Neutralization of inhibitory pathways in lymphocytes

Abstract

To provide a combination product of compositions for use in the treatment of lung cancer in a human individual.SOLUTION: Provided is a combination product, containing: a first pharmaceutical composition containing an antibody that neutralizes the inhibitory activity of NKG2A by binding to human NKG2A and competes with HLA-E for binding to NKG2A, the antibody containing heavy chain CDR1, CDR2 and CDR3 domains having a specific sequence and light chain CDR1, CDR2 and CDR3 domains having a specific sequence; and a second pharmaceutical composition containing an antibody that neutralizes the inhibitory activity of PD-1, the antibody being selected from the group consisting of pembrolizumab, nivolumab, AMP-514, MEDI-4736, CT-011 and MPDL3280A.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 281,217, filed on January 21, 2016, the entire disclosure of which (including all drawings) is incorporated herein by reference.

[0002] Reference to Sequence Listing This application is filed with a sequence listing in electronic form. The sequence listing is provided as a 38 - KB file named "NKG2A - PD1b_ST25" created on January 19, 2017. The electronic form information of the sequence listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to the use of NKG2A neutralizing agents for treating cancers that are less responsive to PD - 1 neutralizing agents, as well as the combined use of NKG2A neutralizing agents and PD - 1 neutralizing agents for treating cancers.

Background Art

[0004] NK cell activity is controlled by a complex mechanism that includes both activating and inhibitory signals. Several distinct NK - specific receptors that play important roles in NK cell - mediated recognition and killing of HLA class I - deficient target cells have been identified. Natural cytotoxic receptors (NCR) refer to the types of activating receptor proteins and the genes that express them (specifically expressed in NK cells). Examples of NCR include NKp30, NKp44, and NKp46 (see, for example, (Non - Patent Document 1), (Non - Patent Document 2), (Non - Patent Document 3), (Non - Patent Document 4), (Non - Patent Document 5); (Non - Patent Document 6), the entire disclosures of which are incorporated herein by reference). These receptors are members of the Ig superfamily, and cross - linking of these induced by specific mAbs results in potent NK cell activation, and as a result, intracellular Ca ++This results in increased levels, induction of cytotoxicity, release of lymphokines, and activation of NK cell toxicity against many types of target cells.

[0005] CD94 / NKG2A is an inhibitory receptor found in a subset of lymphocytes. CD94 / NKG2A restricts the cytokine release and cytotoxic responses of certain lymphocytes to cells expressing the CD94 / NKG2A-ligand HLA-E (see, e.g., Patent Document 1). HLA-E has also been found to be secreted in a soluble form by certain tumor cells (Non-Patent Document 7) and activated endothelial cells (Non-Patent Document 8). Antibodies that inhibit CD94 / NKG2A signaling may increase the cytokine release and cytolytic activity of lymphocytes against HLA-E-positive target cells, such as the response of CD94 / NKG2A-positive NK cells to HLA-E-expressing tumor cells or virus-infected cells. Therefore, therapeutic antibodies that inhibit CD94 / NKG2A but do not induce the death of CD94 / NKG2A-expressing cells (i.e., non-depletion antibodies) may induce control of tumor growth in cancer patients.

[0006] PD-1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed in activated B cells, T cells, and myeloid cells (Non-Patent Literature 9; Non-Patent Literature 10). Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation upon binding to PD-1 (Non-Patent Literature 11; Non-Patent Literature 12; Non-Patent Literature 13). PD-L1 is abundant in various human cancers (Non-Patent Literature 14). The interaction between PD-1 and PD-L1 reduces tumor-infiltrating lymphocytes, decreases T cell receptor-mediated proliferation, and leads to immune evasion by cancer cells. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked. [Prior art documents] [Patent Documents]

[0007]

Patent Document 1

Non-licensed literature

[0008]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0009] PD-1 blockade has yielded impressive antitumor responses in many clinical trials. However, in many cancers (e.g., lung cancer), not all patients respond to treatment with an antitumor response, and some patients experience cancer recurrence after treatment. Therefore, there is a need in this field to improve the benefits for patients treated with PD-1 axis inhibitors. [Means for solving the problem]

[0010] In one embodiment, the present invention provides an improved method for inducing an antitumor immune response in individuals who are poor responders to treatment with PD1 antidotes (e.g., anti-PD-1 or anti-PD-L1 antibodies), in which the individual is treated with an agent that neutralizes the inhibitory receptor NKG2A. Tumor-infiltrating lymphocytes in PD-1 poor responders can express the inhibitory receptor NKG2A after treatment with a PD-1 neutralizing antibody. In PD-1 poor responders, TILs are restricted by the inhibitory NKG2A receptor, so tumors may progress or evade immune surveillance despite treatment with a PD-1 antidote. A PD-1 resistant mouse lymphoma model shows that treatment with an NKG2A antidote induces complete remission in the majority of individuals.

[0011] Accordingly, one embodiment provides a method for treating or preventing cancer in an individual who is a low responder or has a low response to treatment with a drug that neutralizes PD-1 inhibitory activity (e.g., an individual who is observed to be a low responder or has a low response, or is expected to be a low responder or has a low response), the method comprising the step of administering a therapeutically active amount of a compound that inhibits human NKG2A polypeptide to the individual. In one embodiment, the compound that inhibits human NKG2A polypeptide is administered in combination with a compound that inhibits human PD-1 polypeptide. In another embodiment, the compound that inhibits human NKG2A polypeptide is administered without combination treatment with a compound that inhibits human PD-1 polypeptide. In one embodiment, a compound inhibiting human NKG2A polypeptide is administered during (in combination with) or after (post-treatment) treatment with a compound inhibiting human PD-1 polypeptide, and optionally, further, the individual has either developed an incomplete response (or not developed a complete response) or has experienced cancer recurrence or progression during or after treatment with a compound inhibiting human PD-1 polypeptide (e.g., without treatment with a compound inhibiting human NKG2A polypeptide) (e.g., after the first course or cycle of treatment), and optionally, the individual has (e.g., is determined to have) a detectable and / or increased number of NKG2A-expressing NK and / or CD8 T cells and / or increased levels of NKG2A in NK and / or CD8 T cells. In one embodiment, cancer is a hematological malignancy, optionally lymphoma or leukemia. In one embodiment, cancer is a carcinoma. In one embodiment, an individual has a detectable and / or increased number of NKG2A-expressing NK and / or CD8 T cells, as well as an increased level of NKG2A in the NK and / or CD8 T cells.

[0012] In one embodiment, a method for treating or preventing cancer in an individual, (a) A step of administering to an individual a drug that neutralizes the inhibitory activity of PD-1 (for example, a step of administering a cycle or process of drug therapy), (b) If the cancer of the individual in step (a) is determined to be unresponsive to a drug that neutralizes PD-1 inhibitory activity (e.g., advanced, not fully responsive or regressed, or unresponsive), the individual is given a therapeutically active dose of a compound that inhibits human NKG2A polypeptide (optionally, in combination with a drug that neutralizes PD-1 inhibitory activity). A method including this is provided.

[0013] In one embodiment, the compound inhibiting the human NKG2A polypeptide is an antibody that neutralizes the inhibitory activity of NKG2A. In one embodiment, the compound inhibiting the human PD-1 polypeptide is an anti-PD-1 or anti-PD-1 antibody that neutralizes the inhibitory activity of PD-1. The individual may be identified as human.

[0014] In one embodiment, a method is provided for activating CD8+ tumor-infiltrating T cells in an individual having cancer that is less responsive to treatment with a drug that neutralizes PD-1 inhibitory activity (e.g., advanced, not fully responsive or regressive, or unresponsive), the method comprising the step of administering a therapeutically active amount of a compound that inhibits human NKG2A polypeptide to the individual.

[0015] In one aspect of any embodiment described herein, an individual with cancer that is poorly responsive to treatment with a PD-1 inhibitory agent is an individual who is expected to develop, or is highly likely to develop, an incomplete response, a lack of therapeutic response, detectable recurrence or persistence of cancer, and / or progressive disease (e.g., based on one or more prognostic factors) during (or after) treatment with a PD-1 inhibitory agent. In one embodiment, poorly responsive cancer is cancer that is unresponsive, completely unresponsive, detectable or persists, or recurs or progresses despite (e.g., during or after) treatment with a PD-1 inhibitory agent, or cancer that is unresponsive, completely unresponsive, detectable or persists, or is highly likely to recur or progress despite treatment with a PD-1 inhibitory agent (e.g., based on one or more prognostic factors). In one embodiment, a low-responder or an individual with a poorly responsive cancer is an individual who has produced an incomplete response (e.g., not produced a complete response (CR)) during (or after) treatment with a drug that neutralizes PD-1 inhibitory activity. In another embodiment, a low-responder or an individual with a poorly responsive cancer is an individual who has produced at least a partial response (PR) during (or after) treatment with a drug that neutralizes PD-1 inhibitory activity, but whose cancer remains detectable or has relapsed or progressed.

[0016] In one embodiment, an individual who is a low responder or has a cancer with low responsiveness is an individual who has a poor disease prognosis to treatment with an agent that neutralizes PD-1 inhibitory activity (e.g., without treatment with an agent that neutralizes NKG2A inhibitory activity; as monotherapy). An individual with a poor disease prognosis may be determined to have a high or higher risk of cancer progression (e.g., compared to an individual with a good disease prognosis) based on one or more predictors. In one embodiment, predictors include the presence of a detectable and / or increased number of NKG2A-expressing NK and / or CD8 T cells and / or increased levels of NKG2A in NK and / or CD8 T cells. In one embodiment, predictors include the presence or absence of mutations in one or more genes. In one embodiment, mutations define neoepitopes recognized by T cells. In one embodiment, predictors include one or more genes or proteins in tumor cells, e.g., levels of PD-L1 expression, decreased or increased levels of PD-L1 in tumor cells. In one embodiment, the predictor includes the level of expression of one or more genes or proteins, e.g., PD-1, in NK and / or CD8 T cells in the circulating or tumor environment. In one embodiment, the predictor includes the mutational weight in tumor cells, e.g., the number of non-synonymous mutations per exome.

[0017] In one embodiment, individuals who are low-responders or have cancers with low responsiveness are those with cancers known to be low-responder to treatment with compounds that inhibit human PD-1 polypeptides, optionally solid tumors, optionally hematological malignancies, or optionally head and neck squamous cell carcinoma.

[0018] In one embodiment, a composition is provided comprising an antibody inhibiting human NKG2A polypeptide for use in the treatment or prevention of cancer that is poorly responsive to treatment with a compound inhibiting human PD-1 polypeptide (e.g., cancer that has progressed or is expected to progress after treatment with a compound inhibiting human PD-1 polypeptide), wherein optionally the cancer is a solid tumor (e.g., lung cancer), optionally the cancer is a squamous cell carcinoma (e.g., HNSCC), and optionally the cancer is a hematological malignancy (e.g., lymphoma).

[0019] In one embodiment, a human NKG2A inhibitory agent is provided for use in combination with a human PD-1 inhibitory agent for the treatment of cancers that are poorly responsive to treatment with a human PD-1 inhibitory agent. In one embodiment, a human NKG2A inhibitory agent is provided for the treatment of an individual having lung cancer, melanoma, or squamous cell carcinoma (e.g., HNSCC), the treatment comprising the steps of administering to the individual in effective doses of (a) a human NKG2A inhibitory agent, optionally an antibody, and (b) a human PD-1 inhibitory agent, optionally an antibody.

[0020] In one embodiment, a composition is provided comprising an antibody inhibiting human NKG2A polypeptide for use in the treatment or prevention of cancer in an individual who has been or is being treated with a compound that inhibits human PD-1 polypeptide, wherein the individual has a detectable increased and / or increased number of NKG2A-expressing NK and / or CD8 T cells in the tumor environment and / or increased levels of NKG2A expression in NK and / or CD8 T cells in the tumor environment. In one embodiment, the individual has a number of circulating or tumor environment NK and / or CD8 T cells that is increased compared to the number observed before treatment with a compound that inhibits human PD-1 polypeptide and / or increased compared to a baseline (e.g., the number of cells corresponds to a value observed in a patient who develops a low response to a compound that inhibits human PD-1 polypeptide). In one embodiment, the individual has an increased level of NKG2A expression in NK and / or CD8 T cells in the tumor environment compared to the level observed before treatment with a compound that inhibits human PD-1 polypeptide and / or compared to a baseline (e.g., this level corresponds to a value observed in a patient who develops a low response to a compound that inhibits human PD-1 polypeptide). In one embodiment, the anti-NKG2A antibody is administered to a human patient (in vivo) in an amount that results in neutralization of the inhibitory activity of human CD94 / NKG2A, for example, an amount that results in neutralization of the inhibitory activity of human CD94 / NKG2A in CD8 T cells and NK cells in a human patient. In one embodiment, the amount that results in neutralization of the inhibitory activity of human CD94 / NKG2A in a human patient is at least 10 times (e.g., 10-20 times, 10-50 times, 10-100 times, 20-50 times, 20-100 times, 30-100 times, 50-100 times), optionally at least 50 times, 60 times, 80 times, or 100 times, of the lowest concentration required to substantially saturate the NKG2A receptor on the surface of NKG2A+ cells (e.g., in a binding assay where the antibody is escalated against PBMCs). In one embodiment, the anti-NKG2A antibody competes with HLA-E for binding to human NKG2A.

[0021] In one embodiment, combination treatment with an anti-NKG2A antibody and an anti-PD-1 antibody, or an anti-NKG2A antibody and an anti-PD-L1 antibody, is shown herein to be particularly effective in treating cancer. It is also shown herein that treatment with anti-PD1 can cause downregulation of the NKG2A receptor in tumor-infiltrating lymphocytes, as NKG2A can limit the effectiveness of agents that block the PD1 axis. Since both of these receptors can limit the cytotoxic activity of tumor-infiltrating lymphocytes, neutralization of the inhibitory activity of both of these receptors by antibodies allows NKG2A+PD1+ lymphocytes to effectively eradicate cancer cells. In one embodiment, NKG2A+PD1+ lymphocytes are cytotoxic lymphocytes, optionally CD8+ T cells or NK cells.

[0022] In one embodiment, the present invention provides an improved method for promoting an antitumor immune response by combined neutralization of the inhibitory receptor NKG2A and PD-1, for example, by the use of an antibody. The combined treatment can be used to treat individuals with cancer (e.g., cancers known to be poorly responsive to PD-1 neutralizing agents, such as non-small cell lung cancer (NSCLC), kidney cancer, gastrointestinal cancer, pancreatic or esophageal adenocarcinoma, breast cancer, renal cell carcinoma (RCC), melanoma, colorectal cancer, or ovarian cancer), including, for example, individuals who are poorly responsive to treatment with agents that neutralize PD-1 inhibitory activity and individuals who are good responsive to treatment with agents that neutralize PD-1 inhibitory activity, regardless of whether they are poorly responsive to neutralization of PD-1 inhibitory activity.

