Anti-NKG2A antibodies and uses thereof

Abstract

The present invention provides isolated monoclonal antibodies (e.g., humanized and human monoclonal antibodies) or antigen-binding fragments thereof, which specifically bind to human natural killer cell inhibitory receptor group 2A (NKG2A) protein with high affinity and exhibit functional properties desired for treatment, such as for the treatment of, for example, cancer. The present invention also provides immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the anti-NKG2A antibodies of the present invention. In addition, the present invention provides nucleic acid molecules encoding the antibodies, expression vectors, host cells and methods of using the antibodies to treat, for example, cancer. The present invention also provides combination therapies in which the anti-NKG2A antibodies of the present invention are co-administered with at least one additional agent, such as another antibody (e.g., anti-PD-1, anti-PD-L1 and / or anti-CTLA-4 antibodies).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 768,471, filed on November 16, 2018, and priority to U.S. Provisional Application No. 62 / 927,211, filed on October 29, 2019, both of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to anti-NKG2A (natural killer cell inhibitory receptor group 2A) antibodies and pharmaceutical compositions thereof. The present invention also relates to methods of using such antibodies, including methods of treating diseases such as cancer by administering anti-NKG2A antibodies and pharmaceutical compositions thereof.

[0004] References to sequence listings submitted electronically via EFS-WEB

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on November 4, 2019, named 13119-WO-PCT_SL.txt, 273,467 bytes in size. Background Art

[0006] Cancer is a global epidemic. According to the World Health Data Exchange, cancer is one of the leading causes of disease and the second leading cause of death, accounting for about 17% of deaths worldwide. (Hannah Ritchie and Max Roser, "Causes of Death-Share of deaths by cause, World, 2017", OurWorldInData.org, 2018, available from https: / / ourworldindata.org / grapher / According to the World Health Organization, even in 2010, the economic impact of cancer was $1.16 trillion; in 2018, cancer caused an estimated 9.6 million deaths worldwide. (“Cancer.” World Health Organization. World Health Organization, 2018, available from https: / / www.who.int / news-room / fact-sheets / detail / cancer). According to the National Cancer Institute, in 2019, more than 1.7 million new patients will be diagnosed with cancer and more than 600,000 patients will die from cancer in the United States. ("Cancer Stat Facts: Cancer of Any Site." SEER Training Modules, US National Institutes of Health, National Cancer Institute, 2019<https: / / seer.cancer.gov / statfacts / html / all.html> ).

[0007] Traditional cancer treatments include surgery, radiotherapy and chemotherapy, among other therapies. In recent years, tumor immunity or immunotherapy has become a new option for using the body's immune system to treat cancer. Tumor immunity is different from traditional cancer treatments, which, for example, attempt to directly target tumors and / or destroy tumor blood supply. Alternatively, tumor immunity is designed to use the patient's own immune system to help restore or enhance the patient's anti-tumor immune response. In the absence of tumor immunity methods, the patient's own immune response often fails to prevent tumor growth for various reasons. For example, many tumors have developed specific mechanisms to escape 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, the rapid growth of tumors can even overwhelm the ability of the immune system to effectively control tumors. (Abbas et al., "Chap. 18: Immunity to Tumors", Cellular and Molecular Immunology, 9th Edition Elsevier, Inc., (2018)). Understanding how the immune system affects cancer development and how the immune system can be used to treat cancer has raised challenging and multifaceted questions. For example, many patients do not respond to existing tumor immunotherapies, and some patients develop resistance mechanisms such as T cell exhaustion, when T cells (a specific type of white blood cell) no longer function normally. (Dempke et al., Eur. J. of Cancer, 74:55-72 (2017)).

[0008] Patients need improved treatments for diseases such as cancer to improve traditional therapies, including currently available cancer immunotherapies. There is an urgent need for new tumor immunotherapies that can be used alone or in combination with existing agents to improve patient response rates and overcome drug resistance. Summary of the invention

[0009] In some aspects, the present invention provides isolated monoclonal antibodies (e.g., humanized and human monoclonal antibodies) that bind to human NKG2A protein (SEQ ID NO: 1), i.e., anti-NKG2A antibodies, including anti-hNKG2A antibodies that exhibit desired functional properties. In one aspect, the desired functional properties of the anti-NKG2A antibodies disclosed herein include stimulating an immune response, such as treating cancer. In another aspect, the desired functional properties of the anti-NKG2A antibodies disclosed herein include treating individuals with viral infections, including human patients. In some embodiments, the anti-NKG2A antibodies disclosed herein treat infectious diseases. In another aspect, the anti-NKG2A antibodies disclosed herein treat autoimmune conditions. In other embodiments, the anti-NKG2A antibodies of the present invention are used as antagonistic anti-NKG2A antibodies to stimulate and / or enhance the immune response of an individual, such as stimulating and / or enhancing the anti-tumor response of the immune system (including natural killer cells and / or T cells). In other embodiments, the anti-NKG2A antibodies of the present invention are used in combination with other antibodies to treat various conditions, including cancer, infectious diseases (including viral infections), and autoimmune diseases. Thus, in some embodiments, the anti-NKG2A antibodies disclosed herein, alone or in combination with other treatments (such as other tumor immunotherapy and / or chemotherapy and / or surgery), are used to treat various conditions or diseases, including cancer and viral infections. In other embodiments, the anti-NKG2A antibodies disclosed herein are used in a method for detecting NKG2A protein in a sample.

[0010] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to human NKG2A and exhibits at least one of the following properties:

[0011] (a) reducing (e.g., blocking) the binding and / or interaction between NKG2A ligands (e.g., HLA-E in humans) and human NKG2A proteins;

[0012] (b) Reversal of NKG2A-mediated inhibitory signaling;

[0013] (c) does not bind, or binds with low affinity, to human NKG2C protein;

[0014] (d) binds to human and / or cynomolgus NKG2A;

[0015] (e) Enhanced natural killer cell responses;

[0016] (f) Enhance the functional activity of T cells;

[0017] (g) reduced binding to human Fcγ receptors (FcγR);

[0018] (h) inducing and / or enhancing anti-tumor immune responses;

[0019] (i) inducing and / or enhancing antiviral immune responses;

[0020] and / or

[0021] (j) have low immunogenicity in individuals, including human individuals.

[0022] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof has one or more of the following properties:

[0023] (a) EC binding to human NKG2A protein as measured by cell binding assay 50 The value is about 0.6 nM or lower;

[0024] (b) EC binding to human NKG2C protein as measured by cell binding assay 50 The value is about 9.0 nM or higher;

[0025] (c) EC binding to human NKG2A protein 50 The second EC binding to human NKG2C protein 50 The value is about 15 times smaller;

[0026] (d) IC for reducing the binding and / or interaction of HLA-E with human NKG2A protein as measured by a cell blocking assay 50 The value is about 1.0 nM or less;

[0027] (e) K of binding to human NKG2A protein as measured by Scatchard analysis D is about 0.4 nM or less;

[0028] (f) K binding to human NKG2A as measured by surface plasmon resonance D is about 61 nM or less;

[0029] (g) K binding to cynomolgus monkey NKG2A as measured by Scatchard analysis D is about 1.0 nM or less;

[0030] (h) internalization after binding to cells expressing NKG2A;

[0031] (i) Increased interferon-γ (IFNγ) production;

[0032] (j) with an EC of about 0.5 nM or less 50 Internalization of values; and / or

[0033] (k) wherein the half-life of the anti-NKG2A antibody:NKG2A complex is about 40 seconds or longer. In some embodiments, the half-life of the anti-NKG2A antibody:NKG2A complex is measured using surface plasmon resonance analysis.

[0034] In one embodiment, the anti-NKG2A antibodies or antigen-binding fragments thereof disclosed herein reduce (eg, block) the interaction of human NKG2A protein with human NKG2A ligand (ie, HLA-E).

[0035] In another aspect, the present invention provides an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human NKG2A protein, wherein the antibody comprises:

[0036] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 10, 11, 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 13, 14, and 15;

[0037] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 10, 11 and 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 154, 14 and 15; or

[0038] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 10, 11 and 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 155, 14 and 15.

[0039] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein and comprises heavy chain and light chain variable regions, wherein

[0040] (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:167; and / or

[0041] (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:9, SEQ ID NO:164, or SEQ ID NO:169.

[0042] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[0043] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:164.

[0044] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:167, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:169.

[0045] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human NKG2A protein, wherein the heavy chain and the light chain consist essentially of:

[0046] (a) the amino acid sequences of SEQ ID NOs: 7 and 5, respectively;

[0047] (b) the amino acid sequences of SEQ ID NOs: 7 and 19, respectively; or

[0048] (c) the amino acid sequences of SEQ ID NOs: 35 and 36, respectively.

[0049] In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof competes with an anti-NKG2A antibody disclosed herein for binding to a NKG2A protein or binds to the same epitope as an anti-NKG2A antibody disclosed herein.

[0050] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, which, when bound to human NKG2A protein, specifically binds to the following amino acid residues as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS):

[0051] (a) LSIDNEEMKF (SEQ ID NO: 156);

[0052] (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157);

[0053] (c) LAFKHEIKDSDN (SEQ ID NO: 158); and

[0054] (d) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159).

[0055] In some embodiments, the monoclonal antibody blocks the binding of a NKG2A ligand (eg, HLA-E in humans) to a human NKG2A protein.

[0056] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, which, when bound to human NKG2A, specifically binds to the following amino acid residues as determined by HDX-MS and / or fast photochemical oxidation of protein (FPOP) epitope mapping:

[0057] (a) LSIDNEEMKF (SEQ ID NO: 156)

[0058] (b)PSSWIGVFRNSSHHPW(SEQ ID NO:157)

[0059] (c)LAFKHEIKDSDN(SEQ ID NO:158)

[0060] (d) L; and

[0061] (e) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159).

[0062] In some embodiments, the antibody blocks the binding of a NKG2A ligand (eg, HLA-E in humans) to a human NKG2A protein.

[0063] 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, humanized or chimeric antibody.

[0064] In one aspect, the present invention provides an isolated full-length monoclonal antibody that specifically binds to human NKG2A protein, wherein

[0065] (a) the heavy chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:7, and / or

[0066] (b) the light chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% identical to the amino acid sequence of SEQ ID NO:5.

[0067] In some embodiments, the heavy chain of the isolated full length monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:7, and the light chain comprises the amino acid sequence shown in SEQ ID NO:5.

[0068] In another aspect, the isolated full-length monoclonal antibody specifically binds to human NKG2A protein, wherein the heavy chain consists essentially of the amino acid sequence shown in SEQ ID NO:7, and the light chain consists essentially of the amino acid sequence shown in SEQ ID NO:5.

[0069] In another aspect, the invention provides an isolated nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the nucleic acid molecule is a complementary DNA (cDNA).

[0070] In another aspect, the present invention provides an expression vector comprising a nucleic acid molecule described herein. In another aspect, the present invention provides a host cell transformed with an expression vector described herein.

[0071] In another aspect, the invention provides an immunoconjugate comprising an antibody described herein linked to an agent.

[0072] In another aspect, the present invention provides a method of producing an antibody, comprising culturing a host cell as described herein. In some embodiments, the method further comprises recovering the antibody from the host cell.

[0073] In another aspect, the invention provides a bispecific molecule comprising an anti-NKG2A antibody described herein linked to a second functional moiety.

[0074] In another aspect, the present invention provides a composition comprising an anti-NKG2A antibody as described herein, or a bispecific molecule as described herein and a pharmaceutically acceptable carrier and / or a soluble neutral active hyaluronidase glycoprotein. In some embodiments, the composition further comprises an additional therapeutic agent. In other embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. In other embodiments, the anti-PD-1 antibody is nivolumab, and the anti-CTLA antibody is ipilimumab. In other embodiments, the antibodies described herein are used as drugs for treating cancer.

[0075] In some embodiments, the cancer treated using the anti-NKG2A antibodies, immunoconjugates, bispecific molecules, and compositions described herein is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, gastric cancer, melanoma, kidney cancer, urothelial carcinoma, glioblastoma multiforme, or a virus-related cancer. In other embodiments, the cancer is cervical cancer, head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, non-small cell lung cancer-adenocarcinoma (NSCLC-AD), non-small cell lung cancer-squamous cell (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 multiforme, prostate cancer (also known as prostate adenocarcinoma or PRC), or non-Hodgkin lymphoma.

[0076] In some embodiments, the anti-NKG2A antibody is used to enhance an immune response. In other embodiments, the invention provides the use of the anti-NKG2A antibody described herein for the manufacture of a medicament for treating cancer.

[0077] In one aspect, the invention provides a method of treating cancer in a human subject or delaying the progression of the cancer, 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 cancer / 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 cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, gastric cancer, melanoma, kidney cancer, urothelial carcinoma, glioblastoma multiforme or virus-related cancer. In other embodiments, the cancer is cervical cancer, head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, non-small cell lung cancer-adenocarcinoma (NSCLC-AD), non-small cell lung cancer-squamous cell (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 multiforme, prostate cancer (also known as prostate adenocarcinoma or PRC), or non-Hodgkin lymphoma.

[0078] In other embodiments, the method further comprises administering one or more additional therapeutic agents to the human subject. In some embodiments, the one or more additional therapeutic agents are chemotherapeutic agents, radiotherapeutic agents, and / or immunotherapeutic agents. In other embodiments, the one or more additional therapeutic agents are 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 ipilimumab.

[0079] In another aspect, the invention provides a method of stimulating an immune response in a human subject, comprising administering to the human subject an effective amount of an anti-NKG2A antibody, bispecific molecule, or composition described herein. In some embodiments, the human subject has a tumor and an anti-tumor immune response is stimulated. In other embodiments, the human subject has a chronic viral infection and an anti-viral immune response is stimulated.

[0080] In another aspect, 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 thereof disclosed herein under conditions that allow for the formation of a complex between the antibody or antigen binding fragment thereof and the NKG2A protein, and detecting the formation of a complex. In some embodiments of the method, the antibody or antigen binding fragment thereof forms a complex with the NKG2A protein 15 times faster than with the NKG2C protein.

[0081] Other features and advantages of the present invention will be apparent from the following detailed description and examples, which should not be considered restrictive. The contents of the references, GenBank and other sequence entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1A -C illustrates three methods used to discover and test the anti-NKG2A antibodies disclosed herein. Figure 1A The hybridoma method and the steps for high-level discovery and development of anti-NKG2A antibodies are described. Figure 1B Methods for generating antibody libraries for antibody discovery are described. Figure 1C Describe the B cell monoclonal (SBCC) method.

[0084] Figure 2 The invention describes a mutation scanning analysis for optimizing the discovered anti-NKG2A antibodies. Specifically, Figure 2The steps to generate variants of the 13F3.A4 I107T antibody and characterize their binding to the NKG2A protein are shown. This analysis allowed the inventors to generate variants of the 13F3.A4 I107T antibody with improved properties and provided a rich set of information on the effects of single amino acid substitutions on the binding of the 13F3.A4 I107T antibody to the NKG2A protein. Figure 2 SEQ ID NOs: 183-190 are shown from top to bottom in order of appearance, respectively.

[0085] Figure 3 Figure 2 is an exemplary heat map generated using mutation scanning analysis and can be used to interpret sequence-activity relationships for single amino acid substitutions. Figure 3 SEQ ID NO: 191 (germline) and SEQ ID NO: 192 (parental) are disclosed. Figure 4 Shown are the CDR positions analyzed using mutation scanning analysis for the 13F3.A4 I107T anti-NKG2A antibody. Figure 4 SEQ ID NOs: 30, 15, 192, 11 and 193 are shown in order of appearance, respectively.

[0086] Figure 5A -E is a mutation scanning analysis using 13F3.A4 I107T antibody targeting LCDR1 substitutions (SEQ ID NOs: 194 and 30, germline and parental, respectively) ( Figure 5A ); LCDR3 substitution (SEQ ID NO: 195 and 15, germline and parent, respectively) ( Figure 5B ); HCDR1 substitution (SEQ ID NO: 196 and 192, germline and parental, respectively) ( Figure 5C ); HCDR2 substitution (SEQ ID NO: 42 and 11, germline and parent, respectively) ( Figure 5D ); and HCDR3 substitution (SEQ ID NO: 193 and 193, germline and parental, respectively) ( Figure 5E ) generated by the heat map.

[0087] Figure 6 An alignment of representative sequences of full-length human NKG2A (SEQ ID NO: 182) and human NKG2C (SEQ ID NO: 3) amino acid sequences is shown. Approximately 76% of the amino acid residues (177 of 233 amino acid residues) are conserved, approximately 6% of the amino acid residues (14 of 233 amino acid residues) are similar, and only approximately 18% of the amino acid residues (42 of 233 amino acid residues) differ between human NKG2A and human NKG2C proteins.

[0088] Fig. 7AThe 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 are shown. The amino acid sequences of the VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) sequences are shown in gray boxes.

[0089] Figure 7B 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 are shown. The amino acid sequences of the VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are shown in gray boxes.

[0090] Fig. 8A 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 are shown. The amino acid sequences of the VH CDR1 (SEQ ID NO: 55), VH CDR2 (SEQ ID NO: 56) and VH CDR3 (SEQ ID NO: 57) sequences are shown in grey boxes.

[0091] Figure 8B 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 are shown. The amino acid sequences of the VL CDR1 (SEQ ID NO: 58), VL CDR2 (SEQ ID NO: 59) and VL CDR3 (SEQ ID NO: 60) sequences are shown in grey boxes.

[0092] Fig. 9A 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 are shown. The amino acid sequences of the VH CDR1 (SEQ ID NO: 41), VH CDR2 (SEQ ID NO: 42), and VH CDR3 (SEQ ID NO: 43) sequences are shown in gray boxes.

[0093] Fig. 9BThe 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 are shown. The amino acid sequences of VL CDR1 (SEQ ID NO: 44), VL CDR2 (SEQ ID NO: 45) and VL CDR3 (SEQ ID NO: 46) sequences are shown in grey boxes.

[0094] Fig. 10A 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 are shown. The amino acid sequences of the VH CDR1 (SEQ ID NO: 69), VH CDR2 (SEQ ID NO: 70) and VH CDR3 (SEQ ID NO: 71) sequences are shown in grey boxes.

[0095] Fig. 10B The nucleotide sequence (SEQ ID NO: 176) and amino acid sequence (SEQ ID NO: 177) of the VL of the 4G5.D1 antibody are shown. The amino acid sequences of the VL CDR1 (SEQ ID NO: 72), VL CDR2 (SEQ ID NO: 73), and VL CDR3 (SEQ ID NO: 74) sequences are shown in grey boxes.

[0096] Fig.11A 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 are shown. The amino acid sequences of the VH CDR1 (SEQ ID NO: 83), VH CDR2 (SEQ ID NO: 84) and VH CDR3 (SEQ ID NO: 85) sequences are shown in grey boxes.

[0097] Fig. 11B The nucleotide sequence (SEQ ID NO: 180) and amino acid sequence (SEQ ID NO: 181) of the VL region of the 1G5.B2 antibody are shown. The amino acid sequences of the VL CDR1 (SEQ ID NO: 86), VL CDR2 (SEQ ID NO: 87) and VL CDR3 (SEQ ID NO: 88) sequences are shown in grey boxes.

[0098] Fig.12The amino acid sequence liabilities evaluated in the 13F3.A4 antibody are shown. The V, D and J germline origins are indicated. The amino acid sequence of the heavy chain variable region of the 13F3.A4 antibody (SEQ ID NO: 167) is shown on the left, and the amino acid sequence of the light chain variable region of the 13F3.A4 antibody (SEQ ID NO: 169) is shown on the right. The amino acid sequences of the VL CDR1 (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 31), and VL CDR3 (SEQ ID NO: 32) sequences are underlined. The amino acid sequences of the VH CDR1 (SEQ ID NO: 27), VH CDR2 (SEQ ID NO: 28), and VH CDR3 (SEQ ID NO: 29) sequences are underlined. The sequence liabilities evaluated are circled and annotated.

[0099] Fig.13 The full length amino acid sequence of the anti-NKG2A antibody NKG2A.9 is shown. 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 (the terminal lysine is shown, which is absent in another embodiment). Fig.13 Two mutations made to the 13F3.A4 VL sequence (N30S) and VH sequence (I107T) to generate the NKG2A.9 sequence are marked with ellipses. The N-glycosylation motif is marked with a dotted ellipse. Three mutations made to the Fc region (L234A, L235E and G273A) are shown in bold italics. The terminal amino acid lysine in the heavy chain sequence is removed from the mature sequence. The amino acid sequences of the VL CDR1 (SEQ ID NO: 13), VL CDR2 (SEQ ID NO: 14) and VL CDR3 (SEQ ID NO: 15) sequences are in bold and underlined form. The amino acid sequences of the VH CDR1 (SEQ ID NO: 10), VH CDR2 (SEQ ID NO: 11) and VHCDR3 (SEQ ID NO: 12) sequences are in bold and underlined form.

[0100] Fig.14 The full-length amino acid sequence of the anti-NKG2A antibody NKG2A.9 is shown. 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.

[0101] Fig.15 The full-length amino acid sequence of the anti-NKG2A antibody NKG2A.11 is shown. 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.

[0102] Fig.16 Circular diagram showing the results of epitope grouping (epitopebinning) of exemplary antibodies produced by the B cell monoclonal method (SBCC), and showing the diversity of these antibodies. Antibodies that cross-block each other are connected by lines. Sample antibodies with similar blocking properties compared to benchmark antibodies (13F3.A4, Z270, RD-ahNKG2a (clone 131411, catalog number MAB 1059) and RD-ahCD94 (clone 131412, catalog number MAB 1058)) are grouped together and divided into groups 1-4, 6-7 and 9. Benchmark antibodies with similar blocking properties to the sample antibody group are also grouped together and divided into groups 5, 8 and 10.

[0103] Fig.17A An assay is shown that evaluates the ability of anti-human NKG2A antibodies generated using the SBCC method to block NKG2A / HLA-E interactions. Fig. 17B It was shown that the sample antibodies only partially blocked or did not block the NKG2A / HLA-E interaction compared to the positive control (NKG2A.9 antibody) and the negative control (isotype).

[0104] Fig.18A -C shows the binding analysis method ( Fig.18A ), which was used to evaluate the interaction of anti-NKG2A antibodies with expression of human NK2GA ( Fig.18B ) and CHO cells expressing NKG2C ( Fig. 18C ). Fig.18B As shown in , 13F3.A4, 11H9.A1 and 2EB.B1 antibodies showed specific binding to CHO cells expressing human NKG2A. Fig.18D -F Display blocking analysis method ( Fig.18D ), which was used to evaluate the effect of anti-NKG2A antibodies on NKG2A / HLA-E interaction ( Fig.18E ) and NKG2C / HLA-E interaction ( Fig.18F ). 13F3.A4 and 11H9.A1 antibodies showed specific binding to CHO cells expressing human NKG2A (e.g. Fig.18E ), and does not block NKG2C / HLA-E interaction (as shown in Fig.18F ).

[0105] Fig.19A -B Description of the binding analysis method used ( Fig.19A ), which assessed the ability of anti-NKG2A antibodies to bind to a human NKG2A+ natural killer cell line (NKL) (e.g. Fig.19B ). Fig.19C-D Indicates the blocking analysis method used ( Fig.19C ), which showed that the tested anti-NKG2A antibodies blocked the binding of HLA-E to NKL cells expressing human NKG2A, such as Fig.19D as shown in .

[0106] Fig. 20A -C describes the blocking analysis method used ( Fig. 20A ), which showed that the tested anti-human NKG2A antibodies blocked the NKG2A / HLA-E interaction in CHO cells expressing hNKG2A, such as Fig. 20B -As shown in C.

[0107] Fig.21A -C Describes the binding analysis method used ( Fig.21A ), which showed that the tested anti-NKG2A antibodies bound to CHO cells expressing human NKG2A, such as Fig.21B -As shown in C.

[0108] Fig.22A -C Describes the binding analysis method used ( Fig.22A ), which evaluated the ability of anti-NKG2A antibodies to bind to CHO cells expressing macaque NKG2A (the results are shown in Fig. 22B -C).

