and 193, respectively, in order of appearance. [Figure 5A]Figures 5A-E are heat maps generated using mutation scanning analysis of the 13F3.A4 I107T antibody for LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) (Figure 5A), LCDR3 substitutions (SEQ ID NOs: 195 and 15, germline and parental, respectively) (Figure 5B), HCDR1 substitutions (SEQ ID NOs: 196 and 192, germline and parental, respectively) (Figure 5C), HCDR2 substitutions (SEQ ID NOs: 42 and 11, germline and parental, respectively) (Figure 5D), and HCDR3 substitutions (SEQ ID NOs: 193 and 193, germline and parental, respectively) (Figure 5E). [Figure 5B] Figures 5A-E are heat maps generated using mutation scanning analysis of the 13F3.A4 I107T antibody for LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) (Figure 5A), LCDR3 substitutions (SEQ ID NOs: 195 and 15, germline and parental, respectively) (Figure 5B), HCDR1 substitutions (SEQ ID NOs: 196 and 192, germline and parental, respectively) (Figure 5C), HCDR2 substitutions (SEQ ID NOs: 42 and 11, germline and parental, respectively) (Figure 5D), and HCDR3 substitutions (SEQ ID NOs: 193 and 193, germline and parental, respectively) (Figure 5E). [Figure 5C] Figures 5A-E are heat maps generated using mutation scanning analysis of the 13F3.A4 I107T antibody for LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) (Figure 5A), LCDR3 substitutions (SEQ ID NOs: 195 and 15, germline and parental, respectively) (Figure 5B), HCDR1 substitutions (SEQ ID NOs: 196 and 192, germline and parental, respectively) (Figure 5C), HCDR2 substitutions (SEQ ID NOs: 42 and 11, germline and parental, respectively) (Figure 5D), and HCDR3 substitutions (SEQ ID NOs: 193 and 193, germline and parental, respectively) (Figure 5E). [Figure 5D]Figures 5A-E are heat maps generated using mutation scanning analysis of the 13F3.A4 I107T antibody for LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) (Figure 5A), LCDR3 substitutions (SEQ ID NOs: 195 and 15, germline and parental, respectively) (Figure 5B), HCDR1 substitutions (SEQ ID NOs: 196 and 192, germline and parental, respectively) (Figure 5C), HCDR2 substitutions (SEQ ID NOs: 42 and 11, germline and parental, respectively) (Figure 5D), and HCDR3 substitutions (SEQ ID NOs: 193 and 193, germline and parental, respectively) (Figure 5E). [Figure 5E] Figures 5A-E are heat maps generated using mutation scanning analysis of the 13F3.A4 I107T antibody for LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) (Figure 5A), LCDR3 substitutions (SEQ ID NOs: 195 and 15, germline and parental, respectively) (Figure 5B), HCDR1 substitutions (SEQ ID NOs: 196 and 192, germline and parental, respectively) (Figure 5C), HCDR2 substitutions (SEQ ID NOs: 42 and 11, germline and parental, respectively) (Figure 5D), and HCDR3 substitutions (SEQ ID NOs: 193 and 193, germline and parental, respectively) (Figure 5E). [Figure 6] Figure 6 shows a canonical sequence alignment of the full-length human NKG2A (SEQ ID NO: 182) and human NKG2C (SEQ ID NO: 3) amino acid sequences. Approximately 76% of the amino acid residues (177 of 233 amino acid residues) are conserved, about 6% of the amino acid residues (14 of 233 amino acid residues) are similar, and only about 18% of the amino acid residues (42 of 233 amino acid residues) differ between the human NKG2A and human NKG2C proteins. [Figure 7A]Figure 7A shows the nucleotide sequence (SEQ ID NO: 166) and amino acid sequence (SEQ ID NO: 167, without the signal sequence) of the mature heavy chain variable (VH) region of the anti-NKG2A antibody 13F3.A4. The amino acid sequences of VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) are shown in gray boxes. Figure 7B shows the nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 169) of the light chain variable region of the 13F3.A4 antibody. The amino acid sequences of VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are shown in gray boxes. [Figure 7B] Figure 7A shows the nucleotide sequence (SEQ ID NO: 166) and amino acid sequence (SEQ ID NO: 167, without the signal sequence) of the mature heavy chain variable (VH) region of the anti-NKG2A antibody 13F3.A4. The amino acid sequences of VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) are shown in gray boxes. Figure 7B shows the nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 169) of the light chain variable region of the 13F3.A4 antibody. The amino acid sequences of VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are shown in gray boxes. [Figure 8A] Figure 8A shows the nucleotide sequence (SEQ ID NO:51) and amino acid sequence (SEQ ID NO:52) of the mature VH region of anti-NKG2A antibody 2G6.C2. The amino acid sequences of the VH CDR1 (SEQ ID NO:55), VH CDR2 (SEQ ID NO:56), and VH CDR3 (SEQ ID NO:57) sequences are shown in gray boxes. Figure 8B shows the nucleotide sequence (SEQ ID NO:53) and amino acid sequence (SEQ ID NO:54) of the mature VL region of anti-NKG2A antibody 2G6.C2. The amino acid sequences of the VL CDR1 (SEQ ID NO:58), VL CDR2 (SEQ ID NO:59), and VL CDR3 (SEQ ID NO:60) sequences are shown in gray boxes. [Figure 8B]Figure 8A shows the nucleotide sequence (SEQ ID NO:51) and amino acid sequence (SEQ ID NO:52) of the mature VH region of anti-NKG2A antibody 2G6.C2. The amino acid sequences of the VH CDR1 (SEQ ID NO:55), VH CDR2 (SEQ ID NO:56), and VH CDR3 (SEQ ID NO:57) sequences are shown in gray boxes. Figure 8B shows the nucleotide sequence (SEQ ID NO:53) and amino acid sequence (SEQ ID NO:54) of the mature VL region of anti-NKG2A antibody 2G6.C2. The amino acid sequences of the VL CDR1 (SEQ ID NO:58), VL CDR2 (SEQ ID NO:59), and VL CDR3 (SEQ ID NO:60) sequences are shown in gray boxes. [Figure 9A] Figure 9A shows the nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 171) of the mature heavy chain variable (VH) region of anti-NKG2A antibody 11H9.A1. The amino acid sequences of the VH CDR1 (SEQ ID NO: 41), VH CDR2 (SEQ ID NO: 42), and VH CDR3 (SEQ ID NO: 43) sequences are shown in gray boxes. Figure 9B shows the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 173) of the light chain variable region of mature anti-NKG2A antibody 11H9.A1. The amino acid sequences of the VL CDR1 (SEQ ID NO: 44), VL CDR2 (SEQ ID NO: 45), and VL CDR3 (SEQ ID NO: 46) sequences are shown in gray boxes. [Figure 9B] Figure 9A shows the nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 171) of the mature heavy chain variable (VH) region of anti-NKG2A antibody 11H9.A1. The amino acid sequences of the VH CDR1 (SEQ ID NO: 41), VH CDR2 (SEQ ID NO: 42), and VH CDR3 (SEQ ID NO: 43) sequences are shown in gray boxes. Figure 9B shows the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 173) of the light chain variable region of mature anti-NKG2A antibody 11H9.A1. The amino acid sequences of the VL CDR1 (SEQ ID NO: 44), VL CDR2 (SEQ ID NO: 45), and VL CDR3 (SEQ ID NO: 46) sequences are shown in gray boxes. [Figure 10A]Figure 10A shows the nucleotide sequence (SEQ ID NO: 174) and amino acid sequence (SEQ ID NO: 175) of the mature VH region of the anti-NKG2A antibody 4G5.D1. The amino acid sequences of the VH CDR1 (SEQ ID NO: 69), VH CDR2 (SEQ ID NO: 70), and VH CDR3 (SEQ ID NO: 71) sequences are shown in gray boxes. Figure 10B shows the nucleotide sequence (SEQ ID NO: 176) and amino acid sequence (SEQ ID NO: 177) of the VL of the 4G5.D1 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 72), VL CDR2 (SEQ ID NO: 73), and VL CDR3 (SEQ ID NO: 74) sequences are shown in gray boxes. [Figure 10B] Figure 10A shows the nucleotide sequence (SEQ ID NO: 174) and amino acid sequence (SEQ ID NO: 175) of the mature VH region of the anti-NKG2A antibody 4G5.D1. The amino acid sequences of the VH CDR1 (SEQ ID NO: 69), VH CDR2 (SEQ ID NO: 70), and VH CDR3 (SEQ ID NO: 71) sequences are shown in gray boxes. Figure 10B shows the nucleotide sequence (SEQ ID NO: 176) and amino acid sequence (SEQ ID NO: 177) of the VL of the 4G5.D1 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 72), VL CDR2 (SEQ ID NO: 73), and VL CDR3 (SEQ ID NO: 74) sequences are shown in gray boxes. [Figure 11A] Figure 11A shows the nucleotide sequence (SEQ ID NO: 178) and amino acid sequence (SEQ ID NO: 179) of the mature VH region of the anti-NKG2A antibody 1G5.B2. The amino acid sequences of the VH CDR1 (SEQ ID NO: 83), VH CDR2 (SEQ ID NO: 84), and VH CDR3 (SEQ ID NO: 85) sequences are shown in gray boxes. Figure 11B shows the nucleotide sequence (SEQ ID NO: 180) and amino acid sequence (SEQ ID NO: 181) of the VL region of the 1G5.B2 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 86), VL CDR2 (SEQ ID NO: 87), and VL CDR3 (SEQ ID NO: 88) sequences are shown in gray boxes. [Figure 11B]Figure 11A shows the nucleotide sequence (SEQ ID NO: 178) and amino acid sequence (SEQ ID NO: 179) of the mature VH region of the anti-NKG2A antibody 1G5.B2. The amino acid sequences of the VH CDR1 (SEQ ID NO: 83), VH CDR2 (SEQ ID NO: 84), and VH CDR3 (SEQ ID NO: 85) sequences are shown in gray boxes. Figure 11B shows the nucleotide sequence (SEQ ID NO: 180) and amino acid sequence (SEQ ID NO: 181) of the VL region of the 1G5.B2 antibody. The amino acid sequences of the VL CDR1 (SEQ ID NO: 86), VL CDR2 (SEQ ID NO: 87), and VL CDR3 (SEQ ID NO: 88) sequences are shown in gray boxes. [Figure 12] Figure 12 shows the amino acid sequence trends evaluated for the 13F3.A4 antibody. The V, D, and J germline origins are indicated. The amino acid sequence of the heavy chain variable region of the 13F3.A4 antibody (SEQ ID NO: 167) is shown on the left, and the amino acid sequence of the light chain variable region of the 13F3.A4 antibody (SEQ ID NO: 169) is shown on the right. The amino acid sequences of the VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are underlined. The amino acid sequences of the VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) sequences are underlined. The sequence trends evaluated are circled and labeled. [Figure 13]Figure 13 shows the full-length amino acid sequence of the anti-NKG2A antibody NKG2A.9. The light chain amino acid sequence is set forth in SEQ ID NO:5, and the heavy chain amino acid sequence is set forth in SEQ ID NO:163 (shown with the terminal lysine absent in another embodiment). Figure 13 identifies two mutations, indicated by ovals, that were made to the VL (N30S) and VH (I107T) sequences of 13F3.A4 to obtain the NKG2A.9 sequence. The N-glycosylation motif is indicated by a dashed oval. Three mutations (L234A, L235E, and G273A) made in the Fc region are shown in bold and italic. The terminal amino acid lysine of the heavy chain sequence was removed from the mature sequence. The amino acid sequences of the VL CDR1 (SEQ ID NO:13), VL CDR2 (SEQ ID NO:14), and VL CDR3 (SEQ ID NO:15) sequences are shown in bold and underlined. The amino acid sequences of the VH CDR1 (SEQ ID NO: 10), VH CDR2 (SEQ ID NO: 11), and VH CDR3 (SEQ ID NO: 12) sequences are shown in bold and underlined. [Figure 14] 14 shows the full-length amino acid sequence of the anti-NKG2A antibody NKG2A.9. The light chain amino acid sequence is shown in SEQ ID NO:5, and the heavy chain amino acid sequence is shown in SEQ ID NO:7. [Figure 15] 15 shows the full-length amino acid sequence of the anti-NKG2A antibody NKG2A.11. The light chain amino acid sequence is shown in SEQ ID NO:19, and the heavy chain amino acid sequence is shown in SEQ ID NO:7. [Figure 16] Figure 16 shows a circular plot of epitope binning results for exemplary antibodies generated by the single B cell cloning method (SBCC), demonstrating the diversity of these antibodies. Antibodies that cross-block each other are connected by lines. Sample antibodies with similar blocking profiles compared to the benchmark antibodies (13F3.A4, Z270, RD-ahNKG2a (clone 131411, catalog no. MAB1059), and RD-ahCD94 (clone 131412, catalog no. MAB1058)) are grouped together in groups 1-4, 6-7, and 9. Benchmark antibodies with similar blocking profiles to the sample antibodies are also grouped together in groups 5, 8, and 10. [Figure 17A] Figure 17A shows an assay method for assessing the ability of anti-human NKG2A antibodies generated using the SBCC method to block the NKG2A / HLA-E interaction. Figure 17B shows that the sample antibodies only partially or did not block the NKG2A / HLA-E interaction compared to the positive control (NKG2A.9 antibody) and negative control (isotype). [Figure 17B] Figure 17A shows an assay method for assessing the ability of anti-human NKG2A antibodies generated using the SBCC method to block the NKG2A / HLA-E interaction. Figure 17B shows that the sample antibodies only partially or did not block the NKG2A / HLA-E interaction compared to the positive control (NKG2A.9 antibody) and negative control (isotype). [Figure 18A] Figures 18A-C show a binding assay (Figure 18A) used to evaluate the binding of anti-NKG2A antibodies to human NKG2A-expressing CHO cells (Figure 18B) and NKG2C-expressing CHO cells (Figure 18C). As shown in Figure 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to human NKG2A-expressing CHO cells. Figures 18D-F show a blocking assay (Figure 18D) used to evaluate the blockade of NKG2A / HLA-E interactions (Figure 18E) and NKG2C / HLA-E interactions (Figure 18F) by anti-NKG2A antibodies. The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (shown in Figure 18E) but did not block the NKG2C / HLA-E interaction (shown in Figure 18F). [Figure 18B]Figures 18A-C show a binding assay (Figure 18A) used to evaluate the binding of anti-NKG2A antibodies to human NKG2A-expressing CHO cells (Figure 18B) and NKG2C-expressing CHO cells (Figure 18C). As shown in Figure 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to human NKG2A-expressing CHO cells. Figures 18D-F show a blocking assay (Figure 18D) used to evaluate the blockade of NKG2A / HLA-E interactions (Figure 18E) and NKG2C / HLA-E interactions (Figure 18F) by anti-NKG2A antibodies. The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (shown in Figure 18E) but did not block the NKG2C / HLA-E interaction (shown in Figure 18F). [Figure 18C] Figures 18A-C show a binding assay (Figure 18A) used to evaluate the binding of anti-NKG2A antibodies to human NKG2A-expressing CHO cells (Figure 18B) and NKG2C-expressing CHO cells (Figure 18C). As shown in Figure 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to human NKG2A-expressing CHO cells. Figures 18D-F show a blocking assay (Figure 18D) used to evaluate the blockade of NKG2A / HLA-E interactions (Figure 18E) and NKG2C / HLA-E interactions (Figure 18F) by anti-NKG2A antibodies. The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (shown in Figure 18E) but did not block the NKG2C / HLA-E interaction (shown in Figure 18F). [Figure 18D]Figures 18A-C show a binding assay (Figure 18A) used to evaluate the binding of anti-NKG2A antibodies to human NKG2A-expressing CHO cells (Figure 18B) and NKG2C-expressing CHO cells (Figure 18C). As shown in Figure 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to human NKG2A-expressing CHO cells. Figures 18D-F show a blocking assay (Figure 18D) used to evaluate the blockade of NKG2A / HLA-E interactions (Figure 18E) and NKG2C / HLA-E interactions (Figure 18F) by anti-NKG2A antibodies. The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (shown in Figure 18E) but did not block the NKG2C / HLA-E interaction (shown in Figure 18F). [Figure 18E] Figures 18A-C show a binding assay (Figure 18A) used to evaluate the binding of anti-NKG2A antibodies to human NKG2A-expressing CHO cells (Figure 18B) and NKG2C-expressing CHO cells (Figure 18C). As shown in Figure 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to human NKG2A-expressing CHO cells. Figures 18D-F show a blocking assay (Figure 18D) used to evaluate the blockade of NKG2A / HLA-E interactions (Figure 18E) and NKG2C / HLA-E interactions (Figure 18F) by anti-NKG2A antibodies. The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (shown in Figure 18E) but did not block the NKG2C / HLA-E interaction (shown in Figure 18F). [Figure 18F]Figures 18A-C show a binding assay (Figure 18A) used to evaluate the binding of anti-NKG2A antibodies to human NKG2A-expressing CHO cells (Figure 18B) and NKG2C-expressing CHO cells (Figure 18C). As shown in Figure 18B, the 13F3.A4, 11H9.A1, and 2EB.B1 antibodies showed specific binding to human NKG2A-expressing CHO cells. Figures 18D-F show a blocking assay (Figure 18D) used to evaluate the blockade of NKG2A / HLA-E interactions (Figure 18E) and NKG2C / HLA-E interactions (Figure 18F) by anti-NKG2A antibodies. The 13F3.A4 and 11H9.A1 antibodies showed specific binding to human NKG2A-expressing CHO cells (shown in Figure 18E) but did not block the NKG2C / HLA-E interaction (shown in Figure 18F). [Figure 19A] Figures 19A-B illustrate the binding assay method used (Figure 19A), which assessed the ability of anti-NKG2A antibodies to bind to human NKG2A+ natural killer cell lines (NKL) (shown in Figure 19B). Figures 19C-D illustrate the blocking assay method used (Figure 19C), which demonstrated that the tested anti-NKG2A antibodies blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 19B] Figures 19A-B illustrate the binding assay method used (Figure 19A), which assessed the ability of anti-NKG2A antibodies to bind to human NKG2A+ natural killer cell lines (NKL) (shown in Figure 19B). Figures 19C-D illustrate the blocking assay method used (Figure 19C), which demonstrated that the tested anti-NKG2A antibodies blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 19C]Figures 19A-B illustrate the binding assay method used (Figure 19A), which assessed the ability of anti-NKG2A antibodies to bind to human NKG2A+ natural killer cell lines (NKL) (shown in Figure 19B). Figures 19C-D illustrate the blocking assay method used (Figure 19C), which demonstrated that the tested anti-NKG2A antibodies blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 19D] Figures 19A-B illustrate the binding assay method used (Figure 19A), which assessed the ability of anti-NKG2A antibodies to bind to human NKG2A+ natural killer cell lines (NKL) (shown in Figure 19B). Figures 19C-D illustrate the blocking assay method used (Figure 19C), which demonstrated that the tested anti-NKG2A antibodies blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 20A] Figures 20A-C illustrate the blocking assay method used (Figure 20A), which showed that the anti-human NKG2A antibodies tested blocked NKG2A / HLA-E interactions in hNKG2A-expressing CHO cells, as shown in Figures 20B-C. [Figure 20B] Figures 20A-C illustrate the blocking assay method used (Figure 20A), which showed that the anti-human NKG2A antibodies tested blocked NKG2A / HLA-E interactions in hNKG2A-expressing CHO cells, as shown in Figures 20B-C. [Figure 20C] Figures 20A-C illustrate the blocking assay method used (Figure 20A), which showed that the anti-human NKG2A antibodies tested blocked NKG2A / HLA-E interactions in hNKG2A-expressing CHO cells, as shown in Figures 20B-C. [Figure 21A] Figures 21A-C illustrate the binding assay method used (Figure 21A), which showed that the anti-NKG2A antibodies tested bound to human NKG2A-expressing CHO cells, as shown in Figures 21B-C. [Figure 21B]Figures 21A-C illustrate the binding assay method used (Figure 21A), which showed that the anti-NKG2A antibodies tested bound to human NKG2A-expressing CHO cells, as shown in Figures 21B-C. [Figure 21C] Figures 21A-C illustrate the binding assay method used (Figure 21A), which showed that the anti-NKG2A antibodies tested bound to human NKG2A-expressing CHO cells, as shown in Figures 21B-C. [Figure 22A] Figures 22A-C illustrate the binding assay method used (Figure 22A), which assessed the ability of anti-NKG2A antibodies to bind to cynomolgus NKG2A-expressing CHO cells (results shown in Figures 22B-C). [Figure 22B] Figures 22A-C illustrate the binding assay method used (Figure 22A), which assessed the ability of anti-NKG2A antibodies to bind to cynomolgus NKG2A-expressing CHO cells (results shown in Figures 22B-C). [Figure 22C] Figures 22A-C illustrate the binding assay method used (Figure 22A), which assessed the ability of anti-NKG2A antibodies to bind to cynomolgus NKG2A-expressing CHO cells (results shown in Figures 22B-C). [Figure 23A] Figures 23A-B illustrate the blocking assay method used (Figure 23A) to assess the ability of anti-NKG2A antibodies to desirably block NKG2A / HLA-E interactions (Figure 23B). Figures 23C-D show the binding assay method used (Figure 23C) that demonstrated the ability of anti-NKG2A antibodies to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 23B] Figures 23A-B illustrate the blocking assay method used (Figure 23A) to assess the ability of anti-NKG2A antibodies to desirably block NKG2A / HLA-E interactions (Figure 23B). Figures 23C-D show the binding assay method used (Figure 23C) that demonstrated the ability of anti-NKG2A antibodies to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 23C]Figures 23A-B illustrate the blocking assay method used (Figure 23A) to assess the ability of anti-NKG2A antibodies to desirably block NKG2A / HLA-E interactions (Figure 23B). Figures 23C-D show the binding assay method used (Figure 23C) that demonstrated the ability of anti-NKG2A antibodies to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 23D] Figures 23A-B illustrate the blocking assay method used (Figure 23A) to assess the ability of anti-NKG2A antibodies to desirably block NKG2A / HLA-E interactions (Figure 23B). Figures 23C-D show the binding assay method used (Figure 23C) that demonstrated the ability of anti-NKG2A antibodies to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 24A] Figures 24A-B show the binding assay method (Figure 24A) used to evaluate whether anti-NKG2A antibodies bind to cynomolgus monkey NKG2A+ NK cells (Figure 24B). As shown in Figure 24B, the 11H9.A1 and 4G5.D1 antibodies did not bind to cynomolgus monkey NKG2A+ NK cells, while the 13F3.A4 antibody bound desirably to cynomolgus monkey NKG2A+ NK cells, as indicated by an EC50 of 0.2 nM. [Figure 24B] Figures 24A-B