Anti-NKG2A antibody and its use

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 of related applications This application claims 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 herein by reference in their entirety.

[0002] The present invention relates to anti-NKG2A (natural killer cell inhibitory receptor 2A group) 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] Reference to sequence listings submitted electronically via EFS-WEB This application includes 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 has a size of 273,467 bytes. [Background technology]

[0004] Cancer is a global epidemic. According to the Global Health Data Exchange, cancer is one of the leading causes of disease and the second leading cause of death, accounting for approximately 17% of all 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 estimates by the National Cancer Institute, in 2019, more than 1.7 million new patients will be diagnosed with cancer, and more than 600,000 patients are expected to die from cancer in the United States. ("Cancer Stat Facts: Cancer of Any Site." SEER Training Modules, US National Institutes of Health, National Cancer Institute, 2019<https: / / seer.cancer.gov / statfacts / html / all.html> ).

[0005] Traditional cancer treatments include, among other therapies, surgery, radiation therapy, and chemotherapy. In recent years, immuno-oncology, or immunotherapy, has emerged as a new option for treating cancer using the body's own immune system. Immuno-oncology differs from traditional cancer treatments, which, for example, attempted to directly target tumors and / or disrupt the tumor's blood supply. Instead, immuno-oncology is designed to utilize 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 inhibit 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 lose the expression of antigens that can be recognized by the patient's immune system. In other cases, rapid tumor growth can even overwhelm 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 complex and challenging problem. For example, many patients do not respond to existing immuno-oncology therapies, and some develop resistance mechanisms such as T cell depletion, in which certain types of white blood cells, called T cells, cease to function properly. (Dempke et al., Eur. J. of Cancer, 74: 55-72 (2017)). [Overview of the project] [Problems that the invention aims to solve]

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

[0007] The present invention provides, in several embodiments, 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 exhibiting desirable functional properties. In one embodiment, desirable functional properties of the anti-NKG2A antibodies disclosed herein include stimulating an immune response, e.g., treating cancer. In another embodiment, desirable functional properties of the anti-NKG2A antibodies disclosed herein include treating subjects infected with a virus, including human patients. In some embodiments, the anti-NKG2A antibodies disclosed herein treat infectious diseases. In another embodiment, 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 antitumor response of the immune system, including natural killer cells and / or T cells. In other embodiments, the anti-NKG2A antibody of the present invention is used in combination with other antibodies to treat a variety of conditions, including cancer, infectious diseases including viral infections, and autoimmune diseases. Accordingly, in some embodiments, the anti-NKG2A antibody disclosed herein is used alone or in combination with other therapies, such as other immuno-oncological therapies 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 antibody disclosed herein is used in a method for detecting the NKG2A protein in a sample.

[0008] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, which specifically binds to human NKG2A and has the following properties: (a) Reduce (e.g., block) the binding and / or interaction of NKG2A ligands (e.g., HLA-E in humans) with the human NKG2A protein. (b) Reversing inhibitory signaling mediated by NKG2A, (c) Not binding to human NKG2C protein, or binding with low affinity. (d) To bind to human and / or cynomolgus monkey NKG2A, (e) To enhance the natural killer cell response, (f) To enhance the functional activity of T cells, (g) Reduced binding to the human Fc gamma receptor (FcγR), (h) Inducing and / or enhancing an antitumor immune response, (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 its antigen-binding fragment exhibiting at least one of the following:

[0009] In one embodiment, the isolated monoclonal antibody or its antigen-binding fragment has the following characteristics: (a) When measured by cell binding assay, the EC ratio with respect to human NKG2A protein is approximately 0.6 nM or less. 50 Having a value, (b) When measured by cell binding assay, the EC of approximately 9.0 nM or greater is required for binding to human NKG2C protein. 50 Having a value, (c) Regarding binding to human NKG2A protein, the second EC regarding binding to human NKG2C protein 50 EC is about 15 times lower than the value. 50 Having a value, (d) When measured by a cell-blocking assay, an IC of about 1.0 nM or less with respect to reducing the binding and / or interaction with the human NKG2A protein of HLA-E 50 value, (e) When measured by Scatchard analysis, a K of about 0.4 nM or less D for binding to the human NKG2A protein, (f) When measured by surface plasmon resonance, a K of about 61 nM or less D for binding to human NKG2A, (g) When measured by Scatchard analysis, a K of about 1.0 nM or less D for binding to cynomolgus NKG2A, (h) Being internalized when binding to NKG2A-expressing cells, (i) Increasing interferon-gamma (IFNγ) production, (j) An EC of about 0.5 nM or less 50 value showing internalization, and / or (k) The half-life of the anti-NKG2A antibody:NKG2A complex being about 40 seconds or more having one or more of the above. 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 antibody or antigen-binding fragment thereof disclosed herein reduces (e.g., blocks) the interaction between the human NKG2A protein and the human NKG2A ligand (i.e., HLA-E).

[0011] In another aspect, the invention is an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the 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) Heavy chain variable domains containing the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and light chain variable domains containing the amino acid sequences of SEQ ID NOs: 154, 14, and 15, or (c) Heavy chain variable domains containing the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and light chain variable domains containing the amino acid sequences of SEQ ID NOs: 155, 14, and 15 The present invention provides an isolated monoclonal antibody or its antigen-binding fragment, which includes [the specified substance].

[0012] In another embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, which specifically binds to the human NKG2A protein and comprises heavy chain and light chain variable regions, each, (a) The heavy chain variable region contains 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 contains an amino acid sequence that is at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, approximately 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 164, or SEQ ID NO: 169. This provides isolated monoclonal antibodies or their antigen-binding fragments.

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

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

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

[0016] In another embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to the human NKG2A protein, wherein the heavy chain and light chain are (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) Amino acid sequences of SEQ ID NOs. 35 and 36, respectively This provides an isolated monoclonal antibody or its antigen-binding fragment, which is essentially derived from the above.

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

[0018] In another embodiment, when the present invention is bound to the human NKG2A protein, the following amino acid residues are determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS): (a) LSIDNEEMKF (sequence number 156); (b) PSSWIGVFRNSSHHPW (sequence code 157); (c)LAFKHEIKDSDN(sequence number 158); and (d)QVNRLKSAQQCGSSIIYHC(Sequence ID 159) The present invention provides an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to the antigen.

[0019] In some embodiments, monoclonal antibodies block the binding of NKG2A ligands (e.g., HLA-E in humans) to the human NKG2A protein.

[0020] In another embodiment, the present invention, when bound to human NKG2A, determines by HDX-MS and / or rapid photochemical oxidation (FPOP) epitope mapping of the following amino acid residues: (a) LSIDNEEMKF (Sequence ID 156) (b) PSSWIGVFRNSSHHPW (Sequence ID 157) (c)LAFKHEIKDSDN(Sequence ID 158) (d)L; and (e)QVNRLKSAQQCGSSIIYHC(Sequence ID 159) The present invention provides an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to the antigen.

[0021] In some embodiments, the antibody blocks the binding of an NKG2A ligand (e.g., 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 embodiment, the present invention relates to an isolated full-length monoclonal antibody that specifically binds to the human NKG2A protein, (a) The heavy chain contains 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 contains an amino acid sequence that is at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, approximately 100%, or 100% identical to the amino acid sequence of SEQ ID NO. We provide isolated, full-length monoclonal antibodies.

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

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

[0026] In another embodiment, the present invention provides isolated nucleic acid molecules encoding heavy chain variable regions and / or light chain variable regions of antibodies or antigen-binding fragments 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 as described herein. In yet another aspect, the present invention provides a host cell transformed with an expression vector as described herein.

[0028] In another embodiment, the present invention provides an immunoconjugate comprising an antibody described herein, which is conjugated with a drug.

[0029] In another embodiment, the present invention provides a method for producing antibodies, comprising culturing host cells as described herein. In some embodiments, the method further comprises recovering antibodies from the host cells.

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

[0031] In another embodiment, the present invention provides a composition comprising an anti-NKG2A antibody 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 a further therapeutic agent. In other embodiments, the further 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 intended for use as pharmaceuticals for the treatment of cancer.

[0032] In some embodiments, cancers treated with the anti-NKG2A antibodies, immunoconjugates, bispecific molecules, and compositions described herein include bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell carcinoma, 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, kidney cancer, urothelial carcinoma, glioblastoma pleomorphism, or virus-associated cancers. In other embodiments, cancers include cervical cancer, squamous cell carcinoma of the head and neck (HNSCC), pancreatic cancer, non-small cell lung cancer - adenocarcinoma (NSCLC-AD), non-small cell lung cancer - 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 cancer, glioblastoma pleomorphic, prostate cancer (also known as adenocarcinoma of the prostate or PRC), or non-Hodgkin lymphoma.

[0033] In some embodiments, anti-NKG2A antibodies are intended for use in enhancing the immune response. In other embodiments, the present invention provides the use of anti-NKG2A antibodies described herein in the manufacture of pharmaceuticals for the treatment of cancer.

[0034] In one embodiment, the present 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, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell carcinoma, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, stomach cancer, melanoma, kidney cancer, urothelial carcinoma, glioblastoma pleomorphism, or virus-related cancer. In other embodiments, cancers include cervical cancer, squamous cell carcinoma of the head and neck (HNSCC), pancreatic cancer, non-small cell lung cancer - adenocarcinoma (NSCLC-AD), non-small cell lung cancer - 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 cancer, glioblastoma pleomorphic, prostate cancer (also known as adenocarcinoma of the prostate or PRC), or non-Hodgkin lymphoma.

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

[0036] In another embodiment, the present invention provides a method for 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 antitumor immune response is stimulated. In other embodiments, the human subject has a chronic viral infection, and an antiviral immune response is stimulated.

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

[0038] Other features and advantages of this disclosure will become apparent from the following detailed description and examples, but these shall not be construed as limitations. 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 drawing]