[0023] Accordingly, one embodiment provides a method for treating or preventing cancer that is less responsive to treatment with a compound that inhibits human PD-1 polypeptide, the method comprising the steps of administering to an individual with cancer (a) a therapeutically active amount of a compound that inhibits human NKG2A polypeptide, and (b) a therapeutically active amount of a compound that inhibits human PD-1 polypeptide. In one embodiment, the cancer is a solid tumor, optionally comprising invasive NK and / or CD8 T cells, optionally expressing NKG2A, optionally expressing NKG2A on the surface of at least 10%, 20%, 30%, 40%, or 50% of the NK cells. In one embodiment, the compound that inhibits human NKG2A polypeptide is an antibody that neutralizes the inhibitory activity of NKG2A. In one embodiment, the compound that inhibits human PD-1 polypeptide is an anti-PD-1 or anti-PD-1 antibody that neutralizes the inhibitory activity of PD-1. The individual may be identified as human.

[0024] In one embodiment, a method is provided for activating or promoting the activity of CD8+ tumor-infiltrating T cells in an individual, the method comprising the steps of administering to the individual (a) a therapeutically active amount of a compound that inhibits human NKG2A polypeptide and (b) a therapeutically active amount of a compound that inhibits human PD-1 polypeptide. In one embodiment, the cancer is a solid tumor. Optionally, in any embodiment, the individual has a cancer that is less responsive to treatment with a compound that inhibits human PD-1 polypeptide.

[0025] In one embodiment, the present invention provides a treatment comprising the step of administering a combination of an antibody that neutralizes the inhibitory activity of NKG2A and an antibody that neutralizes the inhibitory activity of PD-1. In one embodiment of any embodiment herein, a composition is provided comprising an antibody that inhibits human NKG2A polypeptide and an antibody that binds to PDL1 or PD1 and neutralizes the inhibitory activity of human PD-1 polypeptide. In one embodiment, this composition is used for the treatment or prevention of cancer.

[0026] In one embodiment, an antibody neutralizing NKG2A inhibitory activity and an antibody neutralizing PD-1 inhibitory activity are used in combination in the treatment or prevention of cancers known to be less responsive to treatment with compounds that inhibit human PD-1 polypeptides, optionally solid tumors, and optionally hematological malignancies. In one embodiment, the cancers known to be less responsive are squamous cell carcinomas, optionally head and neck squamous cell carcinomas.

[0027] In any embodiment of this specification, a compound or agent inhibiting a human PD-1 polypeptide includes, for example, a polypeptide (e.g., an antibody, a polypeptide fused to an Fc domain, an immunoadhesion factor, etc.) that prevents PD-L1-induced PD-1 signaling by blocking the interaction of PD-1 with its native ligand PD-L1 (optionally, by further blocking the interaction of PD-1 with PD-L2). In one embodiment, the polypeptide is an antibody that binds to PD-1 (an anti-PD-1 antibody), and such an antibody can block the interaction of PD-1 with PD-L1 and / or the interaction of PD-1 with PD-L2. In another embodiment, the polypeptide is an antibody that binds to PD-L1 (an anti-PD-L1 antibody), and blocks the interaction of PD-1 with PD-L1. In one embodiment, the antibody that neutralizes the human PD-1 polypeptide is an amount that results in neutralization of the inhibitory activity of human PD-1 in a human patient (in vivo), for example, in a human patient with CD8 The antibody is administered / administered in an amount that results in neutralization of the inhibitory activity of human PD-1 in T cells and NK cells. In one embodiment, the antibody is administered (or is intended for administration) according to a specific clinical dosing regimen, in particular at a specific dose and according to a specific dosing schedule.

[0028] In one aspect of any embodiment described herein, the compound or agent that neutralizes the inhibitory receptor NKG2A is an antibody. In one aspect, the antibody that neutralizes NKG2A is a non-depleting antibody, for example, an antibody that does not kill, eradicate, lyse, or induce such killing, eradication, or lyse, so as not to adversely affect the number of NKG2A-expressing cells present in the sample or subject. In one aspect, the antibody that neutralizes NKG2A is a non-depleting antibody. A non-depleting antibody may, for example, lack an Fc domain or have an Fc domain that binds minimally to or does not bind to one or more Fcγ receptors (e.g., CD16). Examples include antibodies with a constant region from human IgG4 isotype antibodies, and antibodies of any isotype (e.g., IgG1, IgG2, IgG3, IgG4) having a constant region modified to reduce or eliminate binding to one or more Fcγ receptors (e.g., CD16, CD32A, CD32B, and / or CD64).

[0029] In one aspect of any embodiment described herein, the anti-NKG2A antibody is administered in at least one dosing cycle, the dosing cycle comprising at least one first and second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or further) dosing of the anti-NKG2A antibody, and the anti-NKG2A antibody is administered in an amount effective to achieve a continuous (minimum) blood concentration of at least 10 μg / ml (or optionally at least 20, 30, 40, or 50 μg / mL) of the anti-NKG2A antibody between the first and second (and optionally further) dosings. Achieving or maintaining a predetermined continuous blood concentration means that the blood concentration does not substantially fall below a predetermined blood concentration over a predetermined duration (e.g., between two dosings of the antibody, several weeks), i.e., the blood concentration may fluctuate during the predetermined period, but the predetermined blood concentration represents a minimum or "trough" concentration.

[0030] In one embodiment, the anti-NKG2A antibody is administered at the time of administration (e.g., within one or two days of administration) in an amount effective to achieve a peak blood concentration of approximately or at least approximately 50, 60, 70, or 80 μg / ml, optionally at least approximately 100 μg / ml.

[0031] In one embodiment, the anti-NKG2A antibody is administered for at least one week, or at least two weeks, after antibody administration, in an amount effective to achieve a continuous (minimum) blood concentration of approximately or at least approximately 10, 20, 30, 40, 50, 60, 70, or 80 μg / ml, optionally at least approximately 100 μg / ml.

[0032] In one embodiment, the anti-NKG2A antibody is administered in an amount effective to achieve a consecutive (minimum) blood concentration of the anti-NKG2A antibody, approximately or at least approximately 50, 60, 70, or 80 μg / ml, and optionally at least approximately 100 μg / ml, between two consecutive doses. In one embodiment, the first and second doses are separated by a period of approximately two weeks, and optionally approximately one week.

[0033] Anti-NKG2A antibodies may be administered selectively in an effective amount, and at a frequency that achieves a specified continuous (minimum) blood concentration over the entire duration of the administration cycle.

[0034] In one embodiment, an antibody inhibiting human NKG2A polypeptide is administered in a dosing cycle comprising at least two doses of the anti-NKG2A antibody, following the course of treatment (which may be completed entirely or stopped before completion), or following the start of the course of treatment (e.g., in the case of an incomplete response, tumor progression, etc.) together with an antibody neutralizing human PD-1 polypeptide. In another embodiment, an antibody inhibiting human NKG2A polypeptide is administered in combination with an antibody neutralizing human PD-1 polypeptide in a dosing cycle, and the dosing cycle comprises at least two doses of the anti-NKG2A antibody. In one embodiment, the anti-NKG2A antibody is administered at least between two consecutive doses. The anti-NKG2A antibody is administered in an amount effective to achieve a continuous (minimum) concentration in extravascular tissue (e.g., in the tumor environment) of at least 4 μg / mL, or optionally at least 10 μg / mL. Optionally, the anti-NKG2A antibody is administered in an amount effective to achieve a continuous (minimum) concentration in extravascular tissue (e.g., in the tumor environment) of at least 4 μg / mL, or optionally at least 10 μg / mL, over the entire duration of the administration cycle. In one embodiment, the anti-NKG2A antibody is administered between two consecutive administrations or over the duration of the administration cycle in an amount effective to achieve a continuous (minimum) blood concentration of at least 40 μg / mL, or optionally at least 100 μg / mL.

[0035] In one embodiment, cancer is a blood cancer. In one non-limiting embodiment, cancer is a lymphoma or leukemia. In one embodiment, cancer is an advanced and / or refractory solid tumor. In one non-limiting embodiment, cancer (e.g., an advanced, refractory solid tumor) is selected from the group consisting of non-small cell lung cancer (NSCLC), kidney cancer, gastrointestinal cancer, pancreatic or esophageal adenocarcinoma, breast cancer, renal cell carcinoma (RCC), melanoma, colorectal cancer, and ovarian cancer.

[0036] Compounds that inhibit the NKG2A polypeptide (anti-NKG2A agents) are compounds that increase the ability of NKG2A-expressing NK and / or T cells to cause the death of HLA-E-expressing cells. Optionally, compounds that inhibit the NKG2A polypeptide are polypeptides that bind to the NKG2A polypeptide, or optionally, antibodies (e.g., monoclonal antibodies).

[0037] In one embodiment, an anti-NKG2A agent reduces the inhibitory activity of NKG2A by blocking the binding of its ligand, HLA-E. That is, the anti-NKG2A agent interferes with the binding of NKG2A by HLA-E. An antibody having one of the heavy chains of SEQ ID NOs. 4-8 and the light chain of SEQ ID NO. 9 is an example of such an antibody. In another embodiment, an anti-NKG2A agent reduces the inhibitory activity of NKG2A without blocking the binding of its ligand, HLA-E. That is, the anti-NKG2A agent is a non-competitive antagonist and does not interfere with the binding of NKG2A by HLA-E. An antibody having the heavy chain and light chain variable regions of SEQ ID NOs. 10 and 11, respectively, is an example of such an antibody.

[0038] In one embodiment, the anti-NKG2A agent is an antibody that binds to NKG2A with significantly higher affinity than to one or more activated NKG2 receptors. For example, in one embodiment, the agent is an antibody that binds to NKG2A with significantly higher affinity than to NKG2C. In additional or alternative embodiments, the agent is an antibody that binds to NKG2A with significantly higher affinity than to NKG2E. In additional or alternative embodiments, the agent is an antibody that binds to NKG2A with significantly higher affinity than to NKG2H.

[0039] In one embodiment, the anti-NKG2A agent competes for binding to CD94 / NKG2A with antibodies having the heavy and light chains of SEQ ID NOs. 4-8 and 9, respectively, with antibodies having the light chain of SEQ ID NOs. 7, or antibodies having the heavy and light chain variable regions of SEQ ID NOs. 10 and 11, respectively. The agent may be, for example, a human or humanized anti-NKG2A antibody.

[0040] In one embodiment, the anti-NKG2A antibody is a humanized antibody having a heavy chain CDR of any of the heavy chains of SEQ ID NOs: 4-8 and a light chain CDR of the light chain of SEQ ID NO: 9. In one embodiment, the anti-NKG2A antibody is a humanized antibody having a heavy chain variable region of any of the heavy chains of any of the heavy chains of SEQ ID NOs: 4-8 and a light chain variable region of the light chain of SEQ ID NO: 9. Exemplary complementarity-determining region (CDR) residues or sequences and / or sites for amino acid substitutions in the framework region (FR) of such a humanized antibody having improved properties (e.g., lower immunogenicity, improved antigen-binding or other functional properties) and / or improved physicochemical properties (e.g., better stability).

[0041] In some optional embodiments, a method is provided for identifying individuals with cancer who are poor responders to treatment with agents that neutralize the inhibitory activity of human PD-1, and this method a) In individuals treated with an agent that neutralizes the inhibitory activity of human PD-1 (e.g., received at least one dose of this agent), the levels of NKG2A (and optionally further PD-1) expression in NK and / or CD8 T cells, as well as / or NKG2A + (Optionally select NKG2A) + PD1 + ) A step of determining the number of NK and / or CD8 T cells, b) An individual has increased levels of NKG2A expression and / or an increased number of NKG2A in NK and / or CD8 T cells (optionally selected PD-1 expressing NK or T cells). + (Optionally select NKG2A) + PD1 + )If it is determined that the individual has NK and / or CD8 T cells (e.g., increased compared to reference values, increased compared to reference values, increased compared to values ​​observed before drug treatment), the individual is identified as a low responder to treatment with a drug that neutralizes human PD-1 inhibitory activity. Includes.

[0042] In some optional embodiments, a patient may be identified for treatment with an anti-NKG2A agent by evaluating the presence of NKG2A expression in NK and / or CD8+ T cells in a tumor sample (e.g., tumor tissue and / or adjacent tumor tissue). In any embodiment of the therapeutic use or method of treating or preventing cancer described herein, the treatment or prevention of cancer in an individual is a) A step of determining the level of NKG2A expression in NK and / or CD8 T cells and / or the number of NKG2A-expressing NK and / or CD8 T cells in an individual with cancer who has been treated with or is being treated with an agent that neutralizes PD-1 inhibitory activity, b) If it is determined that the individual has increased levels of NKG2A expression in NK and / or CD8 T cells and / or an increased number of NKG2A-expressing NK and / or CD8 T cells, the individual is administered a compound that neutralizes the inhibitory activity of human NKG2A polypeptide. Includes.

[0043] In any embodiment of the present method, the step of determining the level of NKG2A expression in NKG2A+ NK and / or CD8 T cells and / or the number of NKG2A+ NK and / or CD8 T cells includes the step of determining the level of NKG2A nucleic acid or polypeptide expression in NK and / or CD8 T cells and / or determining the number of NKG2A+ NK and / or CD8 T cells in a biological sample and comparing this level to a reference level (e.g., a value, weak or strong cell surface staining). The reference level may correspond to, for example, a healthy person, an individual who is responsive or unresponsive to treatment with a human PD-1 polypeptide inhibitor, an individual who does not / has little clinical benefit from treatment with an anti-NKG2A antibody, or an individual who obtains substantial clinical benefit from treatment with an anti-NKG2A antibody (optionally in combination with a human PD-1 polypeptide inhibitor). A determination that a biological sample contains an increased number of NKG2A-expressing NK and / or CD8 T cells (e.g., a number corresponding to individuals who do not receive sufficient clinical benefit from treatment with human PD-1 polypeptide inhibitors, a number corresponding to individuals who receive substantial clinical benefit from treatment with anti-NKG2A antibodies) indicates that the individual has cancer that can be treated with anti-NKG2A antibodies, for example, according to the treatment methods described herein. A determination that a biological sample expresses NKG2A nucleic acid or polypeptide at increased levels (e.g., high levels, strong surface staining, levels corresponding to individuals who do not receive sufficient clinical benefit from treatment with human PD-1 polypeptide inhibitors, levels corresponding to individuals who receive substantial clinical benefit from treatment with anti-NKG2A antibodies, levels higher than those corresponding to individuals who receive no / low clinical benefit from treatment with anti-NKG2A antibodies, etc.) indicates that the individual has cancer that can be treated with anti-NKG2A antibodies, for example, according to the treatment methods described herein.

[0044] In one embodiment, the CD8 T cells are tumor-infiltrating CD8 T cells. In one embodiment, the NK cells are tumor-infiltrating NK cells. In one embodiment, at least 15%, 20%, 25% or 30% of the NK and / or CD8 T cells are PD1 + NKG2A + positive.

[0045] In one embodiment, an individual has a cancer comprising malignant cells that express HLA-E polypeptide on their surface. In one embodiment, the method of treatment further comprises determining the status of the HLA-E polypeptide of malignant cells (e.g., tumor cells) in an individual having cancer, and determination that the malignant cells express HLA-E nucleic acid or polypeptide indicates that the individual has a cancer that can be treated with an agent that inhibits NKG2A.

[0046] In other embodiments, pharmaceutical compositions and kits, and methods of using them are provided. In one embodiment, a pharmaceutical composition is provided that comprises a compound that neutralizes the inhibitory activity of the human NKG2A polypeptide. In one embodiment, a kit is provided that comprises a compound that neutralizes the inhibitory activity of the human NKG2A polypeptide and an agent (e.g., an antibody or other NKG2A binding agent conjugated to a detectable moiety) capable of detecting the expression of NKG2A on the surface of NK and / or CD8 T cells.