show the binding assay method (Figure 24A) used to evaluate whether anti-NKG2A antibodies bind to cynomolgus monkey NKG2A+ NK cells (Figure 24B). As shown in Figure 24B, the 11H9.A1 and 4G5.D1 antibodies did not bind to cynomolgus monkey NKG2A+ NK cells, while the 13F3.A4 antibody bound desirably to cynomolgus monkey NKG2A+ NK cells, as indicated by an EC50 of 0.2 nM. [Figure 25A]Figure 25A illustrates the in vitro method used to evaluate whether anti-NKG2A antibodies enhanced NK cell degranulation. Figures 25B-C are graphs of flow cytometry analysis results, showing that all tested anti-NKG2A antibodies (NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2, and 4G5.D1 antibodies) enhanced NK cell degranulation (as measured by fold change in CD107a compared to isotype control) in NKG2A+ NK cells. The 25E7.G8 clone (shown in Figure 25B) is a non-blocking antibody used as a negative control. [Figure 25B] Figure 25A illustrates the in vitro method used to evaluate whether anti-NKG2A antibodies enhanced NK cell degranulation. Figures 25B-C are graphs of flow cytometry analysis results, showing that all tested anti-NKG2A antibodies (NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2, and 4G5.D1 antibodies) enhanced NK cell degranulation (as measured by fold change in CD107a compared to isotype control) in NKG2A+ NK cells. The 25E7.G8 clone (shown in Figure 25B) is a non-blocking antibody used as a negative control. [Figure 25C] Figure 25A illustrates the in vitro method used to evaluate whether anti-NKG2A antibodies enhanced NK cell degranulation. Figures 25B-C are graphs of flow cytometry analysis results, showing that all tested anti-NKG2A antibodies (NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2, and 4G5.D1 antibodies) enhanced NK cell degranulation (as measured by fold change in CD107a compared to isotype control) in NKG2A+ NK cells. The 25E7.G8 clone (shown in Figure 25B) is a non-blocking antibody used as a negative control. [Figure 26A] Figure 26A illustrates an in vitro experiment using NKL cells and CHO / MICA / HLA-E showing that anti-NKG2A antibodies blocked NKG2A / HLA-E interactions and increased IFN-γ production in activated NK cells. Figure 26B shows that the 13F3.A4, 11H9.A1, and 4G5.D1 antibodies favorably increased IFN-γ production compared to the isotype control (human IgG1.3 antibody). The 25E7.G8 clone was used as a negative control. [Figure 26B] Figure 26A illustrates an in vitro experiment using NKL cells and CHO / MICA / HLA-E showing that anti-NKG2A antibodies blocked NKG2A / HLA-E interactions and increased IFN-γ production in activated NK cells. Figure 26B shows that the 13F3.A4, 11H9.A1, and 4G5.D1 antibodies favorably increased IFN-γ production compared to the isotype control (human IgG1.3 antibody). The 25E7.G8 clone was used as a negative control. [Figure 27A] Figure 27A illustrates an in vitro experiment used to evaluate whether anti-NKG2A antibodies enhanced CD8+ T cell responses, resulting in increased IFNγ production, in Hs766T target cells, a pancreatic cancer cell line. Figure 27B is a graphical representation of the assay results, showing that the 13F3.A4 and 11H9.A1 antibodies increased IFN-γ production compared to the isotype control. The 25E7.G8 clone was used as a negative control. [Figure 27B] Figure 27A illustrates an in vitro experiment used to evaluate whether anti-NKG2A antibodies enhanced CD8+ T cell responses, resulting in increased IFNγ production, in Hs766T target cells, a pancreatic cancer cell line. Figure 27B is a graphical representation of the assay results, showing that the 13F3.A4 and 11H9.A1 antibodies increased IFN-γ production compared to the isotype control. The 25E7.G8 clone was used as a negative control. [Figure 28A]Figures 28A-B show the results of differential hydrogen-deuterium exchange (HDX) analysis of mFc-hNKG2A-hCD94 upon interaction with the NKG2A.9 antibody (Figure 28A) and the 13F3.A4 antibody (Figure 28B), respectively. Epitope sequences are labeled in Figures 28A-B (SEQ ID NOs: 119-122, disclosed from left to right along the analysis). [Figure 28B] Figures 28A-B show the results of differential hydrogen-deuterium exchange (HDX) analysis of mFc-hNKG2A-hCD94 upon interaction with the NKG2A.9 antibody (Figure 28A) and the 13F3.A4 antibody (Figure 28B), respectively. Epitope sequences are labeled in Figures 28A-B (SEQ ID NOs: 119-122, disclosed from left to right along the analysis). [Figure 29A] Figures 29A-B show the FPOP protection percentage in hNKG2A upon interaction with the NKG2A.9 antibody (Figure 29A) and the 13F3.A4 antibody (Figure 29B), respectively, for four residues (M163, F179, H184, L206). [Figure 29B] Figures 29A-B show the FPOP protection percentage in hNKG2A upon interaction with the NKG2A.9 antibody (Figure 29A) and the 13F3.A4 antibody (Figure 29B), respectively, for four residues (M163, F179, H184, L206). [Figure 30] Figure 30 shows the epitopes of the NKG2A.9 and 13F3.A4 antibodies mapped to the mFc-hNKG2A-hCD94 sequence (SEQ ID NO: 125) as determined by HDX-MS and FPOP. Epitopes shown in bold and underlined were determined by HDX-MS, and circled epitopes were determined by FPOP analysis. [Figure 31] Figure 31 shows the epitopes of anti-NKG2A antibodies (e.g., of the NKG2A.9 and 13F3.A4 antibodies) visualized in the NKG2A / CD94 / HLA-E crystal structure. The anti-NKG2A antibody epitopes are shown in black. [Figure 32A]Figures 32A-B show an alignment of the VH regions (SEQ ID NOS: 167, 8, and 8, respectively, in order of appearance from top to bottom) (Figure 32A) and the VL regions (SEQ ID NOS: 169, 9, and 164, in order of appearance from top to bottom) (Figure 32B) of certain portions of anti-NKG2A antibodies (13F3.A4, NKG2A.9, and NKG2A.11). This alignment led to the discovery of anti-NKG2A antibodies with consensus CDR sequences shown in boxes. [Figure 32B] Figures 32A-B show an alignment of the VH regions (SEQ ID NOS: 167, 8, and 8, respectively, in order of appearance from top to bottom) (Figure 32A) and the VL regions (SEQ ID NOS: 169, 9, and 164, in order of appearance from top to bottom) (Figure 32B) of certain portions of anti-NKG2A antibodies (13F3.A4, NKG2A.9, and NKG2A.11). This alignment led to the discovery of anti-NKG2A antibodies with consensus CDR sequences shown in boxes. [Figure 33] FIG. 33 shows the results of an assay in which the NKG2A.9 antibody, compared to isotype, reversed the inhibition of NK-κB signaling in NKG2A-expressing Jurkat T cells stimulated by CHO / scOKT3 / HLA-E. [Figure 34A] Figure 34A illustrates the experimental method used to analyze the effects of NKG2A.9 and anti-PD-L1 antibodies, either alone or in combination, on IFN-γ induction by NKG2A+ CD8+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs). Figure 34B shows the results of this method, in which the combination of NKG2A.9 and anti-PD-L1 antibodies enhanced IFN-γ production by NKG2A+ CD8+ T cells in a dose-dependent manner. [Figure 34B]Figure 34A illustrates the experimental method used to analyze the effects of NKG2A.9 and anti-PD-L1 antibodies, either alone or in combination, on IFN-γ induction by NKG2A+ CD8+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs). Figure 34B shows the results of this method, in which the combination of NKG2A.9 and anti-PD-L1 antibodies enhanced IFN-γ production by NKG2A+ CD8+ T cells in a dose-dependent manner. [Figure 35A] Figures 35A-B illustrate the assay method (Figure 35A) used to show that NKG2A.9 and / or anti-PD-L1 antibodies enhanced IFN-γ production in NKG2A+ CD8+ T cells isolated from human tumors co-cultured with CHO / scOKT3 / HLA-E / PD-L1 (Figure 35B). [Figure 35B] Figures 35A-B illustrate the assay method (Figure 35A) used to show that NKG2A.9 and / or anti-PD-L1 antibodies enhanced IFN-γ production in NKG2A+ CD8+ T cells isolated from human tumors co-cultured with CHO / scOKT3 / HLA-E / PD-L1 (Figure 35B). [Figure 36A] Figure 36A is a graph showing that the NKG2A.9 antibody was internalized after binding to NKG2A-expressing cells. Figure 36B is a graph showing the binding kinetics of the NKG2A.9 antibody, showing that the NKG2A.9 antibody was internalized in a dose-dependent manner with an EC50 of 0.5 nM. [Figure 36B] Figure 36A is a graph showing that the NKG2A.9 antibody was internalized after binding to NKG2A-expressing cells. Figure 36B is a graph showing the binding kinetics of the NKG2A.9 antibody, showing that the NKG2A.9 antibody was internalized in a dose-dependent manner with an EC50 of 0.5 nM. [Figure 37A]Figures 37A-B illustrate the methodology used to show that the NKG2A.9 antibody increased NK cell degranulation in a dose-dependent manner compared to the isotype as measured by % CD107a expression by flow cytometry (Figure 37A). Figures 37C-D illustrate the experimental methodology used to show that the NKG2A.9 antibody increased lysis of HLA-E-expressing tumor cells in a dose-dependent manner compared to the isotype (Figure 37D). [Figure 37B] Figures 37A-B illustrate the methodology used to show that the NKG2A.9 antibody increased NK cell degranulation in a dose-dependent manner compared to the isotype as measured by % CD107a expression by flow cytometry (Figure 37A). Figures 37C-D illustrate the experimental methodology used to show that the NKG2A.9 antibody increased lysis of HLA-E-expressing tumor cells in a dose-dependent manner compared to the isotype (Figure 37D). [Figure 37C] Figures 37A-B illustrate the methodology used to show that the NKG2A.9 antibody increased NK cell degranulation in a dose-dependent manner compared to the isotype as measured by % CD107a expression by flow cytometry (Figure 37A). Figures 37C-D illustrate the experimental methodology used to show that the NKG2A.9 antibody increased lysis of HLA-E-expressing tumor cells in a dose-dependent manner compared to the isotype (Figure 37D). [Figure 37D] Figures 37A-B illustrate the methodology used to show that the NKG2A.9 antibody increased NK cell degranulation in a dose-dependent manner compared to the isotype as measured by % CD107a expression by flow cytometry (Figure 37A). Figures 37C-D illustrate the experimental methodology used to show that the NKG2A.9 antibody increased lysis of HLA-E-expressing tumor cells in a dose-dependent manner compared to the isotype (Figure 37D). [Figure 38A]Figure 38A illustrates the method used to measure the effect of the 13F3.A4 antibody on IFN-γ production in NKLs co-cultured with CHO / MICA / HLA-E. Figure 38B shows that the 13F3.A4 antibody increased IFN-γ production in NKLs compared to the isotype. [Figure 38B] Figure 38A illustrates the method used to measure the effect of the 13F3.A4 antibody on IFN-γ production in NKLs co-cultured with CHO / MICA / HLA-E. Figure 38B shows that the 13F3.A4 antibody increased IFN-γ production in NKLs compared to the isotype. [Figure 39] Figure 39 shows the results of a dose titration study in which 10 mg / kg, 3 mg / kg, and 1 mg / kg of anti-mNKG2A antibody (NKG2A.3) administered as monotherapy inhibited tumor growth in a colon cancer tumor model, resulting in a 48%, 56%, and 30% reduction in mean tumor volume, respectively. No efficacy was observed at the 0.3 mg / kg dose. [Figure 40A] Figures 40A-E show the results of an in vivo study in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy demonstrated single-agent activity in a CT26 colorectal tumor mouse model. Figures 40A-D show tumor volume at various time points after tumor implantation in mice (n=10 / group) treated with isotype (Figure 40A), anti-mNKG2A antibody alone (Figure 40B), anti-mPD-1 antibody alone (Figure 40C), or a combination of anti-mNKG2A and anti-mPD-1 antibodies (Figure 40D). Figure 40E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies. [Figure 40B]Figures 40A-E show the results of an in vivo study in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy demonstrated single-agent activity in a CT26 colorectal tumor mouse model. Figures 40A-D show tumor volume at various time points after tumor implantation in mice (n=10 / group) treated with isotype (Figure 40A), anti-mNKG2A antibody alone (Figure 40B), anti-mPD-1 antibody alone (Figure 40C), or a combination of anti-mNKG2A and anti-mPD-1 antibodies (Figure 40D). Figure 40E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies. [Figure 40C] Figures 40A-E show the results of an in vivo study in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy demonstrated single-agent activity in a CT26 colorectal tumor mouse model. Figures 40A-D show tumor volume at various time points after tumor implantation in mice (n=10 / group) treated with isotype (Figure 40A), anti-mNKG2A antibody alone (Figure 40B), anti-mPD-1 antibody alone (Figure 40C), or a combination of anti-mNKG2A and anti-mPD-1 antibodies (Figure 40D). Figure 40E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies. [Figure 40D]Figures 40A-E show the results of an in vivo study in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy demonstrated single-agent activity in a CT26 colorectal tumor mouse model. Figures 40A-D show tumor volume at various time points after tumor implantation in mice (n=10 / group) treated with isotype (Figure 40A), anti-mNKG2A antibody alone (Figure 40B), anti-mPD-1 antibody alone (Figure 40C), or a combination of anti-mNKG2A and anti-mPD-1 antibodies (Figure 40D). Figure 40E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies. [Figure 40E] Figures 40A-E show the results of an in vivo study in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy demonstrated single-agent activity in a CT26 colorectal tumor mouse model. Figures 40A-D show tumor volume at various time points after tumor implantation in mice (n=10 / group) treated with isotype (Figure 40A), anti-mNKG2A antibody alone (Figure 40B), anti-mPD-1 antibody alone (Figure 40C), or a combination of anti-mNKG2A and anti-mPD-1 antibodies (Figure 40D). Figure 40E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mNKG2A antibody alone, anti-mPD-1 antibody alone, or a combination of anti-mNKG2A and anti-mPD-1 antibodies. [Figure 41A] Figures 41A-C are graphs showing the results of an in vivo study in which anti-NKG2A and anti-PD-1 antibodies increased NK (Figure 41A) and tumor-specific CD8+ T cell cytotoxicity and IFN-γ (Figures 41B-C) in a mouse colon cancer model. [Figure 41B]Figures 41A-C are graphs showing the results of an in vivo study in which anti-NKG2A and anti-PD-1 antibodies increased NK (Figure 41A) and tumor-specific CD8+ T cell cytotoxicity and IFN-γ (Figures 41B-C) in a mouse colon cancer model. [Figure 41C] Figures 41A-C are graphs showing the results of an in vivo study in which anti-NKG2A and anti-PD-1 antibodies increased NK (Figure 41A) and tumor-specific CD8+ T cell cytotoxicity and IFN-γ (Figures 41B-C) in a mouse colon cancer model. [Figure 42A] Figures 42A-E show the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies, either alone or in combination, in the 1956 mouse sarcoma model. Figures 42A-D show tumor volume at various time points after tumor implantation in mice treated with isotype (Figure 42A), anti-mCTLA-4 antibody (Figure 42B, CTLA-4 IgG2a, 0.1 mg / kg), anti-mNKG2A antibody (Figure 42C, 10 mg / kg), or the combination of anti-mNKG2A and anti-mCTLA-4 (Figure 42D). Figure 42E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or the combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42B] Figures 42A-E show the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies, either alone or in combination, in the 1956 mouse sarcoma model. Figures 42A-D show tumor volume at various time points after tumor implantation in mice treated with isotype (Figure 42A), anti-mCTLA-4 antibody (Figure 42B, CTLA-4 IgG2a, 0.1 mg / kg), anti-mNKG2A antibody (Figure 42C, 10 mg / kg), or the combination of anti-mNKG2A and anti-mCTLA-4 (Figure 42D). Figure 42E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or the combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42C]Figures 42A-E show the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies, either alone or in combination, in the 1956 mouse sarcoma model. Figures 42A-D show tumor volume at various time points after tumor implantation in mice treated with isotype (Figure 42A), anti-mCTLA-4 antibody (Figure 42B, CTLA-4 IgG2a, 0.1 mg / kg), anti-mNKG2A antibody (Figure 42C, 10 mg / kg), or the combination of anti-mNKG2A and anti-mCTLA-4 (Figure 42D). Figure 42E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or the combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42D] Figures 42A-E show the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies, either alone or in combination, in the 1956 mouse sarcoma model. Figures 42A-D show tumor volume at various time points after tumor implantation in mice treated with isotype (Figure 42A), anti-mCTLA-4 antibody (Figure 42B, CTLA-4 IgG2a, 0.1 mg / kg), anti-mNKG2A antibody (Figure 42C, 10 mg / kg), or the combination of anti-mNKG2A and anti-mCTLA-4 (Figure 42D). Figure 42E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or the combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42E]Figures 42A-E show the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies, either alone or in combination, in the 1956 mouse sarcoma model. Figures 42A-D show tumor volume at various time points after tumor implantation in mice treated with isotype (Figure 42A), anti-mCTLA-4 antibody (Figure 42B, CTLA-4 IgG2a, 0.1 mg / kg), anti-mNKG2A antibody (Figure 42C, 10 mg / kg), or the combination of anti-mNKG2A and anti-mCTLA-4 (Figure 42D). Figure 42E shows the mean tumor volume as a function of time (days after tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or the combination of anti-mNKG2A and anti-mCTLA-4. [Figure 43] Figure 43 shows the anti-tumor activity of anti-NKG2A, anti-PD-1, and anti-mLAG-3 antibodies, as well as their combination, in a mouse lymphoma model. Administration of anti-NKG2A antibody alone provided a survival benefit with a 10% survival rate. Combination therapy of anti-NKG2A antibody with either anti-mPD-1 or anti-mLAG-3 antibody extended survival by 50% and 70%, respectively. The triple combination of anti-mNKG2A, anti-mPD-1, and anti-mLAG-3 antibodies provided the greatest benefit with an 80% survival rate. [Figure 44] FIG. 44 shows that NKG2A expression levels were reduced relative to the isotype in both splenic and tumor-infiltrating lymphocyte (TIL) NK cells after treatment with anti-mNKG2A antibody in a murine CT26 colon cancer model. [Figure 45] Figure 45 shows the results of NKG2A expression assessed by immunohistochemistry in different tumor types. [Figure 46] FIG. 46 shows the binding profile of FITC-conjugated NKG2A.6 antibody in multiple tumors. [Figure 47] Figure 47 shows the results of HLA-E expression in seven different tumor types as assessed by immunohistochemistry. [Figure 48] Figure 48 shows representative images of HLA-E expression in different tumor types as assessed by immunohistochemistry. [Figure 49A] Figures 49A-B show the levels of soluble HLA-E levels across healthy control and cancer patients. [Figure 49B] Figures 49A-B show the levels of soluble HLA-E levels across healthy control and cancer patients. [Figure 50] FIG. 50 shows the clinical development plan for the anti-NKG2A antibodies discussed herein, including the patient selection process and combination therapy with the anti-NKG2A antibodies described herein. [Figure 51] Figures 51A-D are graphs showing the kinetics and binding affinity of the NKG2A.9 antibody as determined by Biacore analysis. [Figure 52A] Figures 52A-B show the kinetics and binding affinity of NKG2A.9 as measured by Scatchard analysis. [Figure 52B] Figures 52A-B show the kinetics and binding affinity of NKG2A.9 as measured by Scatchard analysis. [Figure 53] Figure 53 shows the results of an NK degranulation assay comparing P1-069366 to NKG2A.9 and isotype. The NKG2A.9 antibody demonstrated functionality in the NK degranulation assay, whereas the P1-069366 antibody did not. [Figure 54A] Figures 54A-C show the results of analyzing the 13F3.A4 antibody using an in silico HLA binding tool for undesirable binding clusters. Figures 54A-C disclose SEQ ID NOs: 197-204, respectively, in order of appearance from top to bottom. [Figure 54BC] Figures 54A-C show the results of analyzing the 13F3.A4 antibody using an in silico HLA binding tool for undesirable binding clusters. Figures 54A-C disclose SEQ ID NOs: 197-204, respectively, in order of appearance from top to bottom. [Figure 55] FIG. 55 shows the results of an in vitro DC:T cell proliferation assay demonstrating the low immunogenicity risk of anti-NKG2A antibodies, particularly the NKG2A.6, NKG2A.9, and NKG2A.11 antibodies. [Figure 56]Figures 56A-D show the binding affinity of the NKG2A.9 antibody to human NKG2A-CD94 (Figures 56A-B) and NKG2C-CD94 heterodimers (Figures 56C-D) at 37° C. as determined by Biacore using both single-cycle (Figures 56A and 56C) and multiple-cycle (Figures 56B and 56D) kinetics. SPR responses are shown as a function of analyte binding and dissociation. [Figure 57] Figure 57 shows a box plot of the total HLA-E positivity score across 16 different tumor types as assessed by immunohistochemistry. The total HLA-E score is defined as the combined percentage of cytoplasmic and / or membrane HLA-E positivity in tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0040] In some aspects, the present invention provides isolated antibodies, e.g., monoclonal antibodies, e.g., humanized, human, and chimeric monoclonal antibodies, that specifically bind to human NKG2A ("hNKG2A") and have antagonist activity that stimulates an anti-tumor immune response. In some embodiments, the anti-NKG2A antibodies described herein comprise particular structural features, e.g., CDR regions comprising particular amino acid sequences. In other embodiments, the anti-NKG2A antibodies compete with the anti-NKG2A antibodies of the present invention for binding to human NKG2A protein or bind to the same or similar epitope as the anti-NKG2A antibodies of the present invention.