[0039] [Figure 1A] Figures 1A–C illustrate three methods used to discover and test the anti-NKG2A antibodies disclosed herein. Figure 1A illustrates the hybridoma method and the anti-NKG2A antibody discovery and development process at a high level. 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 the hybridoma method and the anti-NKG2A antibody discovery and development process at a high level. 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 the hybridoma method and the anti-NKG2A antibody discovery and development process at a high level. 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 scan analysis used to optimize the discovered anti-NKG2A antibody. Specifically, Figure 2 shows the steps for generating a variant of the 13F3.A4 I107T antibody and characterizing its binding to the NKG2A protein. This analysis enabled the inventors to generate a variant 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 sequence numbers 183-190, respectively, in order of appearance from top to bottom. [Figure 3] Figure 3 is an exemplary heatmap that allows for the interpretation of sequence-activity relationships of single amino acid substitutions, generated using mutation scan analysis. Figure 3 discloses Sequence ID No. 191 (germline) and Sequence ID No. 192 (parent). [Figure 4] Figure 4 shows the CDR locations analyzed for the 13F3.A4 I107T anti-NKG2A antibody using mutation scan analysis. Figure 4 discloses sequence numbers 30, 15, 192, 11, and 193, respectively, in order of appearance. [Figure 5A]Figures 5A-E are heatmaps generated using mutation scan 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 heatmaps generated using mutation scan 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 heatmaps generated using mutation scan 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 heatmaps generated using mutation scan 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 heatmaps generated using mutation scan 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 the 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 out of 233) are conserved, approximately 6% (14 out of 233) are similar, and only approximately 18% (42 out of 233) 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 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) 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 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) 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 the anti-NKG2A antibody 2G6.C2. The amino acid sequences of VH CDR1 (SEQ ID NO: 55), VH CDR2 (SEQ ID NO: 56), and VH CDR3 (SEQ ID NO: 57) 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 the anti-NKG2A antibody 2G6.C2. The amino acid sequences of VL CDR1 (SEQ ID NO: 58), VL CDR2 (SEQ ID NO: 59), and VL CDR3 (SEQ ID NO: 60) 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 the anti-NKG2A antibody 2G6.C2. The amino acid sequences of VH CDR1 (SEQ ID NO: 55), VH CDR2 (SEQ ID NO: 56), and VH CDR3 (SEQ ID NO: 57) 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 the anti-NKG2A antibody 2G6.C2. The amino acid sequences of VL CDR1 (SEQ ID NO: 58), VL CDR2 (SEQ ID NO: 59), and VL CDR3 (SEQ ID NO: 60) 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 the anti-NKG2A antibody 11H9.A1. The amino acid sequences of VH CDR1 (SEQ ID NO: 41), VH CDR2 (SEQ ID NO: 42), and VH CDR3 (SEQ ID NO: 43) 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 the mature anti-NKG2A antibody 11H9.A1. The amino acid sequences of VL CDR1 (SEQ ID NO: 44), VL CDR2 (SEQ ID NO: 45), and VL CDR3 (SEQ ID NO: 46) 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 the anti-NKG2A antibody 11H9.A1. The amino acid sequences of VH CDR1 (SEQ ID NO: 41), VH CDR2 (SEQ ID NO: 42), and VH CDR3 (SEQ ID NO: 43) 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 the mature anti-NKG2A antibody 11H9.A1. The amino acid sequences of VL CDR1 (SEQ ID NO: 44), VL CDR2 (SEQ ID NO: 45), and VL CDR3 (SEQ ID NO: 46) 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 VH CDR1 (SEQ ID NO: 69), VH CDR2 (SEQ ID NO: 70), and VH CDR3 (SEQ ID NO: 71) 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 region of the 4G5.D1 antibody. The amino acid sequences of VL CDR1 (SEQ ID NO: 72), VL CDR2 (SEQ ID NO: 73), and VL CDR3 (SEQ ID NO: 74) 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 VH CDR1 (SEQ ID NO: 69), VH CDR2 (SEQ ID NO: 70), and VH CDR3 (SEQ ID NO: 71) 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 region of the 4G5.D1 antibody. The amino acid sequences of VL CDR1 (SEQ ID NO: 72), VL CDR2 (SEQ ID NO: 73), and VL CDR3 (SEQ ID NO: 74) 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 VH CDR1 (SEQ ID NO: 83), VH CDR2 (SEQ ID NO: 84), and VH CDR3 (SEQ ID NO: 85) 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 VL CDR1 (SEQ ID NO: 86), VL CDR2 (SEQ ID NO: 87), and VL CDR3 (SEQ ID NO: 88) 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 VH CDR1 (SEQ ID NO: 83), VH CDR2 (SEQ ID NO: 84), and VH CDR3 (SEQ ID NO: 85) 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 VL CDR1 (SEQ ID NO: 86), VL CDR2 (SEQ ID NO: 87), and VL CDR3 (SEQ ID NO: 88) are shown in gray boxes. [Figure 12] Figure 12 shows the amino acid sequence trends evaluated for the 13F3.A4 antibody. 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 VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) are underlined. 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 also underlined. The evaluated sequence trends are indicated by circles 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 shown in SEQ ID NO: 5, and the heavy-chain amino acid sequence is shown in SEQ ID NO: 163 (shown with terminal lysine, which is absent in another embodiment). Figure 13 identifies two mutations, indicated by ellipses, made against the VL sequence (N30S) and VH sequence (I107T) of 13F3.A4 to obtain the NKG2A.9 sequence. The N-glycosylation motif is indicated by a dashed ellipse. Three mutations made in the Fc region (L234A, L235E, and G273A) are shown in bold italics. The terminal amino acid lysine of the heavy-chain sequence was removed from the mature sequence. The amino acid sequences of VL CDR1 (SEQ ID NO: 13), VL CDR2 (SEQ ID NO: 14), and VL CDR3 (SEQ ID NO: 15) are shown in bold and underlined. The amino acid sequences of VH CDR1 (SEQ ID NO: 10), VH CDR2 (SEQ ID NO: 11), and VH CDR3 (SEQ ID NO: 12) are shown in bold and underlined. [Figure 14] Figure 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] Figure 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 produced by the single B cell cloning method (SBCC), illustrating the diversity of these antibodies. Antibodies that cross-block each other are connected by lines. Sample antibodies with similar blocking profiles to benchmark antibodies (13F3.A4, Z270, RD-ahNKG2a (clone 131411, catalog number MAB1059), and RD-ahCD94 (clone 131412, catalog number MAB1058)) are grouped together into groups 1-4, 6-7, and 9. Benchmark antibodies with similar blocking profiles to the sample antibodies are also grouped together into groups 5, 8, and 10. [Figure 17A] Figure 17A shows an assay method for evaluating the ability of anti-human NKG2A antibodies produced using the SBCC method to block NKG2A / HLA-E interactions. Figure 17B shows that the sample antibodies partially blocked or did not block NKG2A / HLA-E interactions compared to the positive control (NKG2A.9 antibody) and the negative control (isotype). [Figure 17B] Figure 17A shows an assay method for evaluating the ability of anti-human NKG2A antibodies produced using the SBCC method to block NKG2A / HLA-E interactions. Figure 17B shows that the sample antibodies partially blocked or did not block NKG2A / HLA-E interactions compared to the positive control (NKG2A.9 antibody) and the negative control (isotype). [Figure 18A] Figures 18A-C show binding assay methods (Figure 18A) to evaluate the binding of anti-NKG2A antibodies to human NK2GA-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 blocking assay methods (Figure 18D) used to evaluate the blocking 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) and did not block NKG2C / HLA-E interactions (shown in Figure 18F). [Figure 18B]Figures 18A-C show binding assay methods (Figure 18A) to evaluate the binding of anti-NKG2A antibodies to human NK2GA-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 blocking assay methods (Figure 18D) used to evaluate the blocking 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) and did not block NKG2C / HLA-E interactions (shown in Figure 18F). [Figure 18C] Figures 18A-C show binding assay methods (Figure 18A) to evaluate the binding of anti-NKG2A antibodies to human NK2GA-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 blocking assay methods (Figure 18D) used to evaluate the blocking 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) and did not block NKG2C / HLA-E interactions (shown in Figure 18F). [Figure 18D]Figures 18A-C show binding assay methods (Figure 18A) to evaluate the binding of anti-NKG2A antibodies to human NK2GA-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 blocking assay methods (Figure 18D) used to evaluate the blocking 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) and did not block NKG2C / HLA-E interactions (shown in Figure 18F). [Figure 18E] Figures 18A-C show binding assay methods (Figure 18A) to evaluate the binding of anti-NKG2A antibodies to human NK2GA-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 blocking assay methods (Figure 18D) used to evaluate the blocking 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) and did not block NKG2C / HLA-E interactions (shown in Figure 18F). [Figure 18F]Figures 18A-C show binding assay methods (Figure 18A) to evaluate the binding of anti-NKG2A antibodies to human NK2GA-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 blocking assay methods (Figure 18D) used to evaluate the blocking 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) and did not block NKG2C / HLA-E interactions (shown in Figure 18F). [Figure 19A] Figures 19A and 19B illustrate the binding assay method used (Figure 19A), which evaluated the ability of the anti-NKG2A antibody to bind to human NKG2A+ natural killer (NKL) cell lines (shown in Figure 19B). Figures 19C and 19D illustrate the blocking assay method used (Figure 19C), which showed that the tested anti-NKG2A antibody blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 19B] Figures 19A and 19B illustrate the binding assay method used (Figure 19A), which evaluated the ability of the anti-NKG2A antibody to bind to human NKG2A+ natural killer (NKL) cell lines (shown in Figure 19B). Figures 19C and 19D illustrate the blocking assay method used (Figure 19C), which showed that the tested anti-NKG2A antibody blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 19C]Figures 19A and 19B illustrate the binding assay method used (Figure 19A), which evaluated the ability of the anti-NKG2A antibody to bind to human NKG2A+ natural killer (NKL) cell lines (shown in Figure 19B). Figures 19C and 19D illustrate the blocking assay method used (Figure 19C), which showed that the tested anti-NKG2A antibody blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 19D] Figures 19A and 19B illustrate the binding assay method used (Figure 19A), which evaluated the ability of the anti-NKG2A antibody to bind to human NKG2A+ natural killer (NKL) cell lines (shown in Figure 19B). Figures 19C and 19D illustrate the blocking assay method used (Figure 19C), which showed that the tested anti-NKG2A antibody blocked the binding of HLA-E to human NKG2A-expressing NKL cells, as shown in Figure 19D. [Figure 20A] Figures 20A-20C illustrate the blockade assay method used (Figure 20A), which demonstrated that the tested anti-human NKG2A antibody blocked the NKG2A / HLA-E interaction in hNKG2A-expressing CHO cells, as shown in Figures 20B-20C. [Figure 20B] Figures 20A-20C illustrate the blockade assay method used (Figure 20A), which demonstrated that the tested anti-human NKG2A antibody blocked the NKG2A / HLA-E interaction in hNKG2A-expressing CHO cells, as shown in Figures 20B-20C. [Figure 20C] Figures 20A-20C illustrate the blockade assay method used (Figure 20A), which demonstrated that the tested anti-human NKG2A antibody blocked the NKG2A / HLA-E interaction in hNKG2A-expressing CHO cells, as shown in Figures 20B-20C. [Figure 21A] Figures 21A-C illustrate the binding assay method used (Figure 21A), which shows that the tested anti-NKG2A antibody 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 shows that the tested anti-NKG2A antibody 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 shows that the tested anti-NKG2A antibody bound to human NKG2A-expressing CHO cells, as shown in Figures 21B-C. [Figure 22A] Figures 22A-22C illustrate the binding assay method used (Figure 22A), which evaluated the ability of the anti-NKG2A antibody to bind to cynomolgus monkey NKG2A-expressing CHO cells (the results are shown in Figures 22B-22C). [Figure 22B] Figures 22A-22C illustrate the binding assay method used (Figure 22A), which evaluated the ability of the anti-NKG2A antibody to bind to cynomolgus monkey NKG2A-expressing CHO cells (the results are shown in Figures 22B-22C). [Figure 22C] Figures 22A-22C illustrate the binding assay method used (Figure 22A), which evaluated the ability of the anti-NKG2A antibody to bind to cynomolgus monkey NKG2A-expressing CHO cells (the results are shown in Figures 22B-22C). [Figure 23A] Figures 23A-B illustrate the blocking assay method used to evaluate the ability of the anti-NKG2A antibody to block the NKG2A / HLA-E interaction (Figure 23B) (Figure 23A). Figures 23C-D show the binding assay method used (Figure 23C) to demonstrate the ability of the anti-NKG2A antibody to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 23B] Figures 23A-B illustrate the blocking assay method used to evaluate the ability of the anti-NKG2A antibody to block the NKG2A / HLA-E interaction (Figure 23B) (Figure 23A). Figures 23C-D show the binding assay method used (Figure 23C) to demonstrate the ability of the anti-NKG2A antibody to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 23C]Figures 23A-B illustrate the blocking assay method used to evaluate the ability of the anti-NKG2A antibody to block the NKG2A / HLA-E interaction (Figure 23B) (Figure 23A). Figures 23C-D show the binding assay method used (Figure 23C) to demonstrate the ability of the anti-NKG2A antibody to bind to NKG2A-expressing CHO cells (Figure 23D). [Figure 23D] Figures 23A-B illustrate the blocking assay method used to evaluate the ability of the anti-NKG2A antibody to block the NKG2A / HLA-E interaction (Figure 23B) (Figure 23A). Figures 23C-D show the binding assay method used (Figure 23C) to demonstrate the ability of the anti-NKG2A antibody 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+NKL 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 desirablely 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+NKL 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 desirablely 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-NKGA antibodies increased NK cell degranulation. Figures 25B-C are graphs of flow cytometry 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 (measured by the 50% change in CD107a compared to isotype controls) in NKG2A+ NK cells. The 25E7.G8 clone (shown in Figure 25B) is the non-blocking antibody used as a negative control. [Figure 25B] Figure 25A illustrates the in vitro method used to evaluate whether anti-NKGA antibodies increased NK cell degranulation. Figures 25B-C are graphs of flow cytometry 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 (measured by the 50% change in CD107a compared to isotype controls) in NKG2A+ NK cells. The 25E7.G8 clone (shown in Figure 25B) is the non-blocking antibody used as a negative control. [Figure 25C] Figure 25A illustrates the in vitro method used to evaluate whether anti-NKGA antibodies increased NK cell degranulation. Figures 25B-C are graphs of flow cytometry 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 (measured by the 50% change in CD107a compared to isotype controls) in NKG2A+ NK cells. The 25E7.G8 clone (shown in Figure 25B) is the non-blocking antibody used as a negative control. [Figure 26A] Figure 26A illustrates in vitro experiments using NKL cells and CHO / MICA / HLA-E, demonstrating that anti-NKG2A antibodies blocked NKG2A / HLA-E interactions and increased IFN-γ production in activated NK cells. Figure 26B shows that 13F3.A4, 11H9.A1, and 4G5.D1 antibodies desirablely increased IFN-γ production compared to an isotype control (human IgG1.3 antibody). The 25E7.G8 clone was used as a negative control. [Figure 26B] Figure 26A illustrates in vitro experiments using NKL cells and CHO / MICA / HLA-E, demonstrating that anti-NKG2A antibodies blocked NKG2A / HLA-E interactions and increased IFN-γ production in activated NK cells. Figure 26B shows that 13F3.A4, 11H9.A1, and 4G5.D1 antibodies desirablely increased IFN-γ production compared to an isotype control (human IgG1.3 antibody). The 25E7.G8 clone was used as a negative control. [Figure 27A] Figure 27A illustrates the in vitro experiment used to evaluate whether the anti-NKG2A antibody enhanced the CD8+ T cell response, which is an increased IFNγ, in the pancreatic cancer cell line Hs766T. 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 the in vitro experiment used to evaluate whether the anti-NKG2A antibody enhanced the CD8+ T cell response, which is an increased IFNγ, in the pancreatic cancer cell line Hs766T. 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 and 28B show the results of differential hydrogen-deuterium exchange (HDX) analysis of mFc-hNKG2A-hCD94 in interaction with NKG2A.9 antibody (Figure 28A) and 13F3.A4 antibody (Figure 28B), respectively. Epitope sequences are labeled in Figures 28A and 28B (Sequence IDs 119-122, disclosed from left to right in accordance with the analysis). [Figure 28B] Figures 28A and 28B show the results of differential hydrogen-deuterium exchange (HDX) analysis of mFc-hNKG2A-hCD94 in interaction with NKG2A.9 antibody (Figure 28A) and 13F3.A4 antibody (Figure 28B), respectively. Epitope sequences are labeled in Figures 28A and 28B (Sequence IDs 119-122, disclosed from left to right in accordance with the analysis). [Figure 29A] Figures 29A and 29B show the percentage of FPOP protection in hNKG2A during interaction with the NKG2A.9 antibody (Figure 29A) and the 13F3.A4 antibody (Figure 29B) for four residues (M163, F179, H184, and L206), respectively. [Figure 29B] Figures 29A and 29B show the percentage of FPOP protection in hNKG2A during interaction with the NKG2A.9 antibody (Figure 29A) and the 13F3.A4 antibody (Figure 29B) for four residues (M163, F179, H184, and L206), respectively. [Figure 30] Figure 30 shows the epitopes of 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 epitopes circled were determined by FPOP analysis. [Figure 31] Figure 31 shows the epitopes of anti-NKG2A antibodies (e.g., 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 and 32B show alignments of the VH regions (Sequence IDs 167, 168, and 168, respectively, in top-to-bottom order) and VL regions (Sequence IDs 169, 169, and 164, in top-to-bottom order) of specific portions of anti-NKG2A antibodies (13F3.A4, NKG2A.9, and NKG2A.11) (Figure 32A), as well as alignments of the VL regions (Sequence IDs 169, 169, and 164, in top-to-bottom order) (Figure 32B). This alignment led to the discovery of anti-NKG2A antibodies with the consensus CDR sequence shown within the box. [Figure 32B] Figures 32A and 32B show alignments of the VH regions (Sequence IDs 167, 168, and 168, respectively, in top-to-bottom order) and VL regions (Sequence IDs 169, 169, and 164, in top-to-bottom order) of specific portions of anti-NKG2A antibodies (13F3.A4, NKG2A.9, and NKG2A.11) (Figure 32A), as well as alignments of the VL regions (Sequence IDs 169, 169, and 164, in top-to-bottom order) (Figure 32B). This alignment led to the discovery of anti-NKG2A antibodies with the consensus CDR sequence shown within the box. [Figure 33] Figure 33 shows assay results in which the NKG2A.9 antibody reversed the inhibition of NK-κB signaling in CHO / scOKT3 / HLA-E-stimulated NKG2A-expressing Jurkat T cells compared to the isotype. [Figure 34A] Figure 34A illustrates the experimental method used to analyze the effects of NKG2A.9 and anti-PD-L1 antibodies on IFN-γ induction by NKG2A+ CD8+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs), either individually or in combination. 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 on IFN-γ induction by NKG2A+ CD8+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs), either individually or in combination. 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 demonstrate 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 demonstrate 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 internally transferred to NKG2A-expressing cells after binding. Figure 36B is a graph showing the binding kinetics of the NKG2A.9 antibody, indicating that it was internally transferred in a dose-dependent manner at an EC50 of 0.5 nM. [Figure 36B] Figure 36A is a graph showing that the NKG2A.9 antibody was internally transferred to NKG2A-expressing cells after binding. Figure 36B is a graph showing the binding kinetics of the NKG2A.9 antibody, indicating that it was internally transferred in a dose-dependent manner at an EC50 of 0.5 nM. [Figure 37A]Figures 37A-B illustrate the method used (Figure 37A) to demonstrate 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. Figures 37C-D illustrate the experimental method used (Figure 37C) to demonstrate that the NKG2A.9 antibody increased the 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 method used (Figure 37A) to demonstrate 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. Figures 37C-D illustrate the experimental method used (Figure 37C) to demonstrate that the NKG2A.9 antibody increased the 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 method used (Figure 37A) to demonstrate 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. Figures 37C-D illustrate the experimental method used (Figure 37C) to demonstrate that the NKG2A.9 antibody increased the 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 method used (Figure 37A) to demonstrate 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. Figures 37C-D illustrate the experimental method used (Figure 37C) to demonstrate that the NKG2A.9 antibody increased the 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 NKL co-cultured with CHO / MICA / HLA-E. Figure 38B shows that the 13F3.A4 antibody increased IFN-γ production in NKL 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 NKL co-cultured with CHO / MICA / HLA-E. Figure 38B shows that the 13F3.A4 antibody increased IFN-γ production in NKL compared to the isotype. [Figure 39] Figure 39 shows the results of dose titration studies in which anti-mNKG2A antibody (NKG2A.3) at doses of 10 mg / kg, 3 mg / kg, and 1 mg / kg were administered as monotherapy to inhibit 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 a dose of 0.3 mg / kg. [Figure 40A] Figures 40A–E show the results of in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy showed monotherapy 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 mice / 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 in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy showed monotherapy 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 mice / 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 in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy showed monotherapy 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 mice / 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 in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy showed monotherapy 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 mice / 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 in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in a mouse model, and anti-mNKG2A antibody monotherapy showed monotherapy 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 mice / 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 in vivo studies in which anti-NKG2A antibodies 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 in vivo studies in which anti-NKG2A antibodies 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 in vivo studies in which anti-NKG2A antibodies 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 antitumor activity of anti-mNKG2A antibodies 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 a 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 a combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42B] Figures 42A–E show the antitumor activity of anti-mNKG2A antibodies 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 a 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 a combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42C]Figures 42A–E show the antitumor activity of anti-mNKG2A antibodies 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 a 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 a combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42D] Figures 42A–E show the antitumor activity of anti-mNKG2A antibodies 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 a 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 a combination of anti-mNKG2A and anti-mCTLA-4. [Figure 42E]Figures 42A–E show the antitumor activity of anti-mNKG2A antibodies 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 a 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 a combination of anti-mNKG2A and anti-mCTLA-4. [Figure 43] Figure 43 shows the antitumor activity of anti-NKG2A antibody, anti-PD-1 antibody, and anti-LAG3 antibody, as well as combinations thereof, in a mouse lymphoma model. Administration of anti-NKG2A antibody alone provided a survival benefit of 10%. Combination therapy of anti-NKG2A antibody with either anti-mPD-1 antibody or anti-mLAG-3 antibody extended survival by 50% and 70%, respectively. A triple combination of anti-mPD-1 antibody and anti-mLAG-3 antibody provided the greatest benefit, with a survival rate of 80%. [Figure 44] Figure 44 shows that NKG2A expression levels were reduced in both splenic and tumor-infiltrating lymphocytes (TILs) NK cells compared to their isotype after treatment with an anti-mNKG2A antibody in a mouse CT26 colon cancer model. [Figure 45] Figure 45 shows the results of NKG2A expression as assessed by immunohistochemistry in different tumor types. [Figure 46] Figure 46 shows the binding profiles of the 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 evaluated by immunohistochemistry. [Figure 49A] Figures 49A-B show the levels of soluble HLA-E in healthy control patients and cancer patients. [Figure 49B] Figures 49A-B show the levels of soluble HLA-E in healthy control patients and cancer patients. [Figure 50] Figure 50 shows the clinical development plan for the anti-NKG2A antibody discussed herein, including the patient selection process and combination therapy with the anti-NKG2A antibody described herein. [Figure 51] Figures 51A-D are graphs showing the dynamics and binding affinity of the NKG2A.9 antibody as determined by Biacore analysis. [Figure 52A] Figures 52A-B show the dynamics and binding affinity of NKG2A.9 as measured by scatchard analysis. [Figure 52B] Figures 52A-B show the dynamics 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 with NKG2A.9 and its isotype. The NKG2A.9 antibody demonstrated functionality in the NK degranulation assay, while the P1-069366 antibody did not. [Figure 54A] Figures 54A-C show the results of analyzing 13F3.A4 antibodies using an in silico HLA binding tool for undesirable binding clusters. Figures 54A-C disclose sequence numbers 197-204, respectively, in order of appearance from top to bottom. [Figure 54BC] Figures 54A-C show the results of analyzing 13F3.A4 antibodies using an in silico HLA binding tool for undesirable binding clusters. Figures 54A-C disclose sequence numbers 197-204, respectively, in order of appearance from top to bottom. [Figure 55] Figure 55 shows the results of an in vitro DC:T cell proliferation assay demonstrating a low immunogenicity risk for anti-NKG2A antibodies, particularly NKG2A.6, NKG2A.9, and NKG2A.11 antibodies. [Figure 56]Figures 56A–D show the binding affinity of NKG2A.9 antibodies to human NKG2A-CD94 heterodimers (Figures 56A–B) and NKG2C-CD94 heterodimers (Figures 56C–D) at 37°C, as determined by Biacore using both single-cycle kinetics (Figures 56A and 56C) and multiple-cycle kinetics (Figures 56B and 56D). The SPR response is shown as a function of sample binding and dissociation. [Figure 57] Figure 57 shows box plots of total HLA-E positivity scores across 16 different tumor types, as assessed by immunohistochemistry. Total HLA-E score is defined as the combined percentage of cytoplasmic and / or membrane HLA-E positivity in tumor cells. [Modes for carrying out the invention]

[0040] In some embodiments, the present invention provides isolated antibodies, such as monoclonal antibodies, such as humanized, human, and chimeric monoclonal antibodies, that specifically bind to human NKG2A ("hNKG2A") and have antagonist activity that stimulates an antitumor immune response. In some embodiments, the anti-NKG2A antibodies described herein include specific structural features, such as a CDR region containing a specific amino acid sequence. In other embodiments, the anti-NKG2A antibodies compete with the anti-NKG2A antibodies of the present invention for binding to the human NKG2A protein, or bind to the same or similar epitopes.

[0041] In some embodiments, the present invention provides such anti-NKG2A antibodies, immunoconjugates comprising such anti-NKG2A antibodies or their antigen-binding fragments, and bispecific molecules, as well as methods for preparing pharmaceutical compositions formulated to include anti-NKG2A antibodies or their antigen-binding fragments. In some embodiments, the present invention provides methods for enhancing the immune response by using anti-NKG2A antibodies alone or in combination with other agents, such as other immuno-oncological agents (e.g., antibodies), chemotherapy, radiotherapy, and / or surgery. Accordingly, in some embodiments, the anti-NKG2A antibodies described herein are used to treat a variety of conditions, including, for example, to safely and effectively treat cancer and / or infections.

[0042] A crucial role of the immune system is its ability to distinguish between normal cells and "foreign" cells. The immune system can therefore attack foreign cells, leaving only normal ones. Tumors express antigens that are recognized as foreign by the host. The immune system uses "checkpoints," which are molecules on certain immune cells that need to be activated or inactivated to initiate an immune response. Tumor cells may be able to use these checkpoints to evade attack by the immune system. Some immuno-oncological 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 that target this PD-1 / PD-L1 interaction, such as monoclonal antibodies, for example, nivolumab (Opdivo®), can block PD-1 / PD-L1 binding and increase the body's immune response to tumor cells.

[0043] The natural killer cell inhibitory receptor 2A group (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 homology in the amino acid sequence of their extracellular domains, while NKG2D is a functionally distinct receptor. NKG2A forms heterodimers with CD94. Among the NKG2 / CD94 heterodimers, 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 non-classical major histocompatibility (MHC) class I molecules, namely 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 times stronger affinity than NKG2C / CD94. Ibid. NKG2A is a natural killer (NK), effector / memory CD8 +NKG2A is expressed on T, NKT, and gamma delta (γδ) T cells. NKG2A expression is induced by binding to the T cell receptor (TCR) 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 binding (Cho, Blood, 118:116-28 (2011)). NKG2A possesses two immunoreceptor tyrosine-dependent 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 therefore act as checkpoint inhibitors.