[0047] These aspects are described in more detail in the description of the invention provided herein, and additional aspects, features, and advantages will be apparent from the description of the invention provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] [Figure 1-1]This shows that PD-L1+Qa-1+RMA-S Qa-1 Qdm B2m and A20 tumor cells are infiltrated by NK cells expressing NKG2A and CD8 T cells expressing NKG2A and / or PD-1. RMA-S Qa-1 Qdm B2m (upper column) and A20 (lower column) tumor-bearing mice were euthanized when the tumor volume was approximately 500 mm3. Tumor cells (Figure 1A) and tumor-infiltrating lymphocytes (TILs) (Figure 1B) were analyzed by flow cytometry for Qa-1 and PDL-1 expression in tumor cells, and for NKG2A / C / E and PD-1 expression in TILs. MFI: Median fluorescence intensity. [Figure 1-2] Same as above. [Figure 2] This study describes the distribution of NKG2A and PD-1 in NK and T cell subsets in mice. Lymphocytes were collected from the spleen, tumor draining lymph nodes, and solid tumors. PD-1 expression was low or absent in all cell subsets from the spleen and lymph nodes, but in tumor-infiltrating lymphocytes (TILs), all cell subsets had a relatively high proportion of PD-1-expressing cells. On the other hand, in the spleen and lymph nodes, NKG2A was found on NK cells but not on T cell subsets, and in tumors, it was found on a significant proportion of TILs, with over 30% of NK cells and over 19% of CD8 T cells being double-positive for NKG2A and PD-1 on average. [Figure 3-1] This figure shows NKG2A and PD-1 expression in tumor-bearing mice. RMA Rae1 (top row), MC38 (middle row), and RMA (bottom row) tumor-bearing mice were euthanized when their tumors reached volumes of 500, 2000, and 800 mm3, respectively. NK cells (Figure 3A) and CD8 T cells (Figure 3B) were analyzed for NKG2A / C / E and PD-1 expression by flow cytometry in the spleen, tumor inflow lymph nodes (LN), and tumors. [Figure 3-2] Same as above. [Figure 4]This study demonstrates that treatment of mice with an anti-PD-1 mAb increases the frequency of NKG2A-expressing TCD8 cells in MC38 tumors. MC38 tumor-bearing mice were treated with either 200 μg of rat IgG2a isotype control (IC) or an anti-mouse PD-1 antibody 11, 14, and 17 days after cell engraftment. Mice were euthanized on day 31, and CD8 T cells were characterized by flow cytometry in the spleen, tumor inflow lymph nodes (LN), and tumors. [Figure 5] The median tumor volume over time is shown for mice treated with isotype controls, anti-mouse NKG2A mAb (200 μg, intravenous (iv)), anti-mouse PD-L1 mAb (200 μg, intraperitoneal (ip)), or anti-mNKG2A / mPDL-1 combination on days 11, 14, and 18. In this model, anti-NKG2A showed only a slight antitumor effect compared to isotype controls, anti-PD-L1 showed a considerable antitumor effect, but tumor volume increased towards day 28, and combined treatment with anti-NKG2A and anti-PD-L1 resulted in complete cessation of tumor growth, with no significant increase in tumor volume observed on day 28. [Figure 6] The median tumor volume over time is shown in mice carrying PD-1 / -L1 resistant A20 lymphoma, treated with isotype controls, neutralizing anti-mouse PD-L1 mAbs, or anti-mouse PD-1. Neither isotype controls, anti-PD1, nor anti-PD-L1 antibodies were effective in controlling tumor growth. [Figure 7] The median tumor volume over time in mice carrying A20 lymphoma treated with an isotype control or a neutralizing anti-mouse PD-1 mAb or anti-mouse PD-1 in combination with neutralizing anti-mouse NKG2A. Neither the isotype control nor the anti-PD1 antibody was effective in controlling tumor growth, but complete tumor regression was observed in the majority of animals (7 / 10) when anti-NKG2A antibody treatment was added. [Figure 8]The median tumor volume over time is shown in mice carrying A20 lymphoma, treated with an isotype control or a neutralizing anti-mouse PD-L1 mAb or anti-mouse PD-L1 in combination with neutralizing anti-mouse NKG2A. Complete tumor regression was observed in the majority of animals (9 / 11) when anti-NKG2A antibody treatment was added to anti-PD-L1 treatment. [Modes for carrying out the invention]

[0049] definition As used herein, “one (a)” or “one (an)” may mean one or more. As used in the claims, when used with the word “including,” the words “one (a)” or “one (an)” may mean one or more. As used herein, “another” may mean at least a second or more.

[0050] When "includes" is used, it can optionally be replaced by "essentially become from" or "consist of."

[0051] NKG2A (OMIM161555, its entire disclosure is incorporated herein by reference) is a member of the NKG2 group of transcripts (Houchins, et al. (1991) J. Exp. Med. 173:1017-1020). NKG2A is encoded by seven exons spanning 25 kb and exhibits some discriminant splicing. Together with CD94, NKG2A forms the heterodimer inhibitory receptor CD94 / NKG2A, found on the surface of NK cells, α / β T cells, γ / δ T cells, and subsets of NKT cells. Like inhibitory KIR receptors, it possesses ITIM in its cytoplasmic domain. As used herein, “NKG2A” refers to any variant, derivative, or isoform of the NKG2A gene or encoding protein. Human NKG2A is sequenced as follows: [ka] It contains 233 amino acids in three domains: the cytoplasmic domain contains residues 1-70, the transmembrane domain contains residues 71-93, and the extracellular domain contains residues 94-233.

[0052] NKG2C (OMIM602891, the entire disclosure is incorporated herein by reference) and NKG2E (OMIM602892, the entire disclosure is incorporated herein by reference) are two other members of the NKG2 group of transcripts (Gilenke, et al. (1998) Immunogenetics 48:163-173). CD94 / NKG2C and CD94 / NKG2E receptors are activating receptors found on the surface of subsets of lymphocytes, such as NK cells and T cells.

[0053] HLA-E (OMIM143010, the entire disclosure of which is incorporated herein by reference) is a non-classical MHC molecule expressed on the cell surface and controlled by the binding of peptides, such as fragments derived from the signal sequences of other MHC class I molecules. A soluble form of HLA-E has also been identified. In addition to its T cell receptor binding properties, HLA-E binds to subsets of natural killer (NK) cells, natural killer T cells (NKT), and T cells (α / β and γ / δ) by specifically binding to CD94 / NKG2A, CD94 / NKG2B, and CD94 / NKG2C (see, for example, Braud et al. (1998) Nature 391:795-799, the entire disclosure of which is incorporated herein by reference). Surface expression of HLA-E protects target cells from lysis by CD94 / NKG2A+NK, T, or NKT cell clones. As used herein, "HLA-E" refers to any variant, derivative, or isoform of the HLA-E gene or encoding protein.

[0054] In relation to the present invention, "NKG2A" or "CD94 / NKG2A-positive lymphocytes" refers to lymphocytes (e.g., NK-, NKT-, and T cells) that express CD94 / NKG2A on their cell surface, which can be detected, for example, by flow cytometry using antibodies that specifically recognize a combined epitope on CD94 and NKG2A, or an epitope on NKG2A alone. "NKG2A-positive lymphocytes" also include lymphocyte-derived immortal cell lines (e.g., NKL, NK-92).

[0055] In relation to the present invention, “reducing the inhibitory activity of NKG2A,” “neutralizing NKG2A,” or “neutralizing the inhibitory activity of NKG2A” refers to the inhibition of CD94 / NKG2A in its ability to adversely affect intracellular processes, resulting in lymphocyte responses such as cytokine release and cytotoxic responses. This can be measured, for example, in NK- or T-cell based cytotoxicity assays, which measure the ability of therapeutic compounds to stimulate the death of HLA-E-positive cells by CD94 / NKG2A-positive lymphocytes. In one embodiment, an antibody preparation induces at least a 10% enhancement of cytotoxicity of CD94 / NKG2A-restricted lymphocytes, optionally at least a 40% or 50% enhancement of lymphocyte cytotoxicity, or optionally at least a 70% enhancement of NK cytotoxicity, as described in the cytotoxicity assay. If an anti-NKG2A antibody reduces or blocks the interaction of CD94 / NKG2A with HLA-E, the cytotoxicity of CD94 / NKG2A-restricted lymphocytes may be increased. This can be evaluated, for example, in a standard 4-hour in vitro cytotoxicity assay using NK cells expressing CD94 / NKG2A and target cells expressing HLA-E. Such NK cells do not efficiently kill HLA-E-expressing targets because CD94 / NKG2A recognizes HLA-E, leading to the initiation and propagation of inhibitory signaling that prevents lymphocyte-mediated cytolysis. Such in vitro cytotoxicity assays can be performed by standard methods well known in the art, such as those described, for example, in Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993).Furthermore, chromium release and / or other parameters for evaluating the ability of antibodies to stimulate lymphocytes to kill target cells such as P815, K562 cells, or appropriate tumor cells are disclosed in Sivori et al., J.Exp.Med.1997;186:1129-1136; Vitale et al., J.Exp.Med.1998;187:2065-2072; Pessino et al. J.Exp.Med.1998;188:953-960; Neri et al. Clin.Diag.Lab.Immun.2001;8:1131-1135; Pende et al. J.Exp.Med.1999;190:1505-1516 (the entirety of each of these disclosures is incorporated herein by reference). Target cells are added before the addition of NK cells. 51 Labeled with Cr, then transferred from the cells to the culture medium as a result of death. 51 Cell death is estimated to be proportional to the release of Cr. Adding an antibody that prevents CD94 / NKG2A from binding to HLA-E consequently prevents the initiation and propagation of inhibitory signaling by CD94 / NKG2A. Therefore, the addition of such a drug results in increased lymphocyte-mediated target cell death. This step involves identifying drugs that prevent CD94 / NKG2A-induced negative signaling, for example, by blocking ligand binding. 51 In creatinine-releasing cytotoxicity assays, CD94 / NKG2A-expressing NK effector cells can kill HLA-E-negative LCL721.221 target cells, but do not kill HLA-E-expressing LCL721.221-Cw3 control cells very effectively. In contrast, CD94 / NKG2A-deficient YTS effector cells efficiently kill both cell lines. Therefore, NK effector cells are necessary for HLA-E-induced inhibitory signaling via CD94 / NKG2A. + It does not kill LCL721.221-Cw3 cells very efficiently. 51In a Cr-releasing cytotoxicity assay, when NK cells are pre-incubated with a blocking anti-CD94 / NKG2A antibody according to the present invention, HLA-E-expressing LCL721.221-Cw3 cells are more efficiently killed in an antibody concentration-dependent manner. The inhibitory activity (i.e., cytotoxicity-enhancing ability) of the anti-NKG2A antibody can also be assessed by its effect on intracellular free calcium in one of several other methods, for example, as described in Sivori et al., J.Exp.Med.1997;186:1129-1136 (the disclosure thereof is incorporated herein by reference). The activation of NK cell cytotoxicity can be assessed, for example, by measuring increased cytokine production (e.g., IFN-γ production) or cytotoxicity markers (e.g., CD107 or CD137 recruitment). In an exemplary protocol, IFN-γ production from PBMCs is assessed after 4 days in culture by cell surface and intracellular staining and flow cytometry analysis. In short, brefelzin A (Sigma Aldrich) is added at a final concentration of 5 μg / ml during the last 4 hours of culture. The cells are then incubated with anti-CD3 and anti-CD56 mAb before permeabilization (IntraPrep®; Beckman Coulter) and staining with PE-anti-IFN-y or PE-IgG1 (Pharmingen). GM-CSF and IFN-y production from polyclonal activated NK cells is measured in the supernatant using ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, MN; IFN-y: OptElA set, Pharmingen).

[0056] As used herein, the term “PD-1” refers to programmed death 1 (PD-1), also known as “programmed cell death 1,” an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. The complete human PD-1 sequence can be found under GenBank accession number U64863 and is shown below. [ka]

[0057] "PD-1" also includes any variants, derivatives, or isoforms of the PD-1 gene or the encoding protein. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The first members of this family, CD28 and ICOS, were discovered by their functional effects on increasing T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260). Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members.

[0058] The complete human PD-L1 sequence can be found in UniProtKB / Swiss-Prot, identifier Q9NZQ7-1, and is shown below. [ka]

[0059] PD-L1 is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 reduces tumor-infiltrating lymphocytes, decreases T-cell receptor-mediated proliferation, and allows cancer cells to evade the immune response (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked.

[0060] In relation to the present invention, “reducing human PD-1 inhibitory activity,” “neutralizing PD-1,” or “neutralizing human PD-1 inhibitory activity” refers to the process by which the signaling capacity of PD-1 is inhibited, resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1 or PD-L2). Agents that neutralize PD-1 inhibitory activity reduce, block, inhibit, suppress, or interfere with the signaling resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2, etc.). Thereafter, such agents can reduce negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes to promote T cell effector functions such as proliferation, cytokine production, and / or cytotoxicity.

[0061] Whenever “treatment of cancer” or similar is referred to in relation to an anti-NKG2A conjugate (e.g., an antibody), it means (a) a method of treating cancer, the method comprising the step of administering an NKG2A conjugate (e.g., together or separately in a pharmaceutically acceptable carrier material) to an individual, mammal, in particular a human, who is in need of such treatment, in a dose (amount) that enables treatment of cancer (a therapeutically effective amount), optionally as specified herein; (b) an anti-NKG2A conjugate for the treatment of cancer (a) the use of anti-NKG2A conjugates for use in the aforementioned treatment (in particular in humans); (c) the use of anti-NKG2A conjugates for the manufacture of a pharmaceutical product for the treatment of cancer, a method of using anti-NKG2A conjugates for the manufacture of a pharmaceutical product for the treatment of cancer, comprising mixing the anti-NKG2A conjugate with a pharmaceutically acceptable carrier, or a pharmaceutical product comprising an effective dose of anti-NKG2A conjugate suitable for the treatment of cancer; or (d) any combination of a), b), and c) relating to patentable subject matter in the country in which this application is filed.

[0062] As used herein, the term "biopsy" is defined as the removal of tissue for the purpose of examination, such as establishing a diagnosis. Examples of types of biopsies include, for example, by aspiration via a needle attached to a syringe; by instrumental removal of tissue fragments; by removal using appropriate instruments via an endoscope; and by surgical excision, such as surgical excision of an entire lesion.

[0063] The term "antibody," as used herein, refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these are further classified into subclasses or isotypes such as IgG1, IgG2, IgG3, and IgG4. The structural unit of an exemplary immunoglobulin (antibody) is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. L ) and variable heavy chain (V H The terms ) refer to these light and heavy chains, respectively. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called "alpha," "delta," "epsilon," "gamma," and "mu," respectively. The subunit structures and three-dimensional arrangements of the various classes of immunoglobulins are well known. IgG is the most common antibody under physiological conditions and is the easiest to produce in a laboratory setting, and is therefore an exemplary type of antibody used herein. Optionally, an antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human, or otherwise human-adapted antibodies. "Antibody" also includes any fragment or derivative of any of the antibodies described herein.