[0041] In some aspects, the invention provides methods of making such anti-NKG2A antibodies, immunoconjugates and bispecific molecules comprising such anti-NKG2A antibodies or antigen-binding fragments thereof, and pharmaceutical compositions formulated with the anti-NKG2A antibodies or antigen-binding fragments thereof. In some aspects, the invention provides methods of using anti-NKG2A antibodies, either alone or in combination with other agents, e.g., other immuno-oncology agents (e.g., antibodies), chemotherapy, radiation therapy, and / or surgery, to enhance immune responses. Thus, in some embodiments, the anti-NKG2A antibodies described herein are used to treat a variety of conditions, including, e.g., to safely and effectively treat cancer and / or infectious diseases.

[0042] A key role of the immune system is its ability to distinguish between normal and "foreign" cells. Therefore, the immune system can attack foreign cells and spare only normal cells. Tumors express antigens that are recognized as foreign by the host. The immune system uses "checkpoints," which are molecules on specific immune cells that must be activated or inactivated to initiate an immune response. Tumor cells may be able to use these checkpoints to avoid being attacked by the immune system. Some immuno-oncology drugs target these checkpoints by acting as checkpoint inhibitors. Programmed death protein 1 (PD-1) is a checkpoint inhibitor that acts as a brake to prevent T cells from attacking other cells in the body. PD-1 does this when it binds to programmed death-ligand 1 (PD-L1), a protein on some normal (and cancer) cells. When PD-1 binds to PD-L1, this interaction signals T cells not to attack other cells. Some cancer cells have large amounts of PD-L1, which helps them evade immune attack. Therapeutic agents such as monoclonal antibodies that target this PD-1 / PD-L1 interaction, such as nivolumab (Opdivo®), can block PD-1 / PD-L1 binding and increase the body's immune response against tumor cells.

[0043] The natural killer cell inhibitory receptor 2A (NKG2A) is a member of the NKG2 lectin receptor family, which also includes NKG2C, NKG2D, and NKG2E (Iwaszko and Bogunia-Kubik, Arch Immunol Ther Exp, 59:353-67 (2011)). NKG2A, NKG2C, and NKG2E share high amino acid sequence homology in their extracellular domains, whereas NKG2D is a functionally distinct receptor. NKG2A forms a heterodimer with CD94. Within the NKG2 / CD94 heterodimer, NKG2A / CD94 is the only receptor with inhibitory function, while NKG2C / CD94 and NKG2E / CD94 are activating receptors. NKG2D is also an activating receptor, but it does not form a heterodimer with CD94, nor does NKG2D bind to HLA-E. The NKG2 / CD94 receptor recognizes nonclassical major histocompatibility (MHC) class I molecules, human leukocyte antigen-E (HLA-E) in humans and Qa-1 in mice (Braud et al, Nature, 391:795-99 (1998), Vance et al., J. Exp. Med. 188:1841-48 (1998)). NKG2A / CD94 binds to HLA-E with approximately six-fold greater affinity than NKG2C / CD94. Ibid. NKG2A is involved in the activation of natural killer (NK) and effector / memory CD8 +It is expressed on T, NKT, and gamma delta (γδ) T cells. NKG2A expression is induced by T cell receptor (TCR) ligation and after stimulation with certain cytokines, including IL-2, IL-10, IL-15, IL-18, and IL-21, although the ability of cytokines to induce NKG2A expression depends on TCR ligation (Cho, Blood, 118:116-28 (2011)). NKG2A has two immunoreceptor tyrosine-based inhibitory motifs (ITIMS) that transmit intracellular inhibitory signals (Kabat et al, J. Immunol, 169:1948-58 (2002); Le Drean El, Eur. J. Immunol 28:264076 (1998)). The anti-NKG2A antibodies described herein inhibit the NKG2A protein and thus act as checkpoint inhibitors.

[0044] definition In order to make the description in this specification easier to understand, certain terms are first defined.Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as that commonly understood by those skilled in the art to which this disclosure pertains.Further definitions are provided throughout the detailed description.The headings provided in this specification do not limit the various aspects of this disclosure that can be understood by referring to this specification as a whole.Therefore, the terms defined immediately below are more fully defined by referring to this specification as a whole.

[0045] As used herein, "NKG2A" refers to the natural killer cell inhibitory receptor 2 protein, which in humans is encoded by the NKG2A gene. NKG2A is also known as, for example, CD159 antigen-like family member A, NK cell receptor A, NKG2A-activating NK receptor, NKG2-A / B-activating NK receptor, killer cell lectin-like receptor C1 (CD159a), and NKG2-A / NKG2-B type II integral membrane protein.

[0046] Three isoforms of the human NKG2A protein have been identified, corresponding to five variants of the mRNA transcript.

[0047] Isoform 1, corresponding to variant 1 (nucleotide sequence set forth in SEQ ID NO: 1 or 209, amino acid sequence set forth in SEQ ID NO: 2) and variant 3 (nucleotide sequence set forth in SEQ ID NO: 212, amino acid sequence set forth in SEQ ID NO: 182), consists of 233 amino acids and represents the canonical NGK2A sequence. Isoform 1 variant 3 is also a naturally occurring variant with a single nucleotide polymorphism (SNP), N29S, in which an asparagine (N) at residue 29 is changed to a serine (S) (SEQ ID NO: 182).

[0048] Isoform 2, corresponding to variant 2 (nucleotide sequence set forth in SEQ ID NO:210, amino acid sequence set forth in SEQ ID NO:206) and variant 4 (nucleotide sequence set forth in SEQ ID NO:211, amino acid sequence set forth in SEQ ID NO:207), lacks an in-frame coding exon and is missing residues 96-113 compared to variant 1 and variant 3, respectively, and is also referred to as NKG2A isoform NKG2-B. Variant 4 has the N29S SNP.

[0049] Isoform 3, corresponding to variant 5 (nucleotide sequence set forth in SEQ ID NO:208 and amino acid sequence set forth in SEQ ID NO:205), consists of 228 amino acids and lacks residues 229-233, which encode the five C-terminal amino acids. This variant also contains the N29S SNP.

[0050] The amino acid sequences of known human NGK2A mutants are shown below. (1) Variant 1: Human NGK2A isoform 1 (nucleotide sequence set forth in SEQ ID NO: 1 or 209 (Accession No. NM 002259.5) and amino acid sequence set forth in SEQ ID NO: 2 (Accession No. NP 002250.2, UniProt ID P26715-1)): [ka] (SEQ ID NO: 2) (2) Variant 2: Human NGK2A isoform 2, also referred to as NKG2A isoform NKG2B (nucleotide sequence set forth in SEQ ID NO: 210 (Accession No. NM 007328.4) and amino acid sequence set forth in SEQ ID NO: 206 (Accession No. NP 015567.2, UniProt ID P26715-2)): [ka] (SEQ ID NO: 206) (3) Variant 3: Human NGK2A isoform 1, with the N29S SNP shown in bold and highlighted (nucleotide sequence set forth in SEQ ID NO: 212 (Accession No. NM 213658.2) and amino acid sequence set forth in SEQ ID NO: 182 (Accession No. NP 998823.1 or AAL65234.1): [ka] (SEQ ID NO: 182) (4) Variant 4: Human NKG2A isoform 2 also corresponds to Variant 4 (nucleotide sequence set forth in SEQ ID NO: 211 (Accession No. NM_213657.2) and amino acid sequence set forth in SEQ ID NO: 207 (Accession No. NP 998822.1)): [ka] (SEQ ID NO: 207) (5) Variant 5: Human NGK2A isoform 3, also referred to as NKG2A isoform C (nucleotide sequence set forth in SEQ ID NO: 208 (Accession No. NM 001304448.1) and amino acid sequence set forth in SEQ ID NO: 205 (Accession No. NM 001291377.1)): [ka] (SEQ ID NO: 205) The following table provides a summary of the accession numbers and corresponding SEQ ID NOs for the DNA and proteins mentioned above.

[0051] [Table A]

[0052] The terms "antibody" and "immunoglobulin" are used interchangeably herein and refer to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies, e.g., naturally occurring antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In certain antibodies, e.g., naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions are referred to as complementarity-determining regions (CDRs), which can be further subdivided into regions of hypervariability interspersed with more conserved regions, referred to as framework regions (FRs). Each V H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the traditional complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable region are numbered using the Kabat numbering system, and amino acids in the constant region are numbered using the EU system. Immunoglobulins can be derived from any of the known isotypes, including IgA, secretory IgA, IgD, IgE, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the anti-NKG2A antibodies described herein are of the IgG1 subtype. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by at most two or three amino acids. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and fully synthetic antibodies.

[0053] As used herein, an "IgG antibody" has the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-NKG2A IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), where these two heavy and light chains are linked by the same number and positions of disulfide bridges as occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds).

[0054] An "antigen" is a molecule or substance that triggers an immune response and to which an antibody binds. An antibody typically binds to its cognate antigen. -5 ~10 -11 The dissociation constant (K D ) but does not bind with high affinity to unrelated antigens. -4 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen is one that binds to the antigen and, in some cases, to a substantially identical antigen. -6 K below M D , 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, or 10 -8 M and 10 -10 K between M and below D "Negative" refers to an antibody that binds with high affinity, meaning that it has high affinity to the antigen, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of the given antigen. For example, an antibody that specifically binds to human NKG2A will, in some embodiments, also cross-react with NKG2A antigens from certain non-human primate species (e.g., cynomolgus monkey NKG2A), but will not cross-react with NKG2A antigens from other species or antigens other than NKG2A.

[0055] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody are used interchangeably herein and refer to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., human NKG2A). It has been shown that the antigen-binding function of an antibody can be performed by a fragment or portion of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody, for example, an anti-NKG2A antibody described herein, include: (1) a Fab fragment (a fragment derived from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains; (2) F(ab')2 fragment (a fragment derived from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (3) V H and an Fd fragment consisting of the CH1 domain; (4) V of a single arm of the antibody L and V H Fv fragments consisting of domains, (5) Single-domain antibody (dAb) fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-46), (6) an isolated complementarity-determining region (CDR); and (7) A combination of two or more isolated CDRs, optionally connected by a synthetic linker. Examples include:

[0056] Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker, allowing the VL and VH region pair to be produced as a single protein chain that forms a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0057] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody that has two different binding specificities, e.g., two different heavy / light chain pairs, resulting in two antigen-binding sites with specificities for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0058] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are substantially similar and bind the same epitope (e.g., the antibody exhibits a single binding specificity and affinity), except for possible variations that may arise during the production of the monoclonal antibody, and such variations are generally present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require production of the antibody by any particular method. The term "human monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies that exhibits a single binding specificity and has variable and, optionally, constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by the hybridoma method. Using the hybridoma method, a transgenic non-human animal, e.g., a transgenic mouse, is exposed to an antigen, and white blood cells known as B cells produce antibodies that bind to the antigen, which are then recovered from the transgenic non-human animal. The isolated B cells are fused with immortalized cells to produce hybrid cell lines called hybridomas. In one embodiment, the hybridoma has a genome comprising human heavy and light chain transgenes fused to an immortalized cell.

[0059] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (1) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (2) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (3) antibodies isolated from recombinant, combinatorial human antibody libraries, and (4) antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), variable regions contain antigen-binding domains encoded by various genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, variable regions can be further modified by multiple single amino acid changes (also called somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant regions further change in response to the antigen (i.e., isotype switching). Thus, rearranged and somatically mutated nucleic acid molecules encoding light and heavy immunoglobulin polypeptides in response to the antigen may not have sequence identity to the original nucleic acid molecule, but instead will be substantially identical or similar (e.g., have at least 80% identity).

[0060] As used herein, a "human antibody" refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The anti-NKG2A antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., by mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another non-human mammalian species, such as a mouse, have been grafted onto human framework sequences. As used herein, the terms "human" and "fully human" antibody are used interchangeably.

[0061] A "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids from a human antibody. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human antibody, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not prevent the antibody from binding to a specific antigen. A "humanized antibody" retains antigen specificity similar to that of the original antibody.

[0062] "Chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0063] As used herein, "isotype" refers to the antibody class (e.g., IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE antibodies) encoded by the antibody's heavy chain constant region genes.

[0064] "Allotype" refers to naturally occurring variants within a particular isotype group, these variants differing by two or three amino acids. (See, e.g., Jefferis et al. (2009) mAbs 1:1.) The anti-NKG2A antibodies described herein can be of any allotype. As used herein, antibodies designated as "IgG1f," "IgG1.1f," or "IgG1.3f" isotypes are IgG1, effectorless IgG1.1, or effectorless IgG1.3 antibodies of allotype "f," respectively.

[0065] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the phrase "antibody that specifically binds to an antigen."

[0066] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other proteins and cellular material.

[0067] As used herein, "effector function" refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the resulting biochemical events. Exemplary "effector functions" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

[0068] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIb, or equivalently, FcγRIIB) receptors. Various exemplary properties of human FcγRs are known in the art. While most innate effector cell types simultaneously express one or more activating FcγRs and the inhibitory FcγRIIb, natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not express the inhibitory FcγRIIb in mice or humans. Human IgG1 binds most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the type of activating Fc receptor it binds.

[0069] As used herein, "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotopes, the Fc region comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and the hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain, as defined herein, may vary, the human IgG heavy chain Fc region is defined as stretching from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy terminus of the heavy chain, this numbering according to the EU index in Kabat (Kabat, et al., 1991). The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain of the Fc region, i.e., the CH3 domain extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG. As used herein, an Fc region can be a native sequence Fc, including any allotypic variant or variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in isolation or in the context of a protein polypeptide comprising an Fc, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0070] A "native sequence Fc region" or "native sequence Fc" has an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fch region, native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0071] The term "epitope" or "antigenic determinant," as defined, for example, by the particular method used to identify it, refers to a site on an antigen (e.g., an hNKG2A protein) to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from both (1) contiguous amino acids (usually linear epitopes) or (2) non-contiguous amino acids juxtaposed by tertiary folding of a protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are usually, but not necessarily, retained upon exposure to denaturing solvents, whereas epitopes formed from tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in a unique spatial conformation.

[0072] The term "epitope mapping" refers to the process of identifying molecular determinants involved in antibody-antigen recognition.Methods for determining the epitope bound by a given antibody (i.e., epitope mapping) are well known in the art, and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or consecutive peptides derived from a protein (e.g., derived from NKG2A) are tested for reactivity with a given antibody (e.g., anti-NKG2A antibody).Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as X-ray crystallography, antigen mutation analysis, two-dimensional nuclear magnetic resonance, yeast display, and hydrogen / deuterium exchange mass spectrometry (HDX-MS) (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0073] With respect to two or more antibodies, the term "binds to the same epitope" means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the "same epitope on NKG2A" using the antibodies described herein include, for example, X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope, HDX-MS, and epitope mapping methods such as rapid photochemical oxidation of proteins (FPOP). Other methods monitor the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or to mutated variations of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is often considered an indication of epitope content, such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081) or yeast display of mutant target sequence variants. Additionally, computational combinatorial methods for epitope mapping may be used. These methods rely on the ability of an antibody of interest to affinity isolate specific short peptides from combinatorial phage-displayed peptide libraries. Antibodies with identical VH and VL or identical CDR1, CDR2, and CDR3 sequences are predicted to bind to the same epitope.

[0074] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target, can be determined using known binding competition experiments, such as Biacore® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with or inhibits the binding of another antibody to the target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb. Protoc. 2006; doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they block each other by at least 50% in both directions, regardless of whether one antibody or the other antibody contacts the antigen first in the competition experiment.

[0075] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include, for example, competition for binding to T cells expressing NKG2A by flow cytometry. Other methods include SPR (e.g., Biacore®), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)), solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), solid-phase direct labeling RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)), solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1986)), and the like. (1990)), and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0076] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to antibody binding to an epitope on a predetermined antigen. In some embodiments, an antibody (1) binds to an epitope of approximately 10, as determined, for example, by SPR technology in a Biacore® SPR instrument using a predetermined antigen, e.g., recombinant human NKG2A, as the analyte and an antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -6 Less than M, e.g., approximately 10 -7 Under M, 10 -8 Under M, 10 -9 Less than M or 10 -10M or even smaller equilibrium dissociation constant (K D ) and (2) binds to a given antigen with an affinity that is at least two-fold greater than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to human NKG2A" is one that binds to a given antigen with an affinity that is at least two-fold greater than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). -6 M or less, e.g., about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than or even smaller than M D The term "antibody that cross-reacts with cynomolgus monkey NKG2A" refers to an antibody that binds to soluble or cell-bound human NKG2A. -6 M or less, e.g., about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K below M or even lower D This refers to an antibody that binds to cynomolgus monkey NKG2A.

[0077] The term "k" a "," k assoc " or "k on " is used interchangeably herein to refer to the binding rate constant of a particular antibody-antigen interaction. d "," k dis " or "k off " is used interchangeably herein to refer to the dissociation rate constant of a particular antibody-antigen interaction. D " as used herein refers to the equilibrium dissociation constant, which is k a k for d The proportion of (i.e., k d / k a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. DAvailable methods for determining include, but are not limited to, surface plasmon resonance (SPR) using biosensor systems such as the Biacore® system, as well as flow cytometry and Scatchard analysis.

[0078] The term “IC 50 " means half maximal inhibitory concentration and measures the potency of a substance, e.g., an antibody, to inhibit a particular biological or biochemical response. In other words, IC 50 is used as a measure of efficacy, and IC 50 The smaller the IC, the more potent the substance. In the context of in vitro or in vivo assays using antibodies or their antigen-binding fragments, the IC 50 refers to the concentration of an antibody or antigen-binding fragment thereof that reduces the maximal biological or biochemical response by 50%.

[0079] The term “EC 50 " means half maximal effective concentration and measures the potency of a substance, e.g., an antibody, to induce a specific biological or biochemical response. IC 50 Similarly, E.C. 50 is used as a measure of efficacy, and EC 50 The smaller the EC is, the more potent the substance. In the context of in vitro or in vivo assays using antibodies or antigen-binding fragments thereof, the EC is 50 refers to the concentration of an antibody or antigen-binding fragment thereof that induces a response that is 50% of the maximal biological or biochemical response.

[0080] As used herein, "receptor occupancy" or "receptor occupancy" refers to the amount of an antibody (e.g., an anti-NKG2A antibody described herein) that binds to an immunostimulatory receptor (e.g., human NKG2A). "Percent (%) receptor occupancy" or "percent (%) receptor occupancy" can be calculated using the following formula: ([Test ΔMFI] / [Total ΔMFI]) × 100. The change in mean fluorescence units (ΔMFI) is calculated by subtracting the MFI of background staining using an isotype control antibody from the MFI from the bound antibody. Total receptor levels are determined by adding a saturating amount of antibody to determine maximum expression, and therefore the MFI, of a particular immunostimulatory receptor. An alternative method for calculating total receptor expression is to use an antibody against the same immunostimulatory receptor that does not compete with the antibody for which receptor occupancy is being calculated.

[0081] As used herein, the term "naturally occurring" as applied to a substance refers to a substance that exists in nature and has not been intentionally modified by humans. For example, naturally occurring polypeptides or polynucleotide sequences present in organisms (including viruses) that can be isolated from sources in nature and have not been intentionally modified by humans in the laboratory.

[0082] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" includes one or more polypeptides.

[0083] The term "nucleic acid molecule" as used herein includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be complementary DNA (cDNA).

[0084] The term "cDNA" or "complementary DNA" refers to a non-naturally occurring nucleic acid molecule made or derived from mRNA, i.e., from which the non-coding regions have been removed.

[0085] As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted nonessential amino acid residue in an anti-NKG2A antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummel et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0086] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids or their designated sequences, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80%, at least about 90%-95%, or at least about 98%-99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the nucleic acid strand.

[0087] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or their designated sequences, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80%, at least about 90%-95%, or at least about 98%-99.5% of the amino acids.

[0088] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology = (number of identical positions) / (total number of positions) x 100), and takes into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0089] The percent identity between two nucleotide sequences can be determined, for example, using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0090] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI), available at https: / / www.ncbi.nlm.nih.gov / ).

[0091] Nucleic acids can be present in whole cells, e.g., host cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. (See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).

[0092] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Expression vectors useful in recombinant DNA technology include plasmids. Because the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0093] The terms "host cell" or "recombinant host cell" are used interchangeably and refer to a cell that contains a nucleic acid that does not naturally occur in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in later generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0094] "Immune response," as understood in the art, generally refers to a biological response in a vertebrate to foreign agents or abnormal, e.g., cancerous, cells, which response protects the vertebrate from these agents and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal cells or tissues, including, e.g., human cells or tissues. The immune response includes, for example, the activation or inhibition of T cells, eg, effector T cells or helper T (Th) cells, such as CD4+ or CD8+ T cells, or the inhibition or depletion of Treg cells.

[0095] "Effector T" ("Teff") cells are T cells with cytolytic activity (e.g., CD4+ and CD8+ T cells), as well as helper T (Th) cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0096] Regulatory T ("Treg") cells are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Memory B cells are a B cell subtype that form within germinal centers after primary infection and are important for generating an accelerated and stronger antibody-mediated immune response in the event of reinfection (also known as a secondary immune response).

[0097] "Natural killer" (NK) cells are important mediators of immune responses against pathogens and tumors and are part of the innate immune system. NK cells also play a role in regulating adaptive immune responses and have been shown to stimulate or inhibit T cell responses in different contexts. NK cells provide a rapid response against virus-infected cells and respond to tumor formation.