[0044] definition To facilitate understanding of the descriptions herein, certain terms are defined first. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those widely understood by those skilled in the art to whom this disclosure relates. Further definitions are provided through the detailed descriptions. The headings provided herein are not intended to limit the various aspects of this disclosure that can be understood by reference to this specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to this specification as a whole.

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

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

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

[0048] Isoform 2, corresponding to mutant 2 (nucleotide sequence described in SEQ ID NO: 210, amino acid sequence described in SEQ ID NO: 206) and mutant 4 (nucleotide sequence described in SEQ ID NO: 211, amino acid sequence described in SEQ ID NO: 207), lacks an in-frame coding exon and is absent from residues 96-113, respectively, compared to mutants 1 and 3, and is also called the NKG2A isoform NKG2-B. Mutant 4 has an N29S SNP.

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

[0050] The amino acid sequences of known human NGK2A variants are shown below. (1) Mutant 1: Human NGK2A isoform 1 (nucleotide sequence is described in SEQ ID NO: 1 or 209 (accession number NM 002259.5), amino acid sequence is described in SEQ ID NO: 2 (accession number NP 002250.2, UniProt ID P26715-1): [ka] (Sequence 2) (2) Mutant 2: Human NGK2A isoform 2 is also called NKG2A isoform NKG2B (nucleotide sequence is described in SEQ ID NO: 210 (accession number NM 007328.4), amino acid sequence is described in SEQ ID NO: 206 (accession number NP 015567.2, UniProt ID P26715-2): [ka] (Sequence ID 206) (3) Mutant 3: Human NGK2A isoform 1, having the N29S SNP shown in bold and emphasized (nucleotide sequence is described in SEQ ID NO: 212 (accession number NM 213658.2), amino acid sequence is described in SEQ ID NO: 182 (accession number NP 998823.1 or AAL65234.1): [ka] (Sequence ID 182) (4) Mutant 4: Human NKG2A isoform 2 also corresponds to mutant 4 (nucleotide sequence is described in SEQ ID NO: 211 (accession number NM_213657.2), amino acid sequence is described in SEQ ID NO: 207 (accession number NP 998822.1)): [ka] (Sequence ID 207) (5) Mutant 5: Human NGK2A isoform 3 is also referred to as NKG2A isoform C (nucleotide sequence is described in SEQ ID NO: 208 (accession number NM 001304448.1), amino acid sequence is described in SEQ ID NO: 205 (accession number NM 001291377.1)): [ka] (Sequence ID 205) The following table provides an overview of the accession numbers and corresponding sequence numbers for the DNA and protein mentioned above.

[0051] [Table A]

[0052] The terms “antibody” or “immunoglobulin” are used synonymously 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 (hereinafter abbreviated as VH) and a heavy chain constant region (hereinafter abbreviated as CH). In certain antibodies, such as naturally occurring antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In certain antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain (hereinafter abbreviated as CL). The VH and VL regions are called complementarity-determining regions (CDRs) and can be further subdivided into more conserved regions called framework regions (FRs) and scattered hypervariable regions. H and V LIt consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the 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, the amino acids of the variable region are numbered using the Kabat numbering system, and the amino acids of the constant region are numbered using the EU system. The immunoglobulin may 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: in humans, IgG1, IgG2, IgG3, and IgG4; and in mice, IgG1, IgG2a, IgG2b, and IgG3. In certain embodiments, the anti-NKG2A antibody described herein is of the IgG1 subclass. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by at most two or three amino acids. "Antibodies" include, as examples, 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] In this specification, “IgG antibody” means having the structure of a naturally occurring IgG antibody; that is, 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 crosslinks as those that occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies (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. Antibodies typically bind to their cognitive antigen and 10 -5 ~10 -11 Dissociation constants (K) less than or equal to M D It binds specifically with high affinity, as reflected by ), but does not bind with high affinity to unrelated antigens. Approximately 10 -4 Any K greater than M D Generally, it is considered to exhibit nonspecific binding. In this specification, an antibody that "specifically binds" to an antigen means an antibody that binds to the antigen and, in some cases, substantially the same antigen, and 10 -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 This refers to an antibody that binds with high affinity to a given antigen, meaning it has high affinity to that antigen, but does not bind with high affinity to an unrelated antigen. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to that given antigen, for example, if it exhibits 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 may, in some embodiments, also cross-react with NKG2A antigens from certain non-human primate species (e.g., cynomolgus monkey NKG2A), but not with NKG2A antigens from other species or with antigens other than NKG2A.

[0055] In this specification, the terms “antigen-binding moiety” or “antigen-binding fragment” of an antibody are used synonymously and refer to one or more portions of an antibody that possess the ability to specifically bind to an antigen (e.g., human NKG2A). It has been shown that the antigen-binding function of an antibody may be carried out by a fragment or portion of a full-length antibody. Examples of binding fragments encompassed within the terms “antigen-binding moiety” or “antigen-binding fragment” of an antibody, e.g., an anti-NKG2A antibody as described herein, include: (1) Fab fragment (fragment derived from papain cleavage) or a similar monovalent fragment consisting of VL, VH, LC, and CH1 domains, (2) F(ab')2 fragment (fragment derived from pepsin cleavage) or a similar divalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region, (3)V H and Fd fragments consisting of CH1 domains, (4) V of a single arm of the antibody L and V H Fv fragment consisting of domains, (5) Single-domain antibody (dAb) fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-46), (6) Isolated complementarity-determining regions (CDRs), and (7) A combination of two or more isolated CDRs, which may be connected by a synthetic linker. These are some examples.

[0056] Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using a recombination method with a synthetic linker, which allows the VL and VH region pair to be produced as a single protein chain forming a monovalent molecule (known as single-stranded 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-stranded antibodies are also encompassed within the term "antigen-binding moiety" 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 usefulness in a similar manner to intact antibodies. Antigen-binding moieties 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 possesses two different binding specificities, for example, two different heavy / light chain pairs, resulting in two antigen-binding sites that are specific to different antigens. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0058] In this specification, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, individual antibodies within the population are substantially similar, except for any variants that may occur during the production of the monoclonal antibody, and bind to the same epitope (e.g., the antibody exhibits a single binding specificity and affinity), and such variants are generally present in small amounts. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and does not require antibody production by any particular method. The term “human monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies that exhibits a single binding specificity and has a variable region derived from a human germline immunoglobulin sequence and, as appropriate, a constant region. In one embodiment, the human monoclonal antibody is produced by a hybridoma method. Using the hybridoma method, a transgenic non-human animal, e.g., a transgenic mouse, is exposed to an antigen, and leukocytes known as B cells produce antibodies that bind to the antigen, which are then recovered from the transgenic non-human animal. Isolated B cells are fused with immortalized cells to produce a hybrid cell line called a hybridoma. In one embodiment, the hybridoma has a genome containing human heavy chain transgenes and light chain transgenes fused to the immortalized cells.

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

[0060] In this specification, “human antibody” refers to an antibody in which both the framework and CDR region have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also derives from a human germline immunoglobulin sequence. Anti-NKG2A antibodies described herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo). However, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another non-human mammalian species, such as mouse, is grafted onto a human framework sequence. In this specification, the terms “human” and “fully human” antibody are used synonymously.

[0061] A "humanized antibody" refers to an antibody in which some, almost all, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from a human antibody. In one embodiment of the humanized form of an antibody, some, almost all, or all of the amino acids outside the CDR domain are replaced with amino acids derived from a human antibody, but some, almost all, 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 acceptable as long as they do not prevent the antibody from binding to a particular antigen. A "humanized antibody" retains similar antigen specificity to that of the original antibody.

[0062] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species, such as an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0063] In this specification, “isotype” refers to the antibody class encoded by the heavy chain constant region gene of the antibody (e.g., IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE antibodies).

[0064] An "allotype" refers to a naturally occurring variant within a specific group of isotypes, which differ by two or three amino acids. (See, for example, Jefferis et al. (2009) mAbs 1:1). The anti-NKG2A antibodies described herein may be of any allotype. In this specification, antibodies referred to as "IgG1f," "IgG1.1f," or "IgG1.3f" isotypes are allotype "f" IgG1, effectorless IgG1.1, or effectorless IgG1.3 antibodies, respectively.

[0065] In this specification, the terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used synonymously with the term "antibody that specifically binds to an antigen."

[0066] In this specification, “isolated antibody” refers to an antibody that substantially contains no other proteins or cellular materials.

[0067] In this specification, “effector function” refers to the interaction between the Fc region of an antibody and an Fc receptor or ligand, or the resulting biochemical event. Exemplary “effector functions” include FcγR-mediated effector functions such as Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), as well as downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require an Fc region 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 the FcγR family of receptors, which include allelic variants and spliced ​​forms of these receptors as an alternative. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mouse; FcγRIA, FcγRIIA, and FcγRIIIA in human) and one inhibitory receptor (FcγRIIb, or equivalently FcγRIIB). Various exemplary properties of human FcγRs are known in the art. Most congenital effector cell types simultaneously express one or more activated FcγR and inhibitory FcγRIIb, while natural killer (NK) cells selectively express one activated Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not express inhibitory FcγRIIb in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activated Fc receptors it binds to.

[0069] In this specification, “Fc region” (fragment crystallizable region), “Fc domain,” or “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins 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 traditional complement system. Thus, the Fc region includes the constant region of the antibody, excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In the antibody isotypes IgG, IgA, and IgD, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody. In the antibody isotopes IgM and IgE, the Fc region comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region includes the immunoglobulin domains CH2 and CH3, as well as the hinge between the CH1 and CH2 domains. While the definition of the boundary of the Fc region of the immunoglobulin heavy chain can vary as defined herein, the human IgG heavy chain Fc region is defined as the stretch from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxyl terminus of the heavy chain, with this numbering being based on Kabat's EU index (Kabat, et al., 1991). The CH2 domain of the human IgG Fc region spans amino acids 237 to 340, and the CH3 domain is located on the C-terminal side of the CH2 domain of the Fc region, i.e., the CH3 domain spans amino acids 341 to 447 or 446 (in the absence of a C-terminal lysine residue) or 445 (in the absence of C-terminal glycine and lysine residues) of IgG. In this specification, the Fc region may be a natural sequence Fc including any allotype variant or mutant Fc (e.g., Fc that does not exist in nature). Fc may also refer to this region in isolation or in relation to an Fc-containing protein polypeptide such as an "Fc-containing binding protein," also called an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

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

[0071] The term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., the hNKG2A protein) to which an immunoglobulin or antibody specifically binds, as defined, for example, by the specific method used to identify it. Epitopes can be formed from both (1) continuous amino acids (usually linear epitopes) or (2) discontinuous amino acids juxtaposed by the three-dimensional folding of the protein (usually conformational epitopes). Epitopes formed from continuous amino acids are usually retained upon exposure to denaturing solvents, though not always, while epitopes formed from three-dimensional folding are usually lost during processing with denaturing solvents. Epitopes typically contain 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 epitopes to which a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which duplicated or sequential peptides derived from a protein (e.g., NKG2A) are tested for reactivity with a given antibody (e.g., anti-NKG2A antibody). Methods for determining the spatial conformation of epitopes include techniques known in the art and those 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] For two or more antibodies, the term "bound to the same epitope" means that the antibody binds to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on NKG2A" using the antibodies described herein include, for example, epitope mapping methods such as X-ray analysis of antigen:antibody complex crystals, HDX-MS, and rapid photochemical oxidation (FPOP) of proteins, which provide atomic dissolution of the epitope. Other methods involve monitoring the binding of the antibody 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 in the antigen sequence is often considered an indicator of the epitope component, such as in alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081) or yeast display of mutant target sequence variants. Furthermore, computational combinatorial methods may be used for epitope mapping. These methods rely on the target antibody's ability to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies with identical VH and VL sequences or identical CDR1, CDR2, and CDR3 sequences are expected to bind to the same epitope.

[0074] An antibody that "competes with another antibody for binding to its target" refers to an antibody that (partially or completely) inhibits the binding of the other antibody to its target. Whether two antibodies compete with each other for binding to their target, that is, whether one antibody inhibits the binding of the other antibody to its target, and to what extent, 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 its target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody initially incubated with the target). Competitive assays can be performed as described, for example, 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 is the first to come into contact with the antigen in the competitive experiment.

[0075] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include, for example, competition for binding to NKG2A-expressing T cells 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 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)), and solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 Examples include (1990)), and directly labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0076] In this specification, the terms “specific binding,” “selective binding,” “selectively binding,” and “specifically binding” refer to antibody binding to an epitope on a given antigen. In some embodiments, the antibody is determined by (1) SPR technique in a Biacore® SPR instrument using a given antigen, e.g., recombinant human NKG2A, as the analyte and the antibody as the ligand, or by scatchard analysis of antibody binding to antigen-positive cells, for example, approximately 10 -6 Less than M, for example, approximately 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, or 10 -10M, or an even smaller equilibrium dissociation constant (K D (2) It binds to the given antigen with an affinity at least twice as great as its affinity for binding to nonspecific antigens other than the given antigen or closely related antigens (e.g., BSA, casein). Therefore, an antibody that "specifically binds to human NKG2A" is 10 -6 M or less, for example, approximately 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than M or even smaller D This refers to antibodies that bind to soluble or cell-bound human NKG2A. Antibodies that "cross-react with cynomolgus monkey NKG2A" are 10 -6 M or less, for example, approximately 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than 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 In this specification, the term "k" is used synonymously to refer to the binding rate constant of a particular antibody-antigen interaction. d "k dis " or "k off In this specification, the term "K" is used synonymously to refer to the dissociation rate constant of a particular antibody-antigen interaction. D In this specification, " refers to the equilibrium dissociation constant, which is k a k for d The proportion (i.e., k d / k a It is obtained from ) and expressed as molar concentration (M). K of the antibody D The value can be determined using methods well established in the art. The K of the antibody DAvailable methods for determining, for example, include surface plasmon resonance (SPR) using a biosensor system such as the Biacore® system, as well as flow cytometry and Scatchard analysis, but are not limited thereto.

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

[0079] The term "EC 50 " means the half-effective concentration and measures the potency of a substance, such as an antibody, to induce a particular biological or biochemical response. Similar to IC 50 , EC 50 is used as a measure of potency, and the smaller the EC 50 , the more potent the substance. In the context of in vitro or in vivo assays using an antibody or its antigen-binding fragment, EC 50 refers to the concentration of the antibody or its antigen-binding fragment that induces a response that is 50% of the maximum biological or biochemical response.

[0080] As used herein, "receptor occupancy" or "occupancy of the receptor" refers to the amount of antibody (e.g., an anti-NKG2A antibody as described herein) that is bound to an immune-stimulatory receptor (e.g., human NKG2A). "Percent receptor occupancy (%)" or "percent occupancy of the receptor (%)" can be calculated using the following formula: ([ΔMFI of the test] / [total ΔMFI])×100. The change in mean fluorescence units (ΔMFI) is calculated by subtracting the MFI of background staining with an isotype control antibody from the MFI from the bound antibody. The total receptor level is determined by adding a saturating amount of antibody to determine the maximum expression, and thus the MFI of a particular immune-stimulatory receptor. An alternative method for calculating total receptor expression is to use an antibody to the same immune-stimulatory 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 has not been intentionally modified by man. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from its natural source in the laboratory and that has not been intentionally modified by man is naturally occurring.

[0082] The term "polypeptide" refers to a chain containing at least two contiguous and linked amino acid residues, and there is no upper limit to the length of the chain. One or more amino acid residues in a protein can contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonding. A "protein" comprises one or more polypeptides.

[0083] The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules as used herein. Nucleic acid molecules can be single-stranded or double-stranded and can be complementary DNA (cDNA).

[0084] The term "cDNA" or "complementary DNA" refers to a nucleic acid molecule that is made or derived from mRNA, i.e., a nucleic acid molecule that does not occur naturally and from which non-coding regions have been removed.

[0085] In this specification, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing an 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. “Conservative amino acid substitution” refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, expected non-essential amino acid residues in the anti-NKG2A antibody are substituted with other amino acid residues derived from the same side chain family. Methods for identifying nucleotide and amino acid-conservative substitutions that do not exclude antigen binding are well known in the art (see, for example, 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 specified sequences are identical, when optimally aligned and compared, using appropriate nucleotide insertions or deletions in at least approximately 80%, at least approximately 90%–95%, or at least approximately 98%–99.5% of the nucleotides. Alternatively, substantial homology exists when the segments hybridize with the complement of the nucleic acid chain under selective hybridization conditions.

[0087] For polypeptides, the term "substantial homology" indicates that two polypeptides or their specified sequences are identical, when optimally aligned and compared, in terms of at least about 80%, at least about 90%–95%, or at least about 98%–99.5% of their amino acids, using appropriate amino acid insertions or deletions.

[0088] The percentage of identity between two types of arrays is a function of the number of identical positions shared by the arrays (i.e., percentage of identity = (number of identical positions) / (total number of positions) × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two types of arrays, and the length of each gap. The comparison of arrays between two types of arrays and the determination of the percentage of identity can be achieved using mathematical algorithms, as described in the following examples (not limited to) below.

[0089] The percentage of identity between two nucleotide sequences can be determined, for example, using the GAP program in the GCG software package, with the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm by E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which is incorporated into the ALIGN program (version 2.0), with the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of 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 the Blossum 62 matrix or the PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

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

[0091] Nucleic acids may exist in whole cells, e.g., in host cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially purified” when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques including alkali / SDS treatment, CsCl band formation, 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] In this specification, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which further DNA segments can be ligated. Another type of vector is a viral vector, in which further DNA segments can be ligated into the viral genome. Certain vectors are self-replicating in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell and thereby replicate with the host genome. Furthermore, certain vectors can direct the expression of a gene to which they are operably ligated. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Plasmids are examples of expression vectors useful in recombinant DNA technology. Since plasmids are the most commonly used form of vectors, “plasmid” and “vector” may be used synonymously herein. However, other forms of expression vectors that perform equivalent functions also include viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0093] The terms “host cell” or “recombinant host cell” are used synonymously and refer to cells that may contain nucleic acids not naturally present in the cell and into which recombinant expression vectors have been introduced. It should be understood that such terms refer not only to specific target cells but also to the offspring of such cells. Since certain modifications can occur in the offspring either by mutation or environmental influences, such offspring may not actually be identical to the parent cells, but for the purposes of this specification, they are still included within the scope of the term “host cell.”