[0064] The term "specifically binding" means that an antibody can bind to a binding partner, preferably NKG2A, PD-1, or PD-L1, in a competitive binding assay, when evaluated using either the recombinant form of the protein, its epitope, or a native protein present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected by physical methods such as radiolabeling or mass spectrometry, or by direct or indirect fluorescent labeling detected using, for example, cell fluorescence analysis (e.g., FACScan). Binding exceeding the amount seen with a nonspecific control drug indicates that the drug binds to its target. Drugs that specifically bind to NKG2A may bind to NKG2A alone or as a dimer with CD94.

[0065] When an antibody is said to "compete" with a particular monoclonal antibody, it means that the antibody competes with the monoclonal antibody in a binding assay using either a recombinant molecule (e.g., NKG2A, PD-1, PD-L1) or a surface-expressed molecule (e.g., NKG2A, PD-1, PD-L1). For example, if a test antibody reduces the binding of an antibody having one of the heavy chains of SEQ ID NOs. 4-8 and the light chain of SEQ ID NO. 9 to NKG2A polypeptide or NKG2A-expressing cells in a binding assay, the antibody is said to "compete" with such antibodies, respectively.

[0066] The term "affinity," as used herein, refers to the strength of an antibody's binding to an epitope. Antibody affinity is given by the dissociation constant Kd, defined as [Ab] × [Ag] / [Ab-Ag] (where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody, and [Ag] is the molar concentration of the unbound antigen). Affinity constant Kd aIt is defined by 1 / Kd. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), these references are fully incorporated herein by reference. One standard method well known in the art for measuring the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (e.g., by analysis using a BIAcore® SPR analyzer).

[0067] In this specification, “determinant” refers to a site of interaction or binding on a polypeptide.

[0068] The term "epitope" refers to an antigenic determinant, a region or area on an antigen to which an antibody binds. Protein epitopes can include amino acid residues directly involved in binding, as well as amino acid residues effectively blocked by specific antigen-binding antibodies or peptides, i.e., amino acid residues within the antibody's "footprint." It is, for example, the simplest form or smallest structural region on a complex antigen molecule that can bind to an antibody or receptor. Epitopes can be linear or steric / structural. The term "linear epitope" is defined as an epitope consisting of consecutive amino acid residues on a linear sequence of amino acids (primary structure). The term "steric or structural epitope" is defined as an epitope consisting of amino acid residues that are not all consecutive and therefore represent separations in a linear sequence of amino acids that are brought into close proximity by molecular folding (secondary, tertiary, and / or quaternary structure). Steric epitopes depend on three-dimensional structure. The term "steric" is therefore often used interchangeably with "structural."

[0069] The term “drug” is used herein to refer to a compound, a mixture of compounds, a biological macromolecule, or an extract made from a biological material. The term “therapeutic drug” refers to a drug that has biological activity.

[0070] For the purposes of this specification, “humanized” or “human” antibody refers to an antibody in which the constant and variable framework regions of one or more human immunoglobulins are fused with the binding region of an animal immunoglobulin, e.g., CDR. Such antibodies retain the binding specificity of the non-human antibody from which the binding region originates, but are designed to avoid immune responses to non-human antibodies. Such antibodies can be obtained from transgenic mice or other animals that have been “engineered” to produce specific human antibodies in response to antigen loading (see, for example, Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teaching of which is incorporated herein by reference). Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display techniques, all of which are known in the art (see, for example, McCafferty et al. (1990) Nature 348:552-553). Human antibodies can also be produced in vitro by activated B cells (see, for example, U.S. Patent Nos. 5,567,610 and 5,229,275, which are incorporated in their entirety by reference).

[0071] A "chimeric antibody" is (a) an antibody molecule in which the constant region or part thereof is modified, substituted, or exchanged so that the antigen-binding site (variable region) is bound to a constant region of a different or modified class, effector function, and / or species, or to a completely different molecule that confers new characteristics to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) an antibody molecule in which the variable region or part thereof is modified, substituted, or exchanged by a variable region having a different or modified antigen specificity.

[0072] The terms “Fc domain,” “Fc portion,” and “Fc region” refer to the C-terminal fragment of an antibody heavy chain, e.g., approximately amino acids (aa)230–450 of a human γ (gamma) heavy chain, or the sequence of its counterpart in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or its naturally occurring allotype. Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0073] The terms “isolated,” “purified,” or “biologically pure” refer to materials that substantially or essentially contain components that would normally accompany them when found in their natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. Proteins, which are the main species present in the preparation, are substantially purified.

[0074] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and naturally occurring amino acid polymers.

[0075] The term "recombinant," when used, for example, in relation to cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins, or by the modification of native nucleic acids or proteins, or that the cells originate from such modified cells. For example, recombinant cells express genes not found in the cell's native (non-recombinant) form, or express native genes that would otherwise be abnormally expressed, low-expressed, or not expressed at all.

[0076] In relation to this specification, the term "antibody that binds to a polypeptide or epitope" refers to an antibody that binds to the determinant by specificity and / or affinity.

[0077] The term “identity” or “identical” refers, when used in relation to the sequences of two or more polypeptides, to the degree of sequence relevance between polypeptides, as determined by the number of matches between the chains of two or more amino acid residues. “Identity” measures the percentage of identical matches between the smaller of two or more sequences by gap alignment (if any), which is addressed by a specific mathematical model or computer program (i.e., “algorithm”). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0078] The method for determining identity is designed to give the maximum match between the sequences being tested. The method for determining identity is described in publicly available computer programs. Examples of computer programs for determining identity between two sequences include the GCG program package, which includes GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., see above). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0079] NKG2A neutralizing agent Anti-NKG2A agents bind to the extracellular portion of the human CD94 / NKG2A receptor, reducing the inhibitory activity of the human CD94 / NKG2A receptor expressed on the surface of CD94 / NKG2A-positive lymphocytes. In one embodiment, the agent competes with HLA-E for binding to CD94 / NKG2A; that is, the agent blocks the interaction between CD94 / NKG2A and its ligand, HLA-E. In another embodiment, the agent does not compete with HLA-E for binding to CD94 / NKG2A; that is, the agent can bind to CD94 / NKG2A simultaneously with HLA-E. Antibodies can bind to a combined epitope on CD94 and NKG2A, or to an epitope on NKG2A alone.

[0080] In one embodiment, the anti-NKG2A agent is an antibody selected from fully human antibodies, humanized antibodies, and chimeric antibodies. In one embodiment, the agent comprises a constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 antibody. In one embodiment, the agent is a fragment of an antibody selected from IgA, IgD, IgG, IgE, and IgM antibodies. In one embodiment, the agent is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, a Fv fragment, a heavy chain Ig (llama or camel Ig), or a V HH The antibody fragment is selected from fragments, single-domain FVs, and single-chain antibody fragments. In one embodiment, the drug is a molecule derived from a synthetic or semi-synthetic antibody selected from scFV, dsFV, minibody, diabody, triabody, kappabody, IgNAR; and polyspecific antibodies.

[0081] Selectively, anti-NKG2A antibodies do not exhibit substantial specific binding to human Fcγ receptors, such as CD16. Selectively, anti-NKG2A antibodies lack substantial specific binding or have low or reduced specific binding to one or more or all of human CD16, CD32A, CD32B, or CD64. Exemplary antibodies may include constant regions of various heavy chains known to have no or low binding to Fcγ receptors. One such example is the human IgG4 constant region. In one embodiment, the IgG4 antibody includes modifications that prevent the formation of a semi-antibody (fab-arm exchange) in vivo, for example, the antibody includes an IgG4 heavy chain with a serine-to-proline mutation at residue 241 corresponding to position 228 according to the EU index (Kabat et al., “Sequences of proteins of immunological interest”, 5 th(ed., NIH, Bethesda, ML, 1991). Such modified IgG4 antibodies retain their original form in vivo and maintain bivalent (high-affinity) binding to NKG2A, in contrast to natural IgG4, which undergoes fab arm exchange in vivo to bind to NKG2A in a monovalent manner that can alter binding affinity. Alternatively, Fc receptor binding can be avoided by using antibody fragments that do not contain a constant region, such as a Fab or F(ab')2 fragment. Fc receptor binding can be evaluated according to methods known in the art, including, for example, testing the binding of an antibody to the Fc receptor protein in a BIACORE assay. Alternatively, any human antibody type (e.g., IgG1, IgG2, IgG3, or IgG4) with the Fc portion modified to minimize or eliminate binding to the Fc receptor can be used (see, for example, International Publication No. 03101485, whose disclosure is incorporated herein by reference). Assays for evaluating Fc receptor binding, such as cell-based assays, are well known in the art and are described, for example, in International Publication No. 03101485.

[0082] Accordingly, the present invention relates to an antibody or other agent that binds to NKG2A. In one embodiment, the antibody binds to NKG2A with a KD at least 100 times lower than that for human NKG2C and / or NKG2E.

[0083] In one aspect of the present invention, the agent reduces CD94 / NKG2A-mediated inhibition of CD94 / NKG2A-expressing lymphocytes by interfering with CD94 / NKG2A signaling, for example by interfering with HLA-E binding by NKG2A, preventing or inducing conformational changes of the CD94 / NKG2A receptor, and / or affecting the dimerization and / or clustering of the CD94 / NKG2A receptor.

[0084] In one aspect of the present invention, the drug binds to the extracellular portion of NKG2A with a KD at least 100 times lower than that for NKG2C. In a more preferred aspect, the drug binds to the extracellular portion of NKG2A with a KD at least 150, 200, 300, 400, or 10,000 times lower than that for NKG2C. In another aspect of the present invention, the drug binds to the extracellular portion of NKG2A with a KD at least 100 times lower than that for NKG2C, NKG2E, and / or NKG2H molecules. In a more preferred aspect, the drug binds to the extracellular portion of NKG2A with a KD at least 150, 200, 300, 400, or 10,000 times lower than that for NKG2C, NKG2C, and / or NKG2H molecules. This can be measured, for example, in a BiaCore experiment, where the ability of a drug to bind to the extracellular portion of immobilized CD94 / NKG2A (e.g., purified from CD94 / NKG2-expressing cells or produced in a biological system) is measured and compared to the binding of the drug to CD94 / NKG2C and / or other CD94 / NKG2 variants similarly produced in the same assay. Alternatively, the binding of a drug to cells that naturally express or overexpress CD94 / NKG2A (e.g., after transient or stable transfection) can be measured and compared to the binding to cells expressing CD94 / NKG2C and / or other CD94 / NKG2 variants. The anti-NKG2A antibody may optionally bind to NKG2B, an NKG2A splice variant that forms an inhibitory receptor with CD94. In one embodiment, affinity can be measured using the method disclosed in U.S. Patent No. 8,206,709, for example, by evaluating binding to an NKG2A-CD94-Fc fusion protein covalently immobilized by Biacore, as shown in Example 8 of U.S. Patent No. 8,206,709 (the disclosure of which is incorporated herein by reference).

[0085] An anti-NKG2A antibody may be a humanized antibody comprising, for example, a VH human acceptor framework from a human acceptor sequence selected from, for example, VH1_18, VH5_a, VH5_51, VH1_f, and VH1_46, and a JH6 J-segment, or other human germline VH framework sequences known in the art. The VL region human acceptor sequence may be, for example, VKI_O2 / JK4.

[0086] In one embodiment, the antibody is a humanized antibody based on antibody Z270. Various humanized Z270VH chains are indicated by SEQ ID NOs: 4-8 (variable region domain amino acids are underlined). humZ270VH6 (SEQ ID NO: 4) is based on VH5_51, humZ270VH1 (SEQ ID NO: 5) is based on VH1_18, humZ270VH5 (SEQ ID NO: 6) is based on VH5_a, humZ270VH7 (SEQ ID NO: 7) is based on VH1_f, and humZ270VH8 (SEQ ID NO: 8) is based on VH1_46, all of which have a JH6 J-segment. Each of these antibodies maintains high affinity binding to NKG2A, and the likelihood of a host immune response to the antibody is low because the six C-terminal amino acid residues of each Kabat CDR-H2 in the humanized construct are identical to the human acceptor framework. Using the alignment program VectorNTI, the following sequence identities were obtained between humZ270VH1 and humZ270VH5, -6, -7, and -8: 78.2% (VH1 vs VH5), 79.0% (VH1 vs VH6), 88.7% (VH1 vs VH7), and 96.0% (VH1 vs VH8).

[0087] In one embodiment, the drug comprises (i) a heavy chain variable region of an amino acid sequence identical to any of SEQ ID NOs: 4-8, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% thereof, and (ii) a light chain variable region of an amino acid sequence identical to SEQ ID NOs: 9, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% thereof. In one embodiment, the drug comprises (i) a heavy chain comprising an amino acid sequence identical to any of SEQ ID NOs: 4-8, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% thereof, and (ii) a light chain comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NOs: 9, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% thereof. An antibody having a heavy chain of any of SEQ ID NOs: 4-8 and a light chain of SEQ ID NO: 9 neutralizes the inhibitory activity of NKG2A but substantially fails to bind to the activating receptors NKG2C, NKGE, or NKG2H. Furthermore, this antibody competes with HLA-E for binding to NKG2A on the cell surface. In one embodiment, the drug comprises HCDR1, HCDR2, and / or HCDR3 sequences derived from a heavy chain having the amino acid sequence of any of SEQ ID NOs: 4-8. In one embodiment of the present invention, the drug comprises LCDR1, LCDR2, and / or LCDR3 sequences derived from a light chain having the amino acid sequence of SEQ ID NO: 9.

[0088] Heavy chain VH6: [ka] VH1: [ka] VH5: [ka] VH7: [ka] VH8: [ka] Light chain [ka]

[0089] In one embodiment, the anti-NKG2A antibody is an antibody comprising CDR-H1 corresponding to residues 31-35 of SEQ ID NOs: 4-8, CDR-H2 corresponding to residues 50-60 (optionally, 50-66 if human-derived amino acids are included) of SEQ ID NOs: 4-8, and CDR-H3 corresponding to residues 99-114 (95-102 according to Kabat) of SEQ ID NOs: 4-8. In one embodiment, CDR-H2 corresponds to residues 50-66 of SEQ ID NOs: 4-8. Optionally, the CDR may contain one, two, three, four, or more amino acid substitutions.

[0090] In one embodiment, the anti-NKG2A antibody is an antibody comprising CDR-L1 corresponding to residues 24-34 of SEQ ID NO: 9, CDR-L2 corresponding to residues 50-56 of SEQ ID NO: 9, and CDR-L3 corresponding to residues 89-97 of SEQ ID NO: 9. Optionally, the CDR may contain one, two, three, four, or more amino acid substitutions.

[0091] In one embodiment, the anti-NKG2A antibody is an antibody comprising CDR-H1 corresponding to residues 31-35 of SEQ ID NOs: 4-8, CDR-H2 corresponding to residues 50-60 (optionally, 50-66) of SEQ ID NOs: 4-8, CDR-H3 corresponding to residues 99-114 (95-102 according to Kabat) of SEQ ID NOs: 4-8, CDR-L1 corresponding to residues 24-34 of SEQ ID NO: 9, CDR-L2 corresponding to residues 50-56 of SEQ ID NO: 9, and CDR-L3 corresponding to residues 89-97 of SEQ ID NO: 9.