[0109] Fig.23A -B Description of blocking analysis method ( Fig.23A ), which is used to assess the ability of anti-NKG2A antibodies to desirably block the NKG2A / HLA-E interaction ( Fig. 23B ). Fig.23C -D Displays the binding analysis method used ( Fig.23C ), which shows the ability of anti-NKG2A antibodies to bind to CHO cells expressing NKG2A ( Fig.23D ).

[0110] Fig.24A -B shows the binding analysis method ( Fig.24A ), which was used to assess whether anti-NKG2A antibodies bind to macaque NKG2A+NKLs ( Fig. 24B ).like Fig. 24B As shown in Figure 2, 11H9.A1 and 4G5.D1 antibodies did not bind to macaque NKG2A+ NK cells, while 13F3.A4 antibody desirably bound to macaque NKG2A+ NK cells, as indicated by an EC of 0.2 nM. 50 Values ​​shown.

[0111] Fig.25A An in vitro method for evaluating whether anti-NKGA antibodies increase NK cell degranulation is described. Fig.25B-C is a graph of the results of flow cytometry analysis and shows that all anti-NKG2A antibodies tested (NKG2A.9, NKG2A.10, NKG2A.11, NKG2A.12, NKG2A.14, NKG2A.15, NKG2A.5, 2G6.C2 and 4G5.D1 antibodies) enhanced NK cell degranulation in NKG2A+ NK cells (measured by the fold change in %CD107a compared to isotype control). 25E7.G8 clone (e.g. Fig.25B ) is a non-blocking antibody used as a negative control.

[0112] Fig.26A In vitro experiments using NKL cells and CHO / MICA / HLA-E are described, showing that anti-NKG2A antibodies blocked NKG2A / HLA-E interaction in activated NK cells and increased IFN-γ production. Fig.26B The 13F3.A4, 11H9.A1 and 4G5.D1 antibodies were shown to desirably increase IFN-γ production compared to the isotype control (human IgG1.3 antibody). The 25E7.G8 clone was used as a negative control.

[0113] Fig.27A Describes an in vitro experiment used to evaluate whether anti-NKG2A antibodies enhance IFNγ-enhancing CD8+ T cell responses in Hs766T target cells, a pancreatic cancer cell line. Fig.27B Figure 1 is a graphical representation of the results of the analysis and shows 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.

[0114] Fig.28A -B shows that, after being treated with NKG2A.9 antibody ( Fig.28A ) and 13F3.A4 antibody ( Fig.28B ) after interaction, the results of differential hydrogen-deuterium exchange (HDX) analysis of mFc-hNKG2A-hCD94. The epitope sequence is annotated at Fig.28A -B (SEQ ID NOs: 119-122, disclosed from left to right along the assay).

[0115] Fig.29A -B shows the results of the comparison with NKG2A.9 antibody ( Fig.29A ) and 13F3.A4 antibody ( Fig.29B ) after interaction with , the FPOP protection percentage of four residues (M163, F179, H184, L206) in hNKG2A.

[0116] Fig.30The 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 by FPOP are shown. The bold, underlined epitopes were determined by HDX-MS, and the circled epitopes were determined by FPOP analysis.

[0117] Fig.31 The epitopes of anti-NKG2A antibodies (e.g., NKG2A.9 and 13F3.A4 antibodies) shown on the NKG2A / CD94 / HLA-E crystal structure are shown. The anti-NKG2A antibody epitopes are shown in black.

[0118] Fig.32A -B shows the VH regions of certain parts of anti-NKG2A antibodies (13F3.A4, NKG2A.9 and NKG2A.11) (SEQ ID NOs: 167, 8 and 8, respectively, from top to bottom in the order of appearance) ( Fig.32A ) and VL regions (SEQ ID NOs: 169, 9 and 164, respectively, in order of appearance) ( Fig.32B ). Through this alignment, it was found that the anti-NKG2A antibodies have the consensus CDR sequences shown in the box.

[0119] Fig.33 Results of the following analysis are shown: NKG2A.9 antibody reverses NK-κB signaling inhibition in CHO / scOKT3 / HLA-E stimulated NKG2A-expressing Jurkat T cells compared to the isotype.

[0120] Fig.34A The experimental method is described for analyzing the effects of NKG2A.9 and anti-PD-L1 antibodies alone or in combination on NKG2A. + Effect of IFN-γ induction by CD8+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs). Fig.34B The results of this method were shown, in which the combination of NKG2A.9 and anti-PD-L1 antibody enhanced the expression of NKG2A in a dose-dependent manner. + IFN-γ production by CD8+ T cells.

[0121] Fig.35A -B Description of the analysis method ( Fig.35A ), which is used to show that NKG2A.9 and / or anti-PD-L1 antibodies enhance NKG2A + IFN-γ production in CD8+ T cells isolated from human tumors co-cultured with CHO / scOKT3 / HLA-E / PD-L1 ( Fig.35B ).

[0122] Fig.36A Figure 2 is a graph showing that the NKG2A.9 antibody is internalized after binding to cells expressing NKG2A. Fig.36B is a graph showing the binding kinetics of the NKG2A.9 antibody and indicates that the NKG2A.9 antibody is internalized in a dose-dependent manner and the EC 50 is 0.5nM.

[0123] Fig.37A -B describes the method used ( Fig.37A ), which showed that the NKG2A.9 antibody increased NK cell degranulation in a dose-dependent manner compared to the isotype, as measured by %CD107a expression measured by flow cytometry. Fig.37C -D Describe the experimental method used ( Fig.37C ), which showed that the NKG2A.9 antibody increased the lysis of HLA-E expressing tumor cells in a dose-dependent manner compared to the isotype ( Fig.37D ).

[0124] Fig.38A Described is a method for measuring the effect of the 13F3.A4 antibody on IFN-γ production in NKL co-cultured with CHO / MICA / HLA-E. Fig.38B The 13F3.A4 antibody was shown to increase IFN-γ production in NKLs compared to the isotype.

[0125] Fig.39 Results of a dose titration study are shown in which 10 mg / kg, 3 mg / kg and 1 mg / kg of an anti-mNKG2A antibody (NKG2A.3) administered as a monotherapy inhibited tumor growth in a colon cancer tumor model with mean tumor volume reductions of 48%, 56% and 30%, respectively. No efficacy was observed for the 0.3 mg / kg dose.

[0126] Fig.40A -E shows the results of in vivo studies in which anti-mNKG2A and anti-mPD-1 antibodies reduced tumor growth in mouse models, with anti-mNKG2A antibody monotherapy showing single-agent activity in the CT26 colorectal tumor mouse model. Fig.40A -D shows that the same type ( Fig.40A ), anti-mNKG2A antibody alone ( Fig.40B ), anti-mPD-1 antibody alone ( Fig.40C ) or a combination of anti-mNKG2A and anti-mPD1 antibodies ( Fig.40D )-treated mice (n=10 / group) at different time points after tumor implantation. Fig.40EShown are mean tumor volumes over 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.

[0127] Fig.41A -C is a graph showing the results of an in vivo study, in which anti-NKG2A and anti-PD-1 antibodies increased NK in a mouse colon cancer model ( Fig.41A ) and tumor-specific CD8+ T cell cytotoxicity and IFN-γ( Fig.41B -C).

[0128] Fig.42A -E shows the anti-tumor activity of anti-mNKG2A and anti-mCTLA-4 antibodies alone or in combination in the 1956 mouse sarcoma model. Fig.42A -D shows that the same type ( Fig.42A )、Anti-mCTLA-4 antibody ( Fig.42B , CTLA-4IgG2a, 0.1mg / kg), anti-mNKG2A antibody ( Fig.42C , 10 mg / kg), or a combination of anti-mNKG2A and anti-mCTLA-4 ( Fig.42D )-treated mice at different time points after tumor implantation. Fig.42E Shown are mean tumor volumes over time (days post tumor implantation) in mice treated with isotype, anti-mCTLA-4 alone, anti-mNKG2A alone, or the combination of anti-mNKG2A and anti-mCTLA-4.

[0129] Fig.43 Anti-NKG2A, anti-PD-1, and anti-LAG3 antibodies and their combinations were shown to have anti-tumor activity in a murine lymphoma model. Administration of anti-NKG2A antibody alone had a survival benefit, with a survival rate of 10%. Combination treatment of anti-NKG2A antibody with anti-mPD-1 or anti-mLAG-3 antibodies increased survival rates to 50% and 70%, respectively. The triple combination of anti-mNKG2A, anti-mPD-1, and anti-mLAG-3 antibodies provided the greatest benefit, with a survival rate of 80%.

[0130] Fig.44 We show that NKG2A expression on both splenic and tumor-infiltrating lymphocyte (TIL) NK cells was reduced after treatment with anti-mNKG2A antibody in a murine CT26 colon cancer model compared with the isotype.

[0131] Fig.45 Shown are the results of NKG2A expression assessed by immunohistochemistry on different tumor types.

[0132] Fig.46The binding of FITC-conjugated NKG2A.6 antibody in various tumors is shown.

[0133] Fig.47 Shown are the results of HLA-E expression in seven different tumor types assessed by immunohistochemistry.

[0134] Fig.48 Shown are representative images of HLA-E expression in different tumor types assessed by immunohistochemistry.

[0135] Fig.49A -B shows the levels of soluble HLA-E in healthy control patients and cancer patients.

[0136] Fig.50 Shown is a clinical development plan for the anti-NKG2A antibodies described herein, including patient selection steps and various combination therapies with the anti-NKG2A antibodies described herein.

[0137] Fig.51A -D is a graph showing the kinetics and binding affinity of the NKG2A.9 antibody determined by Biacore analysis.

[0138] Fig.52A - B shows the kinetics and binding affinity of NKG2A.9 measured by Scatchard analysis.

[0139] Fig.53 Results of NK degranulation assays are shown, comparing P1-069366 to NKG2A.9 and isotypes. The NKG2A.9 antibody was shown to be functional in the NK degranulation assay, while the P1-069366 antibody was not.

[0140] Fig.54A -C shows the results of analyzing the unexpected binding clusters of the 13F3.A4 antibody using the in silico HLA binding tool. Fig.54A -C discloses SEQ ID NOs: 197-204, respectively, in order of appearance from top to bottom.

[0141] Fig.55 Results of in vitro DC:T cell proliferation assays are shown, indicating low immunogenicity risk of anti-NKG2A antibodies, especially NKG2A.6, NKG2A.9 and NKG2A.11 antibodies.

[0142] Fig.56A -D shows the heterodimer of NKG2A.9 antibody and human NKG2A-CD94 ( Fig.56A -B) and NKG2C-CD94 heterodimer ( Fig.56C -D) Binding affinity at 37°C, measured by Biacore using single cycle kinetics ( Fig.56A and 56C ) and multi-cycle dynamics ( Fig.56B and 56D ) determination. The SPR response is shown as a function of analyte association and dissociation.

[0143] Fig.57 Shown are box plots of the total HLA-E positivity scores in 16 different tumor types assessed by immunohistochemistry. The total HLA-E score is defined as the percentage of combined cytoplasmic and / or membrane HLA-E positivity in tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0145] In some aspects, the present invention provides isolated antibodies, such as monoclonal antibodies, e.g., humanized, human and chimeric monoclonal antibodies, that specifically bind to human NKG2A ("hNKG2A") and have antagonistic activity to stimulate anti-tumor immune responses. In some embodiments, the anti-NKG2A antibodies described herein comprise specific structural features, such as CDR regions comprising specific amino acid sequences. In other embodiments, the anti-NKG2A antibodies compete with the anti-NKG2A antibodies of the present invention for binding to human NKG2A protein, or bind to the same or similar epitope as the anti-NKG2A antibodies of the present invention.

[0146] In some aspects, the present invention provides methods for making such anti-NKG2A antibodies, immunoconjugates and bispecific molecules comprising such anti-NKG2A antibodies or antigen-binding fragments thereof, and pharmaceutical compositions formulated to contain anti-NKG2A antibodies or antigen-binding fragments thereof. In some aspects, the present invention provides methods for using anti-NKG2A antibodies alone or in combination with other agents, such as other tumor immunotherapy agents (e.g., antibodies), chemotherapy, radiotherapy, and / or surgery to enhance immune responses. Thus, in some embodiments, the anti-NKG2A antibodies described herein are used to treat a variety of conditions, including, for example, safe and effective treatment of cancer and / or infection.

[0147] An important function of the immune system is its ability to distinguish between normal cells and "foreign" cells. The immune system can therefore attack foreign cells and spare only normal cells. Tumors express antigens that are recognized as foreign by the host. The immune system uses "checkpoints," which are molecules on certain immune cells that need to be activated or inactivated in order to begin an immune response. Tumor cells can sometimes use these checkpoints to avoid being attacked by the immune system. Some immuno-oncology drugs target these checkpoints by acting as checkpoint inhibitors. Programmed death protein 1 (PD-1) is a checkpoint inhibitor that acts as a brake to prevent T cells from attacking other cells in the body. PD-1 does just that 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 the T cells not to attack other cells. Some cancer cells have large amounts of PD-L1, helping them to avoid immune attack. Therapeutic agents that target this PD-1 / PD-L1 interaction, such as monoclonal antibodies, such as nivolumab It can block PD-1 / PD-L1 binding to increase the body's immune response against tumor cells.

[0148] Natural killer cell inhibitory receptor group 2A (NKG2A) is a member of the NKG2 lectin receptor family, which also includes NKG2C, NKG2D and NKG2E. (Iwaszko and Bogunia-Kubik, Arch Immunol Ther Exp, 59: 353-67 (2011)). NKG2A, NKG2C and NKG2E have high homology in the amino acid sequence of their extracellular domains, while NKG2D is a functionally different type of receptor. NKG2A forms a heterodimer with CD94. In the NKG2 / CD94 heterodimer, NKG2A / CD94 is the only receptor with inhibitory function, while NKG2C / CD94 and NKG2E / CD94 are activating receptors. NKG2D is also an activating receptor, but it does not form a heterodimer with CD94, and NKG2D does not bind to HLA-E. The NKG2 / CD94 receptor recognizes nonclassical major histocompatibility (MHC) class I molecules, which are 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. Id. NKG2A is expressed on natural killer (NK) cells, effector / memory CD8 +T, NKT and gamma delta (γδ) T cells. NKG2A expression is induced by T cell receptor (TCR) engagement and after stimulation by certain cytokines (including IL-2, IL-10, IL-15, IL-18, IL-21), but the ability of cytokines to induce NKG2A expression depends on TCR engagement (Cho, Blood, 118: 116-28 (2011). NKG2A has two immunoreceptor tyrosine-based inhibitory motifs (ITIMS) that transmit intracellular inhibitory signals. (Kabat et al., J. Immunol, 169: 1948-58 (2002); Le Dréan El, Eur. J. Immunol 28: 264076 (1998). The anti-NKG2A antibodies described herein inhibit NKG2A protein and can therefore act as checkpoint inhibitors.

[0149] definition

[0150] In order to make the description of the present invention easier to understand, some terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art related to the present invention. In the entire detailed description, other definitions are set forth. The titles provided herein are not limitations of various aspects of the present invention, and the present invention can be understood by reference to the specification as a whole. Therefore, the terms defined below are more fully defined by reference to the entire specification.

[0151] As used herein, "NKG2A" refers to the natural killer cell inhibitory receptor group 2 protein, which is encoded by the NKG2A gene in humans. NKG2A is also known as, for example, CD159 antigen-like family member A, NK cell receptor A, NKG2A activating NK receptor, NKG2-A / B activating NK receptor, killer cell lectin-like receptor C1 (CD159a), and NKG2-A / NKG2-B type II integral membrane protein.

[0152] Three isoforms of the human NKG2A protein have been identified, corresponding to five variants of the mRNA transcript.

[0153] Isoform 1, corresponding to variant 1 (nucleotide sequence set forth in SEQ ID NO: 1 or 209, and amino acid sequence set forth in SEQ ID NO: 2) and variant 3 (nucleotide sequence set forth in SEQ ID NO: 212, and amino acid sequence set forth in SEQ ID NO: 182), consists of 233 amino acids and represents the canonical NGK2A sequence. Isoform 1 variant 3 is also a naturally occurring variant, which is a single nucleotide polymorphism (SNP) at residue 29, wherein asparagine (N) is changed to serine (S), i.e., N29S (SEQ ID NO: 182).

[0154] Isoform 2, corresponding to variant 2 (nucleotide sequence set forth in SEQ ID NO:210, and amino acid sequence set forth in SEQ ID NO:206) and variant 4 (nucleotide sequence set forth in SEQ ID NO:211, and amino acid sequence set forth in SEQ ID NO:207), lacks the in-frame coding exon, resulting in the absence of residues 96-113 compared to variant 1 and variant 3, respectively, and is also referred to as NKG2A isoform NKG2-B. Variant 4 has the N29S SNP.

[0155] Isoform 3, corresponding to variant 5 (nucleotide sequence shown in SEQ ID NO: 208, and amino acid sequence shown in SEQ ID NO: 205), consists of 228 amino acids and lacks residues 229-233 encoding the five C-terminal amino acids. This variant also has the N29S SNP.

[0156] The following are the amino acid sequences of known human NGK2A variants.

[0157] (1) Variant 1: human NGK2A isoform 1 (nucleotide sequence shown in SEQ ID NO: 1 or 209 (accession number NM002259.5), and amino acid sequence shown in SEQ ID NO: 2 (accession number NP 002250.2; UniProt ID P26715-1)):

[0158]

[0159] (2) Variant 2: Human NGK2A isoform 2, also known as NKG2A isoform NKG2B (nucleotide sequence shown in SEQ ID NO: 210 (accession number NM 007328.4), and amino acid sequence shown in SEQ ID NO: 206 (accession number NP015567.2; UniProt ID P26715-2)):

[0160]

[0161] (3) Variant 3: human NGK2A isoform 1 with N29S SNP (bold and highlighted) (nucleotide sequence shown in SEQ ID NO: 212 (accession number NM 213658.2), and amino acid sequence shown in SEQ ID NO: 182 (accession number NP998823.1 or AAL65234.1):

[0162]

[0163] (4) Variant 4: Human NKG2A isoform 2 also corresponds to variant 4 (nucleotide sequence shown in SEQ ID NO: 211 (accession number NM_213657.2), and amino acid sequence shown in SEQ ID NO: 207 (accession number NP998822.1)):

[0164]

[0165] (5) Variant 5: Human NGK2A isoform 3 also known as NKG2A isoform C (nucleotide sequence shown in SEQ ID NO: 208 (accession number NM 001304448.1), and amino acid sequence shown in SEQ ID NO: 205 (accession number NM001291377.1)):

[0166]

[0167] The following table provides a summary of the DNA and protein accession numbers and corresponding SEQ ID NOs described above.

[0168]

[0169] The terms "antibody" or "immunoglobulin" used interchangeably herein refer to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies, such as naturally occurring IgG 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 (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including binding to various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, the amino acids in the variable region are numbered using the Kabat numbering system, while the amino acids in the constant region are numbered using the EU system. Immunoglobulins may be from any of the known isotypes, including IgA, secretory IgA, IgD, IgE, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the anti-NKG2A antibodies described herein are of the IgG1 subtype. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by at most a few amino acids. "Antibodies" include, for example, both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies and fully synthetic antibodies.

[0170] As used herein, an "IgG antibody" has the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy chains 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), wherein the two heavy chains and light chains are linked by disulfide bridges, and the number and position of the disulfide bridges are the same as those in naturally occurring IgG1, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds).

[0171] Antigens are molecules or substances that trigger immune responses and bind to antibodies. Antibodies usually bind with high affinity (10 -5 Up to 10 -11 M or less dissociation constant (K D ) reflects that) binds specifically to its cognate antigen but does not bind unrelated antigens with high affinity. Any -4 M of K D As used herein, an antibody that "specifically binds" to an antigen means that the antibody binds to the antigen and in some cases substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity, wherein high affinity means having a 10 -6 M or lower KD , 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, or 10 -8 M and 10 -10 M or lower K D An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, e.g., 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, in some embodiments, an antibody that specifically binds to human NKG2A also cross-reacts with NKG2A antigens from certain non-human primate species (e.g., macaque NKG2A), but does not cross-react with NKG2A antigens from other species or antigens other than NKG2A.

[0172] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody are used interchangeably herein and refer to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., human NKG2A). It has been shown that the antigen-binding function of an antibody can be performed by a fragment or portion of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody, e.g., an anti-NKG2A antibody described herein, include:

[0173] (1) Fab fragments consisting of VL, VH, LC and CH1 domains (fragments derived from papain cleavage) or similar monovalent fragments;

[0174] (2) F(ab')2 fragments (fragments derived from pepsin cleavage) or similar bivalent fragments, which contain two Fab fragments linked by a disulfide bridge at the hinge region;

[0175] (3) By V H and the Fd fragment composed of the CH1 domain;

[0176] (4) V of one arm of the antibody L and V H Fv fragment composed of domains,

[0177] (5) Single-domain antibody (dAb) fragments (Ward et al., (1989) Nature 341:544-46), which consist of the VH domain;

[0178] (6) isolated complementarity determining regions (CDRs); and

[0179] (7) Combinations of two or more isolated CDRs, which may optionally be connected via a synthetic linker.

[0180] In addition, although the two domains VL and VH of the Fv fragment can be encoded by separate genes, they can also be connected by a linker using recombinant methods, wherein the linker allows the VL and VH to be produced as a single protein chain and paired therein to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also encompassed within the term "antigen binding portion" or "antigen binding fragment" of an antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and available fragments can be screened in the same manner as intact antibodies. The antigen binding portion can be produced by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0181] "Bispecific" or "bifunctional antibodies" are artificial hybrid antibodies that have two different binding specificities, such as two different heavy chain / light chain pairs, thereby generating two antigen binding sites with specificities for different antigens. Bispecific antibodies can be produced by a variety of methods including hybridoma fusion or Fab' fragment ligation. See, e.g., Songsivila and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0182] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody in the population is substantially similar and binds to the same epitope (e.g., the antibody exhibits a single binding specificity and affinity), except for possible variants that may occur during the production of the monoclonal antibody, usually in small amounts. The modifier "monoclonal" refers to the characteristic that the antibody is obtained from a substantially homogeneous antibody population, and does not require that the antibody be produced by any particular method. The term "human monoclonal antibody" refers to an antibody from a substantially homogeneous antibody population that exhibits a single binding specificity and has a variable region and an optional constant region derived from a human germline immunoglobulin sequence. In one embodiment, human monoclonal antibodies are produced using a hybridoma method. Using the hybridoma method, a transgenic non-human animal, such as a transgenic mouse, is exposed to an antigen and white blood cells called B cells produce antibodies that bind to the antigen, which can be harvested from the transgenic non-human animal. The 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 comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0183] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (1) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) for human immunoglobulin genes or hybridomas prepared therefrom; (2) antibodies isolated from host cells transformed to express antibodies (e.g., transfectomas); (3) antibodies isolated from recombinant, combinatorial human antibody libraries; and (4) antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may comprise variable and constant regions that use specific human germline immunoglobulin sequences encoded by germline genes, but may also include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains an antigen binding domain, which may be encoded by various genes that rearrange to form antibodies specific for foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region will change in further response to the antigen (i.e., isotype switching). Therefore, the rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecule, but may be substantially identical or similar (e.g., having at least 80% identity).

[0184] As used herein, "human antibody" refers to an antibody having a variable region of a framework region and a CDR region derived from a human germline immunoglobulin sequence. In addition, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The anti-NKG2A antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced due to random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, the term "human antibody" is not intended to include antibodies to which a CDR sequence derived from the germline of another non-human mammalian species (such as a mouse) has been transplanted onto a human framework sequence. As used herein, the terms "human" and "fully human" antibodies are used interchangeably.

[0185] "Humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody are replaced with the corresponding amino acids from a human antibody. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domain have been replaced with amino acids from a human antibody, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not prevent the antibody from binding to a specific antigen. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody.