[0098] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, such as effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0099] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mediated, for example, by CD8+ T cells.

[0100] An "immunomodulator" or "immunoregulator" refers to an agent, e.g., a component of a signal transduction pathway, that may be involved in the regulation, regulation, or modification of an immune response. "Regulation," "regulation," or "modulation" of an immune response refers to any change in cells of the immune system or in the activity of such cells (e.g., effector T cells, e.g., Th1 cells). Such regulation includes stimulation or suppression of the immune system and may be manifested by an increase or decrease in the numbers of various cell types, an increase or decrease in the activity of these cells, and / or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In some embodiments, the immunomodulator is located on the surface of T cells. An "immunoregulatory target" or "immunoregulatory target" is an immunomodulator that is targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered by the binding of the substance, agent, moiety, compound, or molecule. Immunomodulatory targets include, for example, cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0101] "Immunotherapy" refers to the treatment of a subject, e.g., a human subject, suffering from or at risk of developing or suffering from a recurrence of a disease by methods involving inducing, enhancing, suppressing or otherwise modifying the immune response.

[0102] "Immunostimulating therapy" or "immunostimulatory therapy" refers to a therapy that results in an increase (induction or enhancement) of the immune response in a subject, for example, to treat cancer.

[0103] "Enhancing an endogenous immune response" means increasing the efficacy or potency of an existing immune response in a subject, e.g., a human subject. This increased efficacy and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0104] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinantly fused). Such linkage can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0105] As used herein, "administering" refers to the physical introduction of a therapeutic agent, e.g., a composition comprising an anti-NKG2A antibody, into a subject using any of a variety of methods and delivery systems known to those skilled in the art. "Administering" includes, for example, administration to a human patient by another party, such as one or more medical professionals, and self-administration by a human patient. Various routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, e.g., by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered by a non-parenteral route, such as a topical, epithelial, or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically. The administration can be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0106] As used herein, "adjunctive" or "combined" administration (coadministration) includes simultaneous administration of compounds in the same or different dosage forms or separate administration of compounds (e.g., sequential administration). Thus, a first antibody, e.g., an anti-NKG2A antibody, and a second, third, or more antibodies can be administered simultaneously in a single formulation. Alternatively, the first and second (or more) antibodies can be formulated for separate administration and administered concurrently or sequentially. "Combination" therapy, as used herein, refers to the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous dosing. Specifically, combination therapy encompasses both coadministration (e.g., administration of a coformulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is in some way dependent on the administration of another therapeutic agent. For example, one therapeutic agent can be administered only after a different therapeutic agent has been administered and allowed to act for a predetermined period of time. (See, e.g., Kohrt et al. (2011) Blood 117:2423).

[0107] For example, the anti-NKG2A antibody may be administered first, followed (e.g., immediately following) by the administration of the second antibody, or vice versa. In one embodiment, the anti-NKG2A antibody is administered before the administration of the second antibody. In another embodiment, the anti-NKG2A antibody is administered, for example, within about 30 minutes of the second antibody. Such simultaneous or sequential administration preferably results in both antibodies being present in the treated patient at the same time.

[0108] As used herein, the terms "inhibit" and "block" are used interchangeably and encompass both partial and complete inhibition / blocking. In some embodiments, an anti-NKG2A antibody described herein inhibits NKG2A binding to HLA-E by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, e.g., as determined further herein. In some embodiments, an anti-NKG2A antibody inhibits NKG2A binding to HLA-E by 50% or less, e.g., about 40%, 30%, 20%, 10%, 5%, or 1%, e.g., as determined further herein.

[0109] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell growth or division can lead to the formation of malignant tumors or cells that can infiltrate adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0110] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on a subject or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, or slowing the progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. In contrast, "prophylaxis" or "prevention" refers to administration to a subject not having a disease to prevent the disease from occurring. As used herein, "treat," "treating," and "treatment" do not encompass prophylaxis or prevention.

[0111] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering a disease recurrence, prevents the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or prevent the onset or recurrence of a disease can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0112] Administration of an effective amount of an anti-NKG2A antibody alone, or in combination with, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, according to any of the methods provided herein can result in at least one therapeutic effect, including, for example, a reduction in tumor growth or size, a reduction in the number of metastatic lesions appearing over time, a complete remission, a partial remission, or stable disease. For example, the method of treatment results in a comparative clinical benefit rate (CBR = complete remission (CR) + partial remission (PR) + stable disease (SD) lasting 6 months or more) that is better than that achieved without administration of the anti-NKG2A antibody or than that achieved with administration of any of the combined antibodies alone, e.g., an improvement in clinical benefit rate of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more.

[0113] By way of example, an anti-cancer agent is a drug that slows cancer progression or promotes cancer regression in a subject, including a human subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease symptom-free periods, prevention of functional or disability impairment due to disease affliction, or otherwise amelioration of disease symptoms in a patient. "Pharmacological effectiveness," "efficacy," or "potency" refers to the ability of a drug to promote cancer regression in a patient. "Physiological safety" refers to an acceptably low level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from the administration of a drug.

[0114] By way of example, with respect to tumor treatment, a therapeutically effective amount or dosage of a drug inhibits tumor cell growth by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least more than 70%, at least about 80%, or at least about 90% compared to untreated subjects. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell or tumor growth, i.e., inhibits cell or tumor growth by 100%. The ability of a compound, including an antibody, to inhibit tumor growth can be assessed using the assays described herein. Alternatively, this property of a composition can be assessed by examining the compound's ability to inhibit cell growth, which can be measured in vitro by assays known to those skilled in the art. In some embodiments, tumor growth inhibition may not occur immediately after treatment, but only after a period of time or repeated administration. In other embodiments described herein, tumor regression is observed and continues for at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days, or longer.

[0115] As used herein, the terms "fixed dose," "constant dose," and "fixed fixed dose" are used interchangeably and refer to a dose administered to a patient regardless of the patient's weight or body surface area. A fixed or constant dose is therefore provided as an absolute amount of therapeutic agent, rather than a mg / kg dose.

[0116] As used herein, the term "weight-based" dose or administration means that the dose administered to a patient is calculated based on the patient's weight. For example, if a 60 kg patient requires 3 mg / kg of anti-NKG2A antibody, an appropriate amount of anti-NKG2A antibody (i.e., 180 mg) can be calculated and used for administration.

[0117] The term "patient" includes human and other mammalian subjects receiving either therapeutic or prophylactic treatment.

[0118] The term "subject" includes humans and non-human animals. For example, the methods and compositions disclosed herein can be used to treat subjects with cancer. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human subject.

[0119] As used herein, the term "a" or "an" entity refers to one or more of that entity unless otherwise indicated; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" or "an," "one or more," and "at least one" can be used interchangeably herein.

[0120] As used herein, "and / or" shall be interpreted as a specific disclosure that each of the two specified features or components may or may not involve one another. Thus, the term "and / or," when used in phrases such as "A and / or B," includes "A and B," "A or B," "A" alone, and "B" alone. Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," encompasses each of the following: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B, and C; A alone; B alone; and C alone.

[0121] Where embodiments are described herein using the word "comprising," it is understood that similar embodiments separately described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0122] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI) unless otherwise indicated. Numeric ranges are inclusive of the numbers defining the range. Nucleotide sequences are written left to right in 5' to 3' orientation unless otherwise indicated. Amino acid sequences are written left to right in amino to carboxy orientation.

[0123] As used herein, the term "about" or "approximately" means approximately, roughly, or within a range. When the term "about" is used in conjunction with a numerical range, the term modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above or below the stated value, for example, by 10 percent above or below (higher or lower).

[0124] The headings provided herein are not intended to limit the various aspects of the disclosure, but rather should be read with reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. The various aspects described herein are described in further detail in the following subsections.

[0125] I. Anti-NKG2A antibody In some embodiments, the present disclosure describes anti-NKG2A antibodies, e.g., fully human, humanized, and chimeric antibodies, that have desirable functions or properties. For example, the antibodies specifically bind to human NKG2A protein with high affinity. In certain embodiments, the antibodies are antagonistic antibodies that block or reverse NKG2A-mediated inhibition in immune cells, e.g., T cells and NK cells. In some embodiments, anti-human NKG2A (anti-huNKG2A) antibodies have desirable properties for use as therapeutic agents in treating diseases, e.g., cancer or infection.

[0126] Particular anti-NKG2A antibodies described herein are antibodies having the CDR and / or variable region sequences of the isolated and structurally characterized antibodies 13F3.A4, NKG2A.6, NKG2A.7, NKG2A.8, NKG2A.9, NKG2A.11 described herein, as well as antibodies having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to the amino acid sequences of the anti-NKG2A antibodies described herein. In some embodiments, the anti-NKG2A antibodies described herein have at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to the variable region or CDR sequences of the anti-NKG2A antibodies described herein.

[0127] In some aspects, the antibodies of the present invention are characterized by specific functional features or properties. For example, the antibodies specifically bind to human NKG2A with high affinity. In some embodiments, the anti-NKG2A antibodies inhibit the binding of NKG2A to its ligand, HLA-E, thereby restoring NK and T cell responses against HLA-E-expressing tumors. In other words, the anti-NKG2A antibodies described herein stimulate the anti-tumor responses of T cells and NK cells by inhibiting or blocking the interaction between the NKG2A protein and its ligand, HLA-E.

[0128] In some embodiments, the anti-NKG2A antibodies described herein have the following characteristics: (1) specifically binds to human NKG2A protein; (2) blocking or reducing the binding and / or interaction of an NKG2A ligand (e.g., HLA-E in humans) with a human NKG2A protein (in other embodiments, the anti-NKG2A antibodies described herein block or reduce the binding and / or interaction of an NKG2A ligand with a non-human NKG2A protein); (3) reversing NKG2A-mediated inhibitory signaling; (4) not binding to human NKG2C protein or binding with low affinity; (5) binds to human and cynomolgus monkey NKG2A with high affinity; (6) not binding to mouse or rat NKG2A or exhibiting low affinity therefor; (7) not interfering with the activation signal induced by the binding of HLA-E to the NKG2C protein; (8) Reduced binding to human Fc gamma receptors (FcγRs); (9) Inducing and / or enhancing anti-tumor immune responses; (10) enhancing the functional activity of T cells (in some embodiments, increasing cytotoxic T cell function, e.g., as measured by lysis of HLA-E-expressing tumor cells); (11) enhancing the functional activity of natural killer (NK) cells, for example, by inducing NK cell activation; (12) increasing cytokine, e.g., IFNγ, production; and / or (13) The following amino acid residues, as determined by HDX-MS and / or FPOP epitope mapping: Area 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2, Area 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2, Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2, Region 4: L (amino acid residue 206 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2) specifically binds to an epitope located within a discontinuous region comprising (14) The following amino acid residues as determined by HDX-MS: Area 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2), Area 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2), Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2) specifically binds to an epitope located within a discontinuous region comprising indicates one or more of:

[0129] In some embodiments, the anti-NKG2A antibodies described herein bind with high affinity to human and cynomolgus monkey NKG2A and do not bind, or bind with low affinity, to non-primate NKG2A, e.g., mouse or rat NKG2A. Specifically, in some embodiments, the anti-NKG2A antibodies have the following properties: (a) an EC of about 0.6 nM or less for binding to human NKG2A protein as measured by a cell binding assay 50 having value, (b) an EC of about 9.0 nM or greater for binding to human NKG2C protein as measured by a cell binding assay. 50 having value, (c) an IC of about 1.0 nM or less with respect to reducing the binding and / or interaction of HLA-E with human NKG2A protein as measured by a cell-blocking assay 50 having value, (d) a K of about 0.4 nM or less as measured by Scatchard analysis D and binds to the human NKG2A protein. (e) a K of about 61 nM or less as measured by surface plasmon resonance D and binds to the human NKG2A protein. (f) a K of about 1.0 nM or less as measured by Scatchard analysis D and binds to the cynomolgus monkey NKG2A protein. (g) be internalized upon binding to NKG2A-expressing cells; (h) increasing interferon-gamma (IFNγ) production, and / or (i) The half-life of the anti-NKG2A antibody:NKG2A protein complex is about 40 seconds or longer. indicates one or more of:

[0130] In some embodiments, the anti-NKG2A antibodies described herein block the binding and / or interaction of the NKG2A ligand (HLA-E in humans) with the human NKG2A protein. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit the following properties: a) Anti-NKG2A antibodies have an IC of approximately 0.30 nM for NKL 50 and an IC of approximately 1.0 nM for CHO-hNKG2A cells 50 blocking the binding of HLA-E pentamers to cells expressing human NKG2A; b) Anti-NKG2A antibodies complexed with HNKG2A-CD94-mFC or cynomolgus monkey NKG2A-CD94-mFc proteins block human HLA-E binding. indicates one or more of:

[0131] In some embodiments, the anti-NKG2A antibodies described herein are specific for human NKG2A. Specifically, in some embodiments, the anti-NKG2A antibodies have the following characteristics: a) a second EC for the binding of an anti-NKG2A antibody to human NKG2A, 50 EC values ​​approximately 15 times lower than 50 In some embodiments, the EC value for binding of the anti-NKG2A antibody to human NKG2A is 50 The EC value for binding of anti-NKG2A antibodies to human NKG2C was approximately 0.6 nM. 50 The value was approximately 9.0 nM. b) the absence of specific binding of the anti-NKG2A antibody to human NKG2C based on SPR analysis, and / or c) does not block the interaction of human NKG2C and HLA-E as measured by flow cytometry indicates one or more of:

[0132] In some embodiments, the anti-NKG2A has an inactive Fc (e.g., the antibody is an IgG1 isotype) to reduce or prevent FcγR binding. Without being bound by any theory, NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, so reducing agonism or depletion of NKG2A+ CD8+ T or NK cells is beneficial for anti-tumor function. Therefore, blocking the interaction of NKG2A and HLA-E can be achieved using an anti-NKG2A antibody that does not interact with human FcγR.

[0133] In some embodiments, the anti-NKG2A antibodies described herein enhance the anti-tumor functional activity of T cells. Specifically, in some embodiments, the anti-NKG2A antibodies have the following properties: a) an EC of about 0.2 nM or less 50 reverse the inhibition of NK-κB signaling in NKG2A-expressing Jurkat T cell lines stimulated by CHO / scOKT3 / HLA-E, b) NKG2A isolated from healthy donor PBMCs co-cultured with CHO / scOKT3 / HLA-E / PD-L1 + Induction of interferon-gamma (IFN-γ) in CD8 T cells, c) NKG2A isolated from human tumors co-cultured with CHO / scOKT3 / HLA-E / PD-L1 + Induction of IFN-γ in CD8 T cells indicates one or more of:

[0134] In some embodiments, the anti-NKG2A antibodies described herein enhance the anti-tumor functional activity of NK cells. Specifically, in some embodiments, the anti-NKG2A antibodies have the following properties: a) Increased IFN-γ production in NKL cells co-cultured with CHO / MICA / HLA-E; b) Inducing a dose-dependent increase in NK cell degranulation and lysis of HLA-E-expressing tumor cells indicates one or more of:

[0135] In some embodiments, the anti-NKG2A antibodies described herein are internalized after binding to NKG2A-expressing cells. Specifically, in some embodiments, the anti-NKG2A antibodies have an EC 50 In some embodiments, the anti-NKG2A antibody exhibits internalization at an EC of about 0.5 nM or less. 50 and shows dose-dependent internalization.

[0136] In some embodiments, the anti-NKG2A antibodies of the invention lack sequence propensities that reduce the chemical stability of the antibody. The anti-NKG2A antibodies of the invention have a variety of important uses, for example, for the treatment and / or diagnosis of cancer and other disorders associated with NKG2A expression and / or activity.

[0137] In some embodiments, the anti-NKG2A antibodies disclosed herein by amino acid sequences bind to specific epitopes on human NKG2A, as described in Example 4.

[0138] Binding to human NKG2A can be assessed using one or more techniques well established in the art. For example, in some embodiments, antibodies are tested by flow cytometry assays in which the antibodies are reacted with a cell line that expresses human NKG2A, e.g., CHO cells that have been transfected to express human NKG2A on their cell surface. Additionally, or alternatively, antibody binding can be assessed by measuring binding kinetics (e.g., K D values) can be tested in a Biacore binding assay. Still other suitable binding assays include, for example, ELISA assays using recombinant human NKG2A protein.

[0139] In some embodiments, the anti-NKG2A antibodies or antigen-binding fragments thereof described herein are administered with high affinity, e.g., at concentrations of 1×10 -6 M or less, 1×10 -7M or less, 1×10 -8 M or less, 1×10 -9 M or less, or 10 -10 K below M D It binds to the human NKG2A protein with nanomolar affinity, including

[0140] Some embodiments of the present invention comprise a nucleotide sequence containing the following amino acid residues as determined by HDX-MS and / or FPOP epitope mapping: Area 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2 (native hNKG2A amino acid sequence), Area 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2), Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2), Region 4: L (amino acid residue 206 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2) The present invention relates to an anti-NKG2A monoclonal antibody or antigen-binding portion thereof that specifically binds to an epitope located within a discontinuous region comprising:

[0141] In some embodiments, the present invention provides a method for the preparation of a medicament for the treatment of a medicament comprising administering to a patient a medicament for the treatment of ... Area 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2), Area 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2), Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2) The present invention relates to an anti-NKG2A monoclonal antibody, or antigen-binding portion thereof, that specifically binds to an epitope located within a discontinuous region comprising:

[0142] In some embodiments, the anti-NKG2A antibodies described herein enhance NK cell function by blocking NKG2A / HLA-E-mediated inhibition. In another embodiment, the anti-NKG2A antibodies bind to human NKG2A protein and stimulate an anti-tumor immune response, e.g., an antigen-specific T cell and / or NK cell response. The ability of anti-NKG2A antibodies to stimulate an immune response can be tested by measuring tumor growth, such as in an in vivo tumor graft model, as described in the Examples herein. In other embodiments, the anti-NKG2A antibodies, or antigen-binding portions thereof, increase cytokine production (e.g., interferon-gamma (IFN-γ)) in NKG2A-expressing T cells and / or increase T cell proliferation, including effector T cells and cytotoxic T cells (also known as CD8+ T cells).

[0143] In another embodiment, the anti-NKG2A antibody or antigen-binding fragment thereof binds to human NKG2A and has the following characteristics: a) binds to one or more of the following residues as determined by HDX-MS and / or FPOP epitope mapping: Area 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2 (native hNKG2A amino acid sequence), Area 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2), Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2), Region 4: L (amino acid residue 206 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2), b) binds to the same epitope on human NKG2A as the NKG2A.11 and 13F3.A4 antibodies; c) competes with NKG2A.11 and 13F3.A4 antibodies for binding to human NKG2A; d) an EC of approximately 0.4 nM as measured by Biocore 50 and bind to human NK cells. e) IC of approximately 0.3 nM as measured by Biacore 50 It blocks the binding of human NK cells to HLA-E. f) EC of approximately 1 nM or less 50 and bind to cynomolgus monkey NKG2A-expressing CHO cells. g) EC of approximately 9.0 nM or greater 50 and have low binding to human NKG2C (in other words, the anti-NKG2A antibody does not block the interaction of human NKG2C with HLA-E), and / or h) enhancing the anti-tumor response of CD8+ T cells and NK cells, e.g. i. Increase IFN-γ production in primary T:CHO-OKT3-HLA-E-PDL1 assay; ii. Increase IFN-γ production in T-cell tumor infiltrating lymphocytes (TIL): CHO-OKT3-HLA-E-PDL1 assay, and / or iii. Increase cytotoxicity and IFN-γ production in primary NK cell assays At least one of the following is shown.

[0144] In some embodiments, the anti-NKG2A antibodies of the invention include humanized and fully human monoclonal antibodies. In other embodiments, the antibodies are, for example, chimeric monoclonal antibodies.

[0145] Anti-NKG2A monoclonal antibody In some embodiments, antibodies of the invention are monoclonal antibodies 13F3.A4, NKG2A.9, and NKG2A.11, which have been isolated and structurally characterized as described in the Examples below. The VH and VL amino acid sequences are set forth in the Sequence Listing and Sequence Listing.

[0146] Given that each of these antibodies can bind to human NKG2A, other anti-hNKG2A binding molecules of the invention can be made in which the VH and VL sequences are "mixed and matched." In some embodiments, when VH and VL chains are mixed and matched, a VH from a particular VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, in some embodiments, a VL sequence from a particular VH / VL pairing is replaced with a structurally similar VL sequence. Thus, in one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human NKG2A protein, wherein the light and heavy chain variable regions are (a) the amino acid sequences of SEQ ID NOs: 9 and 8, respectively; (b) the amino acid sequences of SEQ ID NOs: 164 and 8, respectively; or (c) the amino acid sequences of SEQ ID NOs: 169 and 167, respectively. The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

[0147] In another aspect, the present disclosure provides an antibody comprising the heavy and light chain CDR1, CDR2, and CDR3 of the NKG2A.9, NKG2A.11, and 13F3A.4 antibodies. Accordingly, in one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a human NKG2A protein, wherein the antibody comprises: (a) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (b) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 154, 14, and 15, respectively; or (c) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and a light chain variable domain comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 155, 14, and 15, respectively. The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

[0148] It is well known that the CDR3 domain can alone, independently of the CDR1 and / or CDR2 domains, determine the binding specificity of an antibody to its cognate antigen, and that, predictably, multiple antibodies with the same binding specificity can be generated based on a common CDR3 sequence. (See, e.g., Klimka et al., British J. of Cancer 83(2):252-260 (2000). Accordingly, the present disclosure provides monoclonal antibodies comprising one or more heavy and / or light chain CDR3 domains derived from an antibody derived from a human or non-human animal, which can specifically bind to human NKG2A. In certain aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and / or light chain CDR3 domains derived from a non-human antibody, which can specifically bind to human NKG2A. Within some embodiments, such inventive antibodies comprising one or more heavy and / or light chain CDR3 domains derived from a non-human antibody (a) can compete for binding, (b) retain the functional characteristics of, (c) bind to the same epitope, and / or (d) have a similar binding affinity as the corresponding parent non-human antibody.