[0092] In one embodiment, the drug comprises HCDR1, HCDR2, and / or HCDR3 sequences derived from VH having the amino acid sequence of SEQ ID NO: 10. In one embodiment of the present invention, the drug comprises LCDR1, LCDR2, and / or LCDR3 sequences derived from VL having the amino acid sequence of SEQ ID NO: 11. In one embodiment, the drug comprises HCDR1, HCDR2, and / or HCDR3 sequences derived from VH having the amino acid sequence of SEQ ID NO: 10, and LCDR1, LCDR2, and / or LCDR3 sequences derived from VL having the amino acid sequence of SEQ ID NO: 11. The antibody having the heavy chain of SEQ ID NO: 10 and the light chain of SEQ ID NO: 11 neutralizes the inhibitory activity of NKG2A and also binds to the activating receptors NKG2C, NKG2E, or NKG2H. The antibody does not compete with HLA-E for binding to NKG2A on the cell surface (i.e., it is a non-competitive antagonist of NKG2A). [ka]

[0093] In one embodiment, the drug comprises amino acid residues 31-35, 50-60, 62, 64, 66, and 99-108 of a variable weight (VH) domain (SEQ ID NO: 10) and amino acid residues 24-33, 49-55, and 88-96 of a variable light (VL) domain (SEQ ID NO: 11), and optionally has one, two, three, four, or more amino acid substitutions.

[0094] In one embodiment, the drug is a fully human antibody produced against the CD94 / NKG2A epitope to which any of the above antibodies bind.

[0095] While the antibodies described above are usable, it will be understood that other antibodies may also recognize and be produced against any portion of the NKG2A polypeptide, as long as they neutralize the inhibitory activity of the NKG2A. For example, any fragment of NKG2A, preferably (but not exclusively) human NKG2A, or any combination of NKG2A fragments may be used as an immunogen to produce antibodies, which may recognize epitopes at any position within the NKG2A polypeptide, as far as possible, in NKG2A-expressing NK cells as described herein. Optionally, the epitope is one that is specifically recognized by antibodies having the heavy chains of SEQ ID NOs. 4-8 and the light chain of SEQ ID NO. 9.

[0096] In one embodiment, the drug competes with the humZ270 antibody disclosed in U.S. Patent No. 8,206,709 (the disclosure of which is incorporated herein by reference) for binding to the extracellular portion of the human CD94 / NKG2A receptor. Competitive binding can be measured, for example, in a BiaCore experiment, where the drug's ability to bind to the extracellular portion of immobilized CD94 / NKG2A receptors saturated with humZ270 (e.g., purified from CD94 / NKG2-expressing cells or produced in a biological system) is measured. Alternatively, the binding of the drug to cells that naturally express or overexpress (e.g., after transient or stable transfection) the CD94 / NKG2A receptor (these cells are incubated with a saturated dose of Z270) is measured. In one embodiment, competitive binding can be measured using the method disclosed in U.S. Patent No. 8,206,709, for example, by evaluating binding to Ba / F3-CD94-NKG2A cells by flow cytometry, as shown in Example 15 of U.S. Patent No. 8,206,709 (the disclosure of which is incorporated herein by reference).

[0097] PD-1 neutralizing agent Currently, there are at least six drugs that block the PD-1 / PD-L1 pathway, and these drugs are either marketed or undergoing clinical evaluation. One such drug is BMS-936558 (nivolumab / ONO-4538, Bristol-Myers Squibb; formerly MDX-1106). Nivolumab (trade name Opdivo®) is an FDA-approved fully human IgG4 anti-PD-L1 mAb that inhibits the binding of PD-L1 ligands to both PD-1 and CD80 and is described as antibody 5C4 in International Publication No. 2006 / 121168, the disclosure of which is incorporated herein by reference. In patients with melanoma, the most significant OR was observed at a dose of 3 mg / kg, while in other cancer types, it was observed at 10 mg / kg. Nivolumab is generally administered at 10 mg / kg every three weeks until cancer progression.

[0098] MK-3475 (Merck's human IgG4 anti-PD1 mAb) (also known as lambrolizumab or pembrolizumab (trade name Keytruda®)) is approved by the FDA for the treatment of melanoma and is under investigation for other cancers. Pembrolizumab was tested at 2 mg / kg or 10 mg / kg every 2 or 3 weeks until disease progression. DNA constructs encoding the variable regions of the heavy and light chains of the humanized antibody h409All are deposited with the American Type Culture Collection Patent Depository (10801 University Blvd., Manassas, VA). The plasmid containing the DNA encoding the heavy chain of h409A-l 1 was deposited on June 9, 2008, and identified as 081469_SPD-H, and the plasmid containing the DNA encoding the light chain of h409Al 1 was deposited on June 9, 2008, and identified as 0801470_SPD-Ll 1. MK-3475 (also known as Merck 3745 or SCH-900475) is also described in International Publication No. 2009 / 114335.

[0099] MPDL3280A / RG7446 (Roche / Genentech anti-PD-L1) is a human anti-PD-L1 mAb containing a modified Fc domain designed to optimize efficacy and safety by minimizing FcγR binding, resulting in antibody-dependent cell-mediated cytotoxicity (ADCC). MPDL3280A was administered every three weeks for up to one year at doses of 1, 10, 15, and ≤25 mg / kg. In a Phase 3 trial, MPDL3280A was administered intravenously at a dose of 1200 mg every three weeks in patients with NSCLC.

[0100] AMP-224 (Amplimmune and GSK) is an immunoadhesion factor containing a PD-L2 extracellular domain fused to an Fc domain. Other examples of PD-1 neutralizing agents may include antibodies (anti-PD-L2 antibodies) that bind to PD-L2 and block the interaction between PD-1 and PD-L2.

[0101] Pizilizumab (CT-011; CureTech) (a humanized IgG1 anti-PD1 mAb manufactured by CureTech / Teva), pizilizumab (CT-011; CureTech) (see, for example, International Publication No. 2009 / 101611). Thirty patients with rituximab-sensitive relapsed FL were treated with intravenous CT-011 at 3 mg / kg every four weeks for four weeks, in combination with rituximab administered at 375 mg / m2 weekly for four weeks, starting two weeks after the first infusion of CT-011.

[0102] Further known PD-1 antibodies and other PD-1 inhibitors include AMP-224 (GSK-approved B7-DC / IgG1 fusion protein), AMP-514 as described in International Publication No. 2012 / 145493, antibody MEDI-4736 (anti-PD-L1 developed by AstraZeneca / Medimmune) as described in International Publication No. 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559, antibody YW243.55.S70 (anti-PD-L1) as described in International Publication No. 2010 / 077634, and Bristol-Myers as described in International Publication No. 2007 / 005874. Examples include MDX-1105 (also known as BMS-936559), an anti-PD-L1 antibody developed by Squibb, as well as antibodies and inhibitors described in International Publication Nos. 2006 / 121168, 2009 / 014708, 2009 / 114335, and 2013 / 019906, the disclosures of which are incorporated herein by reference. Further examples of anti-PD1 antibodies are disclosed in International Publication No. 2015 / 085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), for example, antibodies having light chain variable domains CDR1, 2, and 3 of SEQ ID NO: 6, SEQ ID NO: 7, and / or SEQ ID NO: 8, respectively, and antibody heavy chain variable domains CDR1, 2, and 3 of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, respectively, where the reference to SEQ ID NOs is the numbering in accordance with International Publication No. 2015 / 085847, whose disclosure is incorporated herein by reference. Antibodies that compete with either of these antibodies for binding to PD-1 or PD-L1 may also be used.

[0103] An exemplary anti-PD-1 antibody is pembrolizumab (see, for example, International Publication No. 2009 / 114335, the disclosure of which is incorporated herein by reference). The anti-PD-1 antibody may be antibody h409Al 1 in International Publication No. 2008 / 156712, comprising a heavy chain variable region encoded by DNA deposited to ATCC as 081469_SPD-H and a light chain variable region encoded by DNA deposited to ATCC as 0801470_SPD-Ll 1. In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of pembrolizumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of pembrolizumab VH encoded by DNA deposited in ATCC as 081469_SPD-H, and the CDR1, CDR2, and CDR3 domains of pembrolizumab VL encoded by DNA deposited in ATCC as 0801470_SPD-Ll 1.

[0104] In some embodiments, the PD-1 neutralizer is an anti-PD-L1 mAb that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 neutralizer is an anti-PD1 mAb that inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 neutralizer is an immunoadhesion factor (e.g., an immunoadhesion factor containing an extracellular or PD-1 binding moiety of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)).

[0105] Another exemplary anti-PD-1 antibody is nivolumab, or its antigen-binding fragments and variants, comprising heavy and light chains having the respective amino acid sequences shown in SEQ ID NOs. 12 and 13, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical thereto. In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of nivolumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy chain of nivolumab having the sequence described in SEQ ID NOs. 12, and the CDR1, CDR2, and CDR3 domains of the light chain of nivolumab having the sequence described in SEQ ID NOs. 13. [ka]

[0106] Exemplary anti-PD-L1 antibodies include heavy chain and light chain variable regions having the respective sequences shown in SEQ ID NOs: 14 and 15, or at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical amino acid sequences thereto, respectively, or their antigen-binding fragments and variants. In other embodiments, the antibody includes the heavy chain and light chain CDR or variable region of MPDL3280A. Thus, in one embodiment, the antibody includes the CDR1, CDR2, and CDR3 domains of the heavy chain having the sequence described in SEQ ID NO: 14, and the CDR1, CDR2, and CDR3 domains of the light chain having the sequence described in SEQ ID NO: 15. [ka]

[0107] Anti-PD-1 or anti-PD-L1 antibodies may be selected from fully human antibodies, humanized antibodies, and chimeric antibodies. In one aspect of the present invention, the drug comprises a constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 antibody. In one aspect of the present invention, the drug is a fragment of an antibody selected from IgA, IgD, IgG, IgE, and IgM antibodies. In one aspect of the present invention, the drug is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, a Fv fragment, a heavy chain Ig (llama or camel Ig), or a V HH The antibody fragment is selected from fragments, single-domain FVs, and single-chain antibody fragments. In one aspect of the present invention, the drug is a molecule derived from a synthetic or semi-synthetic antibody selected from scFVs, dsFVs, minibody, diabody, triabody, kappabody, IgNAR, and polyspecific antibodies.

[0108] Anti-PD-1 or anti-PD-L1 antibodies may lack substantial specific binding to the Fcγ receptor, e.g., CD16. Such antibodies may contain constant regions of various heavy chains known not to bind to the Fc receptor. One such example is the IgG4 constant region. Fc receptor binding can be avoided by using antibody fragments that do not contain a constant region, such as IgG4 or Fab or F(ab')2 fragments. Fc receptor binding can be evaluated according to methods known in the art, including, for example, testing the binding of an antibody to the Fc receptor protein in a BIACORE assay. Alternatively, any human antibody type (e.g., IgG1, IgG2, IgG3, or IgG4) with the Fc portion modified to minimize or eliminate binding to the Fcγ receptor can be used. Thus, anti-PD-1 or anti-PD-L1 antibodies typically have reduced or minimal effector function. In one embodiment, minimal effector function is due to production in prokaryotic cells. In one embodiment, the minimal effector function is due to an "effectorless Fc mutation" or aglycosylation. In yet another embodiment, the effectorless Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0109] Anti-NKG2A agents, or anti-PD-1 agents, or anti-PD-L1 agents (e.g., antibodies) can be incorporated into a pharmaceutical formulation at concentrations of 1 mg / ml to 500 mg / ml, where the formulation has a pH of 2.0 to 10.0. The formulation may further include a buffer system, preservatives, isotonic agents, chelating agents, stabilizers, and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation containing water. Such formulations are typically solutions or suspensions. In further embodiments, the pharmaceutical formulation is an aqueous solution. The term “aqueous formulation” is defined as a formulation containing at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water.

[0110] In another embodiment, the pharmaceutical formulation is a lyophilized formulation to which a solvent and / or diluent is added by a physician or patient before use.

[0111] In another embodiment, the pharmaceutical formulation is a dry formulation (e.g., freeze-dried or spray-dried) that can be used immediately without prior dissolution.

[0112] In a further embodiment, the pharmaceutical preparation comprises an aqueous solution of such antibody and buffer, wherein the antibody is present at a concentration of 1 mg / ml or more, and the preparation has a pH of about 2.0 to about 10.0.

[0113] In another embodiment, the pH of the formulation is within a range selected from the list consisting of approximately 2.0 to approximately 10.0, approximately 3.0 to approximately 9.0, approximately 4.0 to approximately 8.5, approximately 5.0 to approximately 8.0, and approximately 5.5 to approximately 7.5.

[0114] In further embodiments, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the present invention.

[0115] In further embodiments, the formulation further comprises a pharmaceutically acceptable preservative. In further embodiments, the formulation further comprises an isotonic agent. In further embodiments, the formulation also comprises a chelating agent. In further embodiments of the present invention, the formulation further comprises a stabilizer. In further embodiments, the formulation further comprises a surfactant. For convenience, Remington: The Science and Practice of Pharmacy, 19 th See edition, 1995.

[0116] Other components may be present in the peptide pharmaceutical formulation of the present invention. Such additional components may include wetting agents, emulsifiers, antioxidants, bulking agents, osmotic pressure regulators, chelating agents, metal ions, oily media, proteins (e.g., human serum albumin, gelatin, or proteins) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, and histidine). Such additional components should, of course, not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

[0117] The pharmaceutical composition according to the present invention can be administered via several routes of administration (e.g., intravenously). A suitable antibody formulation can also be determined by examining experience with other already developed therapeutic monoclonal antibodies. Several monoclonal antibodies that have shown efficacy in clinical settings, such as Rituxan (rituximab), Herceptin (trastuzumab), Xolair (omalizumab), Bexxar (tositumomab), Campath (aremtuzumab), Zevalin, Oncolym, and similar formulations, can be used in conjunction with the antibody of the present invention. For example, the monoclonal antibody can be supplied at a concentration of 10 mg / mL in a single-use vial of either 100 mg (10 mL) or 500 mg (50 mL), formulated for IV administration in sterile water for injection with 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and the pH is adjusted to 6.5. In another embodiment, the antibody is supplied in a formulation with a pH of about 6.0 containing about 20 mM sodium citrate and about 150 mM NaCl.

[0118] Also provided are kits comprising a pharmaceutical composition containing an anti-NKG2A antibody in a therapeutically effective amount suitable for use in the method described above, and optionally further containing an anti-PD-1 or anti-PD-L1 antibody and a pharmaceutically acceptable carrier. The kit may also optionally include instructions for use (e.g., including an administration schedule) that enable practitioners (e.g., physicians, nurses, or patients) to administer the composition contained in the kit to patients with cancer (e.g., solid tumors). The kit may also include a syringe.

[0119] Optionally, the kit comprises multiple packages of single-dose pharmaceutical compositions each containing an effective amount of anti-NKG2A and, optionally, further anti-PD-1 or PD-L1 antibodies, respectively, for single-dose administration according to the method shown above. Instruments or devices necessary for administering the pharmaceutical compositions may also be included in the kit. For example, the kit may provide one or more pre-filled syringes containing a certain amount of anti-NKG2A, anti-PD-1, or anti-PD-L1 antibodies.