[0186] "Chimeric antibodies" refer to antibodies in which the variable regions are derived from one species and the constant regions are derived from another species, such as antibodies in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0187] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region gene of the antibody (eg, IgG (including IgG1, IgG2, IgG3 and IgG4), IgM, IgA (including IgA1 and IgA2), IgD and IgE antibodies).

[0188] "Allotype" refers to naturally occurring variants within a particular isotype group, wherein the variants differ by a few amino acids. (See, e.g., Jefferis et al. (2009) mAbs 1:1). The anti-NKG2A antibodies described herein may have any allotype. As used herein, antibodies referred to as "IgG1f", "IgG1.1f" or "IgG1.3f" isotypes are IgG1, null IgG1.1 or null IgG1.3 antibodies having allotype "f", respectively.

[0189] The expressions "antibodies that recognize an antigen" and "antibodies specific for an antigen" are used interchangeably herein with the expression "antibodies that specifically bind to an antigen".

[0190] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other proteins and cellular materials.

[0191] As used herein, "effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical events resulting therefrom. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP)), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable region).

[0192] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIb or equivalently FcγRIIB) receptor. Various exemplary properties of human FcγRs are known in the art. In mice and humans, most innate effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIb, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but not inhibitory FcγRIIb. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor it binds.

[0193] As used herein, "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody other than the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments of the second (CH2) and third (CH3) constant domains derived from the two heavy chains of an antibody. In IgM and IgE antibody isotypes, the Fc region comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3 and a hinge between the CH1 and CH2 domains. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be defined differently, as defined herein, a human IgG heavy chain Fc region is defined as extending from amino acid residue D221 for IgG1, from V222 for IgG2, from L221 for IgG3, and from P224 for IgG4 to the carboxyl terminus of the heavy chain, wherein the numbering is according to the EU index in Kabat (Kabat et al., 1991). The CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from amino acid 341 of IgG to amino acid 447 or 446 (if there is no C-terminal lysine residue) or 445 (if there are no C-terminal glycine and lysine residues). As used herein, an Fc region may be a native sequence Fc, including variants of any allotype, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region in isolation, or in a protein polypeptide comprising Fc, such as a "binding protein comprising an Fc region," also known as an "Fc fusion protein" (eg, an antibody or immunoadhesin).

[0194] A "native sequence Fc region" or "native sequence Fc" has an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region and naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc. (See, e.g., Jefferis et al. (2009) mAbs 1:1).

[0195] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., hNKG2A protein) to which an immunoglobulin or antibody specifically binds, such as defined by a particular method for identifying it. An epitope can be formed by (1) contiguous amino acids (usually a linear epitope) or (2) non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed by contiguous amino acids are typically, but not always, retained after exposure to denaturing solvents, while epitopes formed by tertiary folding typically disappear after treatment with denaturing solvents. An epitope typically includes 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.

[0196] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition. Methods for determining the epitope to which a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis, in which overlapping or continuous peptides from a protein (e.g., from NKG2A) are tested for reactivity with a given antibody (e.g., an anti-NKG2A antibody). Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as x-ray crystallography; antigen mutation analysis, two-dimensional nuclear magnetic resonance; yeast display; and hydrogen / deuterium exchange-mass spectrometry (HDX-MS) (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).

[0197] For two or more antibodies, the expression "binding to the same epitope" means that the antibody binds to the amino acid residues of the same segment as determined by a given method. The techniques used to determine whether an antibody binds to the "same epitope on NKG2A" as the antibodies described herein include, for example, epitope mapping methods, such as x-ray crystallography of antigen: antibody complexes (which provide atomic resolution of the epitope), HDX-MS, and protein fast photochemical oxidation (FPOP). Other methods monitor the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or to mutant variant forms of antigens, wherein the loss of binding due to modification of amino acid residues within the antigen sequence is generally considered to indicate an epitope component, such as alanine scanning mutagenesis (Cunningham and Wells (1985) Science 244: 1081) or yeast display of mutant target sequence variants. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the target antibody to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies with the same VH and VL or the same CDR1, CDR2 and CDR3 sequences are expected to bind to the same epitope.

[0198] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, that is, whether one antibody inhibits the binding of another antibody to a target and the extent of inhibition, can be determined using known binding competition assays (e.g. In some embodiments, an antibody competes with another antibody for binding to a target and inhibits its binding by at least 50%, 60%, 70%, 80%, 90%, or 100%. The degree of inhibition or competition may vary depending on which antibody is used as the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competition assays may be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb. Protoc. 2006; doi: 10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies," edited 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 (i.e., regardless of which antibody first contacts the antigen in the competition assay).

[0199] Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include, for example, flow cytometry to determine competitive binding to T cells expressing NKG2A. Other methods include: SPR (e.g. ); 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 I-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0200] As used herein, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope of a predetermined antigen. In some embodiments, the antibody: (1) when detected by, for example, SPR technology When a predetermined antigen, such as recombinant human NKG2A, is used as an analyte in an SPR instrument and an antibody is used as a ligand, or when the binding of an antibody to an antigen-positive cell is determined by Scatchard analysis, the binding of the antibody to the antigen-positive cell is about less than 10 -6 M, such as approximately less than 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K D );and (2) bind to the predetermined antigen with an affinity at least two times greater than its binding affinity to nonspecific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). Therefore, an antibody that "specifically binds to human NKG2A" refers to an antibody that binds to human NKG2A with an affinity of at least 10 -6 M or less, such as approximately less than 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or even lower K D, antibodies that bind to soluble or cell-bound human NKG2A. Antibodies that "cross-react with macaque NKG2A" refer to antibodies that cross-react with 10 -6 M or less, such as approximately less than 10 -7 , 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibody that binds to cynomolgus monkey NKG2A.

[0201] The term "k a ", "k assoc "or" on " are used interchangeably herein to refer to the association rate constant for a particular antibody-antigen interaction. d ", "k dis "or" off "K" are used interchangeably herein to refer to the dissociation rate constant for a particular antibody-antigen interaction. As used herein, the term "K D ” refers to the equilibrium dissociation constant, which is composed of k d With k a ratio (that is, k d / k a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. Determination of the K value of an antibody D Available methods include, but are not limited to, using, for example, biosensor systems (such as system), and flow cytometry and Scatchard analysis.

[0202] The term "IC 50 ” refers to half maximal inhibitory concentration, which measures the potency of a substance (e.g., an antibody) in inhibiting a specific biological or biochemical reaction. In other words, IC 50 Used as a measure of performance; IC 50 The smaller the IC, the more potent the substance. In in vitro or in vivo assays using antibodies or their antigen-binding fragments, IC 50 It refers to the concentration of an antibody or its antigen-binding fragment that reduces the maximum biological or biochemical response by 50%.

[0203] The term "EC 50 " means half maximal effective concentration and measures the potency of a substance (e.g., an antibody) to induce a specific biological or biochemical response. Like IC 50 , E.C. 50 Used as a measure of efficacy; EC 50 The smaller the size, the stronger the substance. In in vitro or in vivo assays using antibodies or their antigen-binding fragments, EC50 It refers to the concentration of an antibody or antigen-binding fragment thereof that induces 50% of the maximum biological or biochemical response.

[0204] As used herein, "receptor occupancy / occupancy of the receptor" refers to the amount of an antibody (e.g., an anti-NKG2A antibody described herein) that binds to an immunostimulatory receptor (e.g., human NKG2A). "Percent (%) receptor occupancy" can be calculated using the following formula: ([ΔMFI of test] / [ΔMFI of total])×100. The mean fluorescence unit change (ΔMFI) is calculated by subtracting the MFI of background staining with an isotype control antibody from the MFI of the bound antibody. Total receptor levels are determined as follows: saturating amounts of antibody are added to determine maximum expression and thereby determine the MFI of a specific immunostimulatory receptor. An alternative method for calculating total receptor expression is to use an antibody against the same immunostimulatory receptor that does not compete with the antibody for which receptor occupancy is calculated.

[0205] As used herein, the term "naturally occurring" as applied to a substance refers to a substance that exists in nature and has not been intentionally modified by man. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and has not been intentionally modified by man in the laboratory and exists in an organism (including a virus) is naturally occurring.

[0206] "Polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. "Protein" includes one or more polypeptides.

[0207] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules are single-stranded or double-stranded, and may be complementary DNA (cDNA).

[0208] The term "cDNA" or "complementary DNA" refers to a non-naturally occurring nucleic acid molecule that is produced or derived from mRNA, ie, from which the non-coding regions have been removed.

[0209] As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted nonessential amino acid residue in an anti-NKG2A antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell 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)).

[0210] With respect to nucleic acids, the term "substantially homologous" means that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists if a segment will hybridize under selective hybridization conditions to the complement of a nucleic acid strand.

[0211] With respect to polypeptides, the term "substantially homologous" means that two polypeptides or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids, with appropriate amino acid insertions or deletions.

[0212] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology = (number of identical positions) / (total number of positions) x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0213] The percent identity between two nucleotide sequences can be determined, for example, using the GAP program in the GCG software package, using the nwsgapdna.cmp matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package, using a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0214] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to search against public databases, for example to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. (See, e.g., National Center for Biotechnology Information (NCBI), at https: / / www.ncbi.nlm.nih.gov / available).

[0215] Nucleic acids can be present in whole cells (e.g., host cells), in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "appear substantially pure" when they are purified and separated from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. (See F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).

[0216] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can be connected to other DNA segments. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and then replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Expression vectors suitable for recombinant DNA technology include plasmids. As used herein, "plasmid" and "vector" are used interchangeably because plasmids are the most common form of vectors. However, other forms of expression vectors that provide equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0217] The terms "host cell" or "recombinant host cell", which are used interchangeably, refer to cells that contain a nucleic acid that is not naturally present in the cell, and may be cells into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, the progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0218] "Immune response" as understood in the art generally refers to a biological response in a vertebrate to a foreign substance or abnormal cell (e.g., a cancer cell), wherein the response protects the vertebrate from such substances and the diseases caused thereby. An immune response may be 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 such cells or the liver, which results in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal cells or tissues, including, for example, human cells or tissues, in the body of the vertebrate. An immune response includes, for example, activation or suppression of T cells, e.g., effector T cells or T helper (Th) cells, such as CD4+ or CD8+ T cells, or suppression or depletion of Treg cells.

[0219] "Effector T" ("Teff") cells are T cells with cytolytic activity (such as CD4+ and CD8+ T cells) and T helper (Th) cells. Th cells secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0220] Regulatory T cells ("Tregs") are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Memory B cells are a subtype of B cells formed in germinal centers after primary infection, which are important for generating accelerated and more stable antibody-mediated immune responses (also known as secondary immune responses) in the event of reinfection.

[0221] 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 situations. NK cells provide a rapid response to virus-infected cells and respond to tumor formation.

[0222] As used herein, the term "T cell-mediated response" refers to responses mediated by T cells, such as effector T cells (eg, CD8+ cells) and helper T cells (eg, CD4+ cells). T cell-mediated responses include, for example, cytotoxicity and proliferation of T cells.

[0223] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL response is mediated by, for example, CD8+ T cells.

[0224] "Immunomodulator" or "immunomodulator" refers to a substance that can participate in regulating, modulating or modifying an immune response, such as a component of a signal transduction pathway. "Regulating", "modulating" or "modifying" an immune response refers to any change in the activity of a cell of the immune system or such a cell (e.g., an effector T cell, such as a Th1 cell). Such regulation includes stimulation or suppression of the immune system, which can be manifested as 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 in the immune system. Inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In some embodiments, the immunomodulator is located on the surface of a T cell. "Immunomodulatory target" or "immunomodulatory 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 a substance, agent, moiety, compound or molecule. Immunomodulatory targets include, for example, receptors on the surface of cells ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0225] "Immunotherapy" refers to the treatment of an individual, such as a human individual, suffering from a disease or at risk of contracting a disease or suffering recurrence of a disease by methods that include inducing, enhancing, suppressing or otherwise modifying an immune response.

[0226] "Immunostimulatory therapy" or "immunostimulatory therapy" refers to therapy that causes an increase (induces or enhances) in a subject's immune response, for example to treat cancer.

[0227] "Enhancing an endogenous immune response" means increasing the effectiveness or potency of an existing immune response of an individual, such as a human individual. This increase in effectiveness and potency can be achieved, for example, by overcoming mechanisms that suppress endogenous host immune responses or by stimulating mechanisms that enhance endogenous host immune responses.

[0228] As used herein, the term "linked" refers to the association of two or more molecules. Linkage can be covalent or non-covalent. Linkage can also be genetic (i.e., fused in a recombinant manner). Such linkage can be achieved using a wide variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0229] As used herein, "administering" refers to the physical introduction of a composition comprising a therapeutic agent, such as an anti-NKG2A antibody, into an individual using any of a variety of methods and delivery systems known to those skilled in the art. "Administering" includes, for example, the administration of a human patient by another (such as one or more) health care provider, as well as self-administration by a human patient. Various routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the expression "parenteral administration" means other modes of administration other than enteral and topical administration, such as modes of administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically. Administration can also be performed, for example, once, multiple times and / or over one or more long periods of time.

[0230] As used herein, "adjuvant" or "combination" administration (co-administration) includes simultaneous administration of compounds in the same or different dosage forms, or separate administration of these compounds (e.g., sequential administration). Thus, a first antibody, such as 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. As used herein, "combination" therapy means administering two or more therapeutic agents in a synergistic manner, and includes, but is not limited to, concurrent administration. Specifically, combination therapy encompasses co-administration (e.g., administration of a co-formulation, or simultaneous administration of separate therapeutic compositions) and continuous or sequential administration, provided that the administration of one therapeutic agent regulates the administration of another therapeutic agent in a certain manner. For example, a therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a specified period of time. (See, e.g., Kohrt et al. (2011) Blood 117:2423).

[0231] For example, the anti-NKG2A antibody may be administered first, followed by (e.g., immediately after) the second antibody, or vice versa. In one embodiment, the anti-NKG2A antibody is administered before the second antibody. In another embodiment, the anti-NKG2A antibody is administered, e.g., within about 30 minutes of the second antibody. Such concurrent or sequential administration preferably allows the two antibodies to be present in the treated patient simultaneously.

[0232] As used herein, the terms "inhibit" or "block" are used interchangeably and encompass partial and complete inhibition / blocking. In some embodiments, the anti-NKG2A antibodies described herein inhibit the binding of NKG2A to HLA-E by at least about 50%, such as about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, for example, as determined further herein. In some embodiments, the anti-NKG2A antibodies inhibit the binding of NKG2A to HLA-E by no more than 50%, such as about 40%, 30%, 20%, 10%, 5%, or 1%, for example, as determined further herein.

[0233] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell growth or division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0234] As used herein, the terms "treat", "treating", or "treatment" refer to any type of intervention or process performed on an individual or the administration of an active agent to an individual with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease. In contrast, "prophylaxis" or "prevention" refers to the administration of medication to an individual who does not have a disease in order to prevent the disease from occurring. As used herein, "treatment" does not include prevention or prophylaxis.

[0235] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from the disease. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to an individual at risk of contracting a disease or suffering a recurrence of a disease, prevents the development or recurrence of a disease. The ability of a therapeutic agent to promote regression of a disease or a prophylactic agent to prevent the development or recurrence of a disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that are predictive of efficacy in humans, or by analyzing the activity of the agent in in vitro assays.

[0236] According to any of the methods provided herein, administering an effective amount of an anti-NKG2A antibody alone, or administering an effective amount of an anti-NKG2A antibody in combination with, for example, an anti-PD-1 antibody, or in combination with an anti-PD-L1 antibody, or in combination with an anti-CTLA-4 antibody, can produce at least one therapeutic effect, including, for example, a reduction in tumor growth or size, a reduction in the number of metastatic lesions that appear over time, a complete remission, a partial remission, or stable disease. For example, the treatment method produces a comparable clinical benefit rate (CBR=complete remission (CR)+partial remission (PR)+stable disease (SD) for ≥6 months), which is better than when no anti-NKG2A antibody is administered, or better than when any of the combination antibodies is administered alone, for example, an improvement in the clinical benefit rate of about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.

[0237] For example, an anticancer agent is a drug that slows the progression of cancer in an individual (including a human individual) or promotes cancer regression. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of achieving cancer clearance. "Promoting cancer regression" means that an effective amount of a drug, administered alone or in combination with an anti-neoplastic agent, causes a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease asymptomatic periods, prevention of damage or disability caused by illness, or otherwise improves disease symptoms. "Pharmacological effectiveness", "effectiveness" or "efficacy" refers to the ability of a drug to promote cancer regression in a patient. "Physiological safety" refers to an acceptable low level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and / or organism level caused by the administration of a drug.

[0238] For example, for tumor treatment, 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 about 70%, at least about 80%, or at least about 90% (relative to an untreated individual). 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, including an antibody, to inhibit tumor growth can be assessed using the assays described herein. Alternatively, this property of a composition can be assessed by examining the ability of a compound to inhibit cell growth; such inhibition can be measured in vitro by assays known to those of ordinary skill in the art. In some embodiments, tumor growth inhibition may not occur immediately after treatment, but may only occur after a period of time or after repeated administration. In other embodiments described herein, tumor regression is observed and lasts 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.

[0239] As used herein, the terms "fixed dose", "flat dose" and "flat-fixed dose" are used interchangeably and refer to a dose that is administered to a patient without regard to the patient's weight or body surface area. A fixed or flat dose is therefore not provided as a mg / kg dose, but rather as an absolute amount of a therapeutic agent.

[0240] As used herein, the term "weight-based" dosage or administration means that the dosage administered to a patient is calculated based on the patient's weight. For example, if a 60 kg patient requires 3 mg / kg of anti-NKG2A antibody, the appropriate amount of anti-NKG2A antibody administered (i.e., 180 mg) can be calculated and used accordingly.

[0241] The term "patient" includes humans and other mammalian subjects receiving therapeutic or prophylactic treatment.

[0242] The term "subject" includes any human or non-human animal. For example, the disclosed methods and compositions can be used to treat a subject suffering from cancer. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human subject.

[0243] Unless otherwise specified, as used herein, the term "a" or "an" entity refers to one or more such entities; for example, "a" nucleotide sequence should be understood to mean one or more nucleotide sequences. Therefore, the terms "a" or "an", "one or more" and "at least one" are used interchangeably herein.

[0244] As used herein, "and / or" should be deemed to encompass specific disclosure of each of the two specified features or components with or without the other. Thus, when used in a phrase such as "A and / or B", the term "and / or" includes "A and B", "A or B", "A alone", and "B alone". Similarly, when used in a phrase such as "A, B, and / or C", the term "and / or" encompasses each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0245] It should be understood that when aspects of the present invention are described herein using the expression “comprising”, similar aspects described using the expressions “consisting of” and / or “consisting essentially of” are also encompassed.

[0246] Unless otherwise specified, units, prefixes and symbols are expressed in their acceptable form for the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, 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 carboxyl direction.

[0247] As used herein, the term "about" or "approximately" means approximately, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it adjusts the range by extending the boundaries above and below the numerical values. In general, the term "about" can modify the numerical value both above and below the numerical value stated, for example, by 10% (higher or lower) above or below the numerical value.

[0248] The titles provided herein are not limitations of the various aspects of the present invention and should be interpreted by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by the entire specification. The various aspects described herein are further described in detail in the following subsections.

[0249] I. Anti-NKG2A Antibody

[0250] In some embodiments, the present invention describes anti-NKG2A antibodies, such as fully human, humanized and chimeric antibodies, having desired functions or properties. For example, the antibody specifically binds to human NKG2A protein with high affinity. In certain embodiments, the antibody is an antagonist antibody that blocks or reverses NKG2A-mediated inhibition of 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 for treating diseases, such as cancer or infection.

[0251] The specific anti-NKG2A antibodies described herein are antibodies having the CDR and / or variable region sequences of antibodies 13F3.A4, NKG2A.6, NKG2A.7, NKG2A.8, NKG2A.9, NKG2A.11 (isolated and structurally characterized as described herein), and antibodies having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to the amino acid sequences of the anti-NKG2A antibodies described herein. In some embodiments, the anti-NKG2A antibodies have at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to the variable region or CDR sequences of the anti-NKG2A antibodies described herein.

[0252] In some aspects, the antibodies of the present invention are characterized by specific functional features or properties. For example, the antibody specifically binds to human NKG2A with high affinity. In some embodiments, the anti-NKG2A antibody inhibits the binding of NKG2A to its ligand HLA-E, which restores the response of NK and T cells to tumors expressing HLA-E. In other words, the anti-NKG2A antibodies described herein stimulate T cells and NK cells to respond to tumors by inhibiting or blocking the interaction between the NKG2A protein and its ligand HLA-E.

[0253] In some embodiments, the anti-NKG2A antibodies described herein exhibit one or more of the following properties:

[0254] (1) Specific binding to human NKG2A protein;

[0255] (2) blocking or reducing the binding and / or interaction between NKG2A ligands (e.g., HLA-E in humans) and human NKG2A proteins; (in other embodiments, the anti-NKG2A antibodies described herein block or reduce the binding and / or interaction between NKG2A ligands and non-human NKG2A proteins);

[0256] (3) Reversal of NKG2A-mediated inhibitory signaling;

[0257] (4) does not bind, or binds with low affinity, to human NKG2C protein;

[0258] (5) binds to human and macaque NKG2A with high affinity;

[0259] (6) does not bind to mouse or rat NKG2A or shows low affinity for it;

[0260] (7) does not interfere with the activation signal triggered by HLA-E binding to NKG2C protein;

[0261] (8) Reduced binding to human Fcγ receptor (FcγR);

[0262] (9) Induce and / or enhance anti-tumor immune response;

[0263] (10) enhancing the functional activity of T cells; (in some embodiments, increasing cytotoxic T cell function, such as measured by lysis of tumor cells expressing HLA-E);

[0264] (11) enhancing the functional activity of natural killer (NK) cells, for example by inducing NK cell activation;

[0265] (12) increase the production of cytokines, such as IFNγ; and / or

[0266] (13) specifically binds to an epitope located within a discrete region comprising the following amino acid residues, as determined by HDX-MS and / or FPOP epitope mapping:

[0267] Region 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2);

[0268] Region 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2);

[0269] Region 3: 192 LAFKHEIKDSDN 203(amino acid residues 192 to 203 of SEQ ID NO: 2);

[0270] Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and

[0271] Zone 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2).

[0272] (14) specifically binds to an epitope located within a discrete region comprising the following amino acid residues, as determined by HDX-MS:

[0273] Region 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2)

[0274] Region 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2);

[0275] Region 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2); and

[0276] Zone 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2).

[0277] In some embodiments, the anti-NKG2A antibodies described herein bind to human and cynomolgus NKG2A with high affinity, and do not bind to, or bind to, non-primate NKG2A, such as mouse or rat NKG2A with low affinity. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

[0278] (a) EC binding to human NKG2A protein as measured by cell binding assay 50 The value is about 0.6 nM or lower;

[0279] (b) EC binding to human NKG2C protein as measured by cell binding assay 50 The value is about 9.0 nM or higher;

[0280] (c) reducing the IC of HLA-E binding and / or interaction with human NKG2A protein as measured by cell blocking assay 50 The value is about 1.0 nM or less;

[0281] (d) with a K of about 0.4 nM or less as measured by Scatchard analysis D Binds to human NKG2A protein;

[0282] (e) with a K of about 61 nM or less as measured by surface plasmon resonance D Binds to human NKG2A protein;

[0283] (f) with a K of about 1.0 nM or less as measured by Scatchard analysis D Binds to macaque NKG2A protein;

[0284] (g) internalization after binding to cells expressing NKG2A;

[0285] (h) increasing interferon-γ (IFNγ) production; and / or

[0286] (i) wherein the half-life of the anti-NKG2A antibody:NKG2A protein complex is about 40 seconds or longer.