[0094] "Immune response," as understood in the art, generally refers to the biological response in vertebrates to foreign agents or abnormal cells, such as cancerous cells, which protect vertebrates 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, damage to, destruction of, and / or elimination from the body of the vertebrate of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal cells or tissues, including, for example, human cells or tissues. The immune response includes, for example, the activation or inhibition of 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. Th cells secrete cytokines that activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0096] Regulatory T ("Treg") cells are a subgroup of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Memory B cells are a subtype of B cells 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 to virus-infected cells and respond to tumorigenesis.

[0098] In this specification, 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] In this specification, 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 that can be involved in the regulation, control, or modification of an immune response, such as a component of a signal transduction pathway. The "regulation", "control", or "modification" 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, such as Th1 cells). Such regulation includes stimulation or suppression of the immune system and can be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, and / or any other change that can occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, and some of them may have enhanced functions in the tumor microenvironment. In some embodiments, the immunomodulator is located on the surface of a T cell. An "immunomodulatory 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, receptors on the surface of cells ("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 having a recurrence of a disease, by a method that includes inducing, enhancing, suppressing, or otherwise modifying an immune response.

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

[0103] "Enhancing the endogenous immune response" means increasing the effectiveness or potency of an existing immune response in a subject, such as a human subject. This increase in effectiveness 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] In this specification, the term “conjugated” refers to the association of two or more molecules. Conjugation may be covalent or non-covalent. Conjugation may also be genetic (i.e., fused by recombination). Such conjugation may be achieved using various techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0105] In this specification, “administering” means the physical introduction of a therapeutic agent, such as a composition containing an anti-NKG2A antibody, to a target using any of the various methods and delivery systems known to those skilled in the art. “Administering” includes, for example, administration to a human patient by another person, such as one or more healthcare workers, and self-administration by a human patient. The various routes of administration of antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. In this specification, the term “parenteral administration” means, but is not limited to, modes of administration other than enteral and topical administration, such as injection, but includes intravenous, intraperitoneal, intramuscular, intraarterial, subarachnoid, lymphatic, intrafocal, intra-articular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered by non-parenteral routes such as local, epithelial, or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administration may be carried out, for example, once, multiple times, and / or over a long period of time, one or more times.

[0106] In this specification, “adjunctive” administration or “combination” administration (co-administration) includes the simultaneous administration of compounds in the same or different dosage forms, or the separate administration of compounds (e.g., sequential administration). Therefore, a first antibody, e.g., an anti-NKG2A antibody, and a second, third, or more antibodies may be administered simultaneously in a single formulation. Alternatively, the first and second (or more) antibodies may be formulated for separate administration and administered in parallel or sequentially. “Combination” therapy, as used herein, means the administration of two or more therapeutic agents in a cooperative manner, and includes, but is not limited to, simultaneous administration. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent somehow influences the administration of another therapeutic agent. For example, one therapeutic agent may only be administered after a different therapeutic agent has been administered and its effects have been tolerated for a predetermined period. (See, for example, Kohrt et al. (2011) Blood 117:2423).

[0107] For example, an anti-NKG2A antibody may be administered first, followed (for example, immediately afterward) by a 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 simultaneously in the patient being treated.

[0108] In this specification, the terms “inhibit” and “block” are used synonymously and encompass both partial and complete inhibition / blockage. In some embodiments, the anti-NKG2A antibodies described herein inhibit the binding of NKG2A to HLA-E by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as determined, for example, as further described herein. In some embodiments, the anti-NKG2A antibodies inhibit the binding of NKG2A to HLA-E by 50% or less, e.g., about 40%, 30%, 20%, 10%, 5%, or 1%, as determined, for example, as further described herein.

[0109] In this specification, “cancer” refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells throughout the body. Uncontrolled cell growth or division can result in the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0110] The terms “to treat,” “to treat,” 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, aimed at reversing, reducing, relieving, inhibiting, or slowing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with the disease. In contrast, “prevention” or “prevention” refers to the administration of an active agent to a subject that does not have the disease, in order to prevent the onset of the disease. In this specification, “to treat,” “to treat,” and “treatment” do not encompass prevention or prevention.

[0111] The term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. A “therapeutic effective dose” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug, used alone or in combination with another therapeutic agent, that promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional impairment or disability due to disease distress. A “prophylactic effective dose” or “prophylactic effective dosage” of a drug is the amount of the drug, used alone or in combination with another therapeutic agent, that prevents the onset or recurrence of disease when administered to a subject at risk of developing or experiencing a relapse of the disease. The ability of a therapeutic agent to promote disease regression or a prophylactic agent to prevent the onset or recurrence of 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 to predict efficacy in humans, or by assaying the activity of the drug in in vitro assays.

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

[0113] For example, anticancer agents are drugs that slow the progression of cancer or promote cancer regression in subjects, including human subjects. In some embodiments, a therapeutically effective amount of the drug promotes cancer regression to the extent that it eliminates the cancer. "Promoting cancer regression" means that administration of an effective amount of the drug, alone or in combination with an antineoplastic agent, results in a patient with reduced tumor growth or size, tumor necrosis, reduced severity of at least one disease symptom, increased frequency and duration of disease-free periods, prevention of functional or disability due to disease distress, or otherwise remission of disease symptoms. "Pharmacological efficacy," "efficacy," or "potency" refers to the ability of a drug to promote cancer regression in a patient. "Physiological safety" refers to an acceptablely low level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or biological level resulting from the administration of the drug.

[0114] For example, with respect to the treatment of tumors, a therapeutically effective amount or dose 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 an untreated subject. In some embodiments, a therapeutically effective amount or dose of a drug completely inhibits cell growth or tumor growth, i.e., 100% inhibition of cell growth or tumor growth. The ability of a compound to inhibit tumor growth, including antibodies, can be evaluated using the assays described herein. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to those skilled in the art. In some embodiments, inhibition of tumor growth may not occur immediately after treatment, but only after a period of time or after 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] In this specification, the terms “fixed dose,” “constant dose,” and “constant fixed dose” are used synonymously and refer to the dose administered to a patient regardless of the patient’s weight or body surface area. Fixed or constant doses are therefore provided as absolute amounts of the therapeutic agent, rather than as mg / kg doses.

[0116] In this specification, the term “body weight-based” dosage or administration means that the dose administered to a patient is calculated based on the patient’s body 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 preventive treatment.

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

[0119] In this specification, the terms “one (a)” or “one (an)” entity refer to one or more such entities unless otherwise indicated; for example, “one nucleotide sequence” is understood to represent one or more nucleotide sequences. Thus, the terms “one (a)” or “one (an),” “one or more,” and “at least one” can be used synonymously in this specification.

[0120] In this specification, “and / or” shall be interpreted as a specific disclosure that each of the two designated features or components may or may not be accompanied by the other. Thus, when the term “and / or” is used in a phrase such as “A and / or B,” it includes “A and B,” “A or B,” “A” alone, and “B” alone. Similarly, when the term “and / or” is used in a phrase such as “A, B, and / or C,” it includes 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 an aspect is described herein using the word “comprising,” it is understood that similar aspects are also provided, which are otherwise described using the terms “consisting of” and / or “essentially consisting of.”

[0122] Units, prefixes, and symbols are given in the form recognized by the International System of Units (SI) unless otherwise specified. Numerical ranges include the number defining the range. Unless otherwise specified, nucleotide sequences are written from left to right, in the 5' to 3' direction. Amino acid sequences are written from left to right, in the amino to carboxy direction.

[0123] In this specification, the terms “about” or “approximately” mean roughly, approximately, or within a range. When the term “about” is used with a numerical range, the term modifies the range by extending the boundary above or below the stated numerical value. Generally, the term “about” may modify a numerical value above or below the stated value, for example, 10 percent above or below (higher or lower).

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

[0125] I. Anti-NKG2A antibody This disclosure describes, in some embodiments, anti-NKG2A antibodies having desirable functions or properties, such as fully human, humanized, and chimeric antibodies. For example, the antibody specifically binds to the human NKG2A protein with high affinity. In certain embodiments, the antibody is an antagonist antibody that blocks or reverses NKG2A-mediated inhibition in immune cells, such as 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, such as cancer or infections.

[0126] The specific 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, and 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 sequence 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 sequence of the anti-NKG2A antibodies described herein.

[0127] In some embodiments, the antibodies of the present invention are characterized by specific functional features or properties. For example, the antibodies bind specifically to human NKG2A with high affinity. In some embodiments, the anti-NKG2A antibody inhibits the binding of NKG2A to its ligand HLA-E, thereby restoring the NK and T cell response to tumors expressing HLA-E. In other words, the anti-NKG2A antibodies described herein stimulate the antitumor response 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 antibody described herein has the following characteristics: (1) Specifically binds to the human NKG2A protein, (2) Blocking or reducing the binding and / or interaction of an NKG2A ligand (e.g., HLA-E in humans) with the 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 the non-human NKG2A protein), (3) Reversing inhibitory signaling mediated by NKG2A, (4) It does not bind to the human NKG2C protein, or it binds to it with low affinity. (5) To bind to human and cynomolgus monkey NKG2A with high affinity, (6) It does not bind to mouse or rat NKG2A, or shows low affinity for it. (7) Do not interfere with the activation signal of HLA-E by binding to the NKG2C protein. (8) Reduced binding to the human Fc gamma receptor (FcγR), (9) Inducing and / or enhancing an antitumor immune response, (10) To enhance the functional activity of T cells (in some embodiments, for example, to increase cytotoxic T cell function as measured by the lysis of HLA-E expressing tumor cells), (11) For example, enhancing the functional activity of NK cells by inducing natural killer (NK) cell activation, (12) to increase cytokine production, e.g., IFNγ production, and / or (13) When determined by HDX-MS and / or FPOP epitope mapping, the following amino acid residues: Area 1: 155 LSIDNEEEMKF 165 (amino acid residues 155-165 of SEQ ID NO: 2, Area 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171-186 of SEQ ID NO: 2) Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192-203 of Sequence ID No. 2, Region 4:L (amino acid residue 206 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (Amino acid residues 212-229 of SEQ ID NO: 2) Specific binding to an epitope located within a discontinuous region containing, (14) When determined by HDX-MS, the following amino acid residues: Area 1: 155 LSIDNEEEMKF 165 (Amino acid residues 155-165 of SEQ ID NO: 2) Area 2: 171 PSSWIGVFRNSSHHPW 186 (Amino acid residues 171-186 of SEQ ID NO: 2) Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192-203 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (Amino acid residues 212-229 of SEQ ID NO: 2) Specific binding to an epitope located within a discontinuous region containing, indicates one or more of the following.

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

[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 have the following properties: a) The anti-NKG2A antibody has an IC 50 of about 0.30 nM for NKL and an IC 50 of about 1.0 nM for CHO-hNKG2A cells, blocking the binding of HLA-E pentamer to cells expressing human NKG2A, b) The anti-NKG2A antibody complexed with HNKG2A-CD94-mFC or cynomolgus NKG2A-CD94-mFc protein blocks human HLA-E binding exhibiting one or more of the above.

[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 properties: a) For the binding of the anti-NKG2A antibody to human NKG2A, the EC 50 value is about 15-fold lower than the second EC 50 value for the binding of the anti-NKG2A antibody to human NKG2C protein. In some embodiments, the EC 50 value for the binding of the anti-NKG2A antibody to human NKG2A is about 0.6 nM, while the EC 50 value for the binding of the anti-NKG2A antibody to human NKG2C is about 9.0 nM. b) Based on SPR analysis, there is no specific binding of the anti-NKG2A antibody to human NKG2C, and / or c) When measured by flow cytometry, it does not block the interaction between human NKG2C and HLA-E exhibiting one or more of the above.

[0132] In some embodiments, anti-NKG2A antibodies have inactive Fc (e.g., the antibody is of the IgG1 isotype) to reduce or prevent FcγR binding. While not bound by any particular theory, since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, reducing agonism or depletion of NKG2A+ CD8+ T or NK cells is beneficial for antitumor function. Therefore, blocking the interaction between NKG2A and HLA-E can be done using anti-NKG2A antibodies that do not interact with human FcγR.

[0133] In some embodiments, the anti-NKG2A antibodies described herein enhance the antitumor activity of T cells. Specifically, in some embodiments, the anti-NKG2A antibodies possess the following properties: a) EC of approximately 0.2 nM or less 50 The value reverses 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 + In the CD8 T cell, the induction of interferon-gamma (IFN-γ) c) NKG2A isolated from human tumors co-cultured with CHO / scOKT3 / HLA-E / PD-L1 + Inducing IFN-γ in CD8 T cells Show one or more of them.

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

[0135] In some embodiments, the anti-NKG2A antibodies described herein are translocated internally after binding to NKG2A-expressing cells. Specifically, in some embodiments, the anti-NKG2A antibodies are translocated to cells with an EC of about 0.5 nM lower or less. 50 Then, internal migration is observed. In some embodiments, the anti-NKG2A antibody has an EC of about 0.5 nM lower or less. 50 It then exhibits dose-dependent internal transfer.

[0136] In some embodiments, the anti-NKG2A antibodies of the present invention lack sequence tendencies that reduce the chemical stability of the antibody. The anti-NKG2A antibodies of the present invention have various important applications, 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 their amino acid sequence bind to specific epitopes on human NKG2A, as described in Example 4.

[0138] Binding to human NKG2A can be evaluated using one or more techniques well established in the art. For example, in some embodiments, the antibody is tested by a flow cytometry assay in which the antibody reacts with a cell line expressing human NKG2A, e.g., CHO cells transfected to express human NKG2A on their cell surface. Furthermore, or or otherwise, antibody binding can be evaluated by its binding kinetics (e.g., K D The Biacore binding assay can be used to test for the following: (value) and others. Other suitable binding assays include, for example, ELISA assays using recombinant human NKG2A protein.

[0139] In some embodiments, the anti-NKG2A antibody or its antigen-binding fragment described herein has high affinity, for example, 1 × 10⁻⁶ -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 contains and binds to the human NKG2A protein with nanomolar-scale affinity.

[0140] Some embodiments of the present invention, as determined by HDX-MS and / or FPOP epitope mapping, include the following amino acid residues: Area 1: 155 LSIDNEEEMKF 165 (Amino acid residues 155-165 of SEQ ID NO: 2 (natural hNKG2A amino acid sequence), Area 2: 171 PSSWIGVFRNSSHHPW 186 (Amino acid residues 171-186 of SEQ ID NO: 2) Area 3: 192 LAFKHEIKDSDN 203 (Amino acid residues 192-203 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 212-229 of SEQ ID NO: 2) This relates to an anti-NKG2A monoclonal antibody or its antigen-binding moiety that specifically binds to an epitope located within a discontinuous region containing [a specific component].

[0141] In some embodiments, the present invention, when determined by HDX-MS, includes the following amino acid residues: Area 1: 155 LSIDNEEEMKF 165 (Amino acid residues 155-165 of SEQ ID NO: 2) Area 2: 171 PSSWIGVFRNSSHHPW 186 (Amino acid residues 171-186 of SEQ ID NO: 2) Area 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192-203 of SEQ ID NO: 2), and Area 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212-229 of SEQ ID NO: 2) The target is an anti-NKG2A monoclonal antibody or its antigen-binding moiety that specifically binds to an epitope located within a discontinuous region containing [the specified element].

[0142] In some embodiments, the anti-NKG2A antibodies described herein enhance NK cell function by blocking NKG2A / HLA-E-mediated inhibition. In other embodiments, the anti-NKG2A antibodies bind to the human NKG2A protein and stimulate an anti-tumor immune response, e.g., 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 antibody or its antigen-binding moiety increases cytokine production (e.g., interferon-gamma (IFN-γ)) in NKG2A-expressing T cells and / or increases T cell proliferation, including effector T cells and cytotoxic T cells (also known as CD8+ T cells).

[0143] In another embodiment, an anti-NKG2A antibody or its antigen-binding fragment binds to human NKG2A and possesses the following properties: a) When determined by HDX-MS and / or FPOP epitope mapping, it must bind to one or more of the following residues: Area 1: 155 LSIDNEEEMKF 165 (Amino acid residues 155-165 of SEQ ID NO: 2 (natural hNKG2A amino acid sequence), Area 2: 171 PSSWIGVFRNSSHHPW 186 (Amino acid residues 171-186 of SEQ ID NO: 2) Area 3: 192 LAFKHEIKDSDN 203 (Amino acid residues 192-203 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 212-229 of SEQ ID NO: 2) b) Binding to the same human NKG2A epitope as NKG2A.11 and 13F3.A4 antibodies, c) Regarding binding to human NKG2A, it competes with NKG2A.11 and 13F3.A4 antibodies. d) When measured by Biocore, the EC is approximately 0.4 nM. 50 And then it binds to human NK cells. e) When measured by Biacore, the IC is approximately 0.3 nM 50 Therefore, blocking the binding of human NK cells to HLA-E, f) EC of approximately 1 nM or less 50 And it binds to cynomolgus monkey NKG2A-expressing CHO cells. g) EC of approximately 9.0 nM or higher 50 Therefore, it has 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 antitumor response of CD8+ T cells and NK cells, for example, i. To increase IFN-γ production in the original T:CHO-OKT3-HLA-E-PDL1 assay, ii. In the T-cell tumor-infiltrating lymphocyte (TIL): CHO-OKT3-HLA-E-PDL1 assay, increase IFN-γ production and / or iii. To increase cytotoxicity and IFN-γ production in primary NK cell assays. Show at least one of them.

[0144] In some embodiments, the anti-NKG2A antibody of the present invention includes humanized and fully human monoclonal antibodies. In other embodiments, the antibody is, for example, a chimeric monoclonal antibody.

[0145] a. Anti-NKG2A monoclonal antibody In some embodiments, the antibodies of the present invention are monoclonal antibodies 13F3.A4, NKG2A.9, and NKG2A.11, which have been isolated and structurally characterized as described in the following examples. The VH and VL amino acid sequences are listed in the sequence listing and sequence list.

[0146] Given that each of these antibodies can bind to human NKG2A, other anti-hNKG2A binding molecules of the present invention can be created by "mixing and matching" the VH and VL sequences. In some embodiments, when the VH and VL chains are mixed and matched, the VH from a particular VH / VL pairing is replaced with a structurally similar VH sequence. Similarly, in some embodiments, the VL sequence from a particular VH / VL pairing is replaced with a structurally similar VL sequence. Thus, in one embodiment, the present disclosure provides an isolated monoclonal antibody or its antigen-binding fragment that binds to the human NKG2A protein, wherein the light chain 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) Amino acid sequences of SEQ ID NOs. 169 and 167, respectively The present invention provides an isolated monoclonal antibody or its antigen-binding fragment, which includes [the specified substance].