[0120] In one embodiment, the present invention is a kit for treating anti-PD-1 / PD-L1 antibody-resistant cancer in human patients, (a) A certain dose of an anti-NKG2A antibody comprising heavy chain CDR1, CDR2, and CDR3 domains having the sequence described in any of SEQ ID NOs: 4-8, and light chain CDR1, CDR2, and CDR3 domains having the sequence described in SEQ ID NO: 9, (b) Selectively administering a certain dose of anti-PD-1 antibody or anti-PD-L1 antibody, (c) Instructions for use of the anti-NKG2A antibody (and optionally anti-PD-1 or PD-L1 antibody) in any of the methods described herein, and We provide a kit that includes this.

[0121] Diagnosis, prognosis, and treatment of malignant tumors The report also describes methods useful in the diagnosis, prognosis, monitoring, treatment, and prevention of cancer in individuals, using, optionally, agents that neutralize NKG2A activity in combination with agents that neutralize PD-1 activity, such as anti-PD-1 antibodies or anti-PD-L1 antibodies. Individuals may optionally be low-responders to treatment with agents that neutralize PD-1 inhibitory activity, for example, individuals who will develop, or are predicted to develop, an incomplete response, lack of therapeutic response, detectable or residual cancer, and / or progressive disease (based on, for example, one or more prognostic factors) during (or after) treatment with agents that neutralize PD-1 inhibitory activity. In one embodiment, an individual's cancer is predicted to not produce, or to have a high probability of not producing, a complete response during (or after) treatment with agents that neutralize PD-1 inhibitory activity. In one embodiment, an individual's cancer progressed (e.g., progressive disease) during (or after) treatment with a drug that neutralizes PD-1 inhibitory activity. In another embodiment, an individual's cancer partially responded or stabilized (partial response or stable disease) during (or after) treatment with a drug that neutralizes PD-1 inhibitory activity, but is expected to progress.

[0122] In one example, an individual has cancer known to be poorly responsive to treatment with agents that neutralize PD-1 inhibitory activity (e.g., monotherapy). In another example, an individual has cancer known to be characterized by tumor-infiltrating NKG2A-expressing CD8+ or NK cells upon treatment with agents that neutralize PD-1 inhibitory activity (e.g., monotherapy). In yet another example, an individual has cancer known to be characterized by HLA-E expression (or increased expression) in cancer cells upon treatment with agents that neutralize PD-1 inhibitory activity (e.g., monotherapy). Optionally, the cancers are head and neck squamous cell carcinoma, non-small cell lung cancer (NSCLC), kidney cancer, gastrointestinal cancer, pancreatic or esophageal adenocarcinoma, breast cancer, renal cell carcinoma (RCC), melanoma, colorectal cancer, or ovarian cancer. Such individuals may, advantageously, be treated with agents that neutralize NKG2A inhibitory activity in combination with agents that neutralize PD-1 inhibitory activity.

[0123] For example, an individual may have a cancer that is unresponsive (or resistant or unresponsive), such as a cancer that recurs or progresses despite treatment with a drug that neutralizes PD-1 inhibitory activity (e.g., during or after treatment). In one embodiment, an individual treated with an anti-NKG2A agent may have an incomplete response (not a complete response (CR)) during (or after) treatment with a drug that neutralizes PD-1 inhibitory activity (e.g., as monotherapy or in combination with a drug other than an NKG2A neutralizer), or may have at least a partial response (PR) during (or after) treatment with a drug that neutralizes PD-1 inhibitory activity, but the cancer has recurred or progressed. In any embodiment herein, treatment response may be defined and / or evaluated according to well-known criteria, such as the Response Evaluation Criteria In Solid Tumors (RECIST) (version 1.1, etc.), see Eisenhauer et al. (2009) Eur.J.Cancer 45:228-247, or see the Immune-Related Response Criteria (irRC), see Wolchock et al. (2009) Clinical Cancer Research 15:7412-7420.

[0124] In one embodiment, an individual who is a low responder (or has a low-response cancer) is an individual who has a poor disease prognosis to treatment with a drug that neutralizes the inhibitory activity of PD-1. An individual with a poor disease prognosis may be determined to have a high or higher risk of cancer progression (compared to, for example, an individual with a good disease prognosis) based on one or more predictors. In one embodiment, the predictors include the presence of an increased number of NKG2A-expressing NK and / or CD8 T cells, and / or an increased level of NKG2A in NK and / or CD8 T cells may indicate that the individual has a poor prognosis to response to treatment with an antibody that neutralizes PD-1. In one embodiment, the predictors include the presence or absence of mutations in one or more genes. In one embodiment, the mutations define neoepitopes recognized by T cells. In one embodiment, the predictors include one or more genes or proteins in tumor cells, e.g., the level of expression of PD-L1, or a decreased or increased level of PD-L1 in tumor cells. In one embodiment, the predictor includes the level of expression of one or more genes or proteins, e.g., PD-1, in NK and / or CD8 T cells in the circulating or tumor environment. In another embodiment, the predictor includes the mutational weight in cancer cells, e.g., the number of non-synonymous mutations per exome.

[0125] The treatment regimens and methods described herein may be used with or without a prior step of detecting PD-L1 expression in cells in a biological sample obtained from an individual (e.g., a biological sample including cancer cells, cancer tissue, or tissue adjacent to cancer). In another embodiment, the disclosure is a method for treating or preventing cancer in an individual in need. a) A step of detecting cells (e.g., tumor cells, tumor-infiltrating immune cells, tumor-infiltrating macrophages) in a sample from an individual expressing PD-L1, b) If, optionally, it is determined that the sample contains cells expressing PD-L1 at a baseline level corresponding to individuals who are unresponsive and / or do not receive substantial benefit from drugs that neutralize PD-1 inhibitory activity, the individual is optionally administered a drug that neutralizes NKG2A inhibitory activity in combination with a drug that neutralizes PD-1 inhibitory activity. The present invention provides a method including the following: PD-L1 reference levels can be characterized by any preferred conventionally used reference levels. For example, using an immunohistochemistry-based assay, if less than 1%, optionally less than 5%, optionally less than 10%, or optionally less than 50% of cells from a tumor cell or tumor tissue sample express PD-L1, the sample may be determined to correspond to an individual who is unresponsive and / or does not derive substantial benefit from drugs that neutralize PD-1 inhibitory activity. An example of such an assay is the PD-L1 IHC 22C3 assay by pharmDx from Dako Denmark A / S. In this assay, the PD-L1 expression level is measured using the tumor percentage score (TPS) and the percentage of tumor cell staining for PD-L1 (0% to 100%). Optionally, the reference level is a level of non-high PD-L1 expression, and optionally, less than 50% of tumor cells express PD-L1 (e.g., the patient has a TPS of less than 50%).

[0126] The treatment regimens and methods described herein may be useful for the treatment of solid tumors and hematological malignancies. The methods and compositions of the present invention include, but are not limited to, squamous cell carcinomas, carcinomas such as those of the bladder, breast, colon, kidney, liver, lung, ovary, head and neck, prostate, pancreas, stomach, neck, thyroid and skin; hematopoietic malignancies of the lymphoid system such as leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma and Burkitt lymphoma and multiple myeloma; and hematopoietic malignancies of the myeloid system such as acute and chronic myeloid leukemia. It is used to treat a variety of cancers and other proliferative disorders, including hematological diseases, promyelocytic leukemia, and myelodysplastic syndromes; mesenchymal tumors, e.g., fibrosarcoma and rhabdomyosarcoma; other tumors, e.g., melanoma, seminomas, teratomas, neuroblastomas, and gliomas; tumors of the central and peripheral nervous systems, e.g., astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors, e.g., fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, e.g., melanoma, xeroderma pigmentosum, keratosacral cell carcinoma, seminomas, and follicular thyroid carcinoma.

[0127] In one embodiment, the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, HNSCC is an oropharyngeal tumor, laryngeal tumor, oral tumor, or hypopharyngeal tumor. In one embodiment, HNSCC is oral squamous cell carcinoma (OCSCC). OCSCC includes squamous cell carcinomas of the lips, anterior two-thirds of the tongue, floor of the mouth, buccal mucosa, gingiva, hard palate, and retromolar triangle. In one embodiment, HNSCC is metastatic cancer.

[0128] When treating individuals with solid tumors, compounds (e.g., antibodies) that neutralize the inhibitory activity of human NKG2A polypeptide can be advantageously administered to individuals with cancer who have not undergone surgery to remove cancer cells, or who are not currently undergoing such surgery, according to the treatment regimens described herein. However, it will be understood that the compounds may also be administered to patients who have undergone or are undergoing surgery to remove cancer cells. When anti-NKG2A compounds are administered to individuals who have not undergone surgical intervention to remove cancer cells (e.g., to remove HNSCC cells), the NKG2A-binding compound may be administered, for example, about 1 to 8 weeks before surgery. In one embodiment, a complete administration cycle of treatment with the anti-NKG2A compound is administered at least once (e.g., once, twice, three times, or more) prior to surgery. In one embodiment, the administration cycle is 2 to 8 weeks long.

[0129] Therapies for treating cancers that are less responsive to PD-1 / PD-L1 as described herein include the administration of a neutralizing anti-NKG2A antibody, either in the absence of or in combination with a PD-1 antagonist, e.g., a neutralizing anti-PD-1 or anti-PD-L1 antibody, to treat subjects with cancer (e.g., advanced, refractory, or progressive solid or hematological malignancies). In one embodiment, the present invention provides an anti-NKG2A antibody, and optionally further combined with an anti-PD-1 antibody, for treating subjects with solid tumors (e.g., solid tumors, advanced, refractory solid tumors) or hematological malignancies. In certain embodiments, the anti-NKG2A antibody comprises the heavy chain of any of SEQ ID NOs: 4-8 and the light chain of SEQ ID NO: 9. In one embodiment, the antibody neutralizing PD-1 inhibitory activity is selected from the group consisting of pembrolizumab, nivolumab, AMP-514, MEDI-4736, CT-011, and MPDL3280A.

[0130] As used herein, adjunctive or concomitant administration (simultaneous administration) includes simultaneous administration of compounds in the same or different dosage forms, or separate administration of compounds (e.g., sequential administration). Therefore, anti-NKG2A and anti-PD-1 or anti-PD-L1 antibodies can be administered simultaneously in a single formulation. Alternatively, anti-NKG2A and anti-PD-1 or anti-PD-L1 antibodies can be formulated for separate administrations, which may be administered simultaneously or sequentially.

[0131] In one embodiment, the cancer treated by the method disclosed herein is characterized by infiltration of NK cells and / or CD8 T cells expressing NKG2A on their surface. In one embodiment, the cancer treated by the method disclosed herein is characterized by NK cell infiltration in which at least 20%, 30%, 40%, or 50% of the NK cells express NKG2A on their surface.

[0132] In one embodiment, the cancer treated by the method disclosed herein is a cancer characterized by high levels of HLA-E. In one embodiment, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC)), renal cell carcinoma (RCC), melanoma, head and neck squamous cell carcinoma (HNSCC), colorectal cancer, and ovarian cancer. It will be understood that a patient with cancer may be treated with an anti-NKG2A agent, with or without the preceding detection step, to assess the expression of HLA-E on the surface of tumor cells. Advantageously, the treatment method may include the step of detecting HLA-E nucleic acid or polypeptide in a biological sample of the tumor from the individual (e.g., on tumor cells). Examples of biological samples include any suitable body fluid (e.g., serum, lymph, blood), cell samples, or tissue samples. For example, a tissue sample may be a sample of tumor tissue or tissue adjacent to the tumor. Optionally, the HLA-E polypeptide is detected on the surface of malignant cells. A determination that a biological sample expresses HLA-E (e.g., significantly expresses it; expresses HLA-E at a high level compared to a reference, or stains with high intensity by an anti-HLA-E antibody) indicates that the individual has cancer that may benefit significantly from treatment with NKG2A-inhibiting agents. In one embodiment, the method includes the step of determining the level of HLA-E nucleic acid or polypeptide expression in a biological sample and comparing this level to a reference level corresponding to a healthy individual (e.g., a value, weak cell surface staining, etc.). A determination that a biological sample expresses HLA-E nucleic acid or polypeptide at an increased level compared to a reference level may indicate that the individual has cancer that may be treated with NKG2A-inhibiting agents.

[0133] In one embodiment, the determination that a biological sample (e.g., a sample containing tumor cells, tumor tissue, and / or tumor-adjacent tissue) significantly expresses an HLA-E nucleic acid or polypeptide indicates that the individual has cancer that can be treated with an NKG2A inhibitor. “Significantly expressed,” when relating to an HLA-E polypeptide, means that the HLA-E polypeptide is expressed in a substantial number of tumor cells taken from a given individual. While the definition of the term “significantly expressed” is not constrained by an exact percentage value, in some examples, the receptor said to be “significantly expressed” would be present in at least 30%, 40%, 50%, 60%, 70%, 80%, or more of the tumor cells (in the sample) taken from the patient.

[0134] Determining whether an individual has cancer cells expressing HLA-E polypeptide may include, for example, obtaining a biological sample from the individual containing cancer cells (e.g., by performing a biopsy), contacting the cells with an antibody that binds to HLA-E polypeptide, and detecting whether the cells express HLA-E on their surface. Optionally, determining whether an individual has cancer cells expressing HLA-E may include performing an immunohistochemical assay. Optionally, determining whether an individual has cancer cells expressing HLA-E may include performing a flow cytometry assay.

[0135] In this treatment method, when anti-NKG2A antibody is administered in combination with anti-PD-1 or anti-PD-L1 antibody, the anti-NKG2A antibody and the anti-PD-1 or anti-PD-L1 antibody may be administered separately, together, sequentially, or in a mixture. In some embodiments, anti-NKG2A is administered before the administration of anti-PD-1 or anti-PD-L1 antibody. For example, anti-NKG2A antibody may be administered approximately 0 to 30 days before the administration of anti-PD-1 or anti-PD-L1 antibody. In some embodiments, anti-NKG2A antibody is administered approximately 30 minutes to 2 weeks, approximately 30 minutes to 1 week, approximately 1 hour to 2 hours, approximately 2 hours to 4 hours, approximately 4 hours to 6 hours, approximately 6 hours to 8 hours, approximately 8 hours to 1 day, or approximately 1 to 5 days before the administration of anti-PD-1 or anti-PD-L1 antibody. In some embodiments, anti-NKG2A antibody is administered simultaneously with the administration of anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the anti-NKG2A antibody is administered after the administration of an anti-PD-1 or anti-PD-L1 antibody. For example, the anti-NKG2A antibody may be administered approximately 0 to 30 days after the administration of the anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the anti-NKG2A antibody is administered approximately 30 minutes to approximately 2 weeks, approximately 30 minutes to approximately 1 week, approximately 1 hour to approximately 2 hours, approximately 2 hours to approximately 4 hours, approximately 4 hours to approximately 6 hours, approximately 6 hours to approximately 8 hours, approximately 8 hours to 1 day, or approximately 1 to 5 days after the administration of the anti-PD-1 or anti-PD-L1 antibody.

[0136] An exemplary treatment protocol for treating a human with an anti-NKG2A antibody includes, for example, the step of administering each of the antibodies that inhibit NKG2A to the patient in an effective dose, the method comprising at least one administration cycle, in which at least one dose of the anti-NKG2A antibody is administered at a dose of 1 to 10 mg / kg body weight. In one embodiment, the administration cycle is 2 to 8 weeks long.