[0287] In some embodiments, the anti-NKG2A antibodies described herein block the binding and / or interaction of NKG2A ligands (HLA-E in humans) with human NKG2A proteins. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

[0288] a) Anti-NKG2A antibodies block HLA-E pentamer binding to cells expressing human NKG2A, and for NKL, IC 50 is about 0.30 nM, and for CHO-hNKG2A cells, IC 50 is about 1.0 nM;

[0289] b) Anti-NKG2A antibodies complexed with HNKG2A-CD94-mFC or cynomolgus NKG2A-CD94-mFc proteins blocked human HLA-E binding.

[0290] In some embodiments, the anti-NKG2A antibodies described herein are specific for human NKG2A. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

[0291] a) Anti-NKG2A antibody binds to the EC of human NKG2A 50 The second EC of anti-NKG2A antibody binding to human NKG2C protein50 In some embodiments, the anti-NKG2A antibody binds to the EC of human NKG2A. 50 The value was about 0.6 nM, while the EC of anti-NKG2A antibody binding to human NKG2C 50 The value is about 9.0 nM.

[0292] b) based on SPR analysis, the anti-NKG2A antibody does not specifically bind to human NKG2C; and / or

[0293] c) does not block the interaction of human NKG2C with HLA-E as measured by flow cytometry.

[0294] In some embodiments, the anti-NKG2A has an inert Fc (e.g., the antibody is of IgG1 isotype) to reduce or prevent FcγR binding. Without being bound by any theory, since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, reducing the excitement or depletion of NKG2A+ CD8+ T or NK cells is beneficial for anti-tumor function. Therefore, blocking NKG2A and HLA-E interactions can be performed by using anti-NKG2A antibodies that do not interact with human FcγRs.

[0295] In some embodiments, the anti-NKG2A antibodies described herein enhance the anti-tumor functional activity of T cells. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

[0296] a) Reversal of inhibition of NK-κB signaling in CHO / scOKT3 / HLA-E stimulated NKG2A-expressing Jurkat T cell line, EC 50 The value is about 0.2 nM or lower.

[0297] b) NKG2A isolated from healthy donor PBMC in co-culture with CHO / scOKT3 / HLA-E / PD-L1 + In CD8 T cells, interferon-γ (IFN-γ) is induced.

[0298] c) NKG2A isolated from human tumors in co-culture with CHO / scOKT3 / HLA-E / PD-L1 + In CD8 T cells, IFN-γ is induced.

[0299] In some embodiments, the anti-NKG2A antibodies described herein enhance the anti-tumor functional activity of NK cells. Specifically, in some embodiments, the anti-NKG2A antibodies exhibit one or more of the following properties:

[0300] a) Increased IFN-γ production in NKL cells co-cultured with CHO / MICA / HLA-E.

[0301] b) Induces a dose-dependent increase in NK cell degranulation and lysis of HLA-E expressing tumor cells.

[0302] In some embodiments, the anti-NKG2A antibodies described herein are internalized after binding to cells expressing NKG2A. Specifically, in some embodiments, the anti-NKG2A antibodies are internalized at an EC of about 0.5 nM or less. 50 In some embodiments, the anti-NKG2A antibody is internalized at an EC of about 0.5 nM or less. 50 Exhibits dose-dependent internalization.

[0303] In some embodiments, the anti-NKG2A antibodies of the invention do not have sequence disadvantages that reduce the chemical stability of the antibody.The anti-NKG2A antibodies of the invention have various important uses, such as for the treatment and / or diagnosis of cancer and other disorders associated with NKG2A expression and / or activity.

[0304] In some embodiments, the anti-NKG2A antibodies disclosed herein bind to a specific epitope on human NKG2A via amino acid sequence, as described in Example 4.

[0305] Binding to human NKG2A can be assessed using one or more techniques established in the art. For example, in some embodiments, the antibodies are tested by flow cytometry analysis, wherein the antibodies react with a cell line expressing human NKG2A, such as CHO cells that have been transfected to express human NKG2A on their cell surface. Additionally or alternatively, antibody binding can be tested in a Biacore binding assay, including binding kinetics (e.g., K D Other suitable binding assays include ELISA assays using, for example, recombinant human NKG2A protein.

[0306] In some embodiments, the anti-NKG2A antibodies or antigen-binding fragments thereof described herein bind with high affinity, such as nanomolar affinity, including 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or lower, or 10 -10 M or lower K D , binds to human NKG2A protein.

[0307] Some embodiments of the invention relate to an anti-NKG2A monoclonal antibody, or antigen binding portion thereof, that specifically binds to an epitope located within a discrete region (as determined by HDX-MS and / or FPOP epitope mapping) comprising the following amino acid residues:

[0308] Region 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2 (native hNKG2A amino acid sequence));

[0309] Region 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2);

[0310] Region 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2);

[0311] Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and

[0312] Zone 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2).

[0313] In some embodiments, the invention relates to an anti-NKG2A monoclonal antibody, or antigen binding portion thereof, that specifically binds to an epitope located within a discrete region comprising the following amino acid residues (as determined by HDX-MS):

[0314] Region 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2);

[0315] Region 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2);

[0316] Region 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2); and

[0317] Zone 5: 212QVNRLKSAQCGSSIIYHC 229 (Amino acid residues 212 to 229 of SEQ ID NO: 2.

[0318] In some embodiments, the anti-NKG2A antibodies described herein enhance NK cell function by blocking NKG2A / HLA-E mediated inhibition. In another embodiment, the anti-NKG2A antibody binds to the human NKG2A protein and stimulates an anti-tumor immune response, such as an antigen-specific T cell and / or NK cell response. The ability of an anti-NKG2A antibody to stimulate an immune response can be tested by measuring tumor growth, such as in an in vivo tumor transplantation model, as described in the Examples herein. In other embodiments, an anti-NKG2A antibody or an antigen-binding portion thereof increases cytokine production (e.g., interferon-γ (IFN-γ)) and / or increases T cell proliferation in T cells expressing NKG2A, including effector T cells and cytotoxic T cells (also known as CD8 + T cells).

[0319] In another embodiment, the anti-NKG2A antibody or antigen-binding fragment thereof binds to human NKG2A and exhibits at least one of the following properties:

[0320] a) binds to one or more of the following residues (as determined by HDX-MS and / or FPOP epitope mapping):

[0321] Region 1: 155 LSIDNEEEMKF 165 (amino acid residues 155 to 165 of SEQ ID NO: 2 (native hNKG2A amino acid sequence);

[0322] Region 2: 171 PSSWIGVFRNSSHHPW 186 (amino acid residues 171 to 186 of SEQ ID NO: 2);

[0323] Region 3: 192 LAFKHEIKDSDN 203 (amino acid residues 192 to 203 of SEQ ID NO: 2);

[0324] Region 4: L (amino acid residue 206 of SEQ ID NO: 2); and

[0325] Zone 5: 212 QVNRLKSAQCGSSIIYHC 229 (amino acid residues 212 to 229 of SEQ ID NO: 2);

[0326] b) binds to the same epitope on human NKG2A as the NKG2A.11 and 13F3.A4 antibodies;

[0327] c) compete with NKG2A.11 and 13F3.A4 antibodies for binding to human NKG2A;

[0328] d) As measured by Biacore, with an EC of approximately 0.4 nM 50 Binds to human NK cells;

[0329] e) As measured by Biacore, with an IC of approximately 0.3 nM 50 Block the binding of human NK cells to HLA-E;

[0330] f) with an EC of about 1 nM or lower 50 Binds to CHO cells expressing macaque NKG2A;

[0331] g) with an EC of about 9.0 nM or higher 50 Binds to human NKG2C at a low level (in other words, the anti-NKG2A antibody does not block the interaction between human NKG2C and HLA-E); and / or

[0332] h) Enhance the anti-tumor response of CD8+ T cells and NK cells. For example:

[0333] i. Increase IFN-γ production in primary T:CHO-OKT3-HLA-E-PDL1 analysis;

[0334] ii. Increase IFN-γ production in T cell tumor infiltrating lymphocytes (TIL): CHO-OKT3-HLA-E-PDL1 assay; and / or

[0335] iii. Increased cytotoxicity and IFN-γ production in primary NK cell assays.

[0336] In some embodiments, the anti-NKG2A antibodies of the invention include humanized and fully human monoclonal antibodies. In other embodiments, the antibody is, for example, a chimeric monoclonal antibody.

[0337] a. Anti-NKG2A monoclonal antibody

[0338] In some embodiments, the antibodies of the invention are monoclonal antibodies 13F3.A4, NKG2A.9 and NKG2A.11, which were isolated and structurally characterized as described in the Examples below. The VH and VL amino acid sequences are shown in the Sequence Table and Sequence Listing.

[0339] Given that each of these antibodies can bind to human NKG2A, the VH and VL sequences can be "mixed and matched" to generate other anti-hNKG2A binding molecules of the present invention. In some embodiments, when the VH and VL chains are mixed and matched, the VH sequence 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. Therefore, in one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a human NKG2A protein, wherein the light chain and heavy chain variable regions comprise:

[0340] (a) the amino acid sequences of SEQ ID NOs: 9 and 8, respectively;

[0341] (b) the amino acid sequences of SEQ ID NOs: 164 and 8, respectively; or

[0342] (c) the amino acid sequences of SEQ ID NOs: 169 and 167, respectively.

[0343] In another aspect, the present invention provides antibodies comprising the heavy and light chain CDR1, CDR2 and CDR3 of NKG2A.9, NKG2A.11 and 13F3A.4 antibodies. Thus, in one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a human NKG2A protein, wherein the antibody comprises:

[0344] (a) a heavy chain variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and a light chain variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively;

[0345] (b) a heavy chain variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and a light chain variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 154, 14 and 15, respectively; or

[0346] (c) a heavy chain variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and a light chain variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 155, 14 and 15, respectively.

[0347] It is well known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can independently determine the binding specificity of an antibody for a cognate antigen, and that multiple antibodies with the same binding specificity can be predictably generated based on a common CDR3 sequence. (See, e.g., Klimka et al., British J. of Cancer 83(2):252-260 (2000). Thus, the present invention provides monoclonal antibodies comprising one or more heavy chain and / or light chain CDR3 domains derived from an antibody of a human or non-human animal, wherein the monoclonal antibodies are capable of specifically binding to human NKG2A. In certain aspects, the present invention provides monoclonal antibodies comprising one or more heavy chain and / or light chain CDR3 domains from a non-human antibody, wherein the monoclonal antibodies are capable of specifically binding to human NKG2A. In some embodiments, such antibodies of the present invention comprising one or more heavy chain and / or light chain CDR3 domains from a non-human antibody, relative to the corresponding parent non-human antibody, (a) are capable of competing for binding; (b) retain functional properties; (c) bind to the same epitope; and / or

[0348] (d) have similar binding affinities.

[0349] In other aspects, the present invention provides monoclonal antibodies comprising one or more heavy chain and / or light chain CDR3 domains from a human antibody (such as a human antibody obtained from a non-human animal), wherein the human antibody is capable of specifically binding to human NKG2A. In other aspects, the present invention provides monoclonal antibodies comprising one or more heavy chain and / or light chain CDR3 domains from a first human antibody (such as a human antibody obtained from a non-human animal), wherein the first human antibody is capable of specifically binding to human NKG2A, and wherein the CDR3 domain from the first human antibody replaces the CDR3 domain in a human antibody lacking binding specificity for NKG2A to produce a second human antibody capable of specifically binding to human NKG2A. In some embodiments, such antibodies of the present invention comprising one or more heavy chain and / or light chain CDR3 domains from a first human antibody, relative to the corresponding parent non-human antibody, (a) are capable of competing for binding; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity.

[0350] In some embodiments, the present invention provides anti-hNKG2A antibodies with inert Fc human IgG1.3 as isotype. In some embodiments, such anti-hNKG2A antibodies with inert Fc exhibit superior efficacy in treating cancer compared to other isotypes.

[0351] b. Antibodies with conservative modifications

[0352] In certain embodiments, the anti-NKG2A antibodies of the present invention contain 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 the CDR sequences comprises a specified amino acid sequence or conservative modifications thereof based on the antibodies described herein (e.g., 13F3.A4, NKG2A.9 and NKG2A.11 antibodies), and wherein the antibodies retain the desired functional properties of the anti-hNKG2A antibodies of the present invention. It is understood in the art that certain conservative sequence modifications that do not remove antigen binding may be made. (See, e.g., Brummell et al. (1993) Biochem 32: 1180-8). Thus, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein:

[0353] (a) the heavy chain variable region comprises a CDR3 sequence comprising the amino acid sequence shown in SEQ ID NO: 12 or a conservative modification thereof; and

[0354] (b) The antibody or antigen-binding portion thereof specifically binds to human NKG2A.

[0355] In other embodiments, the antibodies have 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.

[0356] In some embodiments, the heavy chain variable region comprises a CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 11 or a conservative modification thereof; and the light chain variable region comprises a CDR2 sequence comprising the amino acid sequence shown in SEQ ID NO: 14 or a conservative modification thereof. In another embodiment, the heavy chain variable region comprises a CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 10 or a conservative modification thereof; and the light chain variable region comprises a CDR1 sequence comprising the amino acid sequence shown in SEQ ID NO: 13, 154 or 155 or a conservative modification thereof.

[0357] In various embodiments, the anti-NKG2A antibody is, for example, a human antibody, a humanized antibody, or a chimeric antibody. c. Antibodies that bind to the same epitope as the anti-hNKG2A antibody

[0358] In another embodiment, the present invention provides an antibody that binds to the same epitope on a human NKG2A protein as any of the anti-hNKG2A monoclonal antibodies of the present invention (i.e., an antibody that has the ability to cross-compete with any of the monoclonal antibodies of the present invention for binding to a human NKG2A protein). In some embodiments, the reference antibodies used for cross-competition studies in a standard human NKG2A binding assay are monoclonal antibodies NKG2A.9, NKG2A.11, and 13F3.A4. For example, a standard ELISA assay can be used in which a recombinant human NKG2A protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and the ability of an unlabeled antibody to compete with the labeled antibody for binding is assessed. Additionally or alternatively, a Biacore assay can be used to assess the ability of antibodies to cross-compete. The ability of the test antibody to inhibit, for example, the binding of NKG2A.9, NKG2A.11 and / or 13F3.A4 to human NKG2A indicates that the test antibody is able to compete with NKG2A.9, NKG2A.11 and / or 13F3.A4 for binding to human NKG2A, and thus binds to the same epitope on human NKG2A.9 as NKG2A.9, NKG2A.11 and / or 13F3.A4. In some embodiments, the antibody that binds to the same epitope on human NKG2A as NKG2A.9, NKG2A.11 and / or 13F3.A4 is a humanized or human monoclonal antibody.

[0359] As further discussed in Example 4, the binding of NKG2A.9 and 13F3.A4 has been mapped to specific residues. Thus, in one embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that, when bound to human NKG2A protein, specifically binds to the following amino acid residues (as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS)):

[0360] (e) LSIDNEEMKF (SEQ ID NO: 156);

[0361] (f) PSSWIGVFRNSSHHPW (SEQ ID NO: 157);

[0362] (g) LAFKHEIKDSDN (SEQ ID NO: 158); and

[0363] (h)QVNRLKSAQQCGSSIIYHC (SEQ ID NO:159),

[0364] The monoclonal antibody blocks the binding of NKG2A ligand (eg, HLA-E in humans) to human NKG2A protein.

[0365] In another embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, which, when bound to human NKG2A, specifically binds to the following amino acid residues (as determined by HDX-MS and / or protein fast photochemical oxidation (FPOP) epitope mapping):

[0366] (f) LSIDNEEMKF (SEQ ID NO: 156);

[0367] (g) PSSWIGVFRNSSHHPW (SEQ ID NO: 157);

[0368] (h)LAFKHEIKDSDN (SEQ ID NO: 158);

[0369] (i) L; and

[0370] (j)QVNRLKSAQQCGSSIIYHC (SEQ ID NO:159),

[0371] The monoclonal antibody blocks the binding of NKG2A ligand (eg, HLA-E in humans) to human NKG2A protein.

[0372] Such human or humanized 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 produced using immunization protocols, such as those described herein. The resulting antibodies can be screened for high affinity binding to human NKG2A. The selected antibodies can then be studied in, for example, yeast display assays, where sequence variants of hNKG2A are presented on the surface of yeast cells, or by hydrogen-deuterium exchange experiments and / or FPOP to determine the precise epitope to which the antibody binds.

[0373] Epitope determination can be performed by any method known in the art. In some embodiments, an anti-hNKG2A antibody is considered to bind to the same epitope as an anti-hNKG2A mAb disclosed herein if they contact one or more of the same residues within at least one region of hNKG2A; or if they contact the majority of residues within at least one region of hNKG2A; or if they contact the majority of residues within each region of hNKG2A; or if they contact the majority of contact residues over the entire length of hNKG2A; or if they contact the exact same unique region of hNKG2A; or if they contact all residues of any region on hNKG2A; or if they contact the exact same residues in the exact same region. An epitope "region" is a cluster of residues along the primary sequence, but not necessarily directly adjacent within the primary sequence.

[0374] Techniques for determining antibodies that bind to the same epitope on hNKG2A as the antibodies described herein include x-ray analysis of crystals of the antigen: antibody complex, which provides atomic resolution of the epitope. Other methods monitor the binding of antibodies to antigen fragments or mutant variant forms of the antigen, where the loss of binding caused by amino acid modifications within the antigen sequence indicates the epitope component. The method may also rely on the ability of the target antibody to affinity isolate specific short peptides (in native three-dimensional form or in denatured form) from a combinatorial phage display peptide library or from a protease digest of the target protein. These peptides are considered to be leads for defining the epitopes corresponding to the antibodies that screen the peptide library. For epitope mapping, computational algorithms have also been developed, which have been shown to map conformationally discontinuous epitopes.

[0375] The epitope or region comprising the epitope can also be identified by screening for binding to a series of overlapping peptides across NKG2A. Alternatively, the method of Jespers et al. (1994) Biotechnology 12:899 can be used to guide the selection of antibodies having the same epitope and therefore similar properties as the anti-NKG2A antibodies described herein. Using phage display, first, the heavy chain of the anti-NKG2A antibody is paired with a (e.g., human) light chain library to select NKG2A binding antibodies, and then the new light chain is paired with a (e.g., human) heavy chain library 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.

[0376] Alanine scanning mutagenesis, as described by Cunningham & Wells, Science 244: 1081 (1989), or some other form of amino acid residue point mutagenesis in NKG2A, can also be used to determine the functional epitopes of anti-NKG2A antibodies.

[0377] The epitope or epitope region bound by a specific antibody (an "epitope region" is a region that includes or overlaps with an epitope) can also be determined by evaluating the binding of the antibody to a peptide comprising a fragment of NKG2A. A series of overlapping peptides covering the sequence of NKG2A (e.g., human NKG2A) can be synthesized and screened for binding properties, for example, in a direct ELISA, a competitive ELISA (wherein the peptides are evaluated for their ability to prevent the binding of the antibody to NKG2A bound to a microtiter plate well), or on a chip. Such peptide screening methods may not be able to detect some discontinuous functional epitopes, i.e., functional epitopes involving discontinuous amino acid residues in the primary sequence of the NKG2A polypeptide chain.

[0378] Epitopes can also be identified by MS-based protein footprinting methods, such as HDX-MS and fast photochemical oxidation of proteins (FPOP). HDX-MS can be performed, for example, as further described in Wei et al. (2014) Drug Discovery Today 19:95, the methods of which are specifically incorporated herein by reference. FPOP can be performed, for example, as further described in Hambley and Gross (2005) J. American Soc. Mass Spectrometry 16:2057, the methods of which are specifically incorporated herein by reference.

[0379] The epitope bound by an anti-NKG2A antibody can also be determined by structural methods such as X-ray crystallography (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the HD exchange rate of labile amide hydrogens in NKG2A when free and in a complex with the antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31: 11335; Zinn-Justin et al. (1993) Biochemistry 32: 6884).

[0380] Unless otherwise specified, with respect to the claims, the epitope to which the antibody binds is the epitope determined by the HDX-MS method.

[0381] Anti-NKG2A antibody that binds with high affinity

[0382] In some embodiments, the anti-hNKG2A antibodies of the invention bind to hNKG2A with high affinity, making them effective therapeutic agents. In various embodiments, the anti-hNKG2A antibodies of the invention bind to hNKG2A with a K of less than 10 nM, 5 nM, 2 nM, 1 nM, 300 pM, or 100 pM. D Binding to hNKG2A. Standard assays to assess the ability of antibodies to bind to hNKG2A include ELISA, RIA, Western blot, biolayer interferometry (BLI), and Biacore. TM SPR analysis (see Example 10).

[0383] d. Anti-NKG2A antibody sequence variants

[0384] The anti-NKG2A antibody sequence variants disclosed herein retain the desired functional properties disclosed herein. Unless otherwise specified, CDR regions are delimited using the Kabat system (Kabat et al., 1991). In some embodiments, the present invention further provides human or humanized anti-hNKG2A antibodies comprising CDR sequences that are at least 70%, 75%, 80%, 85%, 90% or 95%, 96%, 97%, 98%, 99% or 99.5% identical to the CDR sequences of the antibodies disclosed herein. The present invention also provides anti-hNKG2A antibodies comprising heavy chain and / or light chain variable region 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 region sequences of the antibodies disclosed herein, as well as 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 the antibodies disclosed herein.

[0385] II. Engineered and Modified Antibodies

[0386] aV H and V L district

[0387] Also provided are engineered and modified antibodies that can be used with V sequences disclosed herein. H and / or V L In some embodiments, antibodies as described herein are prepared by modifying one or more of the variable regions (i.e., V H and / or V L ), such as one or more CDR regions and / or one or more framework regions. Additionally or alternatively, an antibody as described herein is engineered by modifying residues in the constant region, e.g., to alter the effector function(s) of the antibody.

[0388] In one embodiment, variable region engineering includes CDR transplantation. Such transplantation is particularly useful for humanized 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 epitope as the selected 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 hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Expression vectors can be constructed to include CDR sequences from a particular reference (also referred to as a "parent") antibody, wherein the CDR sequences are grafted onto framework sequences from a different antibody (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. USA 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.). In some cases, the resulting recombinant antibody has properties similar to those of the parent antibody. The engineered antibody can then be further modified to obtain properties different from those of the parent antibody. In other cases, the parent CDR sequence is transplanted onto the framework to eliminate certain features of the parent antibody, so that the recombinant antibody no longer has these features. An exemplary feature is the binding affinity for the antigen. In such cases, it may be advantageous to further modify the engineered antibody to restore the desired parent antibody features.

[0389] Such framework sequences can be obtained from public DNA databases or published documents that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and 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 DifferentHypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V HSegments Reveals a Strong Bias in their Usage", Eur. J. Immunol. 24: 827-836; the contents of each of the above documents are expressly incorporated herein by reference.

[0390] In some embodiments, the framework sequences used for the antibodies described herein are framework sequences that are structurally similar to the framework sequences used for the antibodies described herein. H CDR1, 2 and 3 sequences and V L CDR1, 2 and 3 sequences can be transplanted onto framework regions having the same sequence as that found in the germline immunoglobulin gene from which the framework sequences were derived, or CDR sequences can be transplanted onto framework regions that contain up to 20 amino acid substitutions, including conservative amino acid substitutions, compared to the germline sequences. For example, it has been found that in certain cases it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0391] The engineered antibodies described herein include antibodies in which V H and / or V L In some embodiments, the invention relates to modifying the framework residues within an antibody, for example, to improve the properties of the antibody, for example, to reduce the immunogenicity of the antibody. For example, one method is to "revert" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is obtained. In order to restore the framework region sequence to its germline configuration, the somatic mutation can be "reverted" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "reverted" antibodies are also included in the present invention.

[0392] Another type of framework modification involves mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is further described in detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0393] Another type of variable region modification is to mutate the amino acid residues in the CDR region to improve one or more binding properties (e.g., affinity) of the target antibody. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce (one or more) mutations and affect antibody binding or other functional properties of interest. Preferably, conservative modifications are introduced. Mutations can be amino acid additions, deletions, or substitutions. In some embodiments, no more than one, two, three, four, or five residues in the CDR region are changed.