[0149] In other aspects, the disclosure provides monoclonal antibodies comprising one or more heavy and / or light chain CDR3 domains derived from a human antibody, such as a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to human NKG2A. In other aspects, the disclosure provides monoclonal antibodies comprising one or more heavy and / or light chain CDR3 domains derived from a first human antibody, such as a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to human NKG2A, and the CDR3 domains from the first human antibody are replaced with the CDR3 domains of a human antibody that lacks binding specificity for NKG2A, to generate a second human antibody that is capable of specifically binding to human NKG2A. In some embodiments, such inventive antibodies comprising one or more heavy and / or light chain CDR3 domains derived from a first human antibody (a) can compete for binding, (b) retain the functional characteristics, (c) bind to the same epitope, and / or (d) have a similar binding affinity to the corresponding parent non-human antibody.

[0150] In some embodiments, the present invention provides an anti-hNKG2A antibody having an inert Fc human IgG1.3 isotype. In some embodiments, the anti-hNKG2A antibody having such an inert Fc exhibits superior efficacy in cancer treatment compared to other isotypes.

[0151] b. Antibodies with conservative modifications In certain embodiments, the anti-NKG2A antibodies of the present invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprise a designated amino acid sequence or conservative modification thereof based on the antibodies described herein (e.g., 13F3.A4, NKG2A.9, and NKG2A.11 antibodies), which antibodies retain the desired functional properties of the anti-hNKG2A antibodies of the present invention. It is understood in the art that certain conservative sequence modifications that do not eliminate antigen binding may be made. (See, e.g., Brummell et al. (1993) Biochem 32:1180-8.) Accordingly, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, (a) the heavy chain variable region comprises a CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 12, or a conservative modification thereof; (b) the antibody, or antigen-binding portion thereof, specifically binds to human NKG2A; An isolated monoclonal antibody or antigen-binding fragment thereof is provided.

[0152] In further embodiments, the antibody has one or more of the functional properties described herein, such as high affinity binding to human NKG2A and / or the ability to block NKG2A / HLA-E interactions.

[0153] In some embodiments, the heavy chain variable region comprising the CDR2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 11, or a conservative modification thereof, and the light chain variable region comprising the CDR2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 14, or a conservative modification thereof. In another embodiment, the heavy chain variable region comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 10, or a conservative modification thereof, and the light chain variable region comprises a CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 13, 154, or 155, or a conservative modification thereof.

[0154] In various embodiments, the anti-NKG2A antibody is, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0155] c. An antibody that binds to the same epitope as the anti-hNKG2A antibody In another embodiment, the present disclosure provides antibodies that bind to the same epitope on the human NKG2A protein as any of the anti-hNKG2A monoclonal antibodies of the present invention (i.e., antibodies that have the ability to cross-compete with any of the monoclonal antibodies of the present invention for binding to the human NKG2A protein). In some embodiments, the reference antibodies for cross-competition studies are the monoclonal antibodies NKG2A.9, NKG2A.11, and 13F3.A4 in a standard human NKG2A binding assay. For example, a standard ELISA assay can be used in which recombinant human NKG2A protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and the ability of the unlabeled antibody to compete with the binding of the labeled antibody is evaluated. Additionally or alternatively, Biacore analysis can be used to evaluate the ability of antibodies to cross-compete. The ability of the test antibody to inhibit the binding of, for example, NKG2A.9, NKG2A.11, and / or 13F3.A4 to human NKG2A indicates that the test antibody can compete with NKG2A.9, NKG2A.11, and / or 13F3.A4 for binding to human NKG2A and thus binds to the same epitope on human NKG2A.9 as NKG2A.9, NKG2A.11, and / or 13F3.A4. In some embodiments, antibodies that bind to the same epitope on human NKG2A as NKG2A.9, NKG2A.11, and / or 13F3.A4 are humanized or human monoclonal antibodies.

[0156] As discussed further in Example 4, the binding of NKG2A.9 and 13F3.A4 has been mapped to specific residues. Thus, in one embodiment, the present invention provides a method for determining the binding of NKG2A.9 and 13F3.A4 to the following amino acid residues when bound to human NKG2A protein, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS): (e) LSIDNEEMKF (SEQ ID NO: 156); (f) PSSWIGVFRNSSHHPW (SEQ ID NO: 157); (g) LAFKHEIKDSDN (SEQ ID NO: 158); and (h) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to, wherein the monoclonal antibody blocks binding of an NKG2A ligand (e.g., HLA-E in humans) to the human NKG2A protein.

[0157] In another embodiment, the present invention provides a method for the production of NKG2A-binding proteins comprising the following amino acid residues when bound to human NKG2A, as determined by HDX-MS and / or rapid photochemical oxidation (FPOP) epitope mapping of the protein: (f) LSIDNEEMKF (SEQ ID NO: 156); (g) PSSWIGVFRNSSHHPW (SEQ ID NO: 157); (h) LAFKHEIKDSDN (SEQ ID NO: 158); (i) L; and (j) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to, wherein the monoclonal antibody blocks binding of an NKG2A ligand (e.g., HLA-E in humans) to the human NKG2A protein.

[0158] Such humanized or human monoclonal antibodies can be prepared and isolated as described herein. For example, anti-hNKG2A antibodies that bind to the same or similar epitopes as the antibodies disclosed herein can be generated using immunization protocols, such as those described herein. The resulting antibodies can be screened for high-affinity binding to human NKG2A. The selected antibodies can then be studied, for example, in yeast display assays in which sequence variants of hNKG2A are displayed on the surface of yeast cells, or by hydrogen-deuterium exchange experiments and / or FPOP, to determine the exact epitope to which the antibody binds.

[0159] Epitope determination can be performed by any method known in the art. In some embodiments, an anti-hNKG2A antibody is considered to bind to the same epitope as an anti-hNKG2A monoclonal antibody disclosed herein if it contacts one or more of the same residues in at least one region of hNKG2A, if it contacts most of the residues in at least one region of hNKG2A, if it contacts most of the residues in each region of hNKG2A, if it contacts most of the contacts throughout the entire length of hNKG2A, if it contacts all of the same distinct regions of hNKG2A, if it contacts all of the residues in any one region on hNKG2A, or if it contacts all of the same residues in all of the same regions. An epitope "region" is a cluster of residues along, but not necessarily directly adjacent to, the primary sequence.

[0160] Techniques for identifying antibodies that bind to the "same epitope on hNKG2A" using the antibodies described herein include X-ray analysis of crystals of the antigen:antibody complex, which provides atomic resolution of the epitope. Other methods monitor the binding of antibodies to antigen fragments or mutated variations of the antigen, where loss of binding due to amino acid modifications within the antigen sequence indicates epitope components. Methods may also rely on the ability of the antibody of interest to affinity isolate specific short peptides (either in their native three-dimensional form or in denatured forms) from combinatorial phage-display peptide libraries or protease digests of the target protein. The peptides are then considered leads for defining the epitope corresponding to the antibody used to screen the peptide library. Computational algorithms have also been developed for epitope mapping and have been shown to map conformationally discontinuous epitopes.

[0161] Epitopes or regions containing epitopes can also be identified by screening for binding to a series of overlapping peptides spanning NKG2A. Alternatively, the method of Jespers et al. (1994) Biotechnology;12:899 may be used to guide the selection of antibodies that have the same epitope and therefore similar properties to the anti-NKG2A antibodies described herein. Using phage display, the heavy chain of an anti-NKG2A antibody is first paired with a repertoire of (e.g., human) light chains to select for NKG2A-binding antibodies, and then the new light chain is paired with a repertoire of (e.g., human) heavy chains to select for (e.g., human) NKG2A-binding antibodies that have the same epitope or epitope region as the anti-NKG2A antibodies described herein. Alternatively, variants of the antibodies described herein can be obtained by mutagenesis of the cDNA sequences encoding the heavy and light chains of the antibodies.

[0162] Alanine scanning mutagenesis as described by Cunningham & Wells, Science 244: 1081 (1989) or some other form of point mutagenesis of amino acid residues in NKG2A may be used to probe the functional epitope of an anti-NKG2A antibody.

[0163] The epitope or epitope region bound by a specific antibody (an "epitope region" is a region that includes or overlaps with the epitope) may also be determined by assessing the binding of the antibody to peptides comprising NKG2A fragments. A series of overlapping peptides encompassing the NKG2A sequence (e.g., human NKG2A) may be synthesized and screened for binding, for example, in direct ELISA, competitive ELISA (in which peptides are assessed for their ability to prevent antibody binding to NKG2A bound to wells of a microtiter plate), or on a chip. Such peptide screening methods may not be able to detect some discontinuous functional epitopes, i.e., functional epitopes that comprise amino acid residues that are not contiguous along the primary sequence of the NKG2A polypeptide chain.

[0164] Epitopes can also be identified by MS-based protein footprinting methods, such as HDX-MS and fast photochemical oxidation of proteins (FPOP). HDX-MS can be carried out, for example, as further described in Wei et al. (2014) Drug Discovery Today 19:95, which method is specifically incorporated herein by reference. FPOP can be carried out, for example, as described in Hambley & Gross (2005) J. American Soc. Mass Spectrometry 16:2057, which method is specifically incorporated herein by reference.

[0165] The epitope bound by an anti-NKG2A antibody may also be determined by structural methods such as X-ray crystal structure determinations (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the HD exchange rate of the labile amide hydrogen in NKG2A when free and when bound in complex with the antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31:11335, Zinn-Justin et al. (1993) Biochemistry 32:6884).

[0166] Unless otherwise indicated, and with reference to the claims, the epitope to which the antibody binds is the epitope determined by the HDX-MS method.

[0167] High-affinity binding anti-NKG2A antibody In some embodiments, the anti-hNKG2A antibodies of the invention bind to hNKG2A with high affinity, making them effective therapeutic agents. In various embodiments, the anti-hNKG2A antibodies of the invention have a K of less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 300 pM, or less than 100 pM. D Standard assays for assessing the binding ability of an antibody to hNKG2A include ELISA, RIA, Western blot, biolayer interferometry (BLI), and Biacore™ SPR analysis (see Example 10).

[0168] d. Anti-NKG2A antibody sequence variants The anti-NKG2A antibody sequence variants disclosed herein maintain the desirable functional properties disclosed herein. CDR regions are delineated using the Kabat system (Kabat, et al., 1991) unless otherwise indicated. In some embodiments, the present invention further provides human or humanized anti-hNKG2A antibodies comprising CDR sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the CDR sequences of the antibodies disclosed herein. The present invention also provides anti-hNKG2A antibodies comprising heavy and / or light chain variable domain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the heavy and / or light chain variable domain sequences of the antibodies disclosed herein, as well as anti-hNKG2A antibodies comprising full-length heavy and / or light chain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the heavy and / or light chain sequences of the antibodies disclosed herein.

[0169] II. Engineered and Modified Antibodies aV H and V L region To engineer modified antibodies, the V disclosed herein can be used as starting material. H and / or V L Also provided are engineered and modified antibodies that can be prepared using antibodies having one or more of the sequences, which modified antibodies can have altered properties from the starting antibody. In some embodiments, the antibodies described herein have one or both variable regions (i.e., V H and / or V L), e.g., by modifying one or more residues in one or more CDR regions and / or in one or more framework regions. Additionally, or alternatively, the antibodies described herein have been engineered by modifying residues in the constant region(s), e.g., to alter the effector function(s) of the antibody.

[0170] In one embodiment, genetic engineering of the variable region includes CDR grafting. Such grafting is particularly useful in humanizing non-human anti-NKG2A antibodies, for example, anti-HNKG2A antibodies that compete for binding with the anti-hNKG2A antibodies disclosed herein and / or bind to the same epitope as the selective anti-hNKG2A antibodies disclosed herein. Antibodies interact with target antigens primarily through amino acid residues located in the heavy and light chain CDRs. The CDRs are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Expression vectors can be constructed to contain CDR sequences from a particular reference (also referred to as a "parent") antibody grafted onto framework sequences from a different antibody (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. USA 86:10029-10033; Winter, U.S. Pat. No. 5,225,539; and Queen et al., U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370). In some cases, the resulting recombinant antibody has properties similar to those of the parent antibody. The engineered antibody can then be further modified to acquire properties different from those of the parent antibody.In other cases, by grafting the parent CDR sequence onto the framework, certain characteristics of the parent antibody are suppressed, so that the recombinant antibody no longer has these characteristics.One exemplary characteristic is the binding affinity to the antigen.In such cases, it may be advantageous to further modify the engineered antibody to restore the desired characteristics of the parent antibody.

[0171] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the "VBase" human germline sequence database, as well as in Kabat, EA, et al. (1991); Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V H and "Chemical Segments Reveal a Strong Bias in Their Usage," Eur. J. Immunol. 24:827-836, the contents of each of which are expressly incorporated herein by reference.

[0172] In some embodiments, the framework sequences for use in the antibodies described herein are structurally similar to the framework sequences used by the antibodies described herein. H CDR1, 2 and 3 sequences and V LCDR1, 2, and 3 sequences can be grafted onto framework regions that have the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that contain up to 20 amino acid substitutions, including conservative amino acid substitutions, compared to the germline sequences. For example, in certain cases, it has been found to be beneficial to mutate residues within framework regions to maintain or enhance the antigen-binding ability of antibodies (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762, and 6,180,370 to Queen et al.).

[0173] The engineered antibodies described herein may be modified, for example, by adding V to improve the properties of the antibody, e.g., to reduce the immunogenicity of the antibody. H and / or V L These include antibodies in which modifications have been made to framework residues within the framework region. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More particularly, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. Somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis, to return the framework region sequences to their germline configuration. Such "backmutated" antibodies are also encompassed by the present disclosure.

[0174] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T cell epitopes and thereby reduce the immunogenic potential of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0175] Another type of variable region modification is to mutate amino acid residues in the CDR regions to improve one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutation(s) and effect on antibody binding or other functional properties of interest. Conservative modifications are preferably introduced. Mutations can be amino acid additions, deletions, or substitutions. In some embodiments, no more than 1, 2, 3, 4, or 5 residues in the CDR regions are changed.

[0176] The methionine residues in the CDR of an antibody may be oxidized, resulting in the possibility of chemical degradation and the resulting reduction in the efficacy of the antibody.Therefore, also provided herein is an anti-NKG2A antibody in which one or more methionine residues in the heavy and / or light chain CDRs are replaced with amino acid residues that are not susceptible to oxidative degradation.Similarly, deamidation sites, particularly in the CDRs, may also be removed from the anti-NKG2A antibody.Also provided herein is an antibody in which potential glycosylation sites in the antigen-binding domain are eliminated to prevent glycosylation that may interfere with antigen binding.See, for example, U.S. Patent No. 5,714,350.

[0177] b. Antibody masking In some embodiments, the antibodies disclosed herein are modified to limit binding to specific cells and / or tissues. In one embodiment, such antibodies contain a blocking peptide "mask" that specifically binds to the antigen-binding surface of the antibody and interferes with antigen binding. In some embodiments, the mask is linked to each binding arm of the antibody by a protease-cleavable linker. See, for example, U.S. Patent No. 8,518,404 to CytomX. Antibodies with protease-cleavable linkers are useful for treating cancers in which protease levels are significantly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the masking / blocking peptide to be released, allowing antigen binding selectivity in the tumor rather than in peripheral tissues where antigen binding may cause unwanted side effects.

[0178] In another embodiment, a bivalent binding compound ("masking ligand") has been developed that contains two antigen-binding domains and binds to both antigen-binding surfaces of a (bivalent) antibody, interfering with antigen binding. In one embodiment, the two binding domain masks are linked to each other (rather than to the antibody) by a cleavable linker, e.g., a linker cleavable by peptidase. (See, e.g., International Patent Application Publication WO 2010 / 077643 to Tegopharm Corp.) The masking ligand may comprise or be derived from the antigen to which the antibody is intended to bind, or may be independently generated (e.g., an anti-idiotypic binding fragment). Such masking ligands are useful for treating cancers in which protease levels are significantly increased in the tumor microenvironment compared to non-tumor tissue. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to dissociate from each other, reducing the avidity of the antigen-binding surface of the antibody. The resulting dissociation of the masking ligand from the antibody allows for antigen binding selectivity in the tumor rather than in peripheral tissues where antigen binding may cause unwanted side effects.

[0179] c. Fc and modified Fc regions In one embodiment, the antibodies described herein may comprise an Fc region selected based on the biological activity of the antibody. Salfeld, Nat. Biotechnol. 25:1369 (2007). Human IgG can be classified into four subclasses, for example, IgG1, IgG2, IgG3, and IgG4. Each of these subclasses contains an Fc region with a unique profile for binding to one or more of the Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32a,c), FcγRIIIA and FcγRIIIB (CD16a,b), and the inhibitory receptor FcγRIIB (CD32b)), as well as the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to only FcγRIIA. H131 binds to FcγRIIA R131 FcγRIIIA V158 IgG4 has lower affinity for FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 the inhibitory receptor FcγRIIB has lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors (Bruhns et al. (2009) Blood 113:3716). Studies have shown that FcγRI does not bind IgG2, and FcγRIIIB does not bind IgG2 or IgG4. Ibid. Generally, with regard to ADCC activity, human IgG1≧IgG3≫IgG4≧IgG2. In some embodiments, for example, because ADCC is desired, an IgG1 constant domain is selected for use in a therapeutic composition, rather than an IgG2 or IgG4 constant domain.

[0180] The anti-hNKG2A antibody variable regions described herein can be Fc, e.g., IgG1, IgG2, IgG3, or IgG4. It may be linked (e.g., covalently linked or fused) to Fc, which may be, for example, any allotype or isoallotype of IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); any allotype or isoallotype of IgG2: G2m, G2m23(n); any allotype or isoallotype of IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v). (See, e.g., Jefferis et al. (2009) mAbs 1:1). The choice of allotype may be influenced by potential immunogenicity concerns, for example, to minimize the formation of anti-drug antibodies.

[0181] In some embodiments, the anti-NKG2A antibodies of the invention are unable to interact with human FcγR. NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, and therefore NKG2A + Avoiding or reducing the agonism or depletion of CD8+ T or NK cells enhances anti-tumor immunity. Therefore, blocking the NKG2A / HLA-E interaction is desirable for anti-NKG2A antibodies that cannot interact with human FcγRs.

[0182] d.Extended half-life In some embodiments, an anti-NKG2A antibody is modified to increase its biological half-life, e.g., the serum half-life of the antibody. Various approaches are known in the art. In one embodiment, the antibody is altered in the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described by Presta et al. in U.S. Pat. Nos. 5,869,046 and 6,121,022. For example, a combined Fc variant containing M252Y, S254T, and T256E increases half-life by nearly four times. (Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514). Other modifications to increase FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; No. 6,277,375; No. 6,821,505; WO97 / 34631; WO2002 / 060919.

[0183] The serum half-life of the antibodies described herein can also be increased by pegylation. An antibody may be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Preferably, pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an unglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. (See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.).

[0184] In some cases, it may be desirable to decrease rather than increase the half-life of an antibody. In some embodiments, the antibodies described herein contain modifications that decrease their half-life. Modifications such as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A / R I253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in the Fc of human IgG1 can decrease FcRn binding and thus decrease half-life (increase clearance) for use in situations where rapid clearance is preferable, such as medical imaging. (See also Kenanova et al. (2005) Cancer Res. 65:622.) Other means for enhancing clearance include formatting the antigen-binding domain of the present invention as an antibody fragment lacking the ability to bind to FcRn, such as a Fab fragment. Such modifications can, for example, reduce the circulating half-life of an antibody from a few weeks to a few hours. Selective pegylation of the antibody fragment can then be used to increase the half-life of the antibody fragment, if desired. (Chapman et al. (1999) Nat. Biotechnol. 17:780) To increase half-life, the antibody fragment can also be fused with human serum albumin, e.g., to form a fusion protein construct. (Yeh et al. (1992) Proc. Nat'l Acad. Sci. 89:1904) Alternatively, a bispecific antibody can be constructed using a first antigen-binding domain and a second antigen-binding domain of the present invention that bind to human serum albumin (HSA). (See International Patent Application Publication WO2009 / 127691 and the patent references cited therein.) Alternatively, specialized polypeptide sequences, such as "XTEN" polypeptide sequences, can be added to the antibody fragment to increase half-life. (Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; International Patent Application Publication No. WO2010 / 091122).

[0185] e. Further Fc variants In some embodiments, when an IgG1 constant domain is used, potential protease cleavage sites in the hinge of the IgG1 construct can be eliminated by D221G and K222S modifications to increase antibody stability (WO2014 / 043344).

[0186] The affinity and binding properties of an Fc variant for its ligand (Fc receptor) may be determined by a variety of in vitro assay methods (e.g., biochemical or immunological-based assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE® SPR analysis) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may employ labels on one or more components being examined and / or may utilize various detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics, focusing on antibody-immunogen interactions, can be found in Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999).

[0187] In yet other embodiments, the glycosylation of an antibody is modified to increase or decrease effector function. For example, an aglycosylated antibody lacking all effector function can be generated by mutating the conserved asparagine residue at position 297 (e.g., N297A), thus abolishing complement and FcγRI binding. (Bolt et al. (1993) Eur. J. Immunol. 23:403; see also Tao & Morrison (1989) J. Immunol. 143:2595 (using N297Q in IgG1 to eliminate glycosylation at position 297)).

[0188] Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore this function. Aglycosylated antibodies, such as those resulting from N297A / C / D / or H mutations or those produced in a system that does not glycosylate proteins (e.g., E. coli), can be further mutated to restore FcγR binding, for example, S298G and / or T299A / G / or H (WO2009 / 079242) or E382V and M428I (Jung et al. (2010) Proc. Nat'l Acad. Sci (USA) 107:604).