[0147] In another embodiment, the Disclosure provides antibodies comprising the heavy and light chains CDR1, CDR2, and CDR3 of NKG2A.9, NKG2A.11, and 13F3A.4 antibodies. Thus, in one embodiment, the Disclosure provides an isolated monoclonal antibody or its antigen-binding fragment that binds to the human NKG2A protein, wherein the antibody is (a) Heavy chain variable domains containing CDR1, CDR2, and CDR3 regions containing the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively, and light chain variable domains containing CDR1, CDR2, and CDR3 regions containing the amino acid sequences of SEQ ID NOs. 13, 14, and 15, respectively. (b) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively, and light chain variable domains comprising CDR1, CDR2, and CDR3 regions containing the amino acid sequences of SEQ ID NOs. 154, 14, and 15, respectively, or (c) Heavy chain variable domains containing CDR1, CDR2, and CDR3 regions containing the amino acid sequences of SEQ ID NOs. 10, 11, and 12, respectively, and light chain variable domains containing CDR1, CDR2, and CDR3 regions containing the amino acid sequences of SEQ ID NOs. 155, 14, and 15, respectively. The present invention provides an isolated monoclonal antibody or its antigen-binding fragment, which includes [the specified substance].

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

[0149] In other embodiments, the Disclosure provides a monoclonal antibody comprising one or more heavy and / or light chain CDR3 domains derived from a human antibody, for example, a human antibody obtained from a non-human animal, wherein the human antibody can specifically bind to human NKG2A. In other embodiments, the Disclosure provides a monoclonal antibody comprising one or more heavy and / or light chain CDR3 domains derived from a first human antibody, for example, a human antibody obtained from a non-human animal, wherein the first human antibody can specifically bind to human NKG2A, and a second human antibody can be generated in which the CDR3 domain derived from the first human antibody is replaced with a CDR3 domain of a human antibody lacking binding specificity to NKG2A, thereby enabling the second human antibody to specifically bind to human NKG2A. In some embodiments, such an inventive antibody comprising one or more heavy and / or light chain CDR3 domains derived from a first human antibody is capable of (a) competing with a corresponding parental non-human antibody for binding, (b) retaining its functional characteristics, (c) binding to the same epitope, and / or (d) having similar binding affinity.

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

[0151] b. Antibodies with Conservative Modifications In certain embodiments, the anti-NKG2A antibody of the present invention comprises 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 comprises a specified amino acid sequence or a conserved modification thereof based on the antibodies described herein (e.g., 13F3.A4, NKG2A.9, and NKG2A.11 antibodies), and such antibodies retain the desired functional properties of the anti-hNKG2A antibody of the present invention. It is understood in the art that certain conserved sequence modifications that do not remove antigen binding may be performed (see, for example, Brummell et al. (1993) Biochem 32:1180-8). Accordingly, this disclosure relates to an isolated monoclonal antibody or an 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 includes a CDR3 sequence containing the amino acid sequence described in SEQ ID NO: 12 or a conserved modification thereof, (b) The antibody or its antigen-binding portion specifically binds to human NKG2A, This provides isolated monoclonal antibodies or their antigen-binding fragments.

[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 containing the CDR2 sequence includes the amino acid sequence described in SEQ ID NO: 11 or a conserved modification thereof, and the light chain variable region containing the CDR2 sequence includes the amino acid sequence described in SEQ ID NO: 14 or a conserved modification thereof. In another embodiment, the heavy chain variable region includes the CDR1 sequence including the amino acid sequence described in SEQ ID NO: 10 or a conserved modification thereof, and the light chain variable region includes the CDR1 sequence including the amino acid sequence described in SEQ ID NO: 13, 154, or 155 or a conserved 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. Antibodies that bind to the same epitope as anti-hNKG2A antibodies. In another embodiment, the disclosure provides an antibody that binds to the same epitope on the human NKG2A protein as any of the anti-hNKG2A monoclonal antibodies of the present invention (i.e., an antibody having 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 for binding with the labeled antibody can be evaluated. Furthermore, or alternatively, Biacore analysis can be used to evaluate the ability of antibodies to cross-compete. The ability of a 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 may compete with NKG2A.9, NKG2A.11, and / or 13F3.A4 for binding to human NKG2A, and therefore binds to the same epitopes on human NKG2A.9 as NKG2A.9, NKG2A.11, and / or 13F3.A4. In some embodiments, the antibody that binds to the same epitopes on human NKG2A as NKG2A.9, NKG2A.11, and / or 13F3.A4 is a humanized or human monoclonal antibody.

[0156] As will be further discussed in Example 4, the binding of NKG2A.9 and 13F3.A4 is mapped to specific residues. Therefore, in one embodiment, when the present invention binds to the human NKG2A protein, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS), the following amino acid residues are involved: (e)LSIDNEEMKF(sequence number 156); (f)PSSWIGVFRNSSHHPW(sequence code 157); (g)LAFKHEIKDSDN(sequence number 158); and (h)QVNRLKSAQQCGSSIIYHC(Sequence ID 159) The present invention provides an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to a target, wherein the monoclonal antibody blocks the binding of an NKG2A ligand (e.g., HLA-E in humans) to the human NKG2A protein.

[0157] In another embodiment, when the present invention binds to human NKG2A, the following amino acid residues are determined by HDX-MS and / or rapid photochemical oxidation (FPOP) epitope mapping of the protein: (f)LSIDNEEMKF(sequence number 156); (g)PSSWIGVFRNSSHHPW(sequence code 157); (h)LAFKHEIKDSDN(sequence number 158); (i)L; and (j)QVNRLKSAQQCGSSIIYHC(Sequence ID 159) The present invention provides an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to a target, wherein the monoclonal antibody blocks the 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 prepared using an immunotherapy protocol, e.g., one 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 a yeast display assay in which sequence variants of hNKG2A are presented 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 the 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 along 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 region. An epitope “region” is a cluster of residues that aligns with the primary sequence but is not necessarily directly adjacent to it.

[0160] One technique for investigating antibodies that bind to the "identical epitope on hNKG2A" using the antibodies described herein is X-ray analysis of the antigen:antibody complex crystals, which provides atomic dissolution of the epitope. Other methods involve monitoring the binding of the antibody to antigen fragments or mutated variations of the antigen, where loss of binding due to amino acid modifications in the antigen sequence indicates an epitope component. Methods may also rely on the ability to affinity isolate specific short peptides (either in their native three-dimensional form or a denatured form) from the antibody of interest, combinatorial phage display peptide libraries, or protease digests of the target protein. The peptides are then considered leads for defining the epitopes corresponding to the antibodies used to screen the peptide libraries. 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 that extend to NKG2A. Alternatively, the method of Jespers et al. (1994) Biotechnology;12:899 may be used to select antibodies having the same epitope and therefore similar properties to the anti-NKG2A antibodies described herein. Using phage display, NKG2A-binding antibodies can be selected by first pairing the heavy chain of an anti-NKG2A antibody with a repertoire of (e.g., human) light chains, and then by pairing a novel light chain with a repertoire of (e.g., human) heavy chains to select (e.g., human) NKG2A-binding antibodies having 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] Functional epitopes of anti-NKG2A antibodies may be investigated using alanine scanning mutagenesis or several other forms of point mutagenesis of amino acid residues in NKG2A, as described by Cunningham & Wells, Science 244: 1081 (1989).

[0163] The epitope or epitope region to which a specific antibody binds ("epitope region" is a region containing or overlapping with an epitope) may also be determined by evaluating the binding of the antibody to peptides containing the NKG2A fragment. A series of overlapping peptides containing the NKG2A sequence (e.g., human NKG2A) may be synthesized and screened for binding, for example, in direct ELISA, competitive ELISA (where the peptides are evaluated for their ability to prevent the binding of the antibody to NKG2A bound to the 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 containing amino acid residues that are not contiguous along the primary sequence of the NKG2A polypeptide chain.

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

[0165] The epitopes conjugated by anti-NKG2A antibodies may also be determined by structural methods, including X-ray crystallography (e.g., WO2005 / 044853), molecular modeling, and NMR spectroscopy, including nuclear magnetic resonance (NMR) determination of the HD exchange rate of unstable amide hydrogens in NKG2A when free and when conjugated 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 by reference to the claims, the epitope to which the antibody binds is the epitope determined by the HDX-MS method.

[0167] Anti-NKG2A antibody that binds with high affinity In some embodiments, the anti-hNKG2A antibody of the present invention binds to hNKG2A with high affinity, making it an effective therapeutic agent. In various embodiments, the anti-hNKG2A antibody of the present invention has a K content 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 Then, it binds to hNKG2A. Standard assays for evaluating the binding ability of antibodies 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. The CDR region is depicted 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 a CDR sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the CDR sequence of the antibodies disclosed herein. The present invention also provides anti-hNKG2A antibodies comprising heavy chain 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 chain and / or light chain variable domain sequences of antibodies disclosed herein, and anti-hNKG2A antibodies comprising full-length heavy chain 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 chain and / or light chain sequences of antibodies disclosed herein.

[0169] II. Genetically modified and modified antibodies AV H and V L region To genetically modify modified antibodies, V disclosed herein is used as a starting material. H and / or V L Genetically engineered and modified antibodies are also provided, which can be prepared using antibodies having one or more of the sequences, and these modified antibodies may have altered properties from the starting antibody. In some embodiments, the antibodies described herein have one or both of the variable regions (i.e., V H and / or V LThe antibodies were genetically engineered by modifying, for example, one or more residues within one or more CDR regions and / or one or more framework regions. Furthermore, or / or, the antibodies described herein were genetically engineered by modifying residues within a constant region(s) to alter the effector function(s) of the antibody(s).

[0170] In one embodiment, the genetic manipulation of variable regions includes CDR grafting. Such grafting is particularly useful in humanizing non-human anti-NKG2A antibodies, such as anti-HNKG2A antibodies that compete with the anti-hNKG2A antibodies disclosed herein for binding and / or bind to the same epitopes as the selective anti-hNKG2A antibodies disclosed herein. The antibody interacts with the target antigen primarily via amino acid residues located in the heavy and light chain CDRs. The CDRs are sequenced hypervariable and / or form structurally defined loops ("hypervariable loops"). Expression vectors can be constructed to contain a CDR sequence derived from a specific reference antibody (also called the "parent") grafted onto a framework sequence derived from a different antibody (see, for example, 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's U.S. Patent No. 5,225,539, and Queen et al.'s U.S. Patents No. 5,530,101, 5,585,089, 5,693,762, and 6,180,370). In some cases, the resulting recombinant antibody will have properties similar to the parent antibody. Genetically modified antibodies can then be further modified to acquire properties different from those of the parent antibody. In other cases, grafting the parental CDR sequence into a framework can suppress certain features of the parental antibody, resulting in the recombinant antibody no longer possessing those features. One exemplary feature is binding affinity to an antigen. In such cases, it may be advantageous to further modify the genetically modified antibody to regain desired features of the parental antibody.

[0171] Such framework sequences can be obtained from publicly available DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy chain 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 This can be found in “Segments Reveals a Strong Bias in their Usage” Eur. J. Immunol. 24:827–836, the contents of which are explicitly incorporated herein by reference.

[0172] In some embodiments, the framework sequence for use in the antibodies described herein is structurally similar to the framework sequence used by the antibodies described herein. H CDR1, 2, and 3 sequences and V LThe CDR1, 2, and 3 sequences may be grafted onto a framework region having the same sequence as found in the germline immunoglobulin gene from which the framework sequence originates, or the CDR sequences may be grafted onto a framework region containing amino acid substitutions, including up to 20 conserved amino acid substitutions compared to the germline sequence. For example, in certain cases, it has been found that mutating residues within the framework region is beneficial to maintain or enhance the antigen-binding ability of an antibody (see, e.g., Queen et al., U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0173] The genetically modified antibodies described herein are, for example, modified to improve the properties of the antibody, for example, to reduce the immunogenicity of the antibody, H and / or V L This includes antibodies in which modifications have been made to the framework residues within the framework. For example, one approach is to “reverse-mutate” one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from those in the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody originates. To return the framework region sequence to its germline configuration, somatic mutations can be “reverse-mutated” to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such “reverse-mutated” antibodies are also included in this disclosure.

[0174] Another type of framework modification involves mutating one or more residues within a framework region, or even within one or more CDR regions, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach is also called “deimmunization” and is described in more detail in U.S. Patent Publication 20030153043 by Carr et al.

[0175] Another type of variable region modification involves mutating amino acid residues within the CDR region to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations (one or more) and effects on antibody binding or other functional properties of the target. Conservative modifications are preferred. Mutations may be amino acid additions, deletions, or substitutions. In some embodiments, one, two, three, four, or five or fewer residues within the CDR region are modified.

[0176] Methionine residues in the CDR of an antibody can be oxidized, which can result in the possibility of chemical degradation and consequently a reduction in the antibody's potency. Therefore, anti-NKG2A antibodies in which one or more methionine residues in the heavy and / or light chain CDR are substituted with amino acid residues that are not subject to oxidative degradation are also provided herein. Similarly, deamidation sites in the CDR can also be removed from anti-NKG2A antibodies. Antibodies in which potential glycosylation sites within the antigen-binding domain have been eliminated to prevent glycosylation that may interfere with antigen binding are also provided herein. See, for example, U.S. Patent No. 5,714,350.

[0177] b. Antibody masking In some embodiments, the antibodies disclosed herein are modified to restrict binding to specific cells and / or tissues. In one embodiment, such an antibody includes 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 of the antibody's binding arms by a protease-cleavable linker. See, for example, U.S. Patent No. 8,518,404 for 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 for the dissociation of the masking / blocking peptide, which enables antigen-binding selectivity in tumors 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 that bind to both antigen-binding surfaces of a (bivalent) antibody and interfere with antigen binding. In one embodiment, the two binding domain masks are linked to each other (not the antibody) by a cleavable linker, e.g., a peptidase-cleavable linker. (See, for example, Tegopharm Corp.'s International Patent Application Publication WO2010 / 077643.) The masking ligand may contain, or be derived from, the antigen to which the antibody is intended to bind, or may be independently constructed. (e.g., an anti-idiotype 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 tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to dissociate from each other, reducing the binding affinity of the antibody's antigen-binding surface. The resulting dissociation of masking ligands from antibodies enables antigen-binding selectivity in tumors, rather than in peripheral tissues where antigen binding could cause unwanted side effects.

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

[0180] The variable region of the anti-hNKG2A antibody described herein is Fc, for example, IgG1, IgG2, IgG3, or IgG4 Fc can be linked (for example, by covalent bonds or fusion), which may be 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, for example, Jefferis et al. (2009) mAbs 1:1). Allotype selection may be influenced by potential immunogenicity concerns, for example, to minimize the formation of anti-drug antibodies.

[0181] In some embodiments, the anti-NKG2A antibody of the present invention cannot interact with human FcγR. Since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, + Antitumor immunity is enhanced by avoiding or reducing agonism or depletion of CD8+ T or NK cells. Therefore, blocking the NKG2A / HLA-E interaction is desirable for anti-NKG2A antibodies that cannot interact with human FcγR.

[0182] d. Extension of half-life In some embodiments, anti-NKG2A antibodies are modified to increase their biological half-life, for example, the serum half-life of the antibody. Various approaches are known in the art. In one embodiment, the antibody is modified within 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. Patents 5,869,046 and 6,121,022. For example, combined Fc variants including M252Y, S254T, and T256E increase the half-life by nearly fourfold (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; Nos. 6,277,375; 6,821,505; WO97 / 34631; WO2002 / 060919.

[0183] The serum half-life of the antibodies described herein may also be increased by pegylation. Antibodies may be pegylated, for example, to increase their biological (e.g., serum) half-life. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups bind to the antibody or antibody fragment. Preferably, pegylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). In this specification, the term “polyethylene glycol” shall 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 a non-glycosylated antibody. Methods for pegylation of proteins are known in the art and may be applied to the antibodies described herein. (See, for example, EP0154316 by Nishimura et al. and EP0401384 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 include modifications that reduce 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 reduce FcRn binding and, consequently, reduce the half-life (increase clearance) for use in situations where rapid clearance is desirable, such as in medical imaging. (See also Kenanova et al. (2005) Cancer Res. 65:622). Another means of enhancing clearance is to format 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 two or three weeks to several hours. The half-life of the antibody fragment can then be increased, if desired, using selective pegylation of the antibody fragment (Chapman et al. (1999) Nat. Biotechnol. 17:780). To increase the half-life, the antibody fragment may also be fused with human serum albumin to form, for example, a fusion protein construct (Yeh et al. (1992) Proc. Nat'l Acad. Sci. 89:1904). Alternatively, a bispecific antibody may be constructed using the first and second antigen-binding domains of the present invention, which bind to human serum albumin (HSA) (see International Patent Application Publication WO2009 / 127691 and the patent references cited therein). Alternatively, to increase the half-life, a specialized polypeptide sequence, such as the "XTEN" polypeptide sequence, can be added to the antibody fragment. (Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; International Patent Application Publication WO2010 / 091122).

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

[0186] The affinity and binding properties of Fc variants to their ligands (Fc receptors) can be determined by various in vitro assay methods known in the art (e.g., biochemistry or immunology-based assays), including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (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 utilize and / or not utilize various detection methods, including chromogenic, fluorescent, luminescent, or isotopic labeling, with or without limitation using labeling on one or more components being investigated. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), focusing on antibody-immunogen interactions.

[0187] In further embodiments, antibody glycosylation is modified to increase or decrease effector function. For example, by mutating the conserved asparagine residue at position 297 (e.g., N297A), and thereby eliminating complement and FcγRI binding, non-glycosylated antibodies lacking all effector function can be produced. (See also Bolt et al. (1993) Eur. J. Immunol. 23:403; Tao & Morrison (1989) J. Immunol. 143:2595 (Eliminating glycosylation at position 297 using N297Q in IgG1)).

[0188] Non-glycosylated antibodies generally lack effector function, but mutations can be introduced to restore this function. Non-glycosylated antibodies, such as those resulting from the N297A / C / D / or H mutation, or those produced in non-glycosylating systems (e.g., E. coli), can be further mutated to restore FcγR binding, e.g., 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] Glycan manipulation can also be used to modify the anti-inflammatory properties of IgG constructs by altering the α2,6 sialyl content of the carbohydrate chain linked at Asn297 in the Fc region, where an increase in the proportion of the α2,6 sialized form results in enhanced anti-inflammatory effects (see Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513). Conversely, a decrease in the proportion of antibodies with α2,6 sialized hydrocarbons may be useful when anti-inflammatory properties are undesirable. For example, a method for modifying the α2,6 sialized content of an antibody by selective purification of the α2,6 sialized form or by enzymatic modification is provided in U.S. Patent Application Publication 2008 / 0206246. In other embodiments, the amino acid sequence of the Fc region may be modified to mimic the effects of α2,6 sialization, for example, by including the 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 result in increased immunogenicity of the antibody or altered antibody pharmacokinetics due to modified 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 in motifs containing NXS / T sequences. In some embodiments, anti-hNKG2A antibodies do 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 glycosylation region.