[0394] The methionine residues in the CDR of the antibody can be oxidized, causing potential chemical degradation and thus reducing the antibody potency. Therefore, anti-NKG2A antibodies are also provided herein, wherein one or more methionine residues in the heavy chain and / or light chain CDR are replaced with amino acid residues that do not undergo oxidative degradation. Similarly, deamidation sites, especially deamidation sites in CDRs, can be removed from anti-NKG2A antibodies. Antibodies are also provided herein, wherein potential glycosylation sites in the antigen binding domain have been eliminated to prevent glycosylation that may interfere with antigen binding. See, e.g., U.S. Patent No. 5,714,350.

[0395] b. Antibody masking

[0396] In some embodiments, the antibodies disclosed herein are modified to limit their binding to specific cells and / or tissues. In one embodiment, such antibodies comprise 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 connected to each binding arm of the antibody via a protease cleavable linker. See, e.g., U.S. Patent No. 8,518,404 to CytomX. Antibodies with protease cleavable links are useful for treating cancer, where the level of proteases in the tumor microenvironment is greatly increased compared to non-tumor tissues. Selective cleavage of cleavable links in the tumor microenvironment allows the mask / blocking peptide to separate, so that antigen binding can occur selectively in the tumor, rather than in surrounding tissues where antigen binding may cause unwanted side effects.

[0397] In another embodiment, a bivalent binding compound ("shielding ligand") comprising two antigen binding domains is produced that binds to the two antigen binding surfaces of a (bivalent) antibody and interferes with antigen binding. In one embodiment, the two binding domain shields are connected to each other (but not to the antibody) by a cleavable linker (e.g., a linker cleavable by a peptidase). (See, e.g., WO 2010 / 077643 of Tegopharm Corp). The shielding ligand may comprise or be derived from the antigen to which the antibody is intended to bind, or may be produced independently (e.g., an anti-idiotypic binding fragment). Such shielding ligands may be used to treat cancer, where the levels of proteases in the tumor microenvironment are greatly increased compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to separate from each other, reducing the affinity (avidity) for the antigen binding surface of the antibody. The resulting dissociation of the shielding ligand from the antibody allows antigen binding to occur selectively in the tumor, but not in surrounding tissues where antigen binding may cause unwanted side effects.

[0398] c. Fc and modified Fc regions

[0399] In one embodiment, the antibodies described herein may comprise an Fc region selected based on the biological activity of the antibody. Salfeld, Nat. Biotechnol. 25: 1369 (2007). For example, human IgG can be divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. The Fc region contained in each of these subclasses has the unique property of 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)) and the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA H131 , and binds to FcγRIIA with lower affinity R131 , FcγRIIIA V158 IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158; and the inhibitory receptor FcγRIIB has lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. (Bruhns et al. (2009) Blood 113:3716). Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Ibid. In general, with respect to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2. In some embodiments, the IgG1 constant domain is selected rather than IgG2 or IgG4, for example for therapeutic compositions, because ADCC is desired.

[0400] The anti-hNKG2A antibody variable regions described herein can be linked (e.g., covalently linked or fused) to an Fc, such as an IgG1, IgG2, IgG3 or IgG4 Fc, which can be of any allotype or isoallotype, such as for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for 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, eg, Jefferis et al. (2009) mAbs 1:1.) The choice of allotype may be influenced by potential immunogenicity issues, for example to minimize the formation of anti-drug antibodies.

[0401] In some embodiments, the anti-NKG2A antibodies of the present invention cannot interact with human FcγR. Since NKG2A is an inhibitory receptor expressed on CD8+ T and NK cells, avoiding or reducing NKG2A + Stimulation of CD8+ T or NK cells or depletion of these cells can enhance anti-tumor immunity. Therefore, it is desirable to block the NKG2A / HLA-E interaction using anti-NKG2A antibodies that are unable to interact with human FcγRs.

[0402] d. Half-life extension

[0403] In some embodiments, the anti-NKG2A antibody is modified to increase its biological half-life, such as the half-life of the antibody in serum. Various methods are known in the art. In one embodiment, the antibody is changed in the CH1 or CL region to include a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. For example, a combined Fc variant comprising M252Y, S254T and T256E increases the half-life by nearly four times. (Dall'Acqua et al. (2006) J. Biol. Chem. 281: 23514). Other modifications that increase FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO 97 / 34631; WO 2002 / 060919.

[0404] The serum half-life of the antibodies described herein can also be increased by pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. 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 in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any PEG form that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Protein pegylation methods are known in the art and can be applied to the antibodies described herein. (See, e.g., EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0405] In some cases, it may be desirable to reduce the half-life of an antibody rather than increase it. In some embodiments, the antibodies described herein include modifications to reduce their half-life. Modifications in the Fc of human IgG1, 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) can reduce FcRn binding, thereby reducing half-life (increasing clearance rate) for use in situations where rapid clearance is preferred, such as medical imaging. (See also Kenanova et al. (2005) Cancer Res. 65: 622). Other ways to enhance clearance include making the antigen binding domain of the present invention into an antibody fragment form that lacks 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 weeks to hours. Selective pegylation of antibody fragments can then be used to increase the half-life of the antibody fragments if desired. (Chapman et al. (1999) Nat. Biotechnol. 17:780). Antibody fragments can also be fused to human serum albumin (e.g., in a fusion protein construct) to increase half-life. (Yeh et al. (1992) Proc. Nat'l Acad. Sci. 89:1904). Alternatively, bispecific antibodies can be constructed with a first antigen binding domain of the invention and a second antigen binding domain that binds to human serum albumin (HSA). (See WO 2009 / 127691 and patent references cited therein). Alternatively, specialized polypeptide sequences can be added to antibody fragments to increase half-life, such as "XTEN" polypeptide sequences. (Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; International Patent Application Publication WO 2010 / 091122).

[0406] e. Other Fc variants

[0407] In some embodiments, when an IgG1 constant domain is used, the potential protease cleavage site in the hinge of the IgG1 construct can be eliminated by D221G and K222S modifications, thereby increasing the stability of the antibody. (WO 2014 / 043344).

[0408] The affinity and binding properties of an Fc variant for its ligand (Fc receptor) can be determined by a variety of in vitro assays known in the art (e.g., biochemical or immunological based assays), including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetic methods (e.g., 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 use labels on one or more of the components of the assay and / or employ various detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th edition, Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0409] In other embodiments, the glycosylation of the antibody is modified to increase or decrease effector function. For example, an aglycosylated antibody lacking all effector function can be prepared by mutating the conserved asparagine residue at position 297 (e.g., N297A), thereby eliminating complement and FcγRI binding. (Bolt et al. (1993) Eur. J. Immunol. 23: 403; see also Tao and Morrison (1989) J. Immunol. 143: 2595 (using N297Q in IgG1 to eliminate glycosylation at position 297)).

[0410] Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore this function. Aglycosylated antibodies, such as those produced with N297A / C / D / or H mutations or produced in a system that does not glycosylate proteins (e.g., E. coli), can be further mutated to restore FcγR binding, such as S298G and / or T299A / G / or H (WO 2009 / 079242), or E382V and M428I (Jung et al. (2010) Proc. Nat'l Acad. Sci. (USA) 107: 604).

[0411] Glycosyl engineering can also be used to modify the anti-inflammatory properties of IgG constructs by changing the α2,6 sialic acid content in the carbohydrate chain at Asn297 of the Fc region, where an increased proportion of α2,6 sialylated forms results in an enhanced anti-inflammatory effect. (See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513). Conversely, a reduced proportion of antibodies with α2,6 sialylated carbohydrates can be used in situations where anti-inflammatory properties are not desired. U.S. Patent Application Publication No. 2008 / 0206246 provides, for example, methods for changing the α2,6 sialylation content of an antibody by selectively purifying the α2,6 sialylated form or by enzymatic modification. In other embodiments, the amino acid sequence of the Fc region can be modified to mimic the effects of α2,6 sialylation, for example by including a F241A modification. (WO 2013 / 095966).

[0412] III. Antibody Physical Properties

[0413] In certain embodiments, the antibodies described herein contain one or more glycosylation sites in the light chain or heavy chain variable region. Such glycosylation sites may increase the immunogenicity of the antibody or alter the pharmacokinetics of the antibody (due to altered antigen binding) (Marshall et al. (1972) Ann. Rev. Biochem. 41: 673-702; Gala and Morrison (2004) J. Immunol. 172: 5489-94; Wallick et al. (1988) J. Exp. Med. 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. (1985) Nature 316: 452-7; Mimura et al. (2000) Mol Immunol 37: 697-706). Glycosylation is known to occur at a motif containing an NXS / T sequence. In some embodiments, the anti-hNKG2A antibody lacks variable region glycosylation. Such antibodies can be obtained by selecting antibodies that do not have glycosylation motifs in the variable region or by mutating residues within the glycosylation region.

[0414] In certain embodiments, the antibodies described herein do not contain an asparagine isomerism site. Deamidation of asparagine can occur on NG or DG sequences and result in the generation of an isoaspartic acid residue, which introduces a kink into the polypeptide chain, reducing its stability (known as the isoaspartic acid effect).

[0415] In some embodiments, the antibodies described herein have an isoelectric point (pi) within a pH range between 6 and 9.5. In some embodiments, the antibodies described herein have a pi within a pH range of 7-9.5 or 6-8. Antibodies with a pi within a desired pi range can be obtained by selecting an antibody with a pi within that pH range from a panel of candidates or by mutating charged surface residues of a particular antibody.

[0416] In some embodiments, the antibodies described herein are selected and / or engineered to have an onset unfolding temperature (T M1 The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al. (2003) Pharm Res 20: 1952-60; Ghirlando et al. (1999) Immunol Lett. 68: 47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr. Sci. 40: 343-9).

[0417] In some embodiments, the antibodies described herein are selected and / or engineered to have favorable degradation properties, such as slow degradation in vitro and / or in vivo. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem. 67: 3626-32). In some embodiments, the antibodies described herein are selected and / or engineered to have favorable aggregation properties, such as antibodies that show minimal in vitro and / or in vivo aggregation, wherein the aggregation can trigger an unwanted immune response and / or alter or adverse pharmacokinetic properties. In some embodiments, the antibodies described herein show 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less aggregation compared to the aggregation of the parent antibody. Aggregation can be measured by several techniques, including size exclusion columns (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0418] IV. Nucleic Acid Molecules and Recombinant Methods

[0419] Another aspect described herein relates to nucleic acid molecules encoding anti-hNKG2A antibodies described herein. The nucleic acid may be present in whole cells (e.g., host cells), cell lysates, or in partially purified or substantially pure form. The nucleic acid is "isolated" or "appears substantially pure" when it is separated from other cellular components or other impurities, such as other cellular nucleic acids (e.g., other chromosomal DNA, such as chromosomal DNA that is connected to the isolated DNA in nature) or protein purification by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. (See F. Ausubel et al., eds. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). The nucleic acid described herein may, for example, be DNA or RNA and may or may not contain introns. In certain embodiments, the nucleic acid is a cDNA molecule.

[0420] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes as further described below), cDNAs encoding the light and / or heavy chains of the antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding antibodies can be recovered from the libraries.

[0421] Once the code V is obtained H and V L The DNA fragments of the V region can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or a scFv gene. L or V H A DNA fragment encoding a protein (such as an antibody constant region or a flexible linker) is operably linked to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). As used in this context, the term "operably linked" means that the two DNA fragments are linked in such a way that the amino acid sequences encoded by the two DNA fragments remain in the same frame.

[0422] Code V H The isolated DNA encoding the V HThe DNA of the heavy chain constant region can be operably linked to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2 and / or CH3) to convert into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al., 1991), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG (IgG1, IgG2, IgG3 or IgG4), IgA, IgE, IgM or IgD constant region, such as the IgG1 region. For the Fab fragment heavy chain gene, the V H The DNA is operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0423] Code V L The isolated DNA encoding the V L The DNA of the light chain constant region CL can be operably connected to another DNA molecule encoding the light chain constant region CL, and converted into a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is 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 covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0424] To generate scFv genes, the V H and V L The DNA fragment is operably linked to a flexible linker encoding an amino acid sequence (Gly 4 -Ser) 3 (SEQ ID NO: 160), such that V H and V L The sequence can be expressed as a continuous single-chain protein, in which V L and V H The regions are connected by flexible linkers (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0425] V. Antibody Production

[0426] The various antibodies of the present invention, for example, antibodies that bind to the same epitope as the selected anti-hNKG2A antibodies disclosed herein, can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, phage display techniques using human antibody gene libraries, can also be used.

[0427] An exemplary animal system for preparing hybridomas is a murine system. The production of hybridomas in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0428] The chimeric or humanized antibodies described herein can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. Standard molecular biology techniques can be used to obtain DNA encoding heavy and light chain immunoglobulins from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to produce chimeric antibodies, methods known in the art can be used to connect mouse variable regions to human constant regions (see, e.g., U.S. Pat. No. 4,816,567 of Cabilly et al.). In order to produce humanized antibodies, methods known in the art can be used to insert mouse CDR regions into human frameworks (see, e.g., U.S. Pat. No. 5,225,539 of Winter, and U.S. Pat. No. 5,530,101 of Queen et al.; No. 5,585,089; No. 5,693,762 and No. 6,180,370).

[0429] In one embodiment, the antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies against human NKG2A can be produced using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, which are collectively referred to herein as "human Ig mice."

[0430] HuMAb (Medarex, Inc.) contains a human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al., 1994 Nature 368(6474):856-859). Thus, the mice exhibit reduced mouse IgM or κ expression, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications carried by such mice, are further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of which are hereby expressly incorporated by reference in their entirety.(See also, U.S. Pat. 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 to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962 to Lonberg and Kay; and PCT Publication Nos. WO 99 / 45963 to Korman et al. 01 / 14424).

[0431] In certain embodiments, the antibodies described herein are produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying human heavy chain transgenes and human light chain transchromosomes. Such mice (referred to herein as "KM mice") are described in detail in PCT Publication No. WO 02 / 43478 by Ishida et al.

[0432] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are also 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 in, for example, U.S. Pat. Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[0433] In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are also available in the art and can be used to generate anti-NKG2a antibodies described herein. For example, mice carrying human heavy chain transchromosomes and human light chain transchromosomes, called "TC mice," can be used; such mice are described in Tomizuka et al., (2000) Proc. Natl. Acad. Sci. USA 97: 722-727. In addition, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894) and can be used to generate anti-hNKG2A antibodies described herein.

[0434] Other mouse systems described in the art for producing human antibodies, such as human anti-hNKG2A antibodies, include: (i) mice (Regeneron Pharmaceuticals, Inc.) in which the endogenous mouse heavy and light chain variable regions have been replaced by homologous recombination with human heavy and light chain variable regions operably linked to endogenous mouse constant regions, thereby generating chimeric antibodies (human V / mouse C) in the mouse, which can then be subsequently converted to fully human antibodies using standard recombinant DNA techniques; and (ii) Mouse (Merus Biopharmaceuticals, Inc.), wherein the mouse contains an unrearranged human heavy chain variable region and a single rearranged human common light chain variable region. Such mice and uses for producing antibodies are described in, for example, WO 2009 / 15777, US 2010 / 0069614, WO 2011 / 072204, WO 2011 / 097603, WO 2011 / 163311, WO 2011 / 163314, WO 2012 / 148873, US 2012 / 0070861, and US 2012 / 0073004.

[0435] The human monoclonal antibodies described herein can also be prepared by screening human immunoglobulin gene libraries using phage display methods. Such phage display methods for isolating human antibodies are established in the art. (See, e.g., U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.).

[0436] The human monoclonal antibodies described herein can also be prepared using mice with severe combined immunodeficiency (SCID) in which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0437] Immunization

[0438] To generate fully human antibodies against human NKG2A, mice containing human immunoglobulin genes or transgenic or transchromosomal mice (e.g., HCo12, HCo7, or KM mice) can be immunized with purified or enriched preparations of NKG2A antigen and / or cells expressing NKG2A, as described, for example, by Lonberg et al. (1994) Nature 368(6474):856-859; Fishwild et al. (1996) Nature Biotechnology 14:845-851 and WO 98 / 24884 for other antigens. Alternatively, mice can be immunized with DNA encoding human NKG2A. Preferably, mice will be 6-16 weeks old at the time of the first infusion. For example, mice can be immunized intraperitoneally with purified or enriched preparations of recombinant human NKG2A antigen (e.g., 5 μg-50 μg). If immunization with a purified or enriched preparation of NKG2A antigen does not produce antibodies, mice can also be immunized with cells expressing NKG2A, such as a cell line, to boost the immune response.

[0439] HuMAb transgenic mice can first be immunized intraperitoneally or subcutaneously (SC) with antigen in Ribi's adjuvant, followed by IP / SC immunization every other week (up to a total of 10 times) with antigen in Ribi's adjuvant. The immune response can be monitored throughout the immunization regimen, wherein plasma samples are obtained by retroorbital bleeding. 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 antigen for three days, then sacrificed and the spleen and lymph nodes removed. For each immunization, two to three fusions can be performed. For each antigen, 6 to 24 mice can be immunized. In some embodiments, HCo7, HCo12 and KM strains are used. In addition, HCo7 and HCo12 transgenes can be cultivated into a single mouse, and the mouse will have two different human heavy chain transgenes (HCo7 / HCo12).

[0440] Generation of hybridomas producing monoclonal antibodies against NKG2A protein

[0441] To generate hybridomas that produce the monoclonal antibodies described herein, spleen cells and / or lymph node cells can be isolated from immunized mice and fused with an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single cell suspension of spleen lymphocytes from immunized mice can be fused with Sp2 / 0 non-secreting mouse myeloma cells (ATCC, CRL 1581) under 50% PEG. The cells are grown at approximately 2×10 5Plated in flat-bottom microtiter plates, then cultured for two weeks in a selection medium containing 10% fetal bovine serum, 18% "653" conditioned medium, 5% Origen (IGEN), 4mM L-glutamine, 1mM sodium pyruvate, 5mM HEPES, 0.055mM 2-mercaptoethanol, 50 units / ml penicillin, 50mg / ml streptomycin, 50mg / ml gentamicin and 1X HAT (Sigma). After approximately two weeks, cells can be cultured in a medium that replaces HAT with HT. Each well can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, the culture medium can usually be observed after 10-14 days. Antibody-secreting hybridomas can be re-plated, screened again, and if still human IgG-positive, monoclonal antibodies can be subcloned at least twice by limiting dilution. Stable subclones can then be cultured in vitro to produce small amounts of antibodies in tissue culture medium for characterization.

[0442] To purify monoclonal antibodies, selected hybridomas can be grown in two-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography with protein A-agarose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked 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. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0443] VI. Antibody Production

[0444] Generation of transfectomas producing monoclonal antibodies against NKG2A

[0445] The antibodies of the present invention, including the specific antibodies and other related anti-NKG2A antibodies for which sequences are provided, can be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

[0446] For example, in order to express an antibody or its antibody fragment, the DNA encoding part 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 target antibody), and the DNA can be inserted into an expression vector so that the gene is operably linked to the transcription and translation control sequence. In this context, the term "operably linked" is intended to mean that the antibody gene is linked to a vector so that the transcription and translation control sequences in the vector play their intended functions of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences compatible with the expression host cell used are selected. Antibody light chain genes and antibody heavy chain genes can be inserted into separate vectors, or two genes are inserted into the same expression vector. Antibody genes can be inserted into an expression vector by standard methods (e.g., connecting complementary restriction sites on an antibody gene fragment and a vector, or when no restriction sites are present, blunt end connection is performed). The light chain and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting the light chain and heavy chain variable regions of the antibodies described herein into expression vectors that already encode the heavy chain constant region and light chain constant region of the desired isotype, such that V H The fragment is operably linked to the C H Fragment and V L The fragment is operably linked to the C L Fragment. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0447] In addition to the antibody chain genes, the recombinant expression vector may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It should be understood by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on a variety of factors, such as the selection of the host cell to be transformed, the expression amount of the desired protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP), and polyoma virus. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the β-globin promoter. Still further, regulatory elements may be composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0448] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may carry other sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., origin of replication) and selectable marker genes. The selectable marker gene promotes the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017 to Axel et al.). For example, on host cells into which the vector has been introduced, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate. Exemplary selectable marker genes include dihydrofolate reductase (DHFR) genes (for dhfr-host cells and methotrexate selection / amplification) and neo genes (for G418 selection).

[0449] For the expression of light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into the host cells by standard techniques. The various forms of the term "transfection" encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although the antibodies described herein can be expressed in prokaryotic or eukaryotic host cells in theory, since eukaryotic cells and especially mammalian cells are more likely to assemble and secrete correctly folded and immunologically active antibodies than prokaryotic cells, it is most preferred that the antibodies be expressed in eukaryotic cells and most preferably in mammalian host cells. It has been reported that prokaryotic expression of antibody genes is inefficient for high yield 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 sugar-engineered yeast strains (Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24: 210).

[0450] Exemplary mammalian host cells for expressing the recombinant antibodies described herein include CHO cells (including dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a dihydrofolate reductase (DHFR) selectable marker, e.g., as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. Specifically, for use with NSO myeloma cells, another exemplary expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. Standard protein purification methods can be used to recover the antibody from the culture medium.

[0451] Due to commonly observed post-translational modifications, the N- and C-termini of the polypeptide chains of the antibodies of the invention may differ from the expected sequence. For example, the C-terminal lysine residue is often missing in the antibody heavy chain. (Dick et al. (2008) Biotechnol. Bioeng. 100: 1132). On the light and heavy chains of therapeutic antibodies, the N-terminal glutamine residue, and to a lesser extent the glutamic acid residue, is often converted to a pyroglutamic acid residue. (Dick et al. (2007) Biotechnol. Bioeng. 97: 544; Liu et al. (2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286: 11211).

[0452] The amino acid sequences of various anti-hNKG2A antibodies of the present invention are provided in the sequence listing. For the reasons described above, the C-terminal lysine is not included in many sequences of the heavy chain or heavy chain constant domain in the sequence listing. However, in alternative embodiments, the heavy chain of the anti-hNKG2A antibody of the present invention, and / or the genetic construct encoding such an antibody or its heavy chain or light chain, includes this additional lysine residue at the C-terminus of the heavy chain.

[0453] VII. Analytical Tests

[0454] The antibodies described herein can be tested for binding to NKG2A by, for example, standard ELISA. For example, microtiter plates are coated with 1-2 μg / ml of purified NKG2A in PBS, followed by blocking with PBS containing 5% bovine serum albumin. Antibody dilutions (e.g., plasma dilutions from mice immunized with NKG2A) are added to each well and incubated at 37°C for 1 to 2 hours. The plates are washed with PBS / Tween, followed by incubation with a secondary reagent conjugated to horseradish peroxidase (HRP) (e.g., a secondary reagent for human antibodies or antibodies with human heavy chain constant regions, a goat anti-human IgG Fc specific polyclonal reagent) at 37°C for 1 hour. After washing, the microtiter plates are developed with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by spectrophotometer at OD 415-495. Serum from immunized mice was then further screened by flow cytometry for binding to a cell line expressing human NKG2A, but not binding to a control cell line that does not express NKG2A. In short, the binding of anti-NKG2A antibodies was assessed by incubating CHO cells expressing NKG2A with anti-NKG2A antibodies diluted 1:20. The cells were washed and the binding was detected with anti-human IgG Ab labeled with PE. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Preferably, mice producing the highest titer will be used for fusion. If mouse anti-HNKG2A antibodies are to be detected, similar experiments can be performed using anti-mouse detection antibodies.

[0455] For example, as described above, ELISA can be used to screen for antibodies that display positive reactivity with the NKG2A immunogen, and thus screen for hybridomas that produce such antibodies. Hybridomas that produce antibodies that bind NKG2A (preferably with high affinity) can then be subcloned and further characterized. One clone can then be selected from each hybridoma that retains the reactivity of the parental cell (by ELISA) for making a cell bank and for antibody purification.

[0456] To purify anti-NKG2A antibodies, selected hybridomas can be grown in two-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated, followed by affinity chromatography using protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and HPLC to ensure purity. The buffer solution can be replaced with PBS, and the concentration can be determined by OD 280 The assay was performed using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80°C.