[0137] An exemplary treatment protocol for treating a human with an anti-NKG2A antibody includes, for example, the step of administering to the patient effective doses of an antibody that inhibits NKG2A and an antibody that neutralizes the inhibitory activity of human PD-1, the method comprising at least one administration cycle, in which at least one dose of the anti-NKG2A antibody is administered at a dose of 1 to 10 mg / kg body weight and at least one dose of the anti-PD-1 or anti-PD-L1 antibody is administered at a dose of 1 to 20 mg / kg body weight. In one embodiment, the administration cycle is 2 to 8 weeks long.

[0138] In one embodiment, the method comprises at least one administration cycle, the cycle being for a period of 8 weeks or less, and for at least one cycle, two, three, or four doses of anti-NKG2A antibody are administered at a dose of 1 to 10 mg / kg body weight. In one embodiment, each cycle further comprises two, three, or four doses of anti-PD-1 or anti-PD-L1 antibody at a dose of 1 to 20 mg / kg body weight.

[0139] Anti-NKG2A antibodies may be advantageously administered in amounts that achieve a circulating concentration at least 10, 20, or 30 times higher than the concentration required for substantially complete receptor saturation (e.g., 90%, 95%), or optionally in amounts that achieve a concentration in extravascular tissue (e.g., tumor tissue or environment) at least 10, 20, or 30 times higher than the concentration required for substantially complete receptor saturation, when evaluated (e.g., when evaluated for substantially complete receptor saturation in PBMCs by escalating anti-NKG2A antibodies against NKG2A-expressing cells).

[0140] The NKG2A+ NK cell response can be evaluated using suitable assays of the cytotoxic activity of NKG2A-expressing NK cells against HLA-E-expressing target cells. Examples include assays based on markers of NK cell activation, such as CD107 or CD137 expression. The EC assay for the NKG2A+ NK cell response (e.g., when evaluated by a CD107 recruitment assay) of the blocking anti-NKG2A antibody humZ270 (e.g., having a heavy chain of any of SEQ ID NOs. 4-8 and a light chain of SEQ ID NO. 9) used in the examples herein is described below. 50 It is approximately 4 μg / ml, and EC 100 The concentration is approximately 10 μg / ml. Therefore, an amount of anti-NKG2A antibody is administered that maintains a continuous (minimum) blood concentration of at least 4 μg / ml. Advantageously, an amount of anti-NKG2A antibody may be administered that achieves and / or maintains a continuous (minimum) blood concentration of at least 10 μg / ml. For example, the blood concentrations achieved and / or maintained may be 10-12 μg / ml, 10-15 μg / ml, 10-20 μg / ml, 10-30 μg / ml, 10-40 μg / ml, 10-50 μg / ml, 10-70 μg / ml, 10-100 μg / ml, 10-150 μg / ml, or 10-200 μg / ml. When extravascular tissues are targeted (e.g., in the treatment of solid tumors), an amount of anti-NKG2A antibody is administered that achieves and / or maintains a tissue concentration of at least 10 μg / ml, and it is predicted that an amount of anti-NKG2A antibody that achieves a blood concentration of at least 100 μg / ml will achieve a tissue concentration of at least 10 μg / ml. For example, to achieve / maintain 10 μg / ml in tissue, the blood concentrations achieved and / or maintained may be 100-110 μg / ml, 100-120 μg / ml, 100-130 μg / ml, 100-140 μg / ml, 100-150 μg / ml, 100-200 μg / ml, 100-250 μg / ml, or 100-300 μg / ml.

[0141] In some embodiments, at least EC is used for the NKG2A+ lymphocyte response (e.g., NKG2A+ NK cell response). 50 , optionally about or at least about EC100 An amount of anti-NKG2A antibody is administered to obtain the corresponding blood (e.g., serum) concentration. Regarding the NKG2A+ cell response (e.g., NK cell response), an "EC" (Ed. E.K.) is administered. 50 (or "EC 100 ") is 50% (or EC) of the effect on the maximum response or such NKG2A+ cell response (e.g., NK cell response). 100 When referring to 100%, the effective concentration of anti-NKG2A antibody is used. In some embodiments, particularly in the case of treatment of solid tumors, the concentration achieved is the concentration in the tissue (outside the vascular system, e.g., in the tumor environment) that produces at least EC for the NKG2A+ NK cell response. 50 , optionally about the NKG2A+ NK cell response, or at least about EC 100 It is designed to accommodate this.

[0142] EC in response to NKG2A+ NK cell response at approximately 10 μg / ml 100 An exemplary treatment protocol for an anti-NKG2A antibody, such as humZ270, used in the examples herein, comprising at least one administration cycle, wherein at least one dose of anti-NKG2A antibody is administered at a dose of 2–10 mg / kg, optionally 4–10 mg / kg, optionally 6–10 mg / kg, optionally 2–6 mg / kg, optionally 2–8 mg / kg, or optionally 2–4 mg / kg body weight. Optionally, at least 2, 3, 4, 5, 6, 7, or 8 doses of anti-NKG2A antibody are administered. In one embodiment, the administration cycle is 2 to 8 weeks. In one embodiment, the administration cycle is 8 weeks, comprising administering one dose of anti-NKG2A antibody every 2 weeks (i.e., a total of 4 doses).

[0143] In any one embodiment of the embodiments described herein, the anti-NKG2A antibody is administered once every two weeks.

[0144] In particular, exemplary treatment protocols for use with anti-NKG2A antibodies for the treatment of hematopoietic malignancies include, for example, administering anti-NKG2A antibodies to the patient twice a month in amounts effective to maintain a continuous blood concentration of at least 10 μg / ml of anti-NKG2A antibody between at least two consecutive doses of anti-NKG2A antibody, such as 2–10 mg / kg, optionally 2–6 mg / kg, optionally 2–8 mg / kg, optionally 2–4 mg / kg, optionally 2–6 mg / kg, optionally 2–4 mg / kg, and optionally about 4 mg / kg body weight. These doses may be optionally administered to provide a continuous blood concentration of at least 10 μg / ml of anti-NKG2A antibody throughout the entire treatment cycle. Achieving a blood concentration of 10 μg / ml of anti-NKG2A antibody is equivalent to the EC for antibodies such as humanized Z270. 100 It corresponds to.

[0145] In particular, EC of anti-NKG2A antibodies in extravascular tissue (e.g., in tumors or tumor environments) 50 An exemplary treatment protocol for use with anti-NKG2A antibody for the treatment of solid tumors where concentration is required includes, for example, administering anti-NKG2A antibody to the patient twice a month in an amount effective to maintain a continuous blood concentration of at least 40 μg / ml of anti-NKG2A antibody between at least two consecutive doses of anti-NKG2A antibody, which is 2–10 mg / kg, optionally 2–6 mg / kg, optionally 2–4 mg / kg, or optionally about 4 mg / kg body weight. These doses may optionally be administered to provide a continuous blood concentration of at least 40 μg / ml of anti-NKG2A antibody throughout the entire treatment cycle. Achieving a blood concentration of 40 μg / ml of anti-NKG2A antibody is expected to provide a tissue (e.g., extravascular tissue, tumor environment) concentration of about 4 μg / ml, which is therefore EC for antibodies such as humanized Z270. 50 It corresponds to.

[0146] In particular, anti-NKG2A antibody EC in extravascular tissue (e.g., in tumors or tumor environments) 50An exemplary treatment protocol for use with anti-NKG2A antibody for the treatment of solid tumors where concentration is required includes, for example, administering an effective amount of anti-NKG2A antibody to the patient, the antibody being administered twice a month, and effective amounts to maintain a serial blood concentration of at least 100 μg / ml of anti-NKG2A antibody between at least two consecutive administrations of anti-NKG2A antibody are 4–10 mg / kg, optionally 4–6 mg / kg, optionally 4–8 mg / kg, optionally about 4 mg / kg, optionally about 6 mg / kg, optionally about 8 mg / kg, or optionally about 10 mg / kg. These doses may optionally be administered to provide a serial blood concentration of at least 100 μg / ml of anti-NKG2A antibody throughout the entire treatment cycle. Achieving a blood concentration of 100 μg / ml of anti-NKG2A antibody is expected to provide a tissue (e.g., extravascular, tumor environment) concentration of approximately 10 μg / ml, which is therefore equivalent to the EC of antibodies such as humanized Z270. 100 It corresponds to.

[0147] Further exemplary treatment protocols for use with anti-NKG2A antibodies include regimens employing higher-dose loading periods followed by maintenance periods. For example, a loading period may include administering an effective amount of anti-NKG2A antibody to the patient, administered once or multiple times in amounts effective to maintain a continuous blood concentration of at least 100 μg / ml of anti-NKG2A antibody until the first administration of anti-NKG2A antibody in the maintenance regimen. For example, in a single administration, a loading dose of 10 mg / kg of anti-NKG2A antibody may be administered, with the first administration of anti-NKG2A antibody in the maintenance regimen occurring approximately two weeks (or less) after the loading dose. The maintenance regimen can then maintain a continuous blood concentration of at least 100 μg / ml of anti-NKG2A antibody between consecutive administrations in the maintenance regimen using lower doses and / or lower administration frequencies. For example, a maintenance regimen may include administering anti-NKG2A antibody every two weeks at doses of 2-10 mg / kg, optionally 4-10 mg / kg, optionally 2-4 mg / kg, optionally 4-6 mg / kg, optionally 4-8 mg / kg, optionally about 4 mg / kg, optionally about 6 mg / kg, or optionally about 8 mg / kg.

[0148] In certain embodiments, the anti-NKG2A antibody is administered at doses of 4, 6, 8, or 10 mg / kg. In certain embodiments, the anti-PD-1 antibody is administered at doses of 1 to 20 mg / kg, optionally at 10 mg / kg. In certain embodiments, the anti-PD-L1 antibody is administered at doses of 10, 15, 20, or 25 mg / kg, optionally at a total dose of 1200 mg. In certain embodiments, combination therapy allows for the administration of anti-PD-1 or PD-L1 antibodies at lower doses, in one embodiment, each dose of the anti-PD-1 antibody is administered at 2 or 3 mg / kg.

[0149] In one embodiment, the anti-NKG2A antibody and the anti-PD-1 or anti-PD-L1 antibody are administered in the following doses: (a) 1-10 mg / kg of anti-NKG2A antibody and (i) 1-10 mg / kg of anti-PD-1 antibody or (ii) 1-20 mg / kg of anti-PD-L1 antibody, (b) 4, 6, 8 or 10 mg / kg of anti-NKG2A antibody and 10 mg / kg of anti-PD-1 or anti-PD-L1 antibody, (c) 4, 6, 8 or 10 mg / kg of anti-NKG2A antibody and 3 mg / kg of anti-PD-1 antibody, (d) 4, 6, 8 or 10 mg / kg of anti-NKG2A antibody and 2 mg / kg of anti-PD-1 antibody.

[0150] In any one embodiment of the embodiments described herein, the anti-NKG2A antibody is administered once every two weeks. In any one embodiment of the embodiments described herein, the anti-PD-1 or anti-PD-L1 antibody is administered once every three weeks. In any one embodiment of the embodiments described herein, the anti-PD-1 or anti-PD-L1 antibody is administered once every two weeks. In any one embodiment of the embodiments described herein, the anti-PD-1 or anti-PD-L1 antibody is administered once every four weeks.

[0151] In one embodiment, the anti-PD-1 or anti-PD-L1 antibody and / or anti-NKG2A antibody is administered intravenously (iv). In one embodiment, the anti-PD-1 or anti-PD-L1 antibody and / or anti-NKG2A antibody is administered on the same day, and optionally administered again intravenously (iv) once every two weeks.

[0152] In another embodiment, a method for identifying NKG2A+PD1+ NK cells and / or T cells is provided. The co-expression of NKG2A and PD-1 in NK cells and / or T cells can be used in diagnostic or prognostic methods. For example, a biological sample can be obtained from an individual (e.g., from cancer or adjacent tissue obtained from a cancer patient) and analyzed for the presence of NKG2A+PD1+ NK and / or T cells. The expression of both NKG2A and PD-1 in such cells can be used to identify individuals who have tumor-infiltrating NK and / or T cells inhibited, for example, by the NKG2A polypeptide (and optionally further by the PD1 polypeptide). This method may be useful, for example, as a prognostic prediction for response to treatment with an NKG2A-neutralizing agent, as a prognostic prediction for response to treatment with a PD1-neutralizing agent, or as a prognostic prediction for response to combined treatment with an NKG2A-neutralizing agent and a PD1-neutralizing agent.

[0153] In one embodiment, a method is provided for evaluating whether an individual is suitable for treatment with an NKG2A inhibitor and an agent that neutralizes the inhibitory activity of human PD-1, the method comprising the step of detecting lymphocyte populations (e.g., CD8+ T cells, NK cells) expressing both NKG2A nucleic acid or polypeptide and PD-1 nucleic acid or polypeptide in a biological sample from the individual. The determination that an individual has lymphocyte populations expressing both NKG2A nucleic acid or polypeptide and PD-1 nucleic acid or polypeptide indicates that the patient has cancer that can be treated with an NKG2A inhibitor in combination with an agent that neutralizes the inhibitory activity of human PD-1.

[0154] In another embodiment, a method for identifying NKG2A+PD1+ NK cells and / or T cells is provided. The discovery that tumor-infiltrating effector lymphocytes can express both inhibitory receptors NKG2A and PD-1 leads to improved treatment methods and methods for detecting such dual-limiting / inhibitory effector cells, which may be useful for diagnosis and prognosis prediction.

[0155] For example, biological samples can be obtained from individuals (e.g., from cancer or adjacent tissue obtained from cancer patients) and analyzed for the presence of NKG2A+PD1+ NK and / or T cells. Using the expression of both NKG2A and PD-1 in such cells, individuals with tumor-infiltrating NK and / or T cells inhibited by both NKG2A and PD1 polypeptides can be identified. This method may be useful, for example, as a prognostic prediction for response to treatment with NKG2A-neutralizing agents, for response to treatment with PD1-neutralizing agents, or for prognostic prediction for response to combined treatment with NKG2A-neutralizing agents and PD1-neutralizing agents.

[0156] Detecting NKG2A- and PD-1-restricted NK and / or CD8 T cells in biological samples may, more generally, have merit for use in research, evaluation, diagnosis, prognosis, and / or prediction where the characterization of NK and / or CD8 T cells is of interest. For example, prognosis of favorable or unfavorable cancers can be predicted by assessing whether the tumor or tumor-adjacent tissue is characterized by invasive NK and / or CD8 T cells expressing both NKG2A and PD-1.

[0157] For example, anti-NKG2A and anti-PD1 antibodies can be used to identify or evaluate a patient's cancer in order to assess whether tumor-infiltrating NK and / or CD8 T cells are NKG2A+PD1+ (including whether such NK and / or CD8 T cells are present in the peritumoral region (in cancer-adjacent tissue)). This method may be useful in determining whether a patient has a pathology characterized by NK and / or CD8 T cells that are suitable for modulation by therapeutic agents that act directly on NK and / or CD8 T cells (e.g., by binding to NKG2A and / or PD-1, or their respective ligands, HLA-E or PD-L1) or indirectly on NK and / or CD8 T cells (e.g., by producing cytokines or other signaling molecules that can modulate the activity of NK and / or CD8 T cells). Optionally, in any embodiment, the patient is treated with a PD-1 neutralizing agent. The methods described herein may further include, optionally, the step of administering to an individual a therapeutic agent if it is determined that the individual has a pathology suitable for modulation with a therapeutic agent acting on tumor-infiltrating NK and / or CD8 T cells.