[0191] In certain embodiments, the antibodies described herein do not contain asparagine isomerized sites. Deamidation of asparagine can occur in NG or DG sequences, resulting in the introduction of twists 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 having a pI within a desired range can be obtained by either selecting antibodies having a pI within that pH range from a candidate group, or by mutating the charged surface residues of a particular antibody.

[0193] In some embodiments, the antibodies described herein are subjected to the first unfolding temperature (T M1 Antibodies are selected and / or genetically engineered to have a melting point greater than 60°C, greater than 65°C, or greater than 70°C. The melting point of the 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 genetically engineered to have advantageous degradation characteristics, 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 genetically engineered to have desirable aggregation characteristics, e.g., antibodies that exhibit minimal aggregation in vitro and / or in vivo, which may induce undesirable immune responses and / or altered or undesirable pharmacokinetic properties. In some embodiments, the antibodies described herein exhibit aggregation of ≤25%, ≤20%, ≤15%, ≤10%, or ≤5% compared to the aggregation of the parent antibody. Aggregation can be measured by several techniques, including size exclusion columns (SEC), high-performance liquid chromatography (HPLC), and light scattering.

[0195] IV. Nucleic acid molecules and recombination methods Another aspect described herein relates to a nucleic acid molecule encoding the anti-hNKG2A antibody described herein. The nucleic acid may exist in whole cells, e.g., in host cells, in cell lysates, or in partially purified or substantially pure forms. The nucleic acid is “isolated” or “substantially purified” when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to isolated DNA in nature) or proteins, by standard techniques including alkali / SDS treatment, CsCl band formation, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well 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 may 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 further described below), the cDNA encoding the light and / or heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding the antibodies can be recovered from the library.

[0197] V H and V LOnce DNA fragments encoding a segment are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert a variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. These manipulations involve V L or V H The DNA fragment encoding the antibody is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. In this context, the term "operably linked" means that the two DNA fragments are joined together such that the amino acid sequences encoded by both fragments remain in frame.

[0198] V H The isolated DNA encoding the region is V H The DNA encoding the heavy chain can be converted into a full-length heavy chain gene by operably ligating it with 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 containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG (IgG1, IgG2, IgG3, or IgG4), IgA, IgE, IgM, or IgD constant region, e.g., the IgG1 region. For Fab fragment heavy chain genes, see V H The DNA encoding this can be operably ligated with another DNA molecule that encodes only the heavy chain CH1 constant region.

[0199] V L The isolated DNA encoding the region is V LThe DNA encoding the light chain constant region (CL) can be converted into a full-length light chain gene (and Fab light chain gene) by operably ligating it with another DNA molecule encoding the light chain constant region (CL). The sequences of human light chain constant region genes are publicly known in the art (see, for example, 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 containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a κ or λ constant region.

[0200] To construct the scFv gene, DNA fragments encoding VH and VL can be operably linked to another fragment encoding a mobile linker, for example, the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 160), resulting in the expression of a continuous single-strand protein having VL and VH regions linked by the mobile linker (see, for example, 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, for example, those that conjugate to the same epitope as the selected anti-hNKG2A antibody disclosed herein, can be produced using various 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, such as viral or oncogenic transformation of B lymphocytes and phage display techniques using libraries of human antibody genes, can also be used.

[0202] An exemplary animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a well-established procedure. Immunotherapy 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] Chimeric or humanized antibodies described herein can be prepared based on the sequences of mouse monoclonal antibodies prepared as described above. Using standard molecular biology techniques, DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and genetically engineered to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, the mouse variable region can be ligated to the human constant region using methods known in the art (see, for example, U.S. Patent No. 4,816,567 by Cabilly et al.). To produce a humanized antibody, the mouse CDR region can be inserted into the human framework using methods known in the art (see, for example, U.S. Patent No. 5,225,539 by Winter and U.S. Patents No. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 by Queen et al.).

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

[0205] HuMAb mice (registered trademark) (Medarex, Inc.) contain human immunoglobulin gene miniloci encoding unreorganized human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Therefore, 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 produce high-affinity human IgGκ monoclonal antibodies (as outlined in Lonberg, N. et al. (1994), op. cit.; 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).Preparation and use of HuMab mice and the genomic modifications preserved 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: Further details are provided in pp. 579-591 and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are incorporated herein by reference in their entirety. (Also, all of Lonberg and Kay's 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; U.S. Patent No. 5,545,807 by Surani et al.; all of Lonberg and Kay's PCT publication numbers WO92 / 03918, WO93 / 12227, WO94 / 25585, WO97 / 13852, WO98 / 24884 and WO 99 / 45962 and Korman et al.) (See al.'s PCT publication number WO01 / 14424.)

[0206] In certain embodiments, the antibodies described herein are produced using mice that carry human immunoglobulin sequences on transgenes and transchromosomes, for example, mice that carry human heavy chain transgenes and human light chain transchromosomes. Such mice, referred to herein as “KM mice,” are described in detail in PCT Publication WO02 / 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 produce the anti-hNKG2A antibodies described herein. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used, such mice are described, for example, in U.S. Patents 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 by Kucherlapati et al.

[0208] Furthermore, alternative transchromosomic animal lines expressing human immunoglobulin genes are available in the art and can be used to produce the anti-nKG2A antibodies described herein. For example, mice that possess both human heavy chain transchromosomes and human light chain transchromosomes, known as "TC mice," can be used, and such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. In addition, cattle that possess human heavy chain and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to produce the anti-hNKG2A antibodies described herein.

[0209] Further mouse systems described in the art for producing human antibodies, such as human anti-hNKG2A antibodies, include (i) VELOCIMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy chain and light chain variable regions are substituted with human heavy chain and light chain variable regions operably linked to the endogenous mouse constant region by homologous recombination, resulting in the production of a chimeric antibody (human V / mouse C) in the mouse, which is then converted to a fully human antibody using standard recombinant DNA technology; and (ii) MeMo® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains an unreorganized human heavy chain variable region, but a single re-denatured human common light chain variable region. The use of such mice and antibodies for their production is 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 antibodies described herein can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well-established in the art. (See, for example, Ladner et al., U.S. Patents 5,223,409; 5,403,484; and 5,571,698; Dower et al., U.S. Patents 5,427,908 and 5,580,717; McCafferty et al., U.S. Patents 5,969,108 and 6,172,197; and Griffiths et al., U.S. Patents 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081).

[0211] The human monoclonal antibodies described herein can also be prepared using mice with severe combined immunodeficiency (SCID) in which human immune cells are reconstituted to produce a human antibody response during immunization. Such mice are described, for example, in U.S. Patents 5,476,996 and 5,698,767 by Wilson et al.

[0212] Immunotherapy To produce 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 purified or concentrated preparations of NKG2A antigen and / or cells expressing NKG2A, as described for other antigens in, 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, the mice are 6-16 weeks old at the time of the first injection. For example, a purified or concentrated preparation of recombinant human NKG2A antigen (e.g., 5 μg-50 μg) can be used to immunize mice intraperitoneally. If antibody production is not achieved by immunization using purified or concentrated preparations of the NKG2A antigen, the immune response can also be stimulated by immunizing mice with cells expressing NKG2A, such as a cell line.

[0213] HuMAb transgenic mice can be initially immunized intraperitoneally or subcutaneously (SC) with the antigen in the Ribi adjuvant, followed by bi-weekly IP / SC immunization (up to a total of 10 times) with the antigen in the Ribi adjuvant. The immune response can be monitored throughout the immunization protocol using plasma samples obtained by post-orbital hemorrhage. 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 fusion. Mice can be boosted intravenously with the antigen, sacrificed after 3 days, and spleen and lymph nodes can be collected. Two to three fusions may 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 single mice possessing two different human heavy chain transgenes (HCo7 / HCo12).

[0214] Creation of hybridomas that produce monoclonal antibodies against the NKG2A protein. To produce hybridomas that produce the monoclonal antibodies described herein, splenocytes 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 antigen-specific antibody production. 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 to approximately 2 × 10⁶ cells. 5The cells are plated onto flat-bottom microtiter plates and then incubated for 2 weeks in a selective medium containing 10% fetal cloned serum, 18% "653" conditioning 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, the cells can be cultured in medium in which HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. If extensive hybridoma growth occurs, the medium can be observed normally after 10–14 days. Hybridomas secreting antibodies can be replated and screened again, and if still positive for human IgG, the monoclonal antibody can be subcloned at least twice by limiting dilution. Next, stable subclones can be cultured in vitro, and small amounts of antibodies can be produced in tissue culture medium for characterization.

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

[0216] VI. Antibody Manufacturing Creation of a transfectoma that produces a monoclonal antibody against NKG2A The antibodies of the present invention, including both the sequence-provided specific antibody and other related anti-NKG2A antibodies, can be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

[0217] For example, to express an antibody or an antibody fragment, DNA encoding a partial or full-length light chain and heavy chain 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 so that the gene is operably ligated to transcriptional and translational regulatory sequences. In this context, the term “operably ligated” means that the antibody gene is ligated into the vector so that the transcriptional and translational regulatory sequences in the vector perform its intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression regulatory sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and antibody heavy chain gene may be inserted into separate vectors, or both genes may be inserted into the same expression vector. The antibody gene is inserted into the expression vector(s) by standard methods (e.g., complementary restriction sites on the antibody gene fragment and vector ligation, or blunt-end ligation if no restriction sites exist). Using the antibody light chain and heavy chain variable regions described herein, V H The segment is C in the vector H Segments (one or more) are operably connected, V L The segment is C in the vector LFull-length antibody genes of any antibody isotype can be constructed by inserting segments into an expression vector that already encodes the heavy-chain constant and light-chain constant regions of the desired isotype, so that they can be operably linked. Furthermore, recombinant expression vectors may encode signal peptides that promote the secretion of antibody chains from host cells. The antibody chain gene can be cloned into the vector so that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0218] Recombinant expression vectors may contain regulatory sequences that control the expression of the antibody chain gene in host cells, in addition to the antibody chain gene itself. The term “regulatory sequence” includes promoters, enhancers, and other expression regulatory elements that control the transcription or translation of the antibody chain gene (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel's *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 an expression vector, including the selection of regulatory sequences, can vary depending on factors such as the selection of host cells to be transformed, the desired level of protein expression, 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 cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP), and promoters and / or enhancers derived from polyomaviruses). Alternatively, non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters may be used. Furthermore, regulatory elements consisting of sequences derived from different sources, such as the SRα promoter system, containing sequences derived from the SV40 early promoter and long terminal repeat sequences of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0219] Recombinant expression vectors may contain additional sequences, such as antibody chain genes and regulatory sequences, as well as sequences that regulate vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, selection marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Exemplary selection marker genes include the dihydrophorate reductase (DHFR) gene (for use in dhfr-host cells, along with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0220] For the expression of light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term “transfection” encompass a variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. While it is theoretically possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, antibody expression in eukaryotic cells, most preferably mammalian host cells, is most preferred because such eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to fold properly, assemble, and secrete immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be ineffective for the efficient 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 glycosylated yeast strains. (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 dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a dihydrophorate reductase (DHFR) selection marker, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another exemplary expression system is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841. A recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, and the antibody is produced by culturing the host cell for a period of time sufficient to allow antibody expression in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0222] The N and C-terminuses of the antibody polypeptide chains of the present invention may differ from sequences predicted by commonly observed post-translational modifications. For example, the C-terminal lysine residue is often lost from the antibody heavy chain (Dick et al. (2008) Biotechnol. Bioeng. 100:1132). The N-terminal glutamine residue and, to a lesser extent, glutamate residues are frequently converted to pyroglutamate 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, the C-terminal lysine is not included in many of the sequences in the sequence listing for the heavy chain or the heavy chain constant domain. However, in alternative embodiments, each heavy chain of the anti-hNKG2A antibody of the present invention, and / or the gene construct encoding such an antibody or its heavy or light chain, includes 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, a microtiter plate is coated with 1-2 μg / mL of purified NKG2A in PBS, and then blocked with 5% bovine serum albumin in PBS. A diluted antibody (e.g., a diluted plasma obtained from NKG2A-immunized mice) is added to each well and incubated at 37°C for 1-2 hours. The plate is washed with PBS / Tween and then incubated at 37°C for 1 hour with a secondary reagent conjugated with horseradish peroxidase (HRP) (e.g., human antibody, or otherwise an antibody with a human heavy chain constant region, or a goat anti-human IgG Fc-specific polyclonal reagent). After washing, the plate is colored with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by spectrophotometer at OD415-495. Next, serum obtained from immunotreated mice is further screened by flow cytometry for binding to human NKG2A-expressing cell lines, rather than to control cell lines that do not express NKG2A. Briefly, the binding of anti-NKG2A antibodies is evaluated by incubating NKG2A-expressing CHO cells with a 1:20 dilution of anti-NKG2A antibody. After washing the cells, binding is detected using PE-labeled anti-human IgG Ab. Flow cytometry analysis is performed using FACScan flow cytometry (Becton Dickinson, San Jose, CA). It is preferable that the mouse that produces the highest titer is used for fusion. If you wish to detect mouse anti-NKG2A antibodies, a similar experiment may be performed using an anti-mouse detection antibody.

[0225] Using ELISA or similar methods as described above, hybridomas that produce antibodies, and consequently antibodies that show positive reactivity with the NKG2A immunogen, can be screened. Preferably, hybridomas that produce antibodies that bind to NKG2A with high affinity can be subcloned and further characterized. To create a cell bank, one clone can be selected from each hybridoma (by ELISA) that retains the reactivity of the parent cell for antibody purification.

[0226] To purify the anti-NKG2A antibody, selected hybridomas can be grown in a 2-liter spinner flask for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS, and OD analysis can be performed using an extinction coefficient of 1.43. 280 The concentration can be determined by this method. Monoclonal antibodies can be aliquoted and stored at -80°C.

[0227] To investigate whether selected anti-NKG2A monoclonal antibodies bind to specific epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected using streptavidin-labeled probes. As described above, competitive studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be conducted using NKG2A-coated ELISA plates.

[0228] To determine the isotype of purified antibodies, isotype ELISA can be performed using reagents specific to the antibody 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 anti-human immunoglobulin. After blocking with 1% BSA, the plate is reacted with a test monoclonal antibody of 1 μg / mL or less or a purified isotype control at ambient temperature for 1-2 hours. Then, the wells are reacted with either a probe conjugated with human IgG1 or human IgM-specific alkaline phosphatase. The plate is colored as described above and analyzed.

[0229] Flow cytometry can be used to investigate the binding of monoclonal antibodies to viable cells expressing NKG2A. Briefly, a cell line expressing membrane-bound NKG2A (grown under standard growth conditions) is mixed with a certain concentration of monoclonal antibody in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with phycoerythrin (PE)-labeled anti-IgG antibody under the same conditions as primary antibody staining. The samples are analyzed using a FACScan instrument with light and side scattering properties, gated and ungated in single cells, to examine the binding of the labeled antibody. In addition to (or instead of) the flow cytometry assay, an alternative assay using fluorescence microscopy may be used. Cells can be accurately stained as described above and examined by fluorescence microscopy. This method allows for the visualization of individual cells, although they may have reduced sensitivity depending on the antigen density.

[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 antigen is 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 colorimetrically developed using 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 domain 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 domain of human NKG2A and the extracellular domain 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 triplicate molecules. A multispecific antigen-binding molecule, e.g., a multispecific antibody, contains two or more antigen-binding sites, each specific to a different epitope. The different epitopes may be parts of the same antigen or different antigens. In one embodiment, one antigen-binding site is specific to human NKG2A, and the other is specific to a different antigen. In one embodiment, the anti-h NKG2A antibody or its antigen-binding fragment described herein is linked with another antigen-binding molecule having different binding specificity, e.g., another peptide or protein (e.g., another antibody or antibody fragment or receptor ligand), to produce 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 with one or more other antigen-binding molecules to produce a multispecific molecule that binds to two or more different binding sites and / or target molecules. Accordingly, a bispecific molecule comprising a first binding specificity to at least one NKG2A and a second binding specificity to a second target epitope is provided herein. In one embodiment described herein where the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity.

[0234] In one embodiment, the bispecific molecules described herein include, as binding specificity, at least one antibody or an antibody fragment thereof, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv. As described in Ladner et al., U.S. Patent No. 4,946,778, whose disclosure is clearly incorporated by reference, the antibody may also be any smallest fragment thereof, such as a light-chain or heavy-chain dimer or Fv or a single-chain construct.

[0235] Human monoclonal antibodies are preferred, but other antibodies that can be used in the bispecific antibodies described herein include mouse monoclonal antibodies, chimeric monoclonal antibodies, and humanized monoclonal antibodies.

[0236] The bispecific antibodies described herein can be prepared by conjugating their binding specificities as constituent elements using methods known in the art. For example, each binding specificity of a bispecific molecule can be prepared separately and then conjugated to one another. When the binding specificity is a protein or peptide, various coupling or crosslinking agents can be used for covalent conjugation. Examples of crosslinking 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, for example, 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 conjugates include SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

[0237] When the binding specificity is an antibody, they can be conjugated by sulfhydryl linkages in 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 1, prior to conjugation.

[0238] Alternatively, both binding specificities may be encoded in the same vector, expressed in the same host cell, and assembled. This method is particularly useful when the bispecific molecule has a combination of binding specificities such as (mAb × mAb), (mAb × Fab), (Fab × F(ab')2), or (ligand × Fab) fusion proteins. The bispecific molecules described herein may 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 may contain 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] The binding of bispecific molecules to their specific targets can be confirmed using methods recognized in the art, such as ELISA, radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex by using a labeling reagent (e.g., antibody) specific to the complex in question.

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

[0241] Such compositions may also comprise one or a combination of (for example, two or more different) antibodies, immunoconjugates, or bispecific molecules described herein. For example, a pharmaceutical composition described herein may comprise a combination of antibodies (or immunoconjugates or bispecific antibodies) that conjugate to different epitopes on a target antigen or have complementary activity.

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

[0243] In some embodiments, the pharmaceutical compositions disclosed herein may include other compounds, drugs, and / or agents used for the treatment of cancer. Examples of such compounds, drugs, and / or agents include chemotherapeutic agents, small molecule drugs, or antibodies that stimulate an immune response against a given cancer. In some embodiments, the pharmaceutical composition includes a first antibody and a second antibody that are specific to anti-h NKG2A.