[0457] To determine whether the selected anti-NKG2A monoclonal antibodies bind to unique epitopes, each antibody can be biotin-labeled using commercially available reagents (Pierce, Rockford, IL). Biotinylated Mab binding can be detected using a streptavidin-labeled probe. Competition studies of unlabeled and biotinylated monoclonal antibodies can be performed as described above using NKG2A-coated ELISA plates.

[0458] To determine the isotype of purified antibodies, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, microtiter plate wells can be coated overnight at 4°C with 1 μg / ml anti-human immunoglobulin. After blocking with 1% BSA, the plate is reacted with 1 μg / ml or less of the test monoclonal antibody or purified isotype control at ambient temperature for one to two hours. The wells can then be reacted with a probe conjugated to human IgG1 or human IgM specific alkaline phosphatase. The plate is developed and analyzed as described above.

[0459] In order to test the binding of monoclonal antibodies to living cells expressing NKG2A, flow cytometry can be used. Briefly, a cell line expressing membrane-bound NKG2A (grown under standard growth conditions) is mixed with a certain concentration of monoclonal antibodies in PBS containing 0.1% BSA for one hour at 4°C. After washing, the cells are reacted with anti-IgG antibodies labeled with phycoerythrin (PE) under the same conditions as the primary antibody staining. The sample can be analyzed by a FACScan instrument, where single cells are gated using light scattering and side scattering properties, and the binding of the labeled antibody is determined. In conjunction with flow cytometry analysis or in place of flow cytometry analysis, an alternative analysis of fluorescence microscopy can be used. Cells can be stained as described above and examined by fluorescence microscopy. This method allows observation of individual cells, but may have reduced sensitivity depending on the antigen density.

[0460] The reactivity of anti-hNKG2A antibodies to NKG2A antigens can be further tested by Western blotting. Briefly, cell extracts can be prepared from cells expressing NKG2A and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed with BCIP / NBT substrate sheets (Sigma Chem. Co., St. Louis, MO).

[0461] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-NKG2A antibodies include standard analytical methods known in the art, such as Biolayer Interferometry (BLI) analysis, and Biacore SPR analysis using a Biacore SPR instrument.

[0462] In one embodiment, the anti-hNKG2A antibody specifically binds to the extracellular region of human NKG2A. In one embodiment, the antibody binds to a specific domain (e.g., a functional domain) in the extracellular domain of NKG2A. In one embodiment, the anti-hNKG2A antibody specifically binds to the extracellular region of human NKG2A and the extracellular region of macaque NKG2A. In one embodiment, the anti-hNKG2A antibody binds to human NKG2A with high affinity.

[0463] VIII. Multispecific Molecules

[0464] In certain embodiments, the antibodies described herein may be multispecific (e.g., bispecific or trispecific) molecules. Multispecific antigen binding molecules, such as multispecific antibodies, include two or more antigen binding sites, each of which is specific for different epitopes. Different epitopes may be parts of the same or different antigens. In one embodiment, one antigen binding site is specific for human NKG2A and the other is specific for different antigens. In one embodiment, an anti-hNKG2A antibody or an antigen binding fragment thereof as described herein is connected to another antigen binding molecule with different binding specificities, such as another peptide or protein (e.g., another antibody or antibody fragment, or a ligand of a receptor), 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 connected to more than one other antigen binding molecule to produce a multispecific molecule that binds to more than two different binding sites and / or target molecules. Therefore, bispecific molecules comprising at least one first binding specificity for NKG2A and a second binding specificity for a second target epitope are provided herein. In one embodiment in which the bispecific molecule described herein is multispecific, the molecule may further include a third binding specificity.

[0465] In one embodiment, the bispecific molecules described herein comprise at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv as binding specificity. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as the Fv or single-chain constructs described in U.S. Pat. No. 4,946,778 to Ladner et al., the contents of which are expressly incorporated by reference.

[0466] Although human monoclonal antibodies are preferred, other antibodies that can be employed in the bispecific molecules described herein may be murine, chimeric, and humanized monoclonal antibodies.

[0467] The bispecific molecules described herein can be prepared by conjugating the binding specificity components using methods known in the art. For example, each binding specificity of the bispecific molecule can be independently generated and then conjugated to each other. When the binding specificity is a protein or peptide, various coupling agents or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, S-acetyl-N-succinimidyl thioacetate (SATA), 5,5′-disulfide bis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), 3-(2-pyridyldithio)propionic acid N-succinimidyl ester (SPDP), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83) and Glennie et al. (1987) J. Immunol. 139:2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0468] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In an especially preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0469] Alternatively, the two binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This approach is particularly useful where 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 can be single chain molecules comprising one single chain antibody and a binding determinant, or single chain bispecific molecules comprising two binding determinants. The bispecific molecule can comprise at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Pat. No. 5,260,203, U.S. Pat. No. 5,455,030, U.S. Pat. No. 4,881,175, U.S. Pat. No. 5,132,405, U.S. Pat. No. 5,091,513, U.S. Pat. No. 5,476,786, U.S. Pat. No. 5,013,653, U.S. Pat. No. 5,258,498, and U.S. Pat. No. 5,482,858.

[0470] Binding of the bispecific molecule to its specific target can be confirmed using methods recognized in the art, such as using ELISA, radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot analysis. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0471] IX. Composition

[0472] Further provided are compositions, such as pharmaceutical compositions, containing one or more anti-NKG2A antibodies or antigen-binding fragments thereof as described herein formulated with a pharmaceutically acceptable carrier. Thus, the compositions of the invention include human or humanized anti-hNKG2A antibodies (or antigen-binding fragments thereof) having CDR sequences, heavy and / or light chain variable region sequences, or full-length heavy and / or light chain sequences as described herein. The compositions of the invention also include anti-hNKG2A antibodies having variant sequences of the sequences described in the sequence listing. For example, such antibodies may comprise sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99% identical to the CDR sequences, heavy and / or light chain variable region sequences, or full-length heavy and / or light chain sequences shown in the sequence listing.

[0473] Such compositions may also include one antibody or immunoconjugate or bispecific molecule described herein, or a combination of (e.g., two or more different) antibodies or immunoconjugates or bispecific molecules described herein. For example, the pharmaceutical compositions described herein may include a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or have complementary activities.

[0474] The pharmaceutical compositions described herein can also be used as combination therapy, that is, anti-NKG2A antibodies are administered in combination with other agents. For example, combination therapy can include an anti-NKG2A antibody described herein in combination with at least one other anticancer agent and / or T cell stimulator (e.g., activator). Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies described herein.

[0475] In some embodiments, the pharmaceutical compositions disclosed herein may include other compounds, drugs and / or agents for treating cancer. Such compounds, drugs and / or agents may include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some embodiments, the pharmaceutical composition comprises a first antibody and a second antibody specific for anti-hNKG2A.

[0476] In some embodiments, the first antibody and the second antibody are present in the composition at a fixed dose (i.e., a fixed ratio). In other embodiments, the fixed dose is at least about 1:200 to at least about 200:1, at least about 1:150 to at least about 150:1, at least about 1:100 to at least about 100:1, at least about 1:75 to at least about 75:1, at least about 1:50 to at least about 50:1, at least about 1:25 to at least about 25:1, at least about 1:10 to at least about 10:1, at least about 1:5 to at least about 5:1, at least about 1:4 to at least about 4:1, at least about 1:3 to at least about 3:1, or at least about 1:2 to at least about 2:1 of anti-hNKG2A antibody mg: second antibody mg. 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 anti-hNKG2A antibody: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 of first antibody mg: second antibody mg. For example, in one embodiment, an anti-h NKG2A antibody and a second antibody are administered as described in the Examples.

[0477] Additional 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.

[0478] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, the carrier is suitable for intravenous administration. In other embodiments, the carrier is suitable for subcutaneous administration. In some embodiments, the composition comprising the anti-NKG2A antibody uses Halozyme's Drug delivery technology is delivered subcutaneously, and the technology includes recombinant human hyaluronidase (rHuPH20) that temporarily degrades hyaluronic acid. In some embodiments, Drug delivery technology allows for faster subcutaneous administration of compositions compared to intravenous administration. In other embodiments, depending on the route of administration, the active compound, ie, antibody, immunoconjugate or bispecific molecule, can be coated in a material to protect the compound from acids and other natural conditions that can inactivate the compound.

[0479] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the biological activity of the parent compound and do not impart any undesirable toxicological effects (see, e.g., Berge, SM et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like; and salts derived from non-toxic organic acids such as aliphatic monocarboxylic acids and aliphatic dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines such as N,N'-diphenylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0480] The pharmaceutical compositions described herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0481] Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by 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.

[0482] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Preventing the presence of microorganisms can be ensured by sterilization procedures (same as above) and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be necessary to include isotonic agents in the composition, such as sugars, sodium chloride, etc. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including delayed absorption agents (such as aluminum monostearate and gelatin).

[0483] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX TM , Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glucosaminoglycanases, such as chondroitinase.

[0484] 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, it is contemplated for use in the pharmaceutical compositions described herein. Supplementary active compounds may also be incorporated into the compositions.

[0485] The therapeutic composition must typically be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In many cases, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride will preferably be included in the composition. The extended absorption of the injectable composition can be achieved by including a delayed absorption agent (such as monostearate and gelatin) in the composition.

[0486] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into an appropriate solvent with one or a combination of ingredients listed above, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from the above ingredients. In the case where sterile powders are used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produces a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0487] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Based on 100%, this amount can be from about 0.01% to about 99% of the active ingredient, such as from about 0.1% to about 70%, such as from about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0488] In some embodiments, the composition includes an anti-NKG2A antibody, such as NKG2A.9. The composition is a sterile, non-pyrogenic, single-use, preservative-free, isotonic aqueous solution for intravenous administration. The composition can be administered undiluted or further diluted with 0.9% sodium chloride injection to the desired protein concentration prior to infusion. In some embodiments, the anti-NKG2A antibody includes the following excipients: L-histidine, L-histidine hydrochloride monohydrate, sucrose, pentetic acid (also known as diethylenetriaminepentaacetic acid), polysorbate 80, and water for injection.

[0489] The dosage regimen is adjusted to provide the best desired response (e.g., a therapeutic response). For example, a single large dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage forms for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable for use as a single dose in an individual to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with a desired pharmaceutical carrier. The specifications of the unit dosage forms described herein are determined by and directly depend on the following factors: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art when compounding such active compounds for treating sensitive individuals.

[0490] In terms of administering antibodies, the dosage may be about 0.0001 to 100 mg per kg of host body weight, and more generally in the range of 0.01 to 5 mg. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or in the range of 1-10 mg / kg. Alternatively, administering antibodies is a flat dose, which may be in the range of 2 mg to 800 mg, such as a dosage of 25 mg, 80 mg, 200 mg or 400 mg. Exemplary treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a 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 regimen includes an initial dose, and then a maintenance dose of different dosages during intermittent administration.

[0491] In some embodiments, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the range shown. In some embodiments, the therapeutic antibody is administered multiple times. The time interval between single doses may be, for example, weekly, once every three weeks, once every four weeks, monthly, every three months, or annually. The interval may also be irregular, indicated by measuring the blood levels of antibodies to the target antigen in the patient. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods, about 25-300 μg / ml.

[0492] In some embodiments, the antibody can be administered as a sustained release formulation. Administration via a sustained release formulation may require less frequent administration. The dosage and frequency vary depending on the half-life of the antibody in the patient. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In some embodiments, for therapeutic treatment, relatively high dosages are administered at relatively short time intervals. In some embodiments, relatively high dosages are administered until disease progression is reduced or terminated, for example, until the patient shows partial or complete improvement of disease symptoms. In some embodiments, preventive treatment is administered to the patient after therapeutic treatment.

[0493] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition described herein being used, or its ester, salt, or amide; the route of administration; the time of administration; the rate of excretion of the particular compound being used; the duration of treatment; other drugs, compounds, and / or substances used in combination with the particular compound being used; the age, sex, weight, condition, general health, and prior medical history of the patient being treated; and similar factors well known in the medical art.

[0494] A "therapeutically effective dose" of an anti-NKG2A antibody described herein preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of damage or disability resulting from the disease. In the case of cancer, a therapeutically effective dose preferably prevents further worsening of physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, abnormal mole features; changes in the appearance of a mole, including asymmetry, borders, color and / or diameter; new areas of pigmented skin; abnormal moles; darkening of the area under the nails; breast lumps; nipple changes; breast cysts; breast pain; death; weight loss; weakness; excessive fatigue; difficulty eating; loss of appetite; chronic cough; worsening dyspnea; coughing up blood, blood in the urine, blood in the stool, nausea, vomiting, metastasis of liver cancer, metastasis of lung cancer, metastasis of bone cancer, abdominal pain and falling, abdominal distension, peritoneal fluid, vaginal bleeding, constipation, abdominal distension, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, metastasis of colon cancer, metastasis of lung cancer, metastasis of bladder cancer, metastasis of liver cancer, metastasis of bone cancer, metastasis of kidney cancer and pancreatic cancer, difficulty swallowing, etc. The therapeutic efficacy may be observed immediately after the first administration of the anti-hNKG2A monoclonal antibody of the invention, or it may be observed only after a period of time and / or a series of doses. Such delayed efficacy may be observed only after several months of treatment, for example up to 6, 9 or 12 months.

[0495] A therapeutically effective dose can prevent or delay the onset of cancer, such as may be desired when there are early or initial signs of the disease. Thus, it can be determined whether a particular treatment is a therapeutically effective dose for cancer treatment by monitoring any clinical or biochemical analysis of any of the above. One of ordinary skill in the art will be able to determine this amount based on factors such as the individual's body size, the severity of the individual's symptoms, and the particular composition or route of administration selected.

[0496] The compositions described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Exemplary routes of administration of the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.

[0497] Alternatively, the antibodies described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0498] The active compound can be prepared with a carrier that will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations have been patented or are 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.

[0499] The therapeutic composition can be administered with medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition described herein can be administered with a needle-free subcutaneous injection device, such as the device disclosed in U.S. Pat. Nos. 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 implants and modules for use with the anti-hNKG2A antibodies described herein include: U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump that dispenses drugs at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0500] In certain embodiments, the anti-hNKG2A antibodies described herein can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes a variety of highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if necessary), they can be formulated in liposomes, for example. For methods of making liposomes, see, for example, U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes may include one or more moieties that can be selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade, 1989, J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (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); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0501] Kits comprising an antibody composition described herein (e.g., a human antibody, a bispecific or multispecific molecule, or an immunoconjugate) and instructions for use are also within the scope described herein. The kit may further contain at least one additional reagent described herein, or one or more additional human antibodies. The kit may include a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit.

[0502] X. How to use

[0503] The antibodies, antibody compositions and methods described herein have many in vitro and in vivo uses, including, for example, enhancing immune responses by blocking NKG2A / HLA-E interactions. In one embodiment, the anti-NKG2A antibodies described herein are monoclonal human or humanized antibodies. In one embodiment, the anti-hNKG2A antibodies described herein (e.g., 13F3.A4, NKG2A.9, and NKG2A.11) can be administered to cultured cells (in vitro or ex vivo), or to human subjects to enhance immunity to a variety of diseases. In a specific embodiment, the anti-hNKG2A antibody is an antagonist antibody. Provided herein is a method for improving an immune response in an individual, comprising administering an anti-NKG2A antibody or an antigen-binding fragment thereof described herein to the individual to enhance, stimulate, or upregulate the immune response of the individual. In one embodiment, anti-hNKG2A antibodies are administered according to the methods described herein to enhance T cell and / or NK cell responses. In one embodiment, anti-hNKG2A antibodies are administered according to the methods described herein to stimulate, enhance, or upregulate antigen-specific T cell responses against tumors. The T cells may be Teff cells, such as CD4+ Teff cells, CD8+ Teff cells, T helper (T h ) cells and T cell cytotoxicity (T c ) cells. The tumor may be a solid tumor or a liquid tumor, such as 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 individual may also be a virus carrier and the immune response to the virus is enhanced. In one embodiment, an anti-hNKG2A antibody is administered according to the methods described herein to stimulate, enhance or upregulate NK cell responses.

[0504] In one embodiment, the method results in an enhanced immune response in a human subject, wherein such enhancement has a desired effect. In one embodiment, the human subject is a human patient suffering from a condition that can be treated by enhancing an immune response, such as a T cell-mediated immune response. In a specific embodiment, the human patient suffers from cancer. In one embodiment, the anti-hNKG2A antibody described herein can be administered together with an antigen of interest, or the antigen may already be present in the subject to be treated, such as a tumor-bearing or virus-bearing subject. When the anti-NKG2A antibody is administered together with another agent, the two can be administered separately or simultaneously.

[0505] Further provided are methods of inhibiting tumor cell growth in a subject, comprising administering to the subject an anti-hNKG2A antibody described herein, such that tumor cell growth in the subject, such as a human subject, is inhibited. Also provided are methods of treating a chronic viral infection in a subject, comprising administering to the subject an anti-NKG2A antibody described herein, such that a chronic viral infection in the subject, such as a human subject, is treated.

[0506] In some embodiments, anti-NKG2A antibodies are administered to individuals, such as human patients, as adjuvant therapy, adjuvant therapy, or neoadjuvant therapy. In some embodiments, treatment of individuals with cancer with anti-NKG2A antibodies can result in long-term, durable responses compared to current standard care; long-term survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more, recurrence-free survival of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years or more. In certain embodiments, treatment of individuals with cancer with anti-hNKG2A antibodies prevents cancer recurrence or delays cancer recurrence by, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years or more. Anti-NKG2A therapy can be used as first-line, second-line, or subsequent-line therapy.

[0507] These and other methods herein are discussed in further detail below.

[0508] cancer

[0509] Provided herein is a method for treating an individual with cancer, comprising administering to the individual an anti-hNKG2A antibody described herein, such that the individual is treated, for example, cancerous tumor growth is inhibited or reduced and / or the tumor regresses. Anti-NKG2A antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, anti-NKG2A antibodies can be used in combination with another agent as described below, such as other immunogenic agents, standard cancer therapeutics, or other antibodies. Combinations with PD-1 inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies are also provided. Combinations with CTLA-4 inhibitors, such as anti-CTLA-4 antibodies are also provided. Combinations with PD-1 inhibitors and CTLA-4 inhibitors are also provided. Combinations with agonist antibodies of ICOS are also provided.

[0510] In one aspect, provided herein is a method for treating cancer in an individual comprising administering to the individual a therapeutically effective amount of an 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 cancer treatment methods described herein comprise administering an anti-NKG2A antibody that contacts human NKG2A at one or more of the following amino acid residues:

[0511] (a) LSIDNEEMKF (SEQ ID NO: 156);

[0512] (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157);

[0513] (c) LAFKHEIKDSDN (SEQ ID NO: 158); and

[0514] (d) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159).

[0515] In another embodiment, the method of treating cancer described herein comprises administering an anti-NKG2A antibody that contacts human NKG2A at one or more of the following amino acid residues:

[0516] (a) LSIDNEEMKF (SEQ ID NO: 156);

[0517] (b) PSSWIGVFRNSSHHPW (SEQ ID NO: 157);

[0518] (c)LAFKHEIKDSDN (SEQ ID NO: 158);

[0519] (d) L; and

[0520] (e) QVNRLKSAQQCGSSIIYHC (SEQ ID NO: 159).

[0521] In another embodiment, the method comprises administering a 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 a NKG2A.11 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising a NKG2A.9 antibody to treat cancer. In another embodiment, the method comprises administering a composition comprising a 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.

[0522] Examples of cancer include, but are not limited to, squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or carcinomas). Germ cell tumors, pediatric sarcomas, sinonasal natural killer cells, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal tumors (spinal axis tumors), brain cancer (including brain stem gliomas), pituitary adenomas, Kaposi's sarcoma, epidermoid cancers, squamous cell cancers, T-cell lymphomas, environmentally induced cancers (including those induced by asbestos), virus-related cancers (e.g., human papillomavirus (HPV)-related tumors), and hematological malignancies derived from either of the two major blood cell lineages, the myeloid lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute allergic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (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), solitary granulocytic sarcoma and chloroma;Lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoma, lymphoma, T-lymphoblastic lymphoma, and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome syndrome) and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, and smoldering myeloma (also called indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma Tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell disorders, such as T-prolymphocytic leukemia (T-PLL), including small cell and gyrus-like cell types; large granular lymphocytic leukemia (LGL) (preferably of T-cell type); a / d T-NHL hepatosplenic lymphoma; peripheral T-cell lymphoma / post-thymic T-cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, kidney cancer, rectal cancer, thyroid cancer;Acute myeloid lymphoma, and any combination of such cancers. In one embodiment, the methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers that are difficult to treat with previous immunotherapies, such as blocking CTLA-4 and / or PD-1 antibodies), and recurrent cancers. ;

[0523] In one embodiment, the anti-hNKG2A antibody is administered as a monotherapy. In one embodiment, the anti-hNKG2A agonist antibody is administered as the sole immunostimulant. In one embodiment, anti-hNKG2A is administered to the patient together with another agent. In one embodiment, the anti-hNKG2A antibody is administered together with an immunogenic agent. In one embodiment, the anti-hNKG2A antibody is administered together with a cancer vaccine. In some embodiments, the cancer vaccine comprises cancer 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 is a personalized peptide vaccine in some embodiments. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide mixture, 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, the anti-hNKG2A antibody is administered with an adjuvant. Non-limiting examples of tumor vaccines used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. Many experimental strategies for vaccination against tumors have been designed (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; see also Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pages 3023-3043, DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, autologous or allogeneic tumor cells are used to prepare the vaccine. These cellular vaccines have been shown to be most effective when the 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.

[0524] Other cancer vaccines are proteins from viruses involved in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). In some embodiments, another form of tumor-specific antigen used with NKG2A inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. The heat shock proteins contain fragments of proteins from tumor cells and these HSPs can be efficiently delivered to antigen presenting cells to induce tumor immunity (Suot, R and Srivastava, P (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117-120).

[0525] In some embodiments, dendritic cells are powerful antigen presenting cells for inducing antigen-specific responses. Dendritic cells can be produced in vitro and loaded with various protein and peptide antigens and tumor cell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). Dendritic cells can also be transduced by genetic methods to express these tumor antigens. DC has also been directly fused with tumor cells for immune purposes (Kugler et al. (2000) Nature Medicine 6: 332-336). As a method of vaccination, dendritic cell immunization can be effectively combined with anti-NKG2A antibodies to activate (release) a more powerful anti-tumor response.

[0526] In some embodiments, anti-hNKG2A is administered together with standard care treatment, such as surgery, radiotherapy and / or chemotherapy. In some embodiments, anti-hNKG2A antibodies are administered together with chemotherapeutic agents. In some embodiments, anti-hNKG2A antibodies are administered together with one or more of carboplatin, cisplatin, paclitaxel, nab-paclitaxel, gemcitabine or FOLFOX. In some embodiments, anti-hNKG2A antibodies are administered together with carboplatin or nab-paclitaxel. In some embodiments, anti-hNKG2A antibodies are administered together with carboplatin and paclitaxel. In some embodiments, anti-hNKG2A antibodies are administered together with cisplatin and pemetrexed. In some embodiments, anti-hNKG2A antibodies are administered together with cisplatin and gemcitabine. In some embodiments, anti-hNKG2A antibodies are administered together with FOLFOX. In some embodiments, anti-hNKG2A antibodies are administered together with FOLFIRI. In one embodiment, anti-hNKG2A antibodies are administered with dacarbazine to treat melanoma. In some embodiments, cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks. In some embodiments, anti-hNKG2A antibodies are administered together with doxorubicin / adriamycin, cisplatin sulfate bleomycin, carmustine, chlorambucil, dacarbazine and / or cyclophosphamide hydroxyurea. In some embodiments, doxorubicin is administered intravenously at a dose of 60 mg / ml to 75 mg / ml once every 21 days. In one embodiment, anti-hNKG2A antibodies are administered to human patients who are resistant to treatment with at least one drug, wherein the administration of anti-hNKG2A antibodies reduces, alleviates or eliminates resistance to the at least one drug. In some embodiments, anti-hNKG2A antibodies are administered together with agonist antibodies, such as anti-ICOS antibodies.