[0189] Glycoengineering can also be used to modify the anti-inflammatory properties of IgG constructs by altering the α2,6 sialylated content of the carbohydrate chains attached at Asn297 of the Fc region, where increasing the proportion of α2,6 sialylated forms results in enhanced anti-inflammatory effects. (See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513.) Conversely, decreasing the proportion of antibodies with α2,6 sialylated carbohydrates can be useful when anti-inflammatory properties are not desired. For example, methods for modifying the α2,6 sialylated content of antibodies by selective purification of the α2,6 sialylated form or by enzymatic modification are provided in U.S. Patent Application Publication No. 2008 / 0206246. In other embodiments, the amino acid sequence of the Fc region can be modified to mimic the effects of α2,6 sialylation, for example, by including an F241A modification (WO2013 / 095966).

[0190] III. Antibody physical properties In certain embodiments, the antibodies described herein contain one or more glycosylation sites in either the light chain or heavy chain variable region. Such glycosylation sites can increase the immunogenicity of the antibody or alter antibody pharmacokinetics due to altered antigen binding (Marshall et al. (1972) Ann. Rev. Biochem. 41:673-702, Gala and Morrison (2004) J. Immunol. 172:5489-94, Wallick et al. (1988) J. Exp. Med. 168:1099-109, Spiro (2002) Glycobiology 12:43R-56R, Parekh et al. (1985) Nature 316:452-7, Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing the NXS / T sequence. In some embodiments, the anti-hNKG2A antibody does not contain variable region glycosylation. Such antibodies can be obtained by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0191] In certain embodiments, the antibodies described herein do not contain asparagine isomerism sites. Deamidation of asparagine can occur at NG or DG sequences, thereby introducing a kink into the polypeptide chain and reducing its stability (known as the isoaspartate effect).

[0192] In some embodiments, the antibodies described herein have an isoelectric point (pI) in the pH range of 6 to 9.5. In some embodiments, the antibodies described herein have a pI in the pH range of 7 to 9.5 or 6 to 8. Antibodies with a pI within a desired pI range can be obtained either by selecting antibodies from a candidate population with a pI within that pH range, or by mutating charged surface residues of a particular antibody.

[0193] In some embodiments, the antibodies described herein have an initial unfolding temperature (T M1 ) is selected and / or engineered to be greater than 60° C., greater than 65° C., or greater than 70° C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett. 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr. Sci. 40:343-9).

[0194] In some embodiments, the antibodies described herein are selected and / or engineered to have advantageous degradation properties, e.g., slow degradation in vitro and / or in vivo. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32). In some embodiments, the antibodies described herein are selected and / or engineered to have desirable aggregation properties, e.g., antibodies that exhibit minimal aggregation in vitro and / or in vivo, which may elicit an undesirable immune response and / or altered or undesirable pharmacokinetic properties. In some embodiments, the antibodies described herein exhibit 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less aggregation compared to the aggregation of the parent antibody. Aggregation can be measured by several techniques, including size exclusion column (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0195] IV. Nucleic Acid Molecules and Recombinant Methods Another aspect described herein relates to nucleic acid molecules encoding the anti-hNKG2A antibodies described herein. The nucleic acid may be present in whole cells, e.g., host cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA naturally associated with the isolated DNA) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others known in the art. (See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.) The nucleic acids described herein can be, for example, DNA or RNA, and may or may not contain introns. In certain embodiments, the nucleic acid is a cDNA molecule.

[0196] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNA encoding the light and / or heavy chains of the antibody produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibody can be recovered from the library.

[0197] V H and V LOnce DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V H A DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" means that the two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0198] V H The isolated DNA encoding the region is V H A full-length heavy chain gene can be obtained by operably linking the DNA encoding the V to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, et al., 1991), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgE, IgM, or IgD constant region, e.g., an IgG1 region. For a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0199] V L The isolated DNA encoding the region is V LThe DNA encoding the CL can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the CL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0200] To generate an scFv gene, a DNA fragment encoding the VH and VL is operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 160), such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH regions connected by the flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0201] V. Antibody Generation Various antibodies of the present invention, e.g., those that bind to the same epitope as a selected anti-hNKG2A antibody disclosed herein, can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can also be used, e.g., viral or oncogenic transformation of B lymphocytes, and phage display techniques using libraries of human antibody genes.

[0202] An exemplary animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0203] The chimeric or humanized antibodies described herein can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. Using standard molecular biology techniques, DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and genetically engineered to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to create chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To create humanized antibodies, mouse CDR regions can be inserted into human frameworks using methods known in the art (see, for example, U.S. Patent No. 5,225,539 to Winter and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0204] In one embodiment, the antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies directed against human NKG2A can be generated using transgenic or transchromosomic mice that carry parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[0205] HuMAb Mice® (Medarex, Inc.) contain human immunoglobulin gene miniloci encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGκ monoclonal antibodies (reviewed in Lonberg, N. et al. (1994), supra; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546).The preparation and use of HuMab mice and the genomic modifications carried by such mice are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the disclosures of which are incorporated herein by reference in their entireties. (See also U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429, all to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962, all to Lonberg and Kay; and Korman et al. al., PCT Publication No. WO01 / 14424).

[0206] In certain embodiments, the antibodies described herein are produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, e.g., mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in PCT Publication WO 02 / 43478 by Ishida et al.

[0207] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate the anti-hNKG2A antibodies described herein. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used, and such mice are described, for example, in U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

[0208] Additionally, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate the anti-nkg2a antibodies described herein. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Additionally, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to generate the anti-hNKG2a antibodies described herein.

[0209] Additional mouse systems described in the art for producing human antibodies, e.g., human anti-hNKG2A antibodies, include (i) the VELOCIMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions have been replaced by homologous recombination with human heavy and light chain variable regions operably linked to endogenous mouse constant regions, resulting in the generation of a chimeric antibody in the mouse (human V / mouse C), which is then subsequently converted to a fully human antibody using standard recombinant DNA techniques; and (ii) the MeMo® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains unrearranged human heavy chain variable regions but a single rearranged human common light chain variable region. Such mice and their use to generate antibodies are described, for example, in WO2009 / 15777, US2010 / 0069614, WO2011 / 072204, WO2011 / 097603, WO2011 / 163311, WO2011 / 163314, WO2012 / 148873, US2012 / 0070861 and US2012 / 0073004.

[0210] The human monoclonal antibody described herein can also be prepared by using phage display method to screen the library of human immunoglobulin genes.Such phage display method for isolating human antibodies has been established in the art.(For example, see U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.)

[0211] The human monoclonal antibodies described herein can also be prepared using mice with severe combined immunodeficiency (SCID) that have been reconstituted with human immune cells so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0212] Immunization To generate fully human antibodies against human NKG2A, mice containing human immunoglobulin genes or transgenic or transchromosomal mice (e.g., HCo12, HCo7, or KM mice) can be immunized with a purified or enriched preparation of NKG2A antigen and / or cells expressing NKG2A, as described for other antigens by, for example, Lonberg et al. (1994) Nature 368(6474): 856-859, Fishwild et al. (1996) Nature Biotechnology 14: 845-851, and WO98 / 24884. Alternatively, mice can be immunized with DNA encoding human NKG2A. Preferably, mice are 6-16 weeks old at the time of the first injection. For example, a purified or enriched preparation of recombinant human NKG2A antigen (e.g., 5 μg-50 μg) can be used to immunize mice intraperitoneally. If immunization with a purified or enriched preparation of NKG2A antigen does not produce antibodies, mice can also be immunized with cells, e.g., a cell line, that express NKG2A to boost the immune response.

[0213] HuMAb transgenic mice can be initially immunized intraperitoneally or subcutaneously (SC) with antigen in Ribi adjuvant, followed by biweekly IP / SC immunizations (up to a total of 10 immunizations) with antigen in Ribi adjuvant. The immune response can be monitored over the course of the immunization protocol using plasma samples obtained by retro-orbital bleeds. Plasma can be screened by ELISA and FACS (as described below), and mice with sufficient titers of anti-NKG2A human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen and sacrificed 3 days later, and the spleen and lymph nodes can be harvested. Two to three fusions can be performed for each immunization. Between 6 and 24 mice can be immunized for each antigen. In some embodiments, HCo7, HCo12, and KM strains are used. Furthermore, both HCo7 and HCo12 transgenes can be bred together into a single mouse carrying two different human heavy chain transgenes (HCo7 / HCo12).

[0214] Generation of hybridomas producing monoclonal antibodies against NKG2A protein To generate hybridomas producing the monoclonal antibodies described herein, spleen cells and / or lymph node cells can be isolated from immunized mice and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of splenic lymphocytes from immunized mice can be fused with Sp2 / 0 non-secretory mouse myeloma cells (ATCC, CRL 1581) using 50% PEG. The cells can be fused at approximately 2 x 10 5Hybridomas are plated into flat-bottom microtiter plates at 100°C for 2 weeks and subsequently incubated in selective medium containing 10% fetal bovine serum, 18% "653" conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, and 1X HAT (Sigma). After approximately 2 weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Extensive hybridoma growth occurs, and medium can be observed routinely after 10-14 days. Antibody-secreting hybridomas can be replated and screened again; if still positive for human IgG, monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[0215] To purify monoclonal antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using a 1.43 extinction coefficient. Monoclonal antibodies can be aliquoted and stored at -80°C.

[0216] VI. Antibody Production Generation of transfectomas producing monoclonal antibodies against NKG2A Antibodies of the present invention, including both the specific antibodies for which sequences are provided and other related anti-NKG2A antibodies, can be produced, for example, in host cell transfectomas using a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

[0217] For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector(s) by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Using the light and heavy chain variable regions of the antibodies described herein, V H The segment is C in the vector H operably linked to the V segment(s); L The segment is located at C LFull-length antibody genes of any antibody isotype can be produced by inserting the segments operably linked into an expression vector already encoding the heavy and light chain constant regions of the desired isotype. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0218] In addition to the antibody chain genes, the recombinant expression vector can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be apparent to those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can vary depending on, among other factors, the choice of the host cell to be transformed, the level of expression of the desired protein, and other factors. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma virus. Alternatively, nonviral regulatory sequences, such as the ubiquitin promoter or β-globin promoter, may be used. Still further, regulatory elements comprise sequences derived from different sources, such as the SRα promoter system, which contains sequences derived from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0219] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may carry additional sequences, such as sequences regulating replication of the vector in host cells (e.g., origins of replication), and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, the selectable marker gene usually confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0220] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" encompass a variety of commonly used techniques for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. While it is theoretically possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred, as such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be ineffective for high-efficiency production of active antibodies (Boss, MA and Wood, CR (1985) Immunology Today 6:12-13). The antibodies of the present invention can also be produced in glycoengineered strains of yeast. (Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24:210).

[0221] Exemplary mammalian host cells for expressing the recombinant antibodies described herein include CHO cells (e.g., including the dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a dihydrofolate reductase (DHFR) selectable marker, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Another exemplary expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. A recombinant expression vector encoding the antibody genes is introduced into mammalian host cells, and the antibody is produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium that the host cells are grown in. The antibody can be recovered from the culture medium using standard protein purification methods.

[0222] The N- and C-termini of the antibody polypeptide chains of the present invention may differ from the predicted sequences due to commonly observed post-translational modifications. For example, C-terminal lysine residues are often missing from antibody heavy chains (Dick et al. (2008) Biotechnol. Bioeng. 100:1132). N-terminal glutamine residues, and to a lesser extent glutamic acid residues, are frequently converted to pyroglutamic acid residues in both the light and heavy chains of therapeutic antibodies (Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al. (2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211).

[0223] The amino acid sequences of various anti-hNKG2A antibodies of the present invention are provided in the Sequence Listing. For the reasons discussed above, a C-terminal lysine is not included in many of the sequences in the Sequence Listing for the heavy chain or heavy chain constant domain. However, in alternative embodiments, each heavy chain of an anti-hNKG2A antibody of the present invention, and / or a genetic construct encoding such an antibody or its heavy or light chain, comprises this additional lysine residue at the C-terminus of the heavy chain.

[0224] VII. Assay The antibodies described herein can be tested for binding to NKG2A, for example, by standard ELISA. For example, microtiter plates are coated with 1-2 μg / mL of purified NKG2A in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from NKG2A-immunized mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS / Tween and then incubated with a secondary reagent conjugated with horseradish peroxidase (HRP) (e.g., a human antibody, or an antibody otherwise having a human heavy chain constant region, a goat anti-human IgG Fc-specific polyclonal reagent) for 1 hour at 37°C. After washing, the plates are developed with ABTS substrate (Moss Inc., product: ABTS-1000) and analyzed by spectrophotometer at OD 415-495. Sera from immunized mice are then further screened by flow cytometry for binding to cell lines expressing human NKG2A, but not to control cell lines that do not express NKG2A. Briefly, binding of anti-NKG2A antibodies is assessed by incubating NKG2A-expressing CHO cells with anti-NKG2A antibodies at a 1:20 dilution. The cells are washed, and binding is detected using PE-labeled anti-human IgG Ab. Flow cytometry analysis is performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Mice that generate the highest titers are preferably used for fusion. If mouse anti-HNKG2A antibodies are to be detected, similar experiments can be performed using anti-mouse detection antibodies.

[0225] ELISA, such as that described above, can be used to screen for antibodies, and thus hybridomas that produce antibodies that show positive reactivity with the NKG2A immunogen. Hybridomas that produce antibodies that preferably bind to NKG2A with high affinity can be subcloned and further characterized. To generate a cell bank, one clone from each hybridoma that retains the reactivity of the parent cell (by ELISA) can be selected for antibody purification.

[0226] To purify anti-NKG2A antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and OD values ​​can be measured using a 1.43 extinction coefficient. 280 The concentration can be determined by: Monoclonal antibody can be aliquoted and stored at -80°C.

[0227] To determine whether the selected anti-NKG2A monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected using a streptavidin-labeled probe. As described above, NKG2A-coated ELISA plates can be used to perform competition studies using unlabeled and biotinylated monoclonal antibodies.

[0228] To determine the isotype of purified antibodies, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated overnight at 4°C with 1 μg / mL of anti-human immunoglobulin. After blocking with 1% BSA, the plate is reacted with up to 1 μg / mL of the test monoclonal antibody or a purified isotype control for 1-2 hours at ambient temperature. The wells are then reacted with either a human IgG1- or human IgM-specific alkaline phosphatase-conjugated probe. The plate is developed and analyzed as described above.

[0229] Flow cytometry can be used to examine the binding of monoclonal antibodies to live cells expressing NKG2A. Briefly, a cell line expressing membrane-bound NKG2A (grown under standard growth conditions) is mixed with a concentration of monoclonal antibody in PBS containing 0.1% BSA for 1 hour at 4°C. After washing, the cells are reacted with a phycoerythrin (PE)-labeled anti-IgG antibody under conditions identical to those used for primary antibody staining. Samples are analyzed using a FACScan instrument, which uses light and side scatter properties to gate on single cells and examine the binding of the labeled antibody. An alternative assay using a fluorescent microscope may be used (in addition to or instead of) a flow cytometry assay. Cells can be precisely stained as described above and examined by fluorescent microscopy. This method allows visualization of individual cells but may have reduced sensitivity depending on the density of the antigen.

[0230] Anti-hNKG2A antibodies can be further tested for reactivity with the NKG2A antigen by Western blotting. Briefly, cell extracts can be prepared from cells expressing NKG2A and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 20% mouse serum, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0231] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-NKG2A antibodies include standard assays known in the art, such as Biolayer Interferometry (BLI) analysis and Biacore SPR analysis using a Biacore SPR instrument.

[0232] In one embodiment, the anti-hNKG2A antibody specifically binds to the extracellular region of human NKG2A. In one embodiment, the antibody binds to a specific domain (e.g., a functional domain) within the extracellular domain of NKG2A. In one embodiment, the anti-hNKG2A antibody specifically binds to the extracellular region of human NKG2A and the extracellular region of cynomolgus monkey NKG2A. In one embodiment, the anti-hNKG2A antibody binds to human NKG2A with high affinity.

[0233] VIII. Multispecific molecules In certain embodiments, the antibodies described herein are multispecific, e.g., bispecific or trispecific, molecules. Multispecific antigen-binding molecules, e.g., multispecific antibodies, contain two or more antigen-binding sites, each specific for a different epitope. The different epitopes can be part of the same antigen or different antigens. In one embodiment, one antigen-binding site is specific for human NKG2A, and the other is specific for a different antigen. In one embodiment, the anti-hNKG2A antibody or antigen-binding fragment thereof described herein is linked to another antigen-binding molecule with a different binding specificity, e.g., another peptide or protein (e.g., another antibody or antibody fragment, or receptor ligand), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. In one embodiment, the antibodies described herein are derivatized or linked to more than one other antigen-binding molecule to generate a multispecific molecule that binds to more than two different binding sites and / or target molecules. Thus, provided herein are bispecific molecules comprising at least one first binding specificity for NKG2A and a second binding specificity for a second target epitope. In one embodiment described herein in which the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity.

[0234] In one embodiment, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv. An antibody can also be a light or heavy chain dimer or any smallest fragment thereof, such as an Fv or single-chain construct, as described in Ladner et al., U.S. Patent No. 4,946,778, the disclosure of which is expressly incorporated by reference.

[0235] Although human monoclonal antibodies are preferred, other antibodies that can be utilized in the bispecific antibodies described herein include murine, chimeric, and humanized monoclonal antibodies.

[0236] The bispecific antibodies described herein can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Preferred conjugating agents include SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0237] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0238] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule has a combination of binding specificities, such as a (mAb x mAb), (mAb x Fab), (Fab x F(ab')2), or (ligand x Fab) fusion protein. The bispecific molecules described herein can be single-chain molecules containing one single-chain antibody and a binding determinant or single-chain bispecific molecules containing two binding determinants. The bispecific molecule can comprise at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498 and 5,482,858.

[0239] Binding of a bispecific molecule to its specific target can be confirmed using art-recognized methods, such as using ELISA, radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0240] IX. Composition Further provided are compositions, e.g., pharmaceutical compositions, containing one or more anti-NKG2A antibodies or antigen-binding fragment(s) thereof described herein, formulated together with a pharmaceutically acceptable carrier. Thus, the compositions of the present invention include human or humanized anti-hNKG2A antibodies (or antigen-binding fragments thereof) having the CDR sequences, heavy and / or light chain variable region sequences, or full-length heavy and / or light chain sequences described herein. The compositions of the present invention also include anti-hNKG2A antibodies having sequences that are variants of the sequences set forth in the Sequence Listing. For example, such antibodies may comprise sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99% identical to the CDR sequences, heavy and / or light chain variable region sequences, or full-length heavy and / or light chain sequences set forth in the Sequence Listing.

[0241] Such compositions can also include one or a combination of (e.g., two or more different) antibodies, immunoconjugates, or bispecific molecules described herein. For example, the pharmaceutical compositions described herein can include a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or that have complementary activities.

[0242] The pharmaceutical compositions described herein can also be administered as combination therapy, i.e., as an anti-NKG2A antibody in combination with other agents. For example, the combination therapy can include an anti-NKG2A antibody described herein in combination with at least one other anti-cancer agent and / or T cell stimulating (e.g., activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section regarding the use of the antibodies described herein.

[0243] In some embodiments, the pharmaceutical compositions disclosed herein may contain other compounds, drugs and / or agents used for the treatment of cancer.Such compounds, drugs and / or agents include, for example, chemotherapeutic agents, small molecule drugs or antibodies that stimulate immune responses against a given cancer.In some embodiments, the pharmaceutical composition comprises a first antibody and a second antibody specific to anti-hNKG2A.

[0244] In some embodiments, the first antibody and the second antibody are present in the composition at a fixed dose (i.e., a fixed ratio). In other embodiments, the fixed dose is at least about 1:200 to at least about 200:1, at least about 1:150 to at least about 150:1, at least about 1:100 to at least about 100:1, at least about 1:75 to at least about 75:1, at least about 1:50 to at least about 50:1, at least about 1:25 to at least about 25:1, at least about 1:10 to at least about 10:1, at least about 1:5 to at least about 5:1, at least about 1:4 to at least about 4:1, at least about 1:3 to at least about 3:1, or at least about 1:2 to at least about 2:1 mg of anti-hNKG2A antibody to mg of second antibody. In some embodiments, the fixed dose is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, or about 1:200 of anti-hNKG2A antibody to second antibody. In some embodiments, the fixed dose is at least about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 120:1, about 140:1, about 160:1, about 180:1, or about 200:1 mg of first antibody to mg of second antibody. For example, in one embodiment, the anti-hNKG2A antibody and the second antibody are administered as described in the Examples.

[0245] Additional antibodies include, for example, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody, an anti-LAG-3 antibody, an anti-CD73 antibody, an anti-CD137 antibody, an anti-CD27 antibody, or an anti-CSF-1R antibody.

[0246] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the carrier is suitable for intravenous administration. In other embodiments, the carrier is suitable for subcutaneous administration. In some embodiments, compositions comprising anti-NKG2A antibodies are delivered subcutaneously using Halozyme's ENHANZE® Drug Delivery Technology, which includes a recombinant human hyaluronidase enzyme (rHuPH20) that transiently degrades hyaluronan. In some embodiments, ENHANZE® Drug Delivery Technology allows for more rapid subcutaneous administration of the composition compared to intravenous administration. In other embodiments, depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0247] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0248] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc., (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc., and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0249] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0250] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures, as described above, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the compositions. Furthermore, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0251] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX™, Baxter International, Inc.).Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, sHASEGPs are combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0252] The use of such media and agents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is contemplated.Supplementary active compounds can also be incorporated into the compositions.

[0253] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0254] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components as needed, and then by sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components required from above-listed components.For the sterile powder that is used to prepare sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient and any other desired components from the solution that has been previously sterilized and filtered.

[0255] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. In combination with a pharmaceutically acceptable carrier, this amount can range from about 0.01 percent to about 99 percent of the active ingredient, for example, from about 0.1 percent to about 70 percent, for example, from about 1 percent to about 30 percent of the active ingredient, out of 100 percent.