[0244] In some embodiments, the first antibody and the second antibody are present in the composition in fixed doses (i.e., fixed ratios). In other embodiments, the fixed dose is mg of anti-hNKG2A antibody versus mg of the second antibody in a ratio of 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. 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-h NKG2A antibody versus a 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 the first antibody versus mg of the second antibody. For example, in one embodiment, the anti-h NKG2A antibody and the second antibody are administered as described in the examples.

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

[0246] In this specification, "pharmaceutically acceptable carriers" include any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial 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, the composition containing an anti-NKG2A antibody is delivered subcutaneously using Halozyme's ENHANZE® drug delivery technology, which includes recombinant human hyaluronidase enzyme (rHuPH20) that transiently degrades hyaluronan. In some embodiments, the ENHANZE® drug delivery technology allows for faster subcutaneous delivery of the composition compared to intravenous administration. In other embodiments, depending on the route of administration, the compound may be coated on the material with the active compound, i.e., an antibody, an immunocomplex, or a bispecific molecule, to protect the compound from 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” means a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Examples of 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, and phosphorous acid, as well as those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Examples of base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[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, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0249] Suitable aqueous and non-aqueous carriers that may 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. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0250] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial presence can be ensured both by sterilization procedures, as described above, and by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin can induce prolonged absorption of the injectable drug form.

[0251] Examples of pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, e.g., soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more further glycosaminoglycans, e.g., chondroitinase.

[0252] The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is considered. Supplemental active compounds may also be incorporated into the compositions.

[0253] Therapeutic compositions must generally be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Often, it is preferable to include isotonic agents in the composition, such as sugars, mannitol, sorbitol, or polyalcohols such as sodium chloride. Longer absorption of the injectable composition can be induced by including absorption-delaying agents in the composition, such as monostearate and gelatin.

[0254] Sterile injectable solutions can be prepared by incorporating the required amount of active compound in a suitable solvent, along with one or a combination of the components listed above, as needed, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and other necessary components from the above. In the case of sterile powders for preparing sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, from which powders of the active ingredient and any further desired components are obtained from a pre-sterile filtered solution.

[0255] The amount of active ingredient that can be combined with a carrier material to produce a single dose varies depending on the subject being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dose is the amount of the composition that produces the therapeutic effect. In combination with pharmaceutically acceptable carriers, this amount can range from about 0.01 percent to about 99 percent of the active ingredient out of 100 percent, 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.

[0256] In some embodiments, the composition comprises an anti-NKG2A antibody, e.g., NKG2A.9. The composition is a sterile, nonpyrogenic, single-use, preservative-free, isotonic aqueous solution for intravenous administration. The composition may be administered undiluted or further diluted to the protein concentration required by 0.9% sodium chloride injection before 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 administration plan is adjusted to provide the optimal desired response (e.g., a 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 an emergency in the treatment situation. Formulating parenteral compositions into dosage units is particularly advantageous for ease of administration and uniformity of dosage form. In this specification, a dosage unit refers to a physically distinct unit suitable as a unit dose of the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the dosage units described herein are determined and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for the treatment of susceptibility in an individual.

[0258] For antibody administration, the dosage may range from approximately 0.0001 to 100 mg / kg of host body weight, more typically from 0.01 to 5 mg / kg of host body weight. For example, the dosage may 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, antibody administration may be a fixed dose ranging from 2 mg to 800 mg, for example, 25 mg, 80 mg, 200 mg, or 400 mg. An exemplary treatment plan may require administration once a week, once every two weeks, once every three weeks, once every four weeks, once every one month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the treatment plan includes an initial dose, followed by maintenance doses of different doses at intermittent 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 indicated range. In some embodiments, therapeutic antibodies are administered in multiple contexts. The interval between single doses may be, for example, weekly, every three weeks, every four weeks, monthly, every three months, or annually. The interval may also be irregular, as indicated by measuring the blood levels of antibodies against the target antigen in the patient. In some embodiments, the dosage is adjusted to achieve plasma antibody concentrations of approximately 1–1000 μg / mL, and in some methods, approximately 25–300 μg / mL.

[0260] In some embodiments, the antibody may be administered as a sustained-release formulation. Administration with a sustained-release formulation may reduce the frequency of administration required. The dosage and frequency 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 doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for life. In some embodiments, relatively high doses are administered at relatively short intervals for therapeutic treatment. In some embodiments, relatively high doses are administered until the progression of the disease is reduced or stopped, for example, until the patient shows partial or complete remission of the symptoms of the disease. In some embodiments, prophylactic treatment is administered to the patient after therapeutic treatment.

[0261] The actual dose levels of the active ingredients in the pharmaceutical compositions described herein may be modified to obtain an amount of the active ingredient that is not toxic to the patient and is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dose level will vary depending 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 elimination 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, overall health, and prior medical history of the patient being treated, and similar factors well known in the art of medicine.

[0262] The "therapeutically effective doses" of anti-NKG2A antibodies described herein preferably result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease distress. In relation to cancer, therapeutically effective doses preferably prevent further exacerbation of cancer-related physical symptoms. The symptoms of cancer are well known in the art and include, for example, unusual features of moles, changes in the appearance of moles including asymmetry, borders, color and / or diameter, newly colored skin areas, abnormal moles, darkened areas under the nails, 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, hematuria, bloody stools, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal distension, flatulence, fluid in the peritoneal cavity, vaginal bleeding, constipation, abdominal bloating, colonic perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, kidney metastases and pancreatic metastases, and dysphagia. The therapeutic effect may be observed immediately after the first administration of the anti-hNKG2A monoclonal antibody of the present invention, or it may only be observed after a predetermined period and / or a series of doses. Such delayed effects may only be observed several months after treatment, for example, up to 6, 9, or 12 months.

[0263] A therapeutically effective dose, such as one that may be desired when early or preceding signs of the disease are present, may prevent or delay the development of cancer. Therefore, any clinical or biochemical assay monitoring any of the aforementioned factors 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 a dose based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or chosen route of administration.

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

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

[0266] Active compounds can be prepared using carriers that protect them from rapid release, including indwelling agents, transdermal patches, and sustained-release formulations such as microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Numerous methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0267] The therapeutic compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic compositions described herein can be administered using needleless subcutaneous injection devices such as those disclosed in U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implantable agents and modules for use with the anti-NKG2A antibodies described herein include U.S. Patent 4,487,603, which discloses an implantable microinfusion pump for dispensing pharmaceuticals at a controlled rate. Examples include Patent No. 4,486,194 disclosing a therapeutic device for administering a drug through the skin; Patent No. 4,447,233 disclosing a drug infusion pump for delivering a drug at a precise infusion rate; Patent No. 4,447,224 disclosing a variable flow implantable infusion device for continuous drug delivery; Patent No. 4,439,196 disclosing an osmotic drug delivery system having a multi-chamber compartment; and Patent No. 4,475,196 disclosing an osmotic drug delivery system. These patents are incorporated herein by reference. Numerous 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 appropriate distribution in vivo. For example, the blood-brain barrier (BBB) ​​rejects many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein can cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, U.S. Patents 4,522,811, 5,374,548, and 5,399,331. The liposomes may contain one or more moieties that are selectively delivered into specific cells or organs and thus enhance targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 by Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG 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); and p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090), as well as K. Keinanen; ML Laukkanen (1994) FEBS Lett. See also 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0269] Kits, including antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use, are also included within the scope described herein. A kit may further include at least one additional reagent, or one or more additional human antibodies described herein. A kit may include markings indicating the intended use of the kit's contents. Terminology is included in any documents or recording materials supplied on or with the kit, or otherwise accompanying the kit.

[0270] X. How to use The antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo applications, including, for example, enhancement of the immune response by blocking NKG2A / HLA-E interactions. In one embodiment, the anti-NKG2A antibody described herein is a monoclonal human or humanized antibody. In one embodiment, the anti-hNKG2A antibody described herein (e.g., 13F3.A4, NKG2A.9, and NKG2A.11) can be administered to cells in culture medium in vitro or ex vivo, or to human subjects to enhance immunity in various diseases. In a specific embodiment, the anti-hNKG2A antibody is an antagonist antibody. Methods for modifying the immune response in a subject are provided herein, comprising administering to a subject an anti-NKG2A antibody or its antigen-binding fragment described herein such that the immune response in the subject is enhanced, stimulated, or upregulated. In one embodiment, the T cell and / or NK cell response is enhanced by administration of the anti-hNKG2A antibody according to the method described herein. In one embodiment, an antigen-specific T cell response to a tumor is stimulated, enhanced, or upregulated by administering an anti-hNKG2A antibody according to the method described herein. The T cells include Teff cells, e.g., CD4+ Teff cells, CD8+ Teff cells, and helper T(T) cells. h ) cells, and cytotoxic T(T) cThe subject may be a cell. The tumor may be a solid tumor or a liquid tumor, for example, 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 may also be a subject that has a virus and whose immune response to the virus is enhanced. In one embodiment, the NK cell response is stimulated, enhanced, or upregulated by administering an anti-hNKG2A antibody according to the method described herein.

[0271] In one embodiment, the method results in an enhancement of the immune response in a human subject, and such enhancement has a desirable effect. In one embodiment, the human subject is a human patient having a disorder that can be treated by enhancing the immune response, for example, 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 the antigen of the subject, or the antigen may already be present in the subject to be treated, for example, a subject having a tumor or a virus. When the anti-NKG2A antibody is administered together with another drug, the two can be administered separately or simultaneously.

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

[0273] In some embodiments, anti-NKG2A antibodies are administered to subjects, e.g., human patients, as adjuvant therapy, adjuvant therapy, or neoadjuvant therapy. In some embodiments, treatment of subjects with cancer using anti-NKG2A antibodies may result in a long-lasting response compared to current standard of care, long-term survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer, or recurrence-free survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or longer. In certain embodiments, treatment of subjects with cancer using anti-hNKG2A antibodies may prevent or delay cancer recurrence for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or longer. Anti-NKG2A therapy can be used as a first-line, second-line, or subsequent-line treatment.

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

[0275] cancer A method for treating a subject having cancer is provided herein, comprising administering to the subject an anti-hNKG2A antibody as described herein so that the subject is treated, for example, so that the growth of a cancerous tumor is inhibited or reduced and / or the tumor regresses. The anti-NKG2A antibody can be used alone to inhibit the growth of a cancerous tumor. Alternatively, the anti-NKG2A antibody can be used in combination with another agent, for example, other immunogenic agents, standard cancer treatments, or other antibodies, as described below. Combinations with PD-1 inhibitors, for example, anti-PD-1 or anti-PD-L1 antibodies are also provided. Combinations with CTLA-4 inhibitors, for example, 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 embodiment, a method for treating cancer in a subject is provided herein, comprising administering to the subject a therapeutically effective amount of the anti-NKG2A antibody described herein. In one embodiment, the anti-NKG2A antibody may be a chimeric antibody, a human antibody, or a humanized anti-NKG2A antibody. In one embodiment, the method for treating cancer described herein is (a) LSIDNEEMKF (sequence number 156); (b) PSSWIGVFRNSSHHPW (sequence code 157); (c)LAFKHEIKDSDN(sequence number 158); and (d)QVNRLKSAQQCGSSIIYHC(Sequence ID 159) This includes administering an anti-NKG2A antibody that contacts human NKG2A at one or more amino acid residues.

[0277] In another embodiment, the method for treating cancer described herein is: (a) LSIDNEEMKF (sequence number 156); (b) PSSWIGVFRNSSHHPW (sequence code 157); (c)LAFKHEIKDSDN(Sequence ID 158); (d)L; and (e)QVNRLKSAQQCGSSIIYHC(Sequence ID 159) This includes administering an anti-NKG2A antibody that contacts human NKG2A at one or more amino acid residues.

[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 a variant thereof to treat cancer. In another embodiment, the method comprises administering a composition comprising a 13F3.A4 antibody or a 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, kidney 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 adenocarcinoma of the prostate), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (pleomorphic glioblastoma), cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer and head and neck cancer (or carcinoma), gastric cancer (gastric cancer) Cancer, germ cell tumors, pediatric sarcomas, sinus natural killer tumors, melanoma (e.g., metastatic melanoma such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, Cancers of the ureter, carcinomas of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumors of the spinal cord axis, brain cancers such as brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal 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 originating from one of two major hematological cell lineages, namely myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (producing B, T, NK, and plasma cells), such as all types of leukemia, lymphoma, and myeloma, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), and undifferentiated AM L(M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell 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), acute, chronic, lymphocytic and / or myeloid leukemia such as isolated granulocytic sarcoma and chloroplast;Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, undifferentiated (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, vascular central 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, histiocytic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, lymph Lymphomas such as lymphoblastic lymphoma (LBL), lymphoid hematopoietic malignancies, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large B-cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma with Waldenström hypergammaglobulinemia (LPL). Myelomas such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), and hairy cell lymphoma; myeloid hematopoietic malignancies, mesenchymal tumors including fibrosarcoma and rhabdomyosarcoma; central and peripheral nerve tumors including seminoma, teratoma, astrocytoma, and schwannoma; fibrosarcoma, rhabdomyosarcoma Tumors of mesenchymal origin, including domyoscarcoma and osteosarcoma; as well as other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratocarcinoma; hematopoietic malignancies of the lymphoid lineage, including T-cell disorders such as pre-T lymphocytic leukemia (T-PLL), including small cell and cerebral-like 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); vascular centripetal (nasal cavity) T-cell lymphoma; cancers of the head and neck, renal cancer, rectal cancer, thyroid cancer;Examples include, but are not limited to, acute myeloid lymphoma and any combination of the aforementioned cancers. In one embodiment, the methods described herein may also be used for the treatment of metastatic cancer, refractory cancer (e.g., cancer refractory to conventional immunotherapy, e.g., cancer using blocking CTLA-4 and / or PD-1 antibodies), and recurrent cancer.

[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 immunostimulator. In one embodiment, anti-hNKG2A is administered to the patient in combination with another agent. In one embodiment, the anti-hNKG2A antibody is administered in combination with an immunogenic agent. In one embodiment, the anti-hNKG2A antibody is administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine includes 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-chain peptide, a multipeptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, an anti-hNKG2A antibody is administered with an adjuvant. Not limited examples of tumor vaccines used include melanoma antigen peptides such as gp100, MAGE antigen, Trp-2, MART1, and / or tyrosinase peptides, or tumor cells transfected to express the cytokine GM-CSF.Numerous experimental strategies have been devised for the vaccination of tumors (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, the vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF 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 human cancer-related viruses such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). In some embodiments, purified heat shock proteins (HSPs) isolated from tumor tissue itself are used as another form of tumor-specific antigen in conjunction with NKG2A inhibition. These heat shock proteins contain protein fragments derived from tumor cells, and these HSPs are highly efficient in delivery 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 as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). Dendritic cells can also be transduced by genetic means to express these tumor antigens as well. DCs have also been directly fused with tumor cells for immunotherapy purposes (Kugler et al. (2000) Nature Medicine 6: 332-336). As a vaccine method, dendritic cell immunotherapy can be effectively combined with anti-NKG2A antibodies to activate (release) a more potent antitumor response.

[0283] In some embodiments, anti-hNKG2A is administered with standard curative treatment, such as surgery, radiation therapy, and / or chemotherapy. In some embodiments, anti-hNKG2A antibodies are administered with chemotherapeutic agents. In some embodiments, anti-hNKG2A antibodies are administered with one or more of the following: carboplatin, cisplatin, paclitaxel, nab-paclitaxel, gemcitabine, or FOLFOX. In some embodiments, anti-hNKG2A antibodies are administered with carboplatin or nab-paclitaxel. In some embodiments, anti-hNKG2A antibodies are administered with carboplatin and paclitaxel. In some embodiments, anti-hNKG2A antibodies are administered with cisplatin and pemetrexed. In some embodiments, anti-hNKG2A antibodies are administered with cisplatin and gemcitabine. In some embodiments, anti-hNKG2A antibodies are administered 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 sulfate bleomycin, 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, where the administration of the anti-hNKG2A antibody reduces, mitigates, or suppresses resistance to at least one drug. In some embodiments, the anti-hNKG2A antibody is administered together with an agonist antibody, such as an anti-ICOS antibody.

[0284] The combination therapies described above can be administered in various combinations, encompassing combination administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration. In the case of separate administration, the administration of the antibody of the present invention may occur before, simultaneously with, and / or after the administration of further therapeutic agents and / or adjuvants. The antibody of the present invention can also be used in combination with radiotherapy.

[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 anti-hNKG2A antibody and IL-2 is for the treatment of various cancers, including renal cell carcinoma and melanoma. In some embodiments, the anti-hNKG2A antibody discussed herein is combined with an IL-2 pathway agonist for the treatment of various cancers. Examples of combinations include various IL-2 pathway agonists, e.g., those described in WO2012 / 065086 (Nektar Therapeutics) and WO2015 / 125159 (Nektar Therapeutics), the contents of which are incorporated in their entirety by reference. WO2006 / 138572 (Nektar Therapeutics) provides conjugates having degradable linkages and polymerization reagents useful for preparing such conjugates, as well as methods for preparing polymerization reagents and conjugates, the contents of which are incorporated in their entirety by reference.

[0286] In some embodiments, a combination of an anti-hNKG2A antibody described herein, e.g., 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, 20kDa ("mPEG2-C2-fmoc-20K-NHS")):

[0287] [ka] Polyethylene glycol (PEG) reagents based on FMOC (fluorenylmethyloxycarbonyl chloride), on average, have approximately 6 such reagents. It is produced by conjugating a protein having the following 132-amino acid sequence. [ka] (Sequence ID 161)

[0288] WO2012 / 065086 provides a conjugate of an IL-2 moiety with one or more non-peptide water-soluble polymers comprising polyethylene glycol or a derivative thereof. Specifically, Example 2 of WO2012 / 065086 (paragraphs 202-204) describes the pegylation of rIL-2 using mPEG2-C2-fmoc-20K-NHS to obtain the mPEG2-C2-fmoc-20K-NHS structure shown above. Example 1 of WO2015 / 125159 (paragraphs 63-66) describes a scaled-up approach for the pegylation of IL-2 using mPEG2-C2-fmoc-20K-NHS to obtain RSLAIL-2 (NKTR-214). NKTR-214 is a cytokine designed to target CD122 (interleukin-2 receptor beta subunit, also known as IL-2Rβ), a protein found in certain immune cells (e.g., CD8+ T cells and NK cells) to promote the proliferation of these cells in order to enhance antitumor effects.

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

[0290] Other combination therapies that may produce a synergistic effect with the anti-hNKG2A antibodies described herein through cell death include radiation therapy, surgery, and hormone deficiency.