[0527] The combination therapy described above can be administered in various combinations with each other, and encompasses co-administration (wherein two or more therapeutic agents are included in the same or different formulations) and separate administration, in which case administration of the antibody of the present invention can be performed before, simultaneously with, and / or after administration of other therapeutic agents and / or adjuvants. The antibody of the present invention can also be used in combination with radiotherapy.

[0528] In some embodiments, another example of such a combination is the combined administration of an anti-hNKG2A antibody and interleukin-2 (IL-2). In some embodiments, the combination of an anti-hNKG2A antibody and IL-2 is used to treat various cancers, including treatment of renal cell carcinoma and melanoma. In some embodiments, the anti-hNKG2A antibody described herein is combined with an IL-2 pathway agonist to treat various cancers. The combination includes various IL-2 pathway agonists, such as those described in WO 2012 / 065086 (Nektar Therapeutics) and WO 2015 / 125159 (Nektar Therapeutics), the contents of which are incorporated by reference in their entirety. WO 2006 / 138572 (Nektar Therapeutics) provides conjugates with degradable bonds and polymer reagents suitable for preparing such conjugates, as well as methods for making polymer reagents and conjugates, which are incorporated herein by reference in their entirety.

[0529] In some embodiments, a combination of an anti-hNKG2A antibody as described herein, such as NKG2A.9, NKG2A.11, or 13F3.A4 antibody, and an IL-2 pathway agonist, such as NKTR-214, is administered to a patient to treat cancer. As described in more detail below, NKTR-214 is produced as follows: an average of approximately six FMOC (methoxycarbonyl chloride)-based polyethylene glycol (PEG) reagents (mPEG 2 -C2-fomc-20K-N-hydroxysuccinimide derivative, 20 kDa, ("mPEG2-C2-fmoc-20K-NHS"):

[0530]

[0531] Conjugated to a protein having the following 132 amino acid sequence:

[0532]

[0533] WO 2012 / 065086 provides conjugates of IL-2 moieties and one or more non-peptide, water-soluble polymers, including polyethylene glycol or its derivatives. Specifically, Example 2 (paragraphs 202-204) of WO 2012 / 065086 describes rIL-2 PEGylated with mPEG2-C2-fmoc-20K-NHS to produce the above-mentioned mPEG2-C2-fmoc-20K-NHS structure. Example 1 (paragraphs 63-66) of WO 2015 / 125159 describes a scale-up method for IL-2 PEGylated with mPEG2-C2-fmoc-20K-NHS to produce RSLAIL-2 (NKTR-214). NKTR-214 is a cytokine designed to target CD122 (also known as interleukin-2 receptor β subunit, IL-2Rβ), a protein found on certain immune cells (such as CD8+ T cells and NK cells), to expand these cells to promote their anti-tumor effects.

[0534] In some embodiments, an anti-hNKG2A antibody is administered in combination with an anti-angiogenic agent.

[0535] Other combination therapies that may synergize with the anti-hNKG2A antibodies described herein via cell death are radiation, surgery, and hormone deprivation.

[0536] In some embodiments, the anti-hNKG2A antibodies described herein are administered together with bispecific antibodies. Bispecific antibodies can be used to target two different antigens. In some embodiments, anti-hNKG2A antibodies are used in combination with bispecific antibodies targeting effector cells expressing Fcα or Fcγ receptors to treat tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. In some embodiments, the T cell arm of these reactions is increased by the functional activity of anti-hNKG2A antibodies. In some embodiments, antigens are delivered directly to DCs by using bispecific antibodies that bind 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.

[0537] Infectious Diseases

[0538] In another aspect, the invention described herein provides a method of treating an infectious disease in a subject, including a human subject, comprising administering an anti-hNKG2A antibody or antigen-binding fragment thereof to the subject to treat the infectious disease in the subject. In other embodiments, the anti-NKG2A antibody is a chimeric or humanized antibody.

[0539] Similar to tumor treatment as discussed herein, the anti-hNKG2A antibodies described herein can be administered alone, or as an adjuvant, in combination with a vaccine to enhance immune responses against pathogens, toxins, and self-antigens, including treatment of chronic viral infections. Examples of pathogens for which this treatment method is particularly useful include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are not fully effective. Such pathogens include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.

[0540] Examples of pathogenic viruses that cause infections treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus, and arboviral encephalitis virus.

[0541] Examples of pathogenic bacteria that cause infections treatable by the methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.

[0542] Some 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.), Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0543] Examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardialambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, Nippostrongylus brasiliensis.

[0544] The methods described herein of administering an anti-hNKG2A antibody to an individual can be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy (which provides enhanced tumor antigen presentation) (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0545] Autoimmune response

[0546] In some aspects, anti-NKG2A antibodies increase autoimmune responses. Inducing anti-tumor responses using tumor cells and peptide vaccines has shown that many anti-tumor responses involve anti-self reactivity (van Elsas et al. (2001) J. Exp. Med. 194: 481-489; Overwijk et al. (1999) Proc. Natl. Acad. Sci. USA 96: 2982-2987; Hurwitz, (2000) supra; Rosenberg and White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Therefore, anti-NKG2A antibodies are used together with various self-proteins to design vaccination programs to effectively generate immune responses against these self-proteins for disease treatment. For example, Alzheimer's disease involves the inappropriate accumulation of Αβ peptides in amyloid deposits in the brain; antibody responses against amyloid are able to clear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

[0547] Other self-proteins can also be used as targets, such as IgE for allergy and asthma treatment and TNF-α for rheumatoid arthritis treatment. Finally, antibody responses to various hormones can be induced by using 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 specific tumors can be additional vaccination targets.

[0548] Similar approaches to those described above using anti-NKG2A antibodies can be used to induce a therapeutic autoimmune response to treat patients with inappropriate accumulation of other autoantigens, such as amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNF-α, and IgE.

[0549] vaccine

[0550] In some aspects, the anti-NKG2A antibodies described herein are used to stimulate an antigen-specific immune response, wherein the anti-NKG2A antibodies are administered together with an antigen of interest (e.g., a vaccine). Thus, provided herein is a method for enhancing an individual's immune response to an antigen, comprising administering to the individual: (i) an antigen; and (ii) an anti-NKG2A antibody or an antigen-binding fragment thereof, such that the individual's immune response to the antigen is enhanced. The antibody may be a human anti-human NKG2A antibody (such as any of the human anti-NKG2A antibodies described herein). In some embodiments, the anti-NKG2A antibody is a chimeric or humanized antibody. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those described in this section, such as tumor antigens (or tumor vaccines) described herein, or antigens from viruses, bacteria, or other pathogens described herein.

[0551] In certain embodiments, instead of or in addition to the anti-NKG2A antibody, a peptide or fusion protein comprising an epitope bound by the anti-NKG2A antibody is used as a vaccine.

[0552] Suitable routes for in vivo and in vitro administration of the antibody compositions described herein (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) are well known in the art and can be selected by one of ordinary skill in the art. For example, the antibody composition can be administered by intravenous or subcutaneous routes. The suitable dosage of the composition used will depend on the age and weight of the individual and the concentration and / or formulation of the antibody composition.

[0553] As previously described, the anti-NKG2A antibodies described herein may be co-administered with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressants. The anti-NKG2A antibodies may be attached to the agent (in the form of an immune complex) or may be administered separately from the agent. In the latter case (separate administration), the anti-NKG2A antibodies may be administered before, after, or simultaneously with the agent, or may be co-administered with other known therapies, such as anticancer therapies, such as chemotherapy and / or radiation. Such therapeutic agents include, in particular, anti-neoplastic agents, such as doxorubicin / adriamycin, cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which are themselves effective only at toxic or subtoxic levels to the patient. Cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks, and doxorubicin is administered intravenously at a dose of 60-75 mg / ml once every 21 days. The anti-NKG2A antibodies or antigen-binding fragments thereof described herein co-administered with chemotherapeutic agents can provide two anticancer agents that act via different mechanisms to produce cytotoxic effects on human tumor cells. Such co-administration can solve the problem of tumor cells not reacting with antibodies due to drug resistance or changes in the antigenicity of tumor cells.

[0554] Kits comprising an antibody composition described herein (e.g., a human antibody, a bispecific or multispecific molecule, or an immunoconjugate) and instructions for use are also within the scope described herein. The kit may further contain at least one additional reagent described herein, or one or more additional human anti-NKG2A antibodies (e.g., a human antibody with complementary activity that binds to a different NKG2A antigenic epitope than the first human antibody). The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material on or supplied with the kit or otherwise accompanying the kit.

[0555] Combination therapy

[0556] In one aspect, provided herein is a combination therapy, for example, for the treatment of cancer, wherein an anti-hNKG2A antibody is administered in combination with one or more additional agents, such as antibodies, that effectively stimulate an immune response, thereby further enhancing, stimulating or upregulating an immune response in an individual (including a human individual). Provided herein is a method for treating cancer in an individual or delaying the progression of the cancer, comprising administering an anti-hNKG2A antibody (e.g., NKG2A.9, NKG2A.11, and 13F3.A4) to an individual together with another anticancer agent or cancer therapy. In some embodiments, the anti-hNKG2A antibody may be administered together with chemotherapy or a chemotherapeutic agent or with a radiotherapy or radiotherapy agent (as described above). In some embodiments, the anti-hNKG2A antibody may be administered together with an agonist antibody, such as an anti-hICOS antibody. In some embodiments, the anti-hNKG2A antibody may be administered together with a targeted therapy or a targeted therapeutic agent. In some embodiments, the anti-hNKG2A antibody may be administered together with an immunotherapy or an immunotherapeutic agent, such as a monoclonal antibody.

[0557] In some embodiments, the anti-hNKG2A antibodies described herein may be combined with (i) an agonist of another co-stimulatory receptor and / or (ii) an antagonist of an inhibitory signal on a T cell. In some embodiments, a combination therapy comprising an anti-hNKG2A antibody and an agonist and / or an antagonist enhances the antigen-specific T cell response of an individual. In some embodiments, the anti-hNKG2A antibodies described herein may be administered together with an agent targeting a co-stimulatory and co-inhibitory molecule as a member of the immunoglobulin superfamily (IgSF) to improve the immune response. In some embodiments, the anti-hNKG2A antibodies described herein may be administered together with an agent targeting a ligand of a co-stimulatory or co-inhibitory molecule. The membrane-bound ligand family that binds to co-stimulatory or co-inhibitory receptors is the B7 family, which 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 co-stimulatory or co-inhibitory receptors is the TNF family of molecules, which bind to members of the TNF receptor family, including CD40, CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, T WEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1,

[0558] In another aspect, anti-hNKG2A antibodies can be used in combination with antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; or other "immunosuppressive cytokines"), or cytokines that stimulate T cell activation, to stimulate an immune response, e.g., to treat proliferative diseases such as cancer.

[0559] In one aspect, T cell responses are stimulated by a combination of an anti-hNKG2A antibody as described herein and one or more of the following: (i) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4, and (ii) 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.

[0560] Exemplary agents that modulate one of the above proteins and can be used in combination with anti-hNKG2A (such as those described herein) to treat cancer include: / ipilimumab or tremelimumab (targeting CTLA-4), galiximab (targeting B7.1), BMS-936558 (targeting PD-1), pidilizumab / CT-011 (targeting PD-1), / pembrolizumab / MK-3475 (targeting PD-1), AMP224 (targeting B7-DC / PD-L2), BMS-936559 (targeting B7-H1), MPDL3280A (targeting B7-H1), CP-870893 or dacetuzumab / SGN-40 (CD40-Kirkwood et al. (2012) CA Cancer J. Clin. 62:309; Vanderheide and Glennie (2013) Clin. Cancer Res. 19:1035), AMG557 (targeting B7H2), MGA271 (targeting B7H3-WO 11 / 109400), IMP321 (for LAG-3), urelumab / BMS-663513 and PF-05082566 (for CD137 / 4-1BB), varlilumab / CDX-1127 (for CD27), MEDI-6383 and MEDI-6469 (for OX40), RG-7888 (for OX40L-WO 06 / 029879), Atacicept (for TACI), muromonab-CD3 (for CD3), ipilumumab (for CTLA-4). Thus, in one embodiment, an anti-hNKG2A antibody (such as NKG2A.9) is combined with an anti-PD-1 antibody (such as nivolumab) and / or an anti-CTLA-4 antibody (such as ipilimumab).

[0561] Other molecules that can be combined with anti-hNKG2A antibodies for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, anti-hNKG2A antibodies can be combined with KIR antagonists (eg, lirilumab).

[0562] Other agents for use in combination therapy include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO 11 / 70024, WO 11 / 107553, WO 11 / 131407, WO 13 / 87699, WO 13 / 119716, WO 13 / 132044) or FPA-008 (WO 11 / 140249; WO 13 / 169264; WO 14 / 036357).

[0563] In some embodiments, the anti-hNKG2A antibodies described herein are used with one or more of the following: agonists that bind positive co-stimulatory receptors, blockers that attenuate signaling through inhibitory receptors, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome unique immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor binding (e.g., PD-L1 / PD-1 interactions), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibiting metabolic enzymes (such as IDO), or reversing / preventing T cell anergy or exhaustion), and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0564] Provided herein is a method of stimulating an immune response in an individual, comprising administering to an individual an anti-hNKG2A antibody and one or more other immunostimulatory antibodies, such as a PD-1 antagonist (e.g., an antagonist antibody), a PD-L1 antagonist (e.g., an antagonist antibody), a CTLA-4 antagonist (e.g., an antagonist antibody), and / or a LAG3 antagonist (e.g., an antagonist antibody), such that an immune response is stimulated in the individual, for example, to inhibit tumor growth or stimulate an antiviral response. In one embodiment, an anti-hNKG2A antibody and an antagonist anti-PD-1 antibody are administered to an individual. In one embodiment, an anti-hNKG2A antibody and an antagonist anti-PD-L1 antibody are administered to an individual. In one embodiment, an anti-hNKG2A antibody and an antagonist anti-CTLA-4 antibody are administered to an individual. In one embodiment, the at least one other immunostimulatory antibody (e.g., an antagonist anti-PD-1, an antagonist anti-PD-L1, an antagonist anti-CTLA-4, and / or an antagonist anti-LAG3 antibody) is a human antibody. Alternatively, the at least one other immunostimulatory antibody can 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 antibodies).

[0565] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer), comprising administering an anti-hNKG2A antibody and an antagonist PD-1 antibody to an individual. In some embodiments, the cancer is non-small cell lung cancer (NSCLC) or colorectal cancer (CRC). In some embodiments, the cancer is characterized by: a tumor with (i) elevated HLA-E levels; and / or (ii) a higher tumor mutation load. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Also provided herein are methods for altering adverse events associated with immunostimulants for the treatment of hyperproliferative diseases. In one embodiment, the method comprises administering an anti-hNKG2A antibody and a subtherapeutic dose of an anti-PD-1 antibody to an individual. In some embodiments, the individual is human. In some embodiments, the anti-PD-1 antibody is a human monoclonal antibody.

[0566] 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 have been disclosed in U.S. Pat. No. 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. Pat. No. 8,008,449 have been shown to exhibit one or more of the following characteristics: (a) a high affinity of 1×10 -7 M or smaller K D Binds to human PD-1; (b) does not substantially bind to human CD28, CTLA-4 or ICOS; (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and macaque PD-1; (g) inhibits PD-L1 and / or PD-L2 from binding to PD-1; (h) stimulates antigen-specific memory responses; (i) stimulates antibody responses; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments at least five, of the foregoing characteristics.

[0567] Other anti-PD-1 monoclonal antibodies have been described, for example, in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509; U.S. Publication No. 2016 / 0272708; and PCT Publication Nos. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2018 / 19491; and PCT Publication Nos. WO 2014 / 1 No. 2017 / 020858, No. WO 2016 / 197367, No. WO 2017 / 024515, No. WO 2017 / 025051, No. WO 2017 / 123557, No. WO 2016 / 106159, No. WO 2014 / 194302, No. WO No. 2017 / 040790, No. WO 2017 / 133540, No. WO 2017 / 132827, No. WO 2017 / 024465, No. WO 2017 / 025016, No. WO 2017 / 106061, No. WO 2017 / 19846, No. WO No. 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540, each of which is incorporated by reference in its entirety.

[0568] In some embodiments, the anti-PD-1 antibody is nivolumab (also known as 5C4, BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (Merck; also known as lambrolizumab and MK-3475; see WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Pharmaceuticals; see WO 2015 / 35606 and US 2015 / 0079109), Medicine; also known as SHR-1210; see WO 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics, see WO 2017 / 19846) or IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540).

[0569] 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 blocks the interaction with PD-1 ligands (PD-L1 and PD-L2), thereby preventing downregulation of anti-tumor T cell function (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0570] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described in, for example, U.S. Pat. Nos. 8,354,509 and 8,900,587.

[0571] Anti-PD-1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any anti-PD-1 antibody disclosed herein (e.g., nivolumab) for binding to human PD-1 (see, e.g., U.S. Pat. Nos. 8,008,449 and 8,779,105; WO 2013 / 173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as any anti-PD-1 antibody described herein, e.g., nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder other cross-competing antibodies from binding to that specific epitope region. Due to binding to the same epitope region of PD-1, it is expected that these cross-competing antibodies will have very similar functional properties to the 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 analysis, or flow cytometry (see, e.g., WO 2013 / 173223).

[0572] In certain embodiments, the antibody that cross-competes with the human PD-1 antibody nivolumab for binding to human PD-1, or binds to the same epitope region, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0573] Anti-PD-1 antibodies that can be used in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0574] 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 effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 "antibody" includes an antigen binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to the full antibody in inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or its antigen binding portion cross-competes with nivolumab for binding to human PD-1.

[0575] Provided herein is a method for treating a hyperproliferative disease (e.g., cancer), comprising administering an anti-hNKG2A antibody and an antagonist PD-L1 antibody to an individual. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein is a method for changing adverse events associated with immunostimulants for the treatment of hyperproliferative diseases, comprising administering an anti-NKG2A antibody and a subtherapeutic dose of an anti-PD-L1 antibody to an individual. In certain embodiments, the individual 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, such as an antibody comprising the CDR or variable region of an antibody disclosed herein.

[0576] Anti-PD-L1 antibodies known in the art can be used in the methods of the present invention. Examples of anti-PD-L1 antibodies suitable for use in the methods of the present invention include antibodies disclosed in U.S. Pat. No. 9,580,507. The anti-PD-L1 human monoclonal antibodies disclosed in U.S. Pat. No. 9,580,507 have been shown to exhibit one or more of the following characteristics: (a) a 1×10 -7 M or smaller K D Bind to human PD-L1; (b) increase T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increase interferon-γ production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments at least five, of the foregoing characteristics.

[0577] In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as MPDL3280A, RG7446; see US 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(Suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI TM , MEDI-4736; see WO 2011 / 066389), Avelumab (Pfizer; also known as MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), or CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (April 2016)).

[0578] In certain embodiments, the PD-L1 antibody is atezolizumab. ). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0579] In certain embodiments, the PD-L1 antibody is durvalumab (IMFINZI TM Durvalumab is a human IgG1κ monoclonal anti-PD-L1 antibody.

[0580] In certain embodiments, the PD-L1 antibody is avelumab. ). Avelumab is a human IgG1λ monoclonal anti-PD-L1 antibody.

[0581] In other embodiments, the anti-PD-L1 monoclonal antibody is 28-8, 28-1, 28-12, 29-8, 5H1, or any combination thereof.

[0582] Anti-PD-L1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein (e.g., atezolizumab, durvalumab and / or avelumab) for binding to human PD-L1. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein, such as atezolizumab, durvalumab and / or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. Due to binding to the same epitope region of PD-L1, it is expected that the cross-competing antibody will have very similar functional properties to the reference antibody (e.g., atezolizumab and / or avelumab). Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays, such as Biacore analysis, ELISA analysis, or flow cytometry (see, e.g., WO 2013 / 173223).

[0583] In certain embodiments, antibodies that cross-compete with atezolizumab, durvalumab and / or avelumab for binding to human PD-L1, or bind to the same epitope region of human PD-L1 antibodies as atezolizumab, durvalumab and / or avelumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0584] Anti-PD-L1 antibodies useful in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0585] Anti-PD-L1 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any composition or method disclosed herein, the anti-PD-L1 "antibody" includes an antigen binding portion or fragment that binds to PD-L1 and exhibits functional properties similar to the full antibody in inhibiting receptor binding and upregulating the immune system. In certain embodiments, the anti-PD-L1 antibody, or antigen binding portion thereof, cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0586] In one embodiment, an anti-hNKG2A antibody of the invention is combined with a PD-1 / PD-L1 signaling antagonist, such as a PD-1 antagonist (e.g., nivolumab, also known as MDX1106, as described in WO 06 / 121168) or a PD-L1 antagonist, and a third immunotherapeutic agent (e.g., an anti-ICOS antibody, such as ICOS.33 IgG1f S267E (as described in U.S. Pat. No. 10,251,945), in combination with nivolumab and ipilimumab). In one embodiment, the third immunotherapeutic agent is a CTLA-4 antagonist antibody. In certain embodiments, the anti-CTLA-4 antibody is (ipilimumab or antibody 10D1, described in PCT Publication WO 01 / 14424) or tremelimumab (formerly ticilimumab, CP-675,206). In one embodiment, the third immunotherapeutic agent is a GITR antagonist or OX-40 antagonist, such as an anti-GITR or anti-OX40 antibody disclosed herein. In one embodiment, the third immunotherapeutic agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116) and MK-4166 (WO11 / 028683). In one embodiment, the third immunotherapeutic agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod or NLG-919 (WO09 / 73620, WO09 / 1156652, WO11 / 56652, WO12 / 142237).

[0587] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer) comprising administering to an individual an anti-hNKG2A antibody described herein and a CTLA-4 antagonist antibody. In certain embodiments, the anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering adverse events associated with immunostimulant treatment of hyperproliferative diseases, comprising administering to an individual an anti-hNKG2A antibody and a subtherapeutic dose of an anti-CTLA-4 antibody. In certain embodiments, the individual is a human.

[0588] Anti-CTLA-4 antibodies known in the art can be used in the methods of the present invention. The anti-CTLA-4 antibodies of the present invention bind to human CTLA-4 so as to disrupt the interaction between CTLA-4 and human B7 receptors. Because the interaction between CTLA-4 and B7 causes signal transduction, resulting in the inactivation of T cells carrying CTLA-4 receptors, the disruption of this interaction can effectively induce, enhance or prolong the activation of such T cells, thereby inducing, enhancing or prolonging the immune response.

[0589] Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity have been disclosed in U.S. Pat. No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described, for example, in U.S. Pat. Nos. 5,977,318, 6,051,227, 6,682,736, 7,034,121, and International Publication Nos. WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237, and WO 2000 / 037504, all of which are hereby incorporated by reference in their entirety. Anti-CTLA-4 antibodies suitable for use in the present invention include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the aforementioned characteristics.

[0590] In certain embodiments, the CTLA-4 antibody is ipilimumab (also known as MDX-010, 10D1; see U.S. Pat. No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; see WO 2016 / 196237) or tremelimumab (AstraZeneca; also known as teximab, CP-675,206; see WO 2000 / 037504 and Ribas, Update Cancer Ther. 2(3):133-39 (2007)). In certain embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0591] In certain embodiments, the CTLA-4 antibody is ipilimumab for use in the methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0592] In certain embodiments, the CTLA-4 antibody is Tremelimumab.

[0593] In certain embodiments, the CTLA-4 antibody is MK-1308.

[0594] In certain embodiments, the CTLA-4 antibody is AGEN-1884.