[0158] In one embodiment, the inventors provide an in vitro method for detecting NKG2A+ PD-1+ lymphocytes, and optionally NK or CD8+ T cells, comprising the steps of: providing a biological sample containing tumor-infiltrating lymphocytes; and determining whether the lymphocytes express NKG2A and PD-1.

[0159] In one embodiment, a method is provided for evaluating whether an individual is suitable for treatment with an NKG2A inhibitor (and optionally further with an agent that neutralizes human PD-1 inhibitory activity), the method comprising the step of detecting a lymphocyte population (e.g., CD8+ T cells) expressing both NKG2A nucleic acid or polypeptide and PD-1 nucleic acid or polypeptide in a biological sample from the individual. The determination that an individual has a lymphocyte population expressing both NKG2A nucleic acid or polypeptide and PD-1 nucleic acid or polypeptide may indicate that the patient has a cancer that can be treated with an NKG2A inhibitor in combination with an agent that neutralizes human PD-1 inhibitory activity.

[0160] In other embodiments, a method is provided for assessing whether an individual with cancer is a low responder to treatment with a PD-1 neutralizing agent, where the presence and / or number of NKG2A+, and / or NKG2A+PD1+ NK cells, and / or T cells are assessed. Assessing NKG2A expression (and / or co-expression of NKG2A and PD-1) may, for example, involve obtaining a biological sample from an individual (e.g., from cancer or cancer-adjacent tissue obtained from a cancer patient) and analyzing the sample for the presence of NKG2A+ NK and / or CD8 T cells. Expression of NKG2A (and optionally further PD-1) and / or such NKG2A+ and / or NKG2A + PD-1 + An increase in the number of cells allows for the identification of individuals with tumor-infiltrating NK and / or T cells inhibited by both NKG2A and PD1 polypeptides. Optionally, the expression of NKG2A (and optionally further PD-1) and such NKG2A+ and / or NKG2A (compared to, for example, reference values ​​or values ​​before treatment with PD-1 neutralizing agents) can be assessed. + PD-1 + An increase in the number of cells is detected after administration of PD-1 neutralizing agents to individuals. Expression of NKG2A (and optionally further PD-1) and such NKG2A+ and / or NKG2A + PD-1+ Individuals with elevated or increased cell counts may be determined to be low responders to agents that neutralize PD-1 activity. This method may be useful, for example, as a prognostic prediction for response to treatment with NKG2A-neutralizing agents, for response to treatment with PD1-neutralizing agents, or for response to combined treatment with NKG2A-neutralizing agents and PD1-neutralizing agents.

[0161] In any of the methods herein, the step of detecting NKG2A and / or PD1 in T and / or NK cells may include the step of detecting NKG2A and / or PD1 expression in tissue-infiltrating human CD8 T cells and / or NK cells, the method comprising the steps of providing a tumor sample from an individual (e.g., sample or tumor tissue or tissue adjacent to a tumor) and detecting tissue-infiltrating CD8 T cells and / or NK cells in the sample using a monoclonal antibody that specifically binds to human NKG2A polypeptide and a monoclonal antibody that specifically binds to human PD-1 polypeptide in the sample. Optionally, in any embodiment, the patient has been treated with a PD-1 neutralizing agent. In one embodiment, the sample comprises tumor cells, tumor tissue or tissue adjacent to a tumor. In one embodiment, CD8 T cells and / or NK cells are identified using immunohistochemical techniques. In one embodiment, the sample is a paraffin-embedded sample, and optionally, the paraffin-embedded sample is fixed, embedded in paraffin, thinned, deparaffinized, and transferred to a slide before contact with the monoclonal antibody. In one embodiment, CD8 T cells and / or NK cells are identified using flow cytometry. [Examples]

[0162] Example 1 - RMA-S and A20 tumor cells are infiltrated by NK cells expressing NKG2A and CD8 T cells expressing NKG2A and / or PD-1. Lymphocytes are generally known not to co-express NKG2A and PD-1. To investigate the expression of these receptors in tumor-infiltrating lymphocytes, the distribution of NKG2A and PD-1 was studied in NK and T cell subsets in tumors from mice. Lymphocytes were collected from the spleen, tumor-inflowing lymph nodes, and within solid tumors.

[0163] C57 / BL6 mice were engrafted with PDL-1+Qa-1+RMA-S cells (Qa-1, Qdm, B2m) or A20 tumor cells (subcutaneously (sc)). RMA-S Qa-1 Qdm B2m (upper row) and A20 (lower row) tumor-bearing mice had tumor volumes of approximately 500 mm². 3 They were euthanized at that time.

[0164] The results are shown in Figures 1A and 1B. Tumor cells (Figure 1A) and tumor-infiltrating lymphocytes (TILs) (Figure 1B) were analyzed by flow cytometry for the expression of Qa-1 and PDL-1 in tumor cells, and for the expression of NKG2A and PD-1 in TILs. One representative mouse out of three is shown. MFI: Median fluorescence intensity.

[0165] More than half of the invasive NK cells from both tumor types expressed NKG2A, suggesting that tumor-infiltrating NK cells are inhibited by NKG2A. NKG2A+ NK cells generally did not express significant amounts of PD-1. However, CD8 T cells positive for both NKG2A and PD-1 were observed, suggesting that CD8 T cells may be restricted by both inhibitory receptors NKG2A and PD-1.

[0166] Example 2 - NK and T cell subsets from mice carrying rma-rae1 tumors can co-express NKG2A and PD-1. To further investigate the expression of the receptors NKG2A and PD-1, the distribution of NKG2A and PD-1 was studied in NK and T cell subsets in mice. Lymphocytes were collected from the spleen, tumor inflow lymph nodes, and within solid tumors.

[0167] C57 / BL6 mice were engrafted with RMA-Rae clone 6 (2 million cells) subcutaneously (sc). These tumor cells express CD94 / NKG2A ligand and Qa-1. Day 12 (average tumor volume: 723 mm²) 3 SD: 161mm 3 Mice were euthanized (n=4). After preparing cell suspensions from the spleen, linden, and tumor, the cells were stained as follows: CD3e PerCP Cy5.5, NKP46 Alexa 647, NKG2A / C / E FITC, PD1 PE, CD8 Pacific Blue.

[0168] The results are shown in Figure 2 and Tables 1-3.

[0169] In the NK cell subset, cells in both the inflowing lymph nodes and the spleen were approximately half NKG2A-positive and half NKG2A-negative, but in neither case were there significant expressions of PD1. NK cells from lymph nodes were NKG2A + PD-1 - (49.2%) and NKG2A - PD-1 - (49.5%), and less than 1% (on average) of NK cells were NKG2A + PD-1 + The NK cells from the spleen were NKG2A. + PD-1 - (44.1%) and NKG2A - PD-1 - (55.7%), and an average of 0.1% (average) of NK cells were NKG2A + PD-1 + That was the case.

[0170] In the T cell subset, most cells were NKG2A-negative (only 1.1% in lymph nodes and 4.7% in the spleen were NKG2A-negative). + (That is), a very small number of cells are PD-1 + (3.5% in lymph nodes and 10% in the spleen are PD-1 + NKG2A - There were no significant double-positive NKG2A PD-1 cells. Only 0.1% (average) of T cells in the lymph nodes were NKG2A. + PD-1 + Therefore, only 0.4% (on average) of T cells in the spleen are NKG2A + PD-1 + Furthermore, 95.1% of T cells from lymph nodes were double-negative, and 85.6% of T cells from the spleen were double-negative.

[0171] In the CD8 T cell subset, most cells were similarly NKG2A negative (only 1.6% in lymph nodes and 3.9% in the spleen were NKG2A negative). + (That is), a very small number of cells are PD-1 + (1.1% in lymph nodes and 2.5% in the spleen are PD-1 + NKG2A - There were no significant double-positive NKG2A PD-1 cells. Only 0.2% (average) of T cells in the lymph nodes were NKG2A. + PD-1 + Therefore, only 0.3% (on average) of T cells in the spleen are NKG2A + PD-1 + Furthermore, 97.3% of T cells from lymph nodes were double-negative, and 93.6% of T cells from the spleen were double-negative.

[0172] However, within tumor-infiltrating lymphocytes (TILs), all cell subsets contained cells expressing PD-1. NK cells expressing PD-1 in a significant proportion, which had not been previously observed, were identified as NKG2A. + Within the subset, PD-1 positivity was observed in the tumors, and 31.8% (mean) of NK cells were NKG2A. +PD-1 + The results showed that most CD8 T cells outside the tumor did not express NKG2A, while PD-1-expressing CD8 T cells were frequently observed within the tumor (the tumor-infiltrating CD8 T cell subset had an average of 26.3% NKG2A+ positive cells). Furthermore, within this NKG2A-positive CD8 T cell subset, most cells expressed NKG2A + PD-1 + It was (19.4% (average). Furthermore, CD8 - Within the T cell subset, only 5.1% of T cells in tumors expressed NKG2A, and only 3.6% of T cells were double-positive NKG2A PD-1, indicating little difference in NKG2A expression observed between TILs and splenic or lymph node cells.

[0173] [Table 1]

[0174] [Table 2]

[0175] [Table 3]

[0176] Example 3 - Expression of NKG2A and PD-1 in tumor-carrying mice To further investigate NKG2A and PD-1 expression in tumor-bearing mice, C57 / BL6 mice were engrafted with various tumor cells, either RMA-Rae1, MC38, or RMA strains (subcutaneously (sc)). To evaluate the effect of tumor volume, mice were engrafted with tumors of 500, 2000, and 800 mm, respectively. 3 They were euthanized when they reached a certain volume.

[0177] The results for RMA Rae1 (top column), MC38 (middle column), and RMA (bottom column) are shown in Figures 3A and 3B. NK cells (Figure 3A) and CD8 T cells (Figure 3B) were analyzed for NKG2A and PD-1 expression by flow cytometry in the spleen, tumor inflow lymph nodes (LN), and tumors. One representative mouse out of 2-4 is shown.

[0178] In the NK cell subset, cells in tumors, lymph nodes, and spleens were approximately half NKG2A-positive and half NKG2A-negative. NK cells from infiltrating lymph nodes or spleens (despite their NKG2A expression) did not show significant expression of PD1. However, tumor-infiltrating NK cells from RMA-Rae1 and RMA expressed both NKG2A and PD1 at significant levels, particularly in large volumes (2000 mm²). 3 Tumor-infiltrating NK cells from the tumor strain MC38, which were euthanized, expressed NKG2A (50%) but did not significantly express PD1 (3%).

[0179] Unlike NK cells, which express NKG2A in approximately half of the population, CD8 T cells from the spleen and lymph nodes generally did not express either NKG2A or PD1. However, within tumors, the majority of CD8 T cells expressed both NKG2A and PD1 (28% in RMA-Rae1, 43% in MC38, and 40% in RMA were double-positive). The results similarly suggest that tumor-infiltrating CD8 T cells and NK cells may be restricted by both inhibitory receptors NKG2A and PD1, even beyond differences in tumor cell type.

[0180] Example 4 - Increase in NKG2A-expressing tumor-infiltrating CD8 T cells in PD-1 resistant mice To evaluate the effect of anti-PD1 antibody treatment on CD8 T cells, MC38 tumor-bearing mice were treated with either 200 μg of rat IgG2a isotype control (IC) or neutralizing anti-mouse PD-1 monoclonal antibody 11, 14, and 17 days after cell engraftment. Mice (n=3 / group) were euthanized on day 31, and CD8 T cells were characterized by flow cytometry in the spleen, tumor inflow lymph nodes (LN), and tumor. The mean + / -SD (n=3) percentage of CD8 NKG2A+ in CD8 T cells is shown. P<0.005 *** ), P<0.0005( **** Statistical analysis was performed using two-way ANOVA, followed by Tukey's multiple comparison test.

[0181] The results are shown in Figure 4. As observed in other experiments, CD8 T cells from the spleen lymph nodes did not significantly express NKG2A. Administration of anti-PD1 antibody did not produce any change in the level of NKG2A expression in spleen or lymph node T cells. However, in tumor-infiltrating CD8 T cell populations, administration of anti-PD1 antibody resulted in a more than 50% increase in NKG2A-expressing CD8 T cells. The results suggest a significant increase in NKG2A-expressing CD8 T cells in unresponsive mice compared to mice treated with isotype control mAbs. Therefore, the NKG2A receptor may have an increased contribution to inhibiting the CD8 T cell response to tumors in low-responders to anti-PD1. Thus, neutralization of NKG2A may be useful in reversing the inhibition of NKG2A-restricted T cells in individuals treated with PD-1 axis neutralizing agents such as anti-PD1 or PDL1 antibodies.

[0182] Example 5 - Combination of anti-NKG2A / anti-PD1 blockade inhibits tumor growth. To evaluate the efficacy of combined treatment with neutralizing anti-PD1 antibody and neutralizing anti-NKG2A antibody, C57BL / 6 mice were engrafted with RMA-S Qa-1 Qdm B2m tumor cells (subcutaneously (sc)) and treated with a neutralizing anti-PD1 agent (neutralizing anti-PD-L1 antibody) and a neutralizing anti-NKG2A antibody.

[0183] In short, the RMA-S Qa-1 Qdm B2m tumor volume is approximately 85 mm². 3 C57BL / 6 mice (n=8 mice / group) were randomized on day 11 and treated on days 11, 14, and 18 with an isotype control, anti-mouse NKG2A mAb (200 μg, intravenous (iv)), anti-mouse PD-L1 mAb (200 μg, intraperitoneal (ip)), or anti-mNKG2A / mPDL-1 combination. Tumor volume was measured twice weekly, and if the tumor had grown (volume > 2000 mm), it was determined. 3 When ulcers developed or necrosis occurred, the mice were euthanized, and NK and CD8 T cells were characterized by flow cytometry.

[0184] Figure 5 shows the median progression of tumor volume over time. In this model, anti-NKG2A showed only a slight antitumor effect compared to isotype controls, while anti-PD-L1 showed a considerable antitumor effect, but tumor volume increased towards day 28. Combined treatment with anti-NKG2A and anti-PD-L1 resulted in complete cessation of tumor growth, and no significant increase in tumor volume was observed on day 28.

[0185] Example 6 - In vivo model of PD-1 / -L1 resistant cancer The A20 cell line expresses PD-L1, but Qa-1 b It is a mouse B-cell lymphoma line that does not express Qa-1. When subcutaneously injected into Balb / c mice, A20 generates solid tumors, and in these solid tumors, PD-L1 expression is maintained, and Qa-1 b Expression was induced. A20 tumor growth was controlled by NK and CD8 T cells.

[0186] To evaluate the efficacy of combination therapy with neutralizing anti-PD1 antibody or neutralizing anti-PDL1 antibody, Balb / c mice (female, 8-10 weeks old) were given A20 tumor cells (1 × 10⁶). 6Cells) were engrafted (subcutaneous (sc)), and treated with isotype control (IC) or neutralizing anti-mouse PD-1 antibody or neutralizing anti-mouse PD-L1 antibody (200 μg anti-PD1 or anti-PD-L