[0256] In some embodiments, the composition comprises an anti-NKG2A antibody, e.g., NKG2A.9. The composition is a sterile, non-pyrogenic, single-use, preservative-free, isotonic aqueous solution for intravenous administration. The composition may be administered undiluted or further diluted to the required protein concentration in 0.9% sodium chloride injection prior to injection. In some embodiments, the anti-NKG2A antibody comprises the following excipients: L-histine, L-histidine hydrochloride monohydrate, sucrose, pentetic acid (also known as diethylenetriaminepentaacetic acid, polysorbate 80, and water for injection.

[0257] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject being treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms described herein are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active compounds for the treatment of susceptibility in individuals.

[0258] For administration of antibodies, dosages can range from about 0.0001 to 100 mg / kg of host body weight, more usually 0.01 to 5 mg / kg of host body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or within the range of 1 to 10 mg / kg. Alternatively, administration of antibodies is at a fixed dose that can range from 2 mg to 800 mg, e.g., 25 mg, 80 mg, 200 mg, or 400 mg. Exemplary treatment regimens entail administration once per week, once per two weeks, once per three weeks, once per four weeks, once per month, once per two months, once per three months, once per four months, once per five months, or once per six months. In some embodiments, the treatment regimen involves an initial dose followed by maintenance doses of different doses at intermittent dosing intervals.

[0259] In some embodiments, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. In some embodiments, therapeutic antibodies are administered on multiple occasions. The interval between single doses can be, for example, weekly, every three weeks, every four weeks, monthly, every three months, or yearly. Intervals can also be irregular, as indicated by measuring blood levels of antibodies against the target antigen in the patient. In some embodiments, dosages are adjusted to achieve a plasma antibody concentration of about 1-1000 μg / mL, and in some methods, about 25-300 μg / mL.

[0260] In some embodiments, the antibody may be administered as a sustained-release formulation. Administration via a sustained-release formulation may require less frequent administration. The dosage and frequency will vary depending on the half-life of the antibody in the patient. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for life. In some embodiments, relatively high dosages are administered at relatively short intervals for therapeutic treatment. In some embodiments, the relatively high dosages are administered until the progression of the disease is reduced or stopped, for example, until the patient shows partial or complete remission of disease symptoms. In some embodiments, prophylactic treatment is administered to the patient after therapeutic treatment.

[0261] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition described herein or its ester, salt, or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the pharmaceutical arts.

[0262] A "therapeutically effective dose" of an anti-NKG2A antibody described herein preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. In the context of cancer, a therapeutically effective dose preferably prevents further exacerbation of physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, unusual lentigo features, changes in the appearance of lentigines including asymmetry, border, color and / or diameter, newly pigmented skin areas, abnormal lentigines, dark areas under the fingernails, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, eating disorders, loss of appetite, chronic cough, worsening shortness of breath, hemoptysis, blood in the urine, blood in the stool, nausea, vomiting, liver metastasis, lung metastasis, bone metastasis, abdominal bloating, flatulence, fluid in the peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of the colon, acute peritonitis (infection, fever, pain), pain, vomiting blood, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis and pancreatic metastasis, difficulty swallowing, etc. The therapeutic effect may be observable immediately after the first administration of an anti-hNKG2A monoclonal antibody of the invention, or may only be observed after a period and / or series of doses. Such delayed effects may only be observed after several months of treatment, e.g., up to 6, 9, or 12 months.

[0263] A therapeutically effective dose, such as may be desired when early or preceding signs of disease are present, can prevent or delay the onset of cancer. Therefore, any clinical or biochemical assay that monitors any of the above may be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer. Those skilled in the art will be able to determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0264] The compositions described herein can be administered by one or more administration routes, using one or more various methods known in the art.As will be clear to those skilled in the art, the administration route and / or administration mode will vary depending on the desired results.Exemplary administration routes for the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, for example, by injection or infusion.

[0265] Alternatively, the antibodies described herein can be administered by non-parenteral routes such as topical, epithelial or mucosal routes of administration, for example, intranasal, oral, vaginal, rectal, sublingual or topical.

[0266] Active compound can be prepared with carriers that can protect the compound from rapid release, such as sustained release preparations, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used.Many methods for preparing such preparations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0267] Therapeutic compositions can be administered using medical devices known in the art.For example, in preferred embodiments, therapeutic compositions described herein can be administered using needleless hypodermic injection devices, such as the devices disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556.The well-known example of implantable agent and module for use with anti-NKG2A antibody described herein includes U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medicine at a controlled rate. No. 4,486,194, which discloses a therapeutic device for administering medication through the skin; No. 4,447,233, which discloses a medication infusion pump for delivering medication at precise infusion rates; No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; No. 4,439,196, which discloses an osmotic drug delivery system having multiple chamber compartments; and No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0268] In certain embodiments, the anti-hNKG2A antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thus enhancing targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090), and K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; see also JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0269] Also included within the scope of the present disclosure are kits containing the antibody compositions (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) described herein and instructions for use. The kits may further include at least one additional reagent, or one or more additional human antibodies described herein. The kits may include labeling indicating the intended use of the contents of the kit. The term labeling includes any writing or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0270] X. Method of Use The antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo uses, including, for example, enhancing immune responses by blocking NKG2A / HLA-E interactions. In one embodiment, the anti-NKG2A antibodies described herein are monoclonal human or humanized antibodies. In one embodiment, the anti-hNKG2A antibodies described herein (e.g., 13F3.A4, NKG2A.9, and NKG2A.11) can be administered to cells in culture in vitro or ex vivo, or to human subjects to enhance immunity in various diseases. In a specific embodiment, the anti-hNKG2A antibodies are antagonistic antibodies. Provided herein are methods of modifying an immune response in a subject, the method comprising administering to the subject an anti-NKG2A antibody or antigen-binding fragment thereof described herein, such that the immune response in the subject is enhanced, stimulated, or upregulated. In one embodiment, administering an anti-hNKG2A antibody according to the methods described herein enhances T cell and / or NK cell responses. In one embodiment, administration of an anti-hNKG2A antibody according to the methods described herein stimulates, enhances, or upregulates antigen-specific T cell responses against tumors. T cells include Teff cells, e.g., CD4+ Teff cells, CD8+ Teff cells, T helper cells (T h ) cells, and cytotoxic T (T c) cells. The tumor can be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In certain embodiments, the tumor is PD-L1 negative. The subject can also be a subject with a virus, and the immune response to the virus is enhanced. In one embodiment, administering an anti-hNKG2A antibody according to the methods described herein stimulates, enhances, or upregulates an NK cell response.

[0271] In one embodiment, the method results in an enhanced immune response in a human subject, and such enhancement has a desirable effect. In one embodiment, the human subject is a human patient with a disorder that can be treated by enhancing the immune response, e.g., a T cell-mediated immune response. In a specific embodiment, the human patient has cancer. In one embodiment, the anti-hNKG2A antibody described herein can be administered together with an antigen of interest, or the antigen may already be present in the subject to be treated, e.g., a subject with a tumor or a virus. When the anti-NKG2A antibody is administered together with another agent, the two can be administered separately or simultaneously.

[0272] Further provided is a method for inhibiting tumor cell growth in a subject, comprising administering to the subject an anti-hNKG2A antibody described herein, such that tumor cell growth is inhibited in the subject, e.g., a human subject. Also provided is a method for treating a chronic viral infection in a subject, comprising administering to the subject an anti-NKG2A antibody described herein, such that the chronic viral infection is treated in the subject, e.g., a human subject.

[0273] In some embodiments, anti-NKG2A antibodies are administered to a subject, e.g., a human patient, as adjuvant, adjuvant, or neoadjuvant therapy. In some embodiments, treatment of a subject with cancer with an anti-NKG2A antibody can result in a long-term, durable response, prolonged survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years, recurrence-free survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more years, compared to current standard treatments. In certain embodiments, treatment of a subject with cancer with an anti-hNKG2A antibody prevents or delays cancer recurrence, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more years. Anti-NKG2A therapy can be used as a first-line, second-line, or subsequent-line therapy.

[0274] These and other methods described herein are described in further detail below.

[0275] cancer Provided herein are methods for treating a subject with cancer, comprising administering to the subject an anti-hNKG2A antibody described herein such that the subject is treated, e.g., cancerous tumor growth is inhibited or reduced and / or tumor regression occurs. Anti-NKG2A antibodies can be used alone to inhibit cancerous tumor growth. Alternatively, anti-NKG2A antibodies can be used with another agent, e.g., other immunogenic agents, standard cancer treatments, or other antibodies, as described below. Combinations with PD-1 inhibitors, e.g., anti-PD-1 or anti-PD-L1 antibodies, are also provided. Combinations with CTLA-4 inhibitors, e.g., anti-CTLA-4 antibodies, are also provided. Combinations with PD-1 inhibitors and CTLA-4 inhibitors are also provided. Combinations with ICOS agonist antibodies are also provided.

[0276] In one aspect, provided herein is a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an anti-NKG2A antibody described herein. In one embodiment, the anti-NKG2A antibody can be a chimeric antibody, a human antibody, or a humanized anti-NKG2A antibody. In one embodiment, the method of treating cancer described herein comprises: (a) LSIDNEEMKF (SEQ ID NO: 156); (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157); (c) LAFKHEIKDSDN (SEQ ID NO: 158); and (d) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) The method comprises administering an anti-NKG2A antibody that contacts human NKG2A at one or more amino acid residues of

[0277] In another embodiment, the methods of treating cancer described herein include: (a) LSIDNEEMKF (SEQ ID NO: 156); (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157); (c) LAFKHEIKDSDN (SEQ ID NO: 158); (d) L; and (e) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) The method comprises administering an anti-NKG2A antibody that contacts human NKG2A at one or more amino acid residues of

[0278] In another embodiment, the method comprises administering an NKG2A.9 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising a 13F3.A4 antibody to treat cancer. In another embodiment, the method comprises administering an NKG2A.11 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising an NKG2A.9 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising an NKG2A.11 antibody to treat cancer. In another embodiment, the method comprises administering a 13F3.A4 antibody or variant thereof to treat cancer. In another embodiment, the method comprises administering a composition comprising a 13F3.A4 antibody or variant thereof to treat cancer.

[0279] Examples of cancers include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer cancer), germ cell tumors, childhood sarcoma, sinonasal natural killer, melanoma (e.g. metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, Cancers of the ureter, carcinomas of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, tumors of the spinal axis, brain cancers such as brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (producing B, T, NK, and plasma cells), e.g., all types of leukemia, lymphomas, and myelomas, e.g., acute myeloid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AM, acute, chronic, lymphocytic and / or myeloid leukemias, such as L(M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma;Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoma Lymphomas such as lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome) and lymphoplasmacytic lymphoma with Waldenstrom hypergammaglobulinemia (LPL) myelomas, including IgG myeloma, light-chain myeloma, non-secretory myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), and hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nerves, including seminoma, teratocarcinoma, astrocytoma, and schwannoma; fibrosarcoma, rhabdomyosarcoma, and rhabdomyosarcoma. tumors of mesenchymal origin, including thyroid carcinoma (cystic sarcoma) and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, hematopoietic tumors of lymphoid lineage, such as T-cell and B-cell tumors, including T-cell disorders, such as, but not limited to, T-prolymphocytic leukemia (T-PLL), including the small cell and cerebriform cell types; preferably, large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / mature T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head and neck, kidney, rectum, thyroid;These cancers include, but are not limited to, acute myeloid lymphoma and any combination of the foregoing cancers. In one embodiment, the methods described herein may also be used to treat metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., using blocking CTLA-4 and / or PD-1 antibodies), and recurrent cancers.

[0280] In one embodiment, the anti-hNKG2A antibody is administered as monotherapy. In one embodiment, the anti-hNKG2A agonist antibody is administered as the sole immunostimulatory agent. In one embodiment, the anti-hNKG2A is administered to the patient along with another agent. In one embodiment, the anti-hNKG2A antibody is administered along with an immunogenic agent. In one embodiment, the anti-hNKG2A antibody is administered along with a cancer vaccine. In some embodiments, the cancer vaccine comprises cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J. Immunol. 173:4919-28). In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, multiple peptides, peptide cocktail, hybrid peptide, or peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, the anti-hNKG2A antibody is administered with an adjuvant. Non-limiting examples of tumor vaccines used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.Numerous experimental strategies have been devised for tumor vaccination (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition).In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been found to be most effective when tumor cells are transduced to express GM-CSF, which has been shown to be a potent activator of antigen presentation for tumor vaccination. Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-43.

[0281] Other cancer vaccines are proteins derived from viruses implicated in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). In some embodiments, another form of tumor-specific antigen used in conjunction with NKG2A inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are highly efficient at delivering them to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

[0282] In some embodiments, dendritic cells are potent antigen-presenting cells used to prime antigen-specific responses. Dendritic cells can be produced ex vivo and loaded with various protein and peptide antigens and tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). Dendritic cells can also be genetically transduced to express these tumor antigens. DCs have also been directly fused with tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, dendritic cell immunization can be effectively combined with anti-NKG2A antibodies to activate (emit) stronger anti-tumor responses.

[0283] In some embodiments, the anti-hNKG2A antibody is administered in conjunction with standard curative treatment, e.g., surgery, radiation, and / or chemotherapy. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with a chemotherapeutic agent. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with one or more of carboplatin, cisplatin, paclitaxel, nab-paclitaxel, gemcitabine, or FOLFOX. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with carboplatin or nab-paclitaxel. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with carboplatin and paclitaxel. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with cisplatin and pemetrexed. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with cisplatin and gemcitabine. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with FOLFOX. In some embodiments, the anti-hNKG2A antibody is administered with FOLFIRI. In one embodiment, the anti-hNKG2A antibody is administered with dacarbazine for the treatment of melanoma. In some embodiments, cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks. In some embodiments, the anti-hNKG2A antibody is administered with doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and / or cyclophosphamide hydroxyurea. In some embodiments, adriamycin is administered intravenously at a dose of 60 mg / ml to 75 mg / ml once every 21 days. In one embodiment, the anti-hNKG2A antibody is administered to a human patient who is resistant to treatment with at least one drug, wherein administration of the anti-hNKG2A antibody reduces, alleviates, or suppresses resistance to the at least one drug. In some embodiments, the anti-hNKG2A antibody is administered in conjunction with an agonist antibody, such as an anti-ICOS antibody.

[0284] The above-mentioned combination therapies can be administered in various combinations with one another, including combined administration (wherein the two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody of the invention can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibodies of the invention can also be used in combination with radiation therapy.

[0285] In some embodiments, another example of such a combination is an anti-hNKG2A antibody administered in combination with interleukin-2 (IL-2). In some embodiments, the combination of an anti-hNKG2A antibody and IL-2 is for treating various cancers, including for the treatment of renal cell carcinoma and melanoma. In some embodiments, the anti-hNKG2A antibody discussed herein is combined with an IL-2 pathway agonist to treat various cancers. Combinations include various IL-2 pathway agonists, such as those described in WO2012 / 065086 (Nektar Therapeutics) and WO2015 / 125159 (Nektar Therapeutics), the contents of which are incorporated by reference in their entireties. WO2006 / 138572 (Nektar Therapeutics) provides conjugates with degradable linkages and polymerization reagents useful for preparing such conjugates, as well as methods of making the polymerization reagents and conjugates, and is incorporated by reference in its entirety.

[0286] In some embodiments, a combination of an anti-hNKG2A antibody described herein, e.g., an NKG2A.9, NKG2A.11, or 13F3.A4 antibody, and an IL-2 pathway agonist, e.g., NKTR-214, is administered to a patient to treat cancer. As described in more detail below, NKTR-214 has the following structure (mPEG2-C2-fmoc-20K-N-hydroxysuccinimidate derivative, 20 kDa ("mPEG2-C2-fmoc-20K-NHS"):

[0287] [ka] an average of approximately six FMOC (fluorenylmethyloxycarbonyl chloride)-based polyethylene glycol (PEG) reagents having It is produced by conjugating it to a protein having the following 132 amino acid sequence: [ka] (SEQ ID NO: 161)

[0288] WO 2012 / 065086 provides conjugates of an IL-2 moiety and one or more non-peptide water-soluble polymers, including polyethylene glycol or its derivatives. Specifically, Example 2 (paragraphs 202-204) of WO 2012 / 065086 describes the PEGylation of rIL-2 with mPEG2-C2-fmoc-20K-NHS to obtain the mPEG2-C2-fmoc-20K-NHS structure shown above. Example 1 (paragraphs 63-66) of WO 2015 / 125159 describes a scaled-up approach for the PEGylation of IL-2 with mPEG2-C2-fmoc-20K-NHS to obtain RSLAIL-2 (NKTR-214). NKTR-214 is a cytokine designed to target CD122 (also known as interleukin-2 receptor beta subunit, IL-2Rβ), a protein found on certain immune cells (e.g., CD8+ T cells and NK cells) to expand these cells to promote anti-tumor effects.

[0289] In some embodiments, the anti-hNKG2A antibody is administered in combination with an anti-angiogenic agent.

[0290] Other combination therapies that may result in cell death that synergizes with the anti-hNKG2A antibodies described herein include radiation, surgery, and hormone deprivation.

[0291] In some embodiments, the anti-hNKG2A antibodies described herein are administered with a bispecific antibody. Bispecific antibodies can be used to target two different antigens. In some embodiments, anti-hNKG2A antibodies are used in combination with a bispecific antibody that targets effector cells expressing Fcα or Fcγ receptors to treat tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. In some embodiments, the T cell arm of these responses is augmented by the functional activity of the anti-hNKG2A antibody. In some embodiments, antigens are delivered directly to DCs by using a bispecific antibody that binds to a tumor antigen and a dendritic cell-specific cell surface marker. In some embodiments, anti-hNKG2A antibodies are used in combination with antibodies that reduce or inactivate immunosuppressive proteins expressed by tumors, such as anti-TGF-β antibodies, anti-IL-10 antibodies, and anti-Fas ligand antibodies.

[0292] infectious disease In another aspect, the invention described herein provides a method of treating an infectious disease in a subject, including a human subject, comprising administering to the subject an anti-hNKG2A antibody or antigen-binding fragment thereof, such that the subject is treated for the infectious disease. In other embodiments, the anti-NKG2A antibody is a chimeric or humanized antibody.

[0293] Similar to the tumor treatments discussed herein, the anti-hNKG2A antibodies described herein can be administered alone or as adjuvants, or in combination with vaccines, to enhance immune responses to pathogens, toxins, and autoantigens, including for treating chronic viral infections. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which no effective vaccines currently exist or for which conventional vaccines are not completely effective. These pathogens include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, giardia, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0294] Examples of pathogenic viruses causing infections treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0295] Examples of pathogenic bacteria causing infections treatable by the methods described herein include chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.

[0296] Examples of pathogenic fungi causing infections treatable by the methods described herein include Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), genera of the Mucorales order (e.g., mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0297] Examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba species, Giardia lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and the like. gondii) and Brazilian hookworm (Nippostrongylus brasiliensis).

[0298] The methods described herein of administering anti-hNKG2A antibodies to a subject can be combined with cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or other forms of immunotherapy, such as bispecific antibody therapy, which provide enhanced presentation of tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

[0299] Autoimmune reactions In some embodiments, anti-NKG2A antibodies enhance autoimmune responses. Induction of anti-tumor responses using tumor cell and peptide vaccines has shown that many anti-tumor responses involve anti-self reactivity (van Elsas et al. (2001) J. Exp. Med. 194:481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci. USA 96: 2982-2987; Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Therefore, anti-NKG2A antibodies are used in conjunction with these self-proteins to devise vaccination protocols that efficiently generate immune responses against these self-proteins for the treatment of disease. For example, Alzheimer's disease involves the inappropriate accumulation of Aβ peptides in amyloid deposits in the brain, and an antibody response to amyloid can clear these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

[0300] Other self-proteins can also be used as targets, such as IgE for the treatment of allergies and asthma and TNFα for rheumatoid arthritis. Finally, antibody responses to various hormones can be induced by the use of anti-NKG2A antibodies. Neutralization of antibody responses to reproductive hormones can be used for birth control. Neutralization of antibody responses to hormones and other soluble factors required for the growth of certain tumors is an additional vaccination target.

[0301] Similar methods as described above for the use of anti-NKG2A antibodies can be used to induce therapeutic autoimmune responses to treat patients with inappropriate accumulation of amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNFα, and other autoantigens such as IgE.

[0302] In some aspects, the anti-NKG2A antibodies described herein are used to stimulate an antigen-specific immune response by administering the anti-NKG2A antibody together with a subject's antigen (e.g., a vaccine). Thus, provided herein are methods for enhancing an immune response to an antigen in a subject, comprising administering to the subject (i) the antigen and (ii) an anti-NKG2A antibody or antigen-binding fragment thereof, such that the immune response to the antigen in the subject is enhanced. The antibody can be a human anti-human NKG2A antibody (e.g., any of the human anti-NKG2A antibodies described herein). In some embodiments, the anti-NKG2A antibody is a chimeric or humanized antibody. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those discussed in sections herein, such as the tumor antigens (or tumor vaccines) discussed herein or antigens derived from viruses, bacteria, or other pathogens described herein.

[0303] In certain embodiments, peptides or fusion proteins comprising epitopes to which anti-NKG2A antibodies bind are used as vaccines instead of, or in addition to, anti-NKG2A antibodies.

[0304] Suitable routes of administration of the antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) described herein, in vivo and in vitro, are well known in the art and can be selected by one of ordinary skill in the art. For example, antibody compositions can be administered intravenously or subcutaneously. The appropriate dosage of the composition used will vary depending on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0305] As previously described, the anti-NKG2A antibodies described herein can be co-administered with one or more therapeutic agents, e.g., cytotoxic agents, radiotoxic agents, or immunosuppressants. The anti-NKG2A antibodies can be linked to the agents (as immunoconjugates) or administered separately from the agents. In the latter case (separate administration), the anti-NKG2A antibodies can be administered before, after, or simultaneously with the agents, or can be co-administered with other known therapies, e.g., anti-cancer therapies, e.g., chemotherapy and / or radiation. Such therapeutic agents include, among others, anti-tumor, antineoplastic agents such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which, by themselves, are effective only at levels that are toxic or subtoxic to patients. Cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg / ml once every 21 days. Coadministration of the anti-NKG2A antibodies or antigen-binding fragments described herein with chemotherapeutic agents provides two anticancer agents that operate by different mechanisms to exert cytotoxic effects on human tumor cells. Such coadministration may address problems resulting from the development of drug resistance or from antigenic changes in tumor cells that render them unresponsive to antibodies.