[0291] In some embodiments, the anti-hNKG2A antibody described herein is administered in conjunction with a bispecific antibody. The bispecific antibody can be used to target two different antigens. In some embodiments, the anti-hNKG2A antibody is used in combination with a bispecific antibody that targets effector cells expressing Fcα or Fcγ receptors to treat tumor cells (see, for example, U.S. Patents 5,922,845 and 5,837,243). For example, an anti-Fc receptor / antitumor antigen (e.g., Her-2 / neu) bispecific antibody is used to target macrophages to tumor sites. In some embodiments, the T cell arm of these responses is amplified by the functional activity of the anti-hNKG2A antibody. In some embodiments, the antigen is delivered directly to DCs by the use of a bispecific antibody that binds to tumor antigens and dendritic cell-specific cell surface markers. 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 embodiment, the invention described herein provides a method for treating an infectious disease in a subject, including a human subject, comprising administering to the subject an anti-hNKG2A antibody or an antigen-binding fragment thereof, such that the subject is treated for the infectious disease. In other embodiments, the anti-NKG2A antibody is a chimeric antibody or a humanized antibody.

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

[0294] Examples of pathogenic viruses that cause 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), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscum contagiosum viruses, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses.

[0295] Examples of pathogenic bacteria that cause infections treatable by the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus pneumoniae, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis and Lyme disease bacteria.

[0296] Examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), genera of Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, and Paracoccidioides. Examples include *Coccidioides 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, species of the genus Acanthamoeba, Giardia lambia, species of the genus Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, and Toxoplasma Examples include *Nippostrongylus gondii* and *Nippostrongylus brasiliensis*, the Brazilian hookworm.

[0298] The method described herein for administering an anti-hNKG2A antibody to a subject may be combined with cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or other forms of immunotherapy, such as bispecific antibody therapy that enhances the presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, Poljak (1994) Structure 2: 1121-1123).

[0299] Autoimmune reaction In some aspects, anti-NKG2A antibodies enhance autoimmune responses. Induction of antitumor responses using tumor cells and peptide vaccines has shown that anti-autoreactivity is involved in many antitumor responses (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) op. cit., Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Therefore, anti-NKG2A antibodies are used in conjunction with these autoproteins to devise vaccination protocols that efficiently generate immune responses against these autoproteins for the treatment of diseases. For example, Alzheimer's disease involves the inappropriate accumulation of Aβ peptides in amyloid deposition in the brain, and these amyloid deposits can be removed by an antibody response against amyloid (Schenk et al., (1999) Nature 400: 173-177).

[0300] Other autoproteins can also be targeted, such as IgE for the treatment of allergies and asthma, and TNFα for rheumatoid arthritis. Ultimately, antibody responses to various hormones can be induced by the use of anti-NKG2A antibodies. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors required for the growth of certain tumors are further vaccine targets.

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

[0302] In some embodiments, the anti-NKG2A antibodies described herein are used to stimulate an antigen-specific immune response by administering the anti-NKG2A antibody together with a target antigen (e.g., a vaccine). Therefore, a method for enhancing an immune response to an antigen in a target is provided herein, comprising administering to the target (i) an antigen and (ii) an anti-NKG2A antibody or its antigen-binding fragment, such that the immune response to the antigen in the target is enhanced. The antibody may 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 antibody or a humanized antibody. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Examples of such antigens, not limited to those discussed herein, include tumor antigens (or tumor vaccines) or antigens derived from viruses, bacteria, or other pathogens, as discussed in the sections herein.

[0303] In certain embodiments, a peptide or fusion protein containing an epitope to which an anti-NKG2A antibody binds is used as a vaccine, either in place of or in addition to the anti-NKG2A antibody.

[0304] Suitable routes for administering the antibody compositions described herein (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) in vivo and in vitro are well known in the art and can be selected by those skilled 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 administered co-administered with one or more therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressants. The anti-NKG2A antibodies can be conjugated with the agents (as immune complexes) or administered separately from the agents. In the latter case (separate administration), the anti-NKG2A antibodies can be administered before, after, or concurrently with the agents, or co-administered with other known therapies, such as anticancer therapies, such as chemotherapy and / or radiation therapy. Examples of such therapeutic agents include antitumor and antineoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which are effective only at levels that are toxic or quasi-toxic to the patient on their own. 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. The co-administration of the anti-NKG2A antibody or its antigen-binding fragment described herein with a chemotherapeutic agent provides two anticancer agents that act by different mechanisms to induce cytotoxic effects on human tumor cells. Such co-administration can solve problems arising from the development of drug resistance or antigenic changes in tumor cells that make them unresponsive to the antibody.

[0306] Kits, including antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use, are also included within the scope described herein. A kit may further contain at least one additional reagent or one or more additional human anti-NKG2A antibodies described herein (e.g., human antibodies having complementary activity to bind to an epitope in the NKG2A antigen distinct from the first human antibody). A kit typically includes markings indicating the intended use of the kit's contents. Terminology includes any documents or recording materials supplied on or with the kit, or otherwise accompanying the kit.

[0307] Combination therapy In one embodiment, a method of combination therapy, for example, for the treatment of cancer, is provided herein, in which an anti-hNKG2A antibody is administered together with one or more further agents, for example, antibodies effective in stimulating an immune response, thereby enhancing, stimulating, or upregulating an immune response in a subject including a human subject. A method for treating cancer in an individual or delaying its progression is also provided herein, comprising administering an anti-hNKG2A antibody (e.g., NKG2A.9, NKG2A.11, and 13F3.A4) to the individual together with another anticancer agent or cancer therapy. In some embodiments, the anti-hNKG2A antibody may be administered together with chemotherapy or chemotherapeutic agents, or radiotherapy or radiotherapeutic agents, as described above. In some embodiments, the anti-hNKG2A antibody may be administered together with an agonist antibody, for example, an anti-hICOS antibody. In some embodiments, the anti-hNKG2A antibody may be administered together with targeted therapy or targeted therapy agents. In some embodiments, the anti-hNKG2A antibody may be administered together with immunotherapy or an immunotherapy 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, combination therapy comprising an anti-hNKG2A antibody and an agonist and / or antagonist results in an enhancement of the antigen-specific T cell response in the subject. In some embodiments, the anti-hNKG2A antibodies described herein may be administered with agents that target 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 may be administered with agents that target ligands of costimulatory or co-inhibitory molecules. The B7 family is a family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors, and 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 is the TNF family of molecules that bind to allogeneic 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 TRAI 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 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, for example, to stimulate an immune response and treat proliferative disorders such as cancer.

[0310] In one embodiment, the T cell response is (i) an anti-hNKG2A antibody as described herein and (i) a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), e.g., 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 (ii) T cells are stimulated by a combination of (ii) an antagonist of D1H, LAIR1, TIM-1, and TIM-4, and one or more agonists of proteins that stimulate T cell activation, such as 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 can be combined with anti-hNKG2A agents, such as those described herein, to modulate any of the aforementioned proteins and treat cancer include: YERVOY® / ipilimumab or tremelimumab (for CTLA-4), galiximab (for B7.1), BMS-936558 (for PD-1), pidilizumab / CT-011 (for PD-1), KEYTRUDA® / pembrolizumab / MK-3475 (for PD-1), AMP224 (for B7-DC / PD-L2), BMS-936559 (for B7-H1), MPDL3280A (for B7-H1), CP-870893, or dasetuzumab / SGN-40 (CD40) - Kirkwood et al. (2012) CA Cancer J. Clin. 62:309, Vanderheide & Examples include Glennie (2013) Clin. Cancer Res. 19:1035), AMG557 (for B7H2), MGA271 (for B7H3 -WO11 / 109400), IMP321 (for LAG-3), urerumab / BMS-663513 and PF-05082566 (for CD137 / 4-1BB), valrirumab / CDX-1127 (for CD27), MEDI-6383 and MEDI-6469 (for OX40), RG-7888 (for OX40L -WO06 / 029879), atacicept (for TACI), muromonab-CD3 (for CD3), and ipilumumab (for CTLA-4). Therefore, in one embodiment, an anti-hNKG2A antibody (e.g., NKG2A.9) is combined with an anti-PD-1 antibody (e.g., nivolumab) and / or an anti-CTLA-4 antibody (e.g., ipilimumab).

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

[0313] Further drugs for combination therapy include, but are not limited to, drugs that inhibit or deplete macrophages or monocytes, as well as CSF-1R antagonists such as CSF-1R antagonist antibodies, including 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 the following: agonists that ligate positive costimulatory receptors; blockers that attenuate signaling by inhibitory receptors; and agents that systemically increase the frequency of antitumor T cells; agents that overcome distinct immunosuppressive pathways within the tumor microenvironment (e.g., blocking the involvement of inhibitory receptors (e.g., PD-L1 / PD-1 interactions), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by exovivo anti-CD25 bead depletion), inhibiting metabolic enzymes (e.g., IDO), or reversing / preventing T cell anergy or depletion); and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0315] Methods for stimulating an immune response in a subject are provided herein, including administering to a subject an anti-hNKG2A antibody and one or more further immunostimulant 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, for example, 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 further immunostimulatory antibody (e.g., antagonist anti-PD-1, antagonist anti-PD-L1, antagonist anti-CTLA-4, and / or antagonist anti-LAG3 antibody) is a human antibody. Alternatively, at least one further immunostimulatory antibody may be, for example, a chimeric or humanized antibody (e.g., prepared from mouse or hamster anti-PD-1, anti-PD-L1, anti-CTLA-4, and / or anti-LAG3 antibody).

[0316] Methods for treating hyperproliferative disorders (e.g., cancer) comprising administering an anti-hNKG2A antibody and an antagonist PD-1 antibody to a subject are provided herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC) or colorectal cancer (CRC). In some embodiments, the cancer is characterized by (i) elevated HLA-E levels and / or (ii) a tumor with a high tumor mutational burden. In certain embodiments, the anti-hNKG2A antibody is administered in a sub-therapeutic dose, the anti-PD-1 antibody is administered in a sub-therapeutic dose, or both are administered in sub-therapeutic doses. Methods for modifying adverse events associated with the treatment of hyperproliferative disorders using immunostimulants are also provided herein. In one embodiment, the method comprises administering an anti-hNKG2A antibody and a sub-therapeutic dose of an anti-PD-1 antibody to a subject. 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 combination with the anti-NKG2A antibodies described herein in the methods 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) 1 × 10⁻¹⁶ when determined by surface plasmon resonance using a Biacore biosensor system. -7 K below M D(b) binds to human PD-1, (c) substantially does not bind to human CD28, CTLA-4, or ICOS, (d) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay, (e) increases interferon-γ production in an MLR assay, (f) increases IL-2 secretion in an MLR assay, (g) binds to human PD-1 and cynomolgus monkey PD-1, (h) inhibits the binding of PD-L1 and / or PD-L2 to PD-1, (i) stimulates an antigen-specific memory response, (j) stimulates an antibody response, and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies usable in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one of the above-mentioned features, and in some embodiments at least five.

[0318] Other anti-PD-1 monoclonal antibodies include, for example, 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 Publications WO2012 / 145493, WO2008 / 156712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, and WO2016 These are listed in / 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 in its entirety by reference.

[0319] In some embodiments, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 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), semipirimab (Regeneron, also known as REGN-2810, see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA, Si-Yang Liu et al., J. Hematol. See Oncol. 10:136 (2017), BGB-A317 (Beigene, see WO2015 / 35606 and US No. 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, see Si-Yang Liu et al., J. Hematol. Oncol.These include 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. Nivolumab is 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 the downregulation of antitumor 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 targeting 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] Other anti-PD-1 antibodies usable in the disclosed methods include isolated antibodies that specifically bind to human PD-1 and cross-compete with any anti-PD-1 antibodies disclosed herein, e.g., nivolumab, for binding to human PD-1 (see, e.g., U.S. Patents 8,008,449 and 8,779,105, WO2013 / 173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as the anti-PD-1 antibodies described herein, e.g., nivolumab. The ability of antibodies to cross-compete with the antigen indicates that these monoclonal antibodies 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. Given the binding to the same epitope region of PD-1, these cross-competing antibodies are expected to have functional properties very similar to those of a reference antibody, e.g., nivolumab. 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, for example, WO2013 / 173223).

[0323] In certain embodiments, with respect to binding to human PD-1, antibodies that cross-compete with human PD-1 antibodies, nivolumab, or bind to the same epitope region are monoclonal antibodies. With respect to administration to human subjects, these cross-competing antibodies are chimeric antibodies, genetically modified antibodies, or humanized or human antibodies. Such chimeric, genetically modified, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0324] Other anti-PD-1 antibodies usable in the methods of the disclosed invention include the antigen-binding moieties of the aforementioned antibodies. It has been well shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody.

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

[0326] Methods for treating hyperproliferative disorders (e.g., cancer) are provided herein, comprising administering an anti-hNKG2A antibody and an antagonist PD-L1 antibody to a target. In certain embodiments, the anti-hNKG2A antibody is administered in a sub-therapeutic dose, the anti-PD-L1 antibody is administered in a sub-therapeutic dose, or both are administered in sub-therapeutic doses. Methods for modifying adverse events associated with the treatment of hyperproliferative disorders using immunostimulants are also provided herein, comprising administering an anti-NKG2A antibody and a sub-therapeutic dose of the anti-PD-L1 antibody to a target. In certain embodiments, the target is 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 CDR or variable region of the antibody disclosed herein.

[0327] Anti-PD-L1 antibodies known in the art can be used in the methods of this disclosure. Examples of anti-PD-L1 antibodies useful in the methods of this disclosure include the antibody disclosed in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibodies disclosed in U.S. Patent No. 9,580,507 have been demonstrated to exhibit one or more of the following characteristics: (a) When determined by SPR using a Biacore biosensor system, K is ≤1 × 10⁻⁷ M DThe anti-PD-L1 antibodies available for use in this invention include (b) binding to human PD-L1, (c) increasing T cell proliferation in a mixed lymphocyte reaction (MLR) assay, (d) increasing interferon-γ production in an MLR assay, (e) stimulating an antibody response, and (f) reversing the action of regulatory T cells on T cells, effector cells, and / or dendritic cells. Monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one of the above-mentioned features, and in some embodiments at least five.

[0328] In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105, see, for example, U.S. Patent No. 7,943,743 and WO2013 / 173223), and atezolizumab (Roche, TECENTRIQ®, also known as MPDL3280A, RG7446, see, U.S. Patent No. 8,217,149, and Herbst et al. (2013) J Clin Oncol See also 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, for example, WO2017 / 034916), or CK-301 (see Checkpoint Therapeutics, Gorelik et al., AACR:Abstract 4606 (Apr 2016))....

Claims

1. A nucleic acid encoding an antibody or its antigen-binding fragment that binds to human NKG2A, The antibody includes HCDR1 of the amino acid sequence of SEQ ID NO: 10, HCDR2 of the amino acid sequence of SEQ ID NO: 11, HCDR3 of the amino acid sequence of SEQ ID NO: 12, and LCDR1 of the amino acid sequence of SEQ ID NO: 13, LCDR2 of the amino acid sequence of SEQ ID NO: 14, and LCDR3 of the amino acid sequence of SEQ ID NO:

15. Nucleic acid.

2. The nucleic acid according to claim 1, wherein the antibody comprises a VH region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a VL region which is at least 90% identical to the amino acid sequence of SEQ ID NO:

9.

3. The nucleic acid according to claim 1, wherein the antibody or its antigen-binding fragment includes a VH region which is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and a VL region which is at least 98% identical to the amino acid sequence of SEQ ID NO:

9.

4. A nucleic acid encoding an antibody or its antigen-binding fragment that binds to human NKG2A, The antibody comprises the VH amino acid sequence of SEQ ID NO: 8 and the VL amino acid sequence of SEQ ID NO:

9. Nucleic acid.

5. The nucleic acid according to claim 4, wherein the antibody comprises a heavy chain (HC) which is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain (LC) which is at least 90% identical to the amino acid sequence of SEQ ID NO:

5.

6. The nucleic acid according to claim 4, wherein the antibody comprises HC which is at least 98% identical to the amino acid sequence of SEQ ID NO: 7 and LC which is at least 98% identical to the amino acid sequence of SEQ ID NO:

5.

7. A nucleic acid encoding an antibody that binds to human NKG2A, The antibody comprises HC of the amino acid sequence of SEQ ID NO: 7 and LC of the amino acid sequence of SEQ ID NO:

5. Nucleic acid.

8. A nucleic acid encoding an antibody or its antigen-binding fragment that binds to human NKG2A, The antibody includes HCDR1 of the amino acid sequence of SEQ ID NO: 10, HCDR2 of the amino acid sequence of SEQ ID NO: 11, and HCDR3 of the amino acid sequence of SEQ ID NO: 12, as well as LCDR1 of the amino acid sequence of SEQ ID NO: 154, LCDR2 of the amino acid sequence of SEQ ID NO: 14, and LCDR3 of the amino acid sequence of SEQ ID NO:

15. Nucleic acid.

9. The nucleic acid according to claim 8, wherein the antibody comprises a VH region which is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a VL region which is at least 90% identical to the amino acid sequence of SEQ ID NO:

164.

10. The nucleic acid according to claim 8, wherein the antibody or its antigen-binding fragment comprises a VH region which is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and a VL region which is at least 98% identical to the amino acid sequence of SEQ ID NO:

164.

11. A nucleic acid encoding an antibody or its antigen-binding fragment that binds to human NKG2A, The antibody comprises the VH amino acid sequence of SEQ ID NO: 8 and the VL amino acid sequence of SEQ ID NO:

164. Nucleic acid.

12. The nucleic acid according to claim 11, wherein the antibody comprises HC which is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and LC which is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

13. The nucleic acid according to claim 11, wherein the antibody comprises HC which is at least 98% identical to the amino acid sequence of SEQ ID NO: 7 and LC which is at least 98% identical to the amino acid sequence of SEQ ID NO:

19.

14. A nucleic acid encoding an antibody that binds to human NKG2A, The antibody comprises HC of the amino acid sequence of SEQ ID NO: 7 and LC of the amino acid sequence of SEQ ID NO:

19. Nucleic acid.

15. A vector comprising the nucleic acid according to any one of claims 1 to 14.

16. A host cell comprising the vector according to claim 15.

17. The host cell according to claim 16, wherein the cell is a eukaryotic cell.

18. The host cell according to claim 17, wherein the cell is a mammalian cell.

19. The host cell according to claim 18, wherein the mammalian cell is selected from the group consisting of CHO cells, NSO cells, COS cells, and SP2 cells.

20. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in claim 16, wherein the antibody is produced.

21. The method according to claim 20, further comprising recovering an antibody or an antigen-binding fragment thereof from the supernatant of host cells or a host cell culture.

22. The method according to claim 21, wherein recovery includes using affinity chromatography.

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