[0595] Anti-CTLA-4 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any anti-CTLA-4 antibody disclosed herein (e.g., ipilimumab and / or tremelimumab) for binding to human CTLA-4. In some embodiments, the anti-CTLA-4 antibody binds to the same epitope as any anti-CTLA-4 antibody described herein (e.g., ipilimumab and / or tremelimumab). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. Due to binding to the same epitope region of CTLA-4, it is expected that the cross-competing antibody will have very similar functional properties to the reference antibody (e.g., ipilimumab and / or tremelimumab). Cross-competing antibodies can be easily identified in standard CTLA-4 binding assays, such as Biacore analysis, ELISA analysis, or flow cytometry, based on their ability to cross-compete with ipilimumab and / or tremelimumab (see, e.g., WO 2013 / 173223).

[0596] In certain embodiments, antibodies that cross-compete with ipilimumab and / or tremelimumab for binding to human CTLA-4, or that bind to the same epitope region of a human CTLA-4 antibody, are monoclonal antibodies. For administration to human subjects, such cross-competing antibodies may be chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies may be prepared and isolated by methods well known in the art.

[0597] Anti-CTLA-4 antibodies useful in the methods of the disclosed invention also include antigen-binding portions of the above antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0598] Anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and disrupt the interaction of CTLA-4 with the human B7 receptor. In any of the compositions or methods disclosed herein, the anti-CTLA-4 "antibody" includes an antigen binding portion or fragment that binds to CTLA-4 and exhibits functional properties similar to the whole antibody in inhibiting the interaction of CTLA-4 with the human B7 receptor and upregulating the immune system. In certain embodiments, the anti-CTLA-4 antibody or antigen binding portion thereof cross-competes with ipilimumab and / or tremelimumab for binding to human CTLA-4.

[0599] In one embodiment, the anti-hNKG2A antibody of the present invention is combined with an anti-CTLA-4 antibody and a third immunotherapeutic agent. In one embodiment, the third immunotherapeutic agent is a GITR antagonist or an OX-40 antagonist, such as an anti-GITR or anti-OX40 antibody disclosed herein. In one embodiment, the third immunotherapeutic agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116) and MK-4166 (WO11 / 028683). In one embodiment, the third immunotherapeutic agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod or NLG-919 (WO09 / 73620, WO09 / 1156652, WO11 / 56652, WO12 / 142237).

[0600] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer) comprising administering to an individual an anti-hNKG2A antibody and an anti-LAG-3 antibody. In other embodiments, the agonist anti-hNKG2A antibody is administered at a subtherapeutic dose, the anti-LAG-3 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering adverse events associated with immunostimulant treatment of hyperproliferative diseases, comprising administering to an individual an anti-hNKG2A antibody and a subtherapeutic dose of an anti-LAG-3 antibody. In certain embodiments, the individual is human. In certain embodiments, the anti-hNKG2A antibody is a human sequence monoclonal antibody and the anti-hNKG2A antibody is a humanized monoclonal antibody, such as an antibody comprising the CDRs or variable regions of an antibody disclosed herein. Examples of anti-LAG3 antibodies include antibodies comprising the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 described in U.S. Patent Publication Nos. US2011 / 0150892 and WO2014 / 008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other anti-LAG-3 antibodies that may be used include IMP731 or IMP-321 described in US 2011 / 007023. Anti-LAG-3 antibodies that compete with any of these antibodies and / or bind to the same epitope as any of these antibodies may also be used in combination therapy.

[0601] In certain embodiments, the anti-LAG-3 antibody is administered at 5×10 -8 M or lower K D Bind to human LAG-3, at 1×10- 8 M or lower K D Bind to human LAG-3, at 5×10 -9 M or lower K D Bind to human LAG-3, or at 1×10 -8 M and 1×10 -10 M or lower K D Binds to human LAG-3.

[0602] Administration of the anti-hNKG2A antibodies described herein, and other antagonists, e.g., antagonist antibodies, to one or more second target antigens (such as LAG-3 and / or CTLA-4 and / or PD-1 and / or PD-L1) can enhance the patient's immune response against cancer cells. Cancers whose growth can be inhibited using the antibodies of the invention include cancers that are typically responsive to immunotherapy. Examples of cancers treated with the combination therapies described herein include, but are not limited to, those described above for monotherapy with anti-hNKG2A antibodies.

[0603] In certain embodiments, the therapeutic antibody combinations described herein may be administered in parallel in the form of a single composition in a pharmaceutically acceptable carrier, or may be administered in parallel in the form of different separate compositions of each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies may be administered sequentially. For example, anti-CTLA-4 antibodies and anti-hNKG2A antibodies may be administered sequentially, such as first administering anti-CTLA-4 antibodies and then administering anti-hNKG2A antibodies, or first administering anti-hNKG2A antibodies and then administering anti-CTLA-4 antibodies. Additionally or alternatively, anti-PD-1 antibodies and anti-hNKG2A antibodies may be administered sequentially, such as first administering anti-PD-1 antibodies and then administering anti-hNKG2A antibodies, or first administering anti-hNKG2A antibodies and then administering anti-PD-1 antibodies. Additionally or alternatively, anti-PD-L1 antibody and anti-hNKG2A antibody may be administered sequentially, such as anti-PD-L1 antibody is administered first and then anti-hNKG2A antibody is administered, or anti-hNKG2A antibody is administered first and then anti-PD-L1 antibody is administered. Additionally or alternatively, anti-LAG-3 antibody and anti-hNKG2A antibody may be administered sequentially, such as anti-LAG-3 antibody is administered first and then anti-hNKG2A antibody is administered, or anti-hNKG2A antibody is administered first and then anti-LAG-3 antibody is administered.

[0604] In addition, if more than one dose of the combination therapy is administered sequentially, the order of sequential administration may be reversed or remain in the same order at each administration time point, sequential administration may be combined with concurrent administration, or any combination thereof. For example, the first administration of the anti-CTLA-4 antibody and anti-hNKG2A antibody combination may be concurrent administration, the second administration may be sequential administration of anti-CTLA-4 antibody first and anti-hNKG2A antibody second, and the third administration may be sequential administration of anti-hNKG2A antibody first and anti-CTLA-4 antibody second, etc. Alternatively or additionally, the first administration of the anti-PD-1 antibody and anti-hNKG2A antibody combination may be concurrent administration, the second administration may be sequential administration of anti-PD-1 antibody first and anti-hNKG2A antibody second, and the third administration may be sequential administration of anti-hNKG2A antibody first and anti-PD-1 antibody second, etc. Alternatively or additionally, the first administration of the combination of anti-PD-L1 antibody and anti-hNKG2A antibody may be concurrent administration, the second administration may be sequential administration of anti-PD-L1 antibody first and anti-hNKG2A antibody second, and the third administration may be sequential administration of anti-hNKG2A antibody first and anti-PD-L1 antibody second, etc. Alternatively or additionally, the first administration of the combination of anti-LAG-3 antibody and anti-hNKG2A antibody may be concurrent administration, the second administration may be sequential administration of anti-LAG-3 antibody first and anti-hNKG2A antibody second, and the third administration may be sequential administration of anti-hNKG2A antibody first and anti-LAG-3 antibody second, etc. Another representative dosing regimen may include a first administration of sequential administration of anti-hNKG2A first and anti-CTLA-4 antibody (and / or anti-PD-1 antibody and / or anti-PD-L1 antibody and / or anti-LAG-3 antibody) second, and subsequent administrations may be concurrent administrations.

[0605] In one embodiment, anti-hNKG2A antibodies as the sole immunotherapeutic agent, or a combination of anti-hNKG2A antibodies and one or more other immunotherapeutic antibodies (e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies), can be further combined with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells, and cells transfected with cells encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173: 4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below). ICOS agonists and one or more other antibodies (e.g., CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocking) can also be further combined with standard cancer treatments. For example, ICOS agonists and one or more other antibodies (e.g., CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocking) can be combined with chemotherapy regimens. In one embodiment, anti-hNKG2A antibodies are combined with anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies and / or anti-LAG-3 antibodies and dacarbazine and administered to patients to treat melanoma. In one embodiment, anti-hNKG2A antibodies are combined with anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies and / or anti-LAG-3 antibodies and interleukin-2 (IL-2) and administered to patients to treat cancer, including melanoma. Other combination therapies that may produce synergy with anti-hNKG2A antibodies (with or without CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 antagonism) via cytotoxicity include radiation, surgery, or hormone deprivation. In another embodiment, angiogenesis inhibitors may be combined with anti-hNKG2A antibodies and CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 antagonism.

[0606] In one embodiment, anti-hNKG2A antibodies as the sole immunotherapeutic agent, or a combination of anti-hNKG2A antibodies and CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocking antibodies, can also be used in combination with bispecific antibodies, wherein the bispecific antibodies target effector cells expressing Fcα or Fcγ receptors to tumor cells. See, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243. Bispecific antibodies can be used to target two independent antigens.

[0607] In one embodiment, anti-hNKG2A antibodies as the sole immunotherapeutic agent, or in combination with other immunostimulants (e.g., anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies and / or anti-LAG-3 antibodies), can be used together with anti-neoplastic agents (such as (rituximab), (trastuzumab), (tositumomab), (ibritumomab), (alemtuzumab), (eprtuzumab), (bevacizumab) and (erlotinib)). For example, and not wishing to be bound by theory, treatment with an anti-cancer antibody or with an anti-cancer antibody conjugated to a toxin can cause cancer cell death (e.g., tumor cells), which can enhance the immune response mediated by an immunostimulatory agent (e.g., anti-NKG2A antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-ICOS antibody, and / or anti-LAG-3 antibody). In one embodiment, the treatment of a hyperproliferative disease (e.g., a cancer tumor) can include an anti-cancer agent (e.g., an antibody) and an anti-NKG2A antibody and optionally other immunostimulatory agents, such as anti-CTLA-4 antibody and / or anti-PD-1 antibody and / or anti-PD-L1 antibody and / or anti-LAG-3 antibody, in parallel or sequentially or in any combination thereof, which can enhance the host's anti-tumor immune response.

[0608] Provided herein are methods for reducing, ameliorating, or eliminating adverse events associated with immunostimulatory treatment of hyperproliferative diseases (e.g., cancer) comprising administering to an individual an anti-NKG2A antibody (in combination or not with an anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibody). In one embodiment, the method reduces the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to the patient. As used herein, a "non-absorbable steroid" is a glucocorticoid that exhibits significant first-pass metabolism, such that after metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20%. In one embodiment described herein, the non-absorbable steroid is budesonide. Budesonide is a locally acting glucocorticoid that is significantly metabolized, primarily by the liver, after oral administration. ENTOCORT (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and throughout the colon. Approved in the U.S. for the treatment of mild to moderate Crohn's disease involving the ileum and / or ascending colon. ENTOCORT for the treatment of Crohn's disease The usual oral dose is 6 to 9 mg / day. It is released in the intestine and then absorbed and retained in the intestinal mucosa. Once it passes through the intestinal mucosal target tissue, EC is extensively metabolized in the liver by the cytochrome P450 system to metabolites with negligible glucocorticoid activity. Therefore, the bioavailability is low (approximately 10%). The low bioavailability of budesonide results in an improved therapeutic ratio compared with other glucocorticoids that have a lower degree of first-pass metabolism. Budesonide produces fewer adverse effects than systemically acting corticosteroids, including less hypothalamic-pituitary suppression. However, long-term administration of ENTOCORT Can cause systemic glucocorticoid effects such as hyperadrenocorticism and adrenal suppression. See PDR 58th edition 2004; 608-610.

[0609] In one embodiment, the anti-NKG2A antibody, with or without CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 antagonists, used in combination with non-absorbable steroids, may be further combined with salicylates. Salicylates include 5-ASA agents such as sulfasalazine ( Pharmacia & Upjohn); olsalazine ( Pharmacia & UpJohn); balsalazide ( Salix Pharmaceuticals, Inc.); and mesalamine ( Procter & Gamble Pharmaceuticals; Shire US; Axcan Scandipharm, Inc.; Solvay).

[0610] According to the methods described herein, the combination of anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or LAG-3 antibodies, anti-NKG2A antibodies and non-absorbable steroids and salicylates are administered, including any overlap or sequential administration of salicylates and non-absorbable steroids, to achieve the purpose of reducing the incidence of colitis induced by immunostimulatory antibodies. Thus, for example, the methods described herein for reducing the incidence of colitis induced by immunostimulatory antibodies encompass concurrent or sequential administration of salicylates and non-absorbable steroids (e.g., salicylates are administered 6 hours after non-absorbable steroids) or any combination thereof. In addition, salicylates and non-absorbable steroids can be administered by the same route (e.g., both are administered orally) or by different routes (e.g., salicylates are administered orally and non-absorbable steroids are administered rectally), and can be different from the route used to administer anti-NKG2A and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies.

[0611] The anti-NKG2A antibodies and combination antibody therapies described herein can also be used in combination with other well-known therapies, wherein the other resin therapies can be selected according to their specific use in the indications (e.g., cancer) being treated. The combination of anti-NKG2A antibodies described herein can be used sequentially with known pharmaceutically acceptable agents.

[0612] In one embodiment, the anti-NKG2A antibodies and combination antibody therapies described herein can be used in combination (e.g., simultaneously or separately) with other therapies such as radiation, chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu or camptothecin + apo2l / TRAIL (6X combo), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 (IDO1) inhibitors (e.g., INCB24360), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax) or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, small molecule smac mimetics, synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., ), synthetic triterpenoids (see Hyer et al., Cancer Research 2005; 65: 4799-808), c-FLIP (cellular FLICE inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARγ (peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (e.g., Sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors (such as rapamycin and temsirolimus), bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3β inhibitors, IAP inhibitors and / or genotoxic drugs.

[0613] The anti-NKG2A antibodies and combination antibody therapies described herein can be further used in combination with one or more anti-proliferative cytotoxic agents. Classes of compounds that can be used as anti-proliferative cytotoxic agents include (but are not limited to) the following:

[0614] Alkylating agents (including, but not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN TM )fosfamide), Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine and Temozolomide.

[0615] Antimetabolites (including, but not limited to, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

[0616] Suitable anti-proliferative agents for combination with anti-NKG2A antibodies include, but are not limited to, taxanes, paclitaxel (paclitaxel can be TAXOL TMpurchased), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilone, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D (furanoepothilone Epothilone B1,

[17] -dehydrodeoxyepothilone B,

[18] dehydrodeoxyepothilone B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-demethylepothilone B, epothilone B10, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), Halichondrin B ( B), Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cyrptophycin, LY-355703, Maytansine immunoconjugate (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-07 31, STA-5312, eleutherobin, 17β-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides and cryptothilone 1, as well as other tubulin stabilizers known in the art.

[0617] In some embodiments, it may be desirable to render the abnormally proliferative cells quiescent in conjunction with or prior to treatment with an anti-NKG2A antibody described herein, for example by administering to the patient hormones and steroids (including synthetic analogs), such as 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, or both. propionate, testolactone, megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorostenolone, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX TM When using the methods or compositions described herein, other agents used to modulate tumor growth or metastasis in a clinical setting, such as antimimetics, may also be administered as needed.

[0618] Methods for safe and effective administration of chemotherapeutic agents are known to those skilled in th...

Claims

1. An isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human NKG2A protein, wherein the antibody comprises: (a) 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, 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; (b) 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, 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; or (c) 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, LCDR1 of the amino acid sequence of SEQ ID NO: 155, LCDR2 of the amino acid sequence of SEQ ID NO: 14, and LCDR3 of the amino acid sequence of SEQ ID NO:

15.

2. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises: 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.

3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises: 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: 154, LCDR2 of the amino acid sequence of SEQ ID NO: 14, and LCDR3 of the amino acid sequence of SEQ ID NO:

15.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100% or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 167, and / or (b) the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100% or 100% identical to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 164 or SEQ ID NO:

169.

5. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO:

9.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO:

9.

7. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 98% identical to the amino acid sequence of SEQ ID NO:

9.

8. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO:

9.

9. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO:

164.

10. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO:

164.

11. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 98% identical to the amino acid sequence of SEQ ID NO:

164.

12. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO:

164.

13. An isolated antibody or antigen-binding fragment thereof that specifically binds to human NKG2A, wherein the antibody comprises a heavy chain variable region of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region of the amino acid sequence of SEQ ID NO:

9.

14. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody comprises a heavy chain and a light chain, and wherein: The heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100% or 100% identical to the amino acid sequence of SEQ ID NO: 7, and / or The light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100%, or 100% identical to the amino acid sequence of SEQ ID NO:

5.

15. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody comprises a heavy chain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 90% identical to the amino acid sequence of SEQ ID NO:

5.

16. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 95% identical to the amino acid sequence of SEQ ID NO:

5.

17. The antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody comprises a heavy chain that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 98% identical to the amino acid sequence of SEQ ID NO:

5.

18. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody comprises a heavy chain at least 99% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain at least 99% identical to the amino acid sequence of SEQ ID NO:

5.

19. An isolated antibody or antigen-binding fragment thereof that specifically binds to human NKG2A, wherein the antibody comprises a heavy chain of the amino acid sequence of SEQ ID NO: 7 and a light chain of the amino acid sequence of SEQ ID NO:

5.

20. An isolated antibody or antigen-binding fragment thereof that specifically binds to human NKG2A, wherein the antibody comprises a heavy chain variable region of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region of the amino acid sequence of SEQ ID NO:

164.

21. The isolated antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody comprises a heavy chain and a light chain, and wherein: The heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and The light chain is an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100% or 100% identical to the amino acid sequence of SEQ ID NO:

19.

22. The isolated antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody comprises a heavy chain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

23. The isolated antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody comprises a heavy chain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 95% identical to the amino acid sequence of SEQ ID NO:

19.

24. The isolated antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody comprises a heavy chain that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 98% identical to the amino acid sequence of SEQ ID NO:

19.

25. The isolated antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody comprises a heavy chain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain that is at least 99% identical to the amino acid sequence of SEQ ID NO:

19.

26. An isolated antibody or antigen-binding fragment thereof that specifically binds to human NKG2A, wherein the antibody comprises a heavy chain of the amino acid sequence of SEQ ID NO: 7 and a light chain of the amino acid sequence of SEQ ID NO:

19.

27. An isolated antibody or antigen-binding fragment thereof that specifically binds to human NKG2A, wherein the antibody comprises a heavy chain variable region of the amino acid sequence of SEQ ID NO: 167 and a light chain variable region of the amino acid sequence of SEQ ID NO:

169.

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

29. The antibody or antigen-binding fragment thereof of any one of claims 1-28, wherein the antibody is a full-length antibody.

30. The antibody or antigen-binding fragment thereof of any one of claims 1-28, wherein the antibody is an IgG1 antibody.

31. The antibody or antigen-binding fragment thereof of any one of claims 1-28, wherein the antibody is an antibody fragment.

32. The antibody or antigen-binding fragment thereof of claim 31, wherein the antibody fragment is a Fab, Fab', (Fab')2, Fv or scFv fragment.

33. The antibody or antigen-binding fragment thereof of any one of claims 1-28, wherein the antibody is a human, humanized or chimeric antibody.

34. An isolated full-length monoclonal antibody that specifically binds to human NKG2A protein, wherein the antibody consists of a heavy chain having an amino acid sequence as shown in SEQ ID NO: 7 and a light chain having an amino acid sequence as shown in SEQ ID NO:

5.

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

19.

36. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-35.

37. The isolated nucleic acid molecule of claim 36, wherein the heavy chain of the antibody is encoded by SEQ ID NO: 6 and the light chain of the antibody is encoded by SEQ ID NO:

4.

38. The isolated nucleic acid molecule of claim 36, wherein the heavy chain of the antibody is encoded by SEQ ID NO: 6 and the light chain of the antibody is encoded by SEQ ID NO:

18.

39. An expression vector comprising the nucleic acid molecule of claim 36, 37 or 38.

40. A host cell transformed with the expression vector of claim 39.

41. A host cell according to claim 40, wherein the cell is a eukaryotic cell.

42. A host cell according to claim 41, wherein the eukaryotic cell is a mammalian cell.

43. The host cell according to claim 42, wherein the mammalian cell is selected from the group consisting of CHO cells, NSO cells, COS cells and SP2 cells.

44. A host cell according to claim 43, wherein the mammalian cell is a CHO cell.

45. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35 linked to an active agent.

46. ​​A method of producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell of any one of claims 40-44, wherein the antibody is produced.

47. The method of claim 46, further comprising recovering the antibody or antigen-binding fragment thereof from the host cell.

48. A bispecific molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35 linked to a second functional moiety.

49. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 35 or the bispecific molecule of claim 48, and a pharmaceutically acceptable carrier.

50. The composition of claim 49, further comprising an additional therapeutic agent.

51. The composition of claim 50, wherein the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-CTLA-4 antibody.

52. The composition of claim 51, wherein the anti-PD-1 antibody is nivolumab.

53. The composition of claim 51, wherein the anti-CTLA-4 antibody is ipilimumab.

54. The composition of claim 50, wherein the other therapeutic agent is cetuximab.

55. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 35 in the preparation of a medicament for treating cancer, wherein the cancer is bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, melanoma or urothelial carcinoma.

56. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 35 in the preparation of a medicament for treating cancer, wherein the cancer is glioblastoma multiforme.

57. The method of claim 55, wherein the cancer is cervical cancer, head and neck squamous cell carcinoma, pancreatic cancer, non-small cell lung cancer-adenocarcinoma type, non-small cell lung cancer-squamous cell type, gastric cancer, melanoma, colorectal cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, urothelial carcinoma, breast cancer, small cell lung cancer, prostate cancer or non-Hodgkin's lymphoma.

58. Use of an antibody according to any one of claims 1 to 35, an immunoconjugate according to claim 45, a bispecific molecule according to claim 48, or a composition according to any one of claims 49 to 54 in the preparation of a medicament for treating cancer or delaying the progression of cancer in a human subject, wherein the cancer is bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, kidney cancer, head and neck cancer, lung cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, a central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, endometrial cancer, cervical cancer, melanoma, or urothelial cancer.

59. Use of an antibody as claimed in any one of claims 1 to 35, an immunoconjugate as claimed in claim 45, a bispecific molecule as claimed in claim 48, or a composition as claimed in any one of claims 49-54 in the preparation of a medicament for treating cancer or delaying cancer progression in a human subject, wherein the cancer is glioblastoma multiforme.

60. The method of claim 58, wherein the cancer is cervical cancer, head and neck squamous cell carcinoma, pancreatic cancer, non-small cell lung cancer-adenocarcinoma type, non-small cell lung cancer-squamous cell type, gastric cancer, melanoma, colorectal cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, urothelial carcinoma, breast cancer, small cell lung cancer, prostate cancer or non-Hodgkin's lymphoma.

61. The use according to any one of claims 55 to 60, wherein the medicament is formulated for administration in combination with one or more other therapeutic agents.

62. The method of claim 61, wherein the one or more other therapeutic agents are chemotherapeutic agents, radiotherapeutic agents and / or immunotherapeutic agents.

63. The use according to claim 62, wherein the immunotherapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-CTLA-4 antibody.

64. The use of claim 63, wherein the anti-PD-1 antibody is nivolumab, and the anti-CTLA-4 antibody is ipilimumab.

65. The use of claim 63, wherein the other therapeutic agent is cetuximab.

66. Use of an antibody according to any one of claims 1 to 35, an immunoconjugate according to claim 45, a bispecific molecule according to claim 48, or a composition according to any one of claims 49 to 54 in the preparation of a medicament for treating a chronic viral infection in a human subject, wherein the virus is selected from HIV virus, hepatitis virus, herpes virus, Epstein-Barr virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, cossackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arboviral encephalitis virus.

67. A method for detecting the presence of NKG2A protein in a sample in vitro for non-diagnostic purposes, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and the NKG2A protein, and The formation of the complex is detected.

68. The method of claim 67, wherein the antibody or antigen-binding fragment thereof forms a complex with NKG2A protein more rapidly than with NKG2C protein.

69. The method of claim 68, wherein the antibody or antigen-binding fragment thereof forms a complex with NKG2A protein 15 times faster than with NKG2C protein.

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