[0306] Also included within the scope of the present disclosure are kits containing the antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kits may further contain at least one additional reagent or one or more additional human anti-NKG2A antibodies described herein (e.g., human antibodies with complementary activity that bind to an epitope in the NKG2A antigen distinct from that of the first human antibody). The kits will typically include labeling indicating the intended use of the contents of the kit. The term labeling includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit.

[0307] Combination therapy In one aspect, provided herein are methods of combination therapy, e.g., methods for treating cancer, in which an anti-hNKG2A antibody is administered with one or more additional agents, e.g., an antibody effective in stimulating an immune response, thereby enhancing, stimulating, or upregulating the immune response in a subject, including a human subject. Provided herein are methods for treating cancer or delaying its progression in an individual, comprising administering to the individual an anti-hNKG2A antibody (e.g., NKG2A.9, NKG2A.11, and 13F3.A4) with another anti-cancer agent or cancer therapy. In some embodiments, the anti-hNKG2A antibody can be administered with chemotherapy or a chemotherapeutic agent, or radiation therapy or a radiotherapeutic agent, as described above. In some embodiments, the anti-hNKG2A antibody can be administered with an agonist antibody, e.g., an anti-hICOS antibody. In some embodiments, the anti-hNKG2A antibody can be administered with a targeted therapy or a targeted therapeutic agent. In some embodiments, the anti-hNKG2A antibody may be administered in conjunction with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.

[0308] In some embodiments, the anti-hNKG2A antibodies described herein can be combined with (i) an agonist of another costimulatory receptor and / or (ii) an antagonist of an inhibitory signal in T cells. In some embodiments, a combination therapy comprising an anti-hNKG2A antibody and an agonist and / or antagonist results in an enhanced antigen-specific T cell response in a subject. In some embodiments, the anti-hNKG2A antibodies described herein can be administered with an agent that targets costimulatory and co-inhibitory molecules that are members of the immunoglobulin superfamily (IgSF) to enhance the immune response. In some embodiments, the anti-hNKG2A antibodies described herein can be administered with an agent that targets a ligand of a costimulatory or co-inhibitory molecule. The B7 family includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors includes the TNF family of molecules that bind to their cognate TNF receptor family members, including CD40, CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, and TRAILR3. LR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR 3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α Contains 1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0309] In another embodiment, anti-hNKG2A antibodies can be used in combination with antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, or other "immunosuppressive cytokines"), or cytokines that stimulate T cell activation, to stimulate an immune response, e.g., to treat a proliferative disease, e.g., cancer.

[0310] In one embodiment, the T cell response is mediated by a combination of an anti-hNKG2A antibody described herein and (i) a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, P and (ii) a combination of one or more of the following proteins that stimulate T cell activation: antagonists of D1H, LAIR1, TIM-1, and TIM-4, and agonists of B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, DR3, and CD28H.

[0311] Exemplary agents that modulate any of the above-mentioned proteins and can be combined with anti-hNKG2A, such as those described herein, to treat cancer include YERVOY® / ipilimumab or tremelimumab (against CTLA-4), galiximab (against B7.1), BMS-936558 (against PD-1), pidilizumab / CT-011 (against PD-1), KEYTRUDA® / pembrolizumab / MK-3475 (against PD-1), AMP224 (against B7-DC / PD-L2), BMS-936559 (against B7-H1), MPDL3280A (against B7-H1), CP-870893 or dacetuzumab / SGN-40 (against CD40—Kirkwood et al. (2012) CA Cancer J. Clin. 62:309; Vanderheide & Glennie (2013) Clin. Cancer Res. 19:1035), AMG557 (against B7H2), MGA271 (against B7H3 - WO11 / 109400), IMP321 (against LAG-3), urelumab / BMS-663513 and PF-05082566 (against CD137 / 4-1BB), varlilumab / CDX-1127 (against CD27), MEDI-6383 and MEDI-6469 (against OX40), RG-7888 (against OX40L - WO06 / 029879), atacicept (against TACI), muromonab-CD3 (against CD3), ipilumumab (against CTLA-4). Thus, in one embodiment, an anti-hNKG2A antibody (eg, NKG2A.9) is combined with an anti-PD-1 antibody (eg, nivolumab) and / or an anti-CTLA-4 antibody (eg, ipilimumab).

[0312] Other molecules that can be combined with anti-hNKG2A antibodies to treat cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, anti-hNKG2A antibodies can be combined with KIR antagonists (e.g., lirilumab).

[0313] Still other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, CSF-1R antagonists such as CSF-1R antagonist antibodies, including, but not limited to, RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, WO13 / 169264, WO14 / 036357).

[0314] In some embodiments, the anti-hNKG2A antibodies described herein are used in conjunction with one or more of: agonistic agents that ligate positive costimulatory receptors; blocking agents that attenuate signaling by inhibitory receptors; and one or more agents that systemically increase the frequency of anti-tumor T cells; agents that overcome distinct immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interactions); depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies) (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion); inhibiting metabolic enzymes, e.g., IDO; or reversing / preventing T cell anergy or exhaustion); and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0315] Provided herein are methods for stimulating an immune response in a subject, comprising administering to the subject an anti-hNKG2A antibody and one or more additional immunostimulatory antibodies, such as a PD-1 antagonist, e.g., an antagonist antibody, a PD-L1 antagonist, e.g., an antagonist antibody, a CTLA-4 antagonist, e.g., an antagonist antibody, and / or a LAG3 antagonist, e.g., an antagonist antibody, so that an immune response is stimulated in the subject, e.g., to inhibit tumor growth or to stimulate an antiviral response. In one embodiment, the subject is administered an anti-hNKG2A antibody and an antagonist anti-PD-1 antibody. In one embodiment, the subject is administered an anti-hNKG2A antibody and an antagonist anti-PD-L1 antibody. In one embodiment, the subject is administered an anti-hNKG2A antibody and an antagonist anti-CTLA-4 antibody. In one embodiment, at least one additional immunostimulatory antibody (e.g., an antagonist anti-PD-1, antagonist anti-PD-L1, antagonist anti-CTLA-4, and / or antagonist anti-LAG3 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse or hamster anti-PD-1, anti-PD-L1, anti-CTLA-4, and / or anti-LAG3 antibody).

[0316] Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody and an antagonist PD-1 antibody to a subject. In some embodiments, the cancer is non-small cell lung cancer (NSCLC) or colorectal cancer (CRC). In some embodiments, the cancer is characterized by tumors with (i) elevated HLA-E levels and / or (ii) high tumor mutation burden. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. Also provided herein are methods for modifying adverse events associated with treating a hyperproliferative disease with an immunostimulatory agent. In one embodiment, the method comprises administering to a subject an anti-hNKG2A antibody and a subtherapeutic dose of an anti-PD-1 antibody. In some embodiments, the subject is human. In some embodiments, the anti-PD-1 antibody is a human monoclonal antibody.

[0317] In some embodiments, anti-PD-1 antibodies known in the art are used in the methods described herein in combination with the anti-NKG2A antibodies described herein. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. Patent No. 8,008,449 have been demonstrated to exhibit one or more of the following characteristics: (a) a 1×10 -cell count as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D(b) binds to human PD-1 in a mixed lymphocyte reaction (MLR) assay, (b) does not substantially bind to human CD28, CTLA-4, or ICOS, (c) increases T-cell proliferation in a mixed lymphocyte reaction (MLR) assay, (d) increases interferon-γ production in an MLR assay, (e) increases IL-2 secretion in an MLR assay, (f) binds to human PD-1 and cynomolgus PD-1, (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1, (h) stimulates an antigen-specific memory response, (i) stimulates an antibody response, and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments at least five, of the aforementioned characteristics.

[0318] Other anti-PD-1 monoclonal antibodies are described in, e.g., U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO2012 / 145493, WO2008 / 156712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2014 / 194302, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540, each of which is incorporated by reference in its entirety.

[0319] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (OPDIVO®, also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck, also known as KEYTRUDA®, lambrolizumab, and MK-3475, see WO2008 / 156712), PDR001 (Novartis, see WO2015 / 112900), MEDI-0680 (AstraZeneca, also known as AMP-514, see WO2012 / 145493), semipilimab (Regeneron, also known as REGN-2810, see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA, Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene, see WO2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine, also known as SHR-1210, see WO2015 / 085847, Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical, also known as ANB011, see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals, also known as WBP3055, Si-Yang Liu et al., J. Hematol. Oncol.10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics, see WO2014 / 194302), AGEN2034 (Agenus, see WO2017 / 040790), MGA012 (Macrogenics, see WO2017 / 19846), or IBI308 (Innovent, see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540).

[0320] In one embodiment, the anti-PD-1 antibody is nivolumab, a fully human IgG4(S228P)PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0321] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0322] Anti-PD-1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein, e.g., nivolumab, for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105, WO 2013 / 173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as an anti-PD-1 antibody described herein, e.g., nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody, e.g., nivolumab, given their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0323] In certain embodiments, the antibody that cross-competes with the human PD-1 antibody nivolumab for binding to human PD-1 or binds to the same epitope region as nivolumab is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, genetically engineered antibodies, or humanized or human antibodies. Such chimeric, genetically engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0324] Anti-PD-1 antibodies that can be used in the methods of the disclosed invention also include antigen-binding portions of the above-described antibodies. It has been well shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0325] Anti-PD-1 antibodies suitable for use in the disclosed methods or compositions are those that bind to PD-1 with high specificity and affinity, block binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of a full antibody with respect to inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

[0326] Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody and an antagonist PD-L1 antibody to a subject. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. Provided herein are methods for modifying adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-NKG2A antibody and a subtherapeutic dose of an anti-PD-L1 antibody to a subject. In certain embodiments, the subject is a human. In certain embodiments, the anti-PD-L1 antibody is a human-sequence monoclonal antibody, and the anti-hNKG2A antibody is a humanized monoclonal antibody, e.g., an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

[0327] Anti-PD-L1 antibodies known in the art can be used in the methods of the disclosure. Examples of anti-PD-L1 antibodies useful in the methods of the disclosure include the antibodies disclosed in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has been demonstrated to exhibit one or more of the following characteristics: (a) a K of 1×10-7 M or less, as determined by SPR using a Biacore biosensor system; D(b) bind to human PD-L1 in a mixed lymphocyte reaction (MLR) assay, (c) increase interferon-γ production in an MLR assay, (d) increase IL-2 secretion in an MLR assay, (e) stimulate antibody responses, and (f) reverse the effects of regulatory T cells on T cells, effector cells, and / or dendritic cells. Anti-PD-L1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments at least five, of the foregoing characteristics.

[0328] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (12A4, also known as MDX-1105, see, e.g., U.S. Patent No. 7,943,743 and WO2013 / 173223), atezolizumab (Roche, also known as TECENTRIQ®, MPDL3280A, RG7446, see U.S. Patent No. 8,217,149, and Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca, IMFINZI™, also known as MEDI-4736, see WO2011 / 066389), avelumab (Pfizer, BAVENCIO®, also known as MSB-0010718C, see WO2013 / 079174), STI-1014 (Sorrento, see WO2013 / 181634), CX-072 (Cytomx, see WO2016 / 149201), KN035 (3DMed / Alphamab, see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co, see, e.g., WO2017 / 034916), or CK-301 (Checkpoint Therapeutics, see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0329] In a specific embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0330] In a specific embodiment, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0331] In a specific embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0332] In other embodiments, the anti-PD-L1 monoclonal antibody is 28-8, 28-1, 28-12, 29-8, 5H1, or any combination thereof.

[0333] Anti-PD-L1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein, e.g., atezolizumab, durvalumab, and / or avelumab, for binding to human PD-L1. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein, e.g., atezolizumab, durvalumab, and / or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that they bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody, e.g., atezolizumab and / or avelumab, given their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based ...

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, which specifically binds to human Natural Killer Cell Inhibitory Receptor Group 2A (NKG2A) protein and has the following characteristics: (a) reducing the binding and / or interaction of HLA-E with the human NKG2A protein; (b) reversing NKG2A-mediated inhibitory signaling; (c) does not bind, or binds with low affinity, to human NKG2C protein; (d) binding to human or cynomolgus monkey NKG2A protein; (e) enhancing natural killer cell responses; (f) enhancing the functional activity of T cells; (g) reduced binding to human Fc gamma receptors (FcγR); (h) inducing and / or enhancing an anti-tumor immune response, and / or (i) having low immunogenicity in human subjects An isolated monoclonal antibody or antigen-binding fragment thereof, which exhibits at least one of the following:

2. The antibody has the following characteristics: (a) an EC of about 0.6 nM or less for binding to human NKG2A protein as measured by a cell binding assay; ５０ having a value, (b) an EC of about 9.0 nM or greater for binding to human NKG2C protein as measured by a cell binding assay; ５０ having a value, (c) a second EC for binding to human NKG2C protein, with respect to binding to human NKG2A protein; ５０ EC values ​​approximately 15 times lower than ５０ having a value, (d) an IC of about 1.0 nM or less as measured by a cell blocking assay ５０ having a value, (e) a K of about 0.4 nM or less as measured by Scatchard analysis Ｄ and binding to human NKG2A protein; (f) a K of about 61 nM or less as measured by surface plasmon resonance Ｄ and binding to human NKG2A protein; (g) a K of about 1.0 nM or less as measured by Scatchard analysis. Ｄ and binds to cynomolgus monkey NKG2A protein; (h) being internalized upon binding to NKG2A-expressing cells; (i) increasing interferon-gamma (IFNγ) production; (j) the half-life of the anti-NKG2A antibody:NKG2A protein complex is greater than or equal to about 40 seconds; and / or (k) an EC of about 0.5 nM or less ５０ Indicating internal transitions by value The antibody or antigen-binding fragment thereof of claim 1, having one or more of the following:

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody reduces the binding and / or interaction of HLA-E with human NKG2A protein.

4. The antibody or antigen-binding fragment thereof of claim 3 , wherein the antibody binds to human NKG2A protein.

5. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein, (a) a heavy chain variable domain comprising the amino acid sequences of SEQ ID NOs: 10, 11, 12, and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15; (b) a heavy chain variable domain comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 154, 14, and 15; or (c) a heavy chain variable domain comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 155, 14, and 15.

1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising:

6. An isolated monoclonal antibody or antigen-binding fragment thereof, which specifically binds to human NKG2A protein and comprises a heavy chain and a light chain variable region, (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:167; and / or (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:9, SEQ ID NO:164, or SEQ ID NO:169; An isolated monoclonal antibody or antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

9.

8. The antibody or antigen-binding fragment thereof of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

164.

9. The antibody or antigen-binding fragment thereof of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 167 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

169.

10. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein, the heavy and light chains comprising: (a) the amino acid sequences of SEQ ID NOs: 7 and 5, respectively; (b) the amino acid sequences of SEQ ID NOs: 7 and 19, respectively; or (c) the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.

1. An isolated monoclonal antibody or antigen-binding fragment thereof, consisting essentially of:

11. 11. An isolated monoclonal antibody or antigen-binding fragment thereof that competes with or binds to the same epitope as any of the antibodies of any one of claims 1 to 10 for binding to NKG2A protein.

12. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, which, when bound to human NKG2A protein, has the following amino acid residues as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS): (a) LSIDNEEMKF (SEQ ID NO: 156); (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157); (c) LAFKHEIKDSDN (SEQ ID NO: 158); and (d) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) Specific binding to Blocks the binding of HLA-E to human NKG2A protein; An isolated monoclonal antibody or antigen-binding fragment thereof.

13. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, which, when bound to human NKG2A, binds to the following amino acid residues as determined by HDX-MS and / or rapid photochemical oxidation of proteins (FPOP) epitope mapping: (a) LSIDNEEMKF (SEQ ID NO: 156) (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157) (c) LAFKHEIKDSDN (SEQ ID NO: 158) (d) L; and (e) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159) Specific binding to Blocks the binding of HLA-E to human NKG2A protein; An isolated monoclonal antibody or antigen-binding fragment thereof.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antibody is a full-length antibody.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the high length antibody is an IgG1 antibody.

16. 14. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is an antibody fragment.

17. 17. The antigen-binding fragment of claim 16, wherein the antibody fragment is a Fab, Fab', (Fab')2, Fv, or scFv fragment.

18. 18. The antibody or antigen-binding fragment thereof of any one of claims 1 to 17, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

19. An isolated full-length monoclonal antibody that specifically binds to human NKG2A protein, the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and / or the light chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:5; Isolated full-length monoclonal antibodies.

20. An isolated full-length monoclonal antibody that specifically binds to human NKG2A protein, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

19.

21. An isolated full-length monoclonal antibody that specifically binds to human NKG2A, wherein the heavy chain essentially consists of the amino acid sequence set forth in SEQ ID NO:7 and the light chain essentially consists of the amino acid sequence set forth in SEQ ID NO:

19.

22. An isolated full-length monoclonal antibody that specifically binds to human NKG2A protein, the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:7; the light chain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:19; Isolated full-length monoclonal antibodies.

23. 1. An isolated antibody or antigen-binding fragment thereof that binds to human NKG2A protein and cross-competes with a reference antibody for binding to human NKG2A protein, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), (a) the VH comprises the amino acid sequence set forth in SEQ ID NO:8 and the VL comprises the amino acid sequence set forth in SEQ ID NO:9; (b) the VH comprises the amino acid sequence set forth in SEQ ID NO:8 and the VL comprises the amino acid sequence set forth in SEQ ID NO:164; or (c) the VH comprises the amino acid sequence set forth in SEQ ID NO: 167, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 169; An isolated antibody or antigen-binding fragment thereof.

24. 24. An isolated nucleic acid molecule encoding the heavy and / or light chain variable region of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23.

25. 25. The isolated nucleic acid of claim 24, wherein the nucleic acid molecule is a complementary DNA (cDNA).

26. 26. An expression vector comprising the nucleic acid molecule of claim 24 or 25.

27. A host cell transfected with the expression vector of claim 26.

28. 24. An immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 linked to an agent.

29. 30. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 27, wherein the antibody is produced.

30. 30. The method of claim 29, further comprising recovering the antibody or antigen-binding fragment thereof from the host cell.

31. A bispecific molecule comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 linked to a second functional moiety.

32. A composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 23, or a bispecific molecule described in claim 31, and a pharma- ceutically acceptable carrier.

33. 33. The composition of claim 32 further comprising an additional therapeutic agent.

34. The composition of claim 33, wherein the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody.

35. 24. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 for use as a medicament for treating cancer.

36. 36. The antibody or antigen-binding fragment thereof of claim 35, wherein the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, gastric cancer, melanoma, renal cancer, urothelial carcinoma, glioblastoma multiforme, or a virus-associated cancer.

37. 37. The antibody or antigen-binding fragment thereof of claim 36, wherein the cancer is cervical cancer, head and neck squamous cell carcinoma, pancreatic cancer, non-small cell lung carcinoma - adenocarcinoma type, non-small cell lung carcinoma - squamous cell type, gastric cancer, melanoma, colorectal cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, urothelial carcinoma, breast cancer, small cell lung carcinoma, glioblastoma multiforme, prostate cancer, or non-Hodgkin's lymphoma.

38. 24. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 for use in enhancing an immune response.

39. 24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 in the manufacture of a medicament for the treatment of cancer.

40. 35. A method for treating or delaying the progression of cancer in a human subject, comprising administering to the human subject an effective amount of an antibody of any one of claims 1 to 23, an immunoconjugate of claim 28, a bispecific molecule of claim 31, or a composition of claim 33 or 34 to treat or delay the progression of cancer.

41. 41. The method of claim 40, wherein the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, renal cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, gastric cancer, melanoma, renal cancer, urothelial cancer, glioblastoma multiforme, or a virus-associated cancer.

42. 42. The method of claim 41, wherein the cancer is cervical cancer, head and neck squamous cell carcinoma, pancreatic cancer, non-small cell lung carcinoma - adenocarcinoma type, non-small cell lung carcinoma - squamous cell type, gastric cancer, melanoma, colorectal cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, urothelial carcinoma, breast cancer, small cell lung carcinoma, glioblastoma multiforme, prostate cancer, or non-Hodgkin's lymphoma.

43. 43. The method of claim 41 or 42, further comprising administering to the human subject one or more additional therapeutic agents.

44. 44. The method of claim 43, wherein the one or more additional therapeutic agents are a chemotherapeutic agent, a radiotherapeutic agent, and / or an immunotherapeutic agent.

45. 45. The method of claim 44, wherein the immunotherapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody.

46. 46. ​​The method of claim 45, wherein the anti-PD-1 antibody is nivolumab and the anti-CTLA-4 antibody is ipirlimab.

47. 36. A method of stimulating an immune response in a human subject, comprising administering to the human subject an effective amount of an antibody of any one of claims 1 to 23, an immunoconjugate of claim 28, a bispecific molecule of claim 31, or a composition of claim 33, 34, or 35 to stimulate the immune response.

48. 48. The method of claim 47, wherein the human subject has a tumor and an anti-tumor immune response is stimulated.

49. 48. The method of claim 47, wherein the human subject has a chronic viral infection and an anti-viral immune response is stimulated.

50. 1. A method for detecting the presence of NKG2A protein in a sample, comprising: Contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and an NKG2A protein; Detecting the formation of a complex. A method comprising:

51. The method of claim 50, wherein the antibody or antigen-binding fragment thereof forms a complex with NKG2A protein more rapidly than with NKG2C protein.

52. The method of claim 51, wherein the antibody or antigen-binding fragment thereof forms a complex with NKG2A protein 15 times more rapidly than with NKG2C protein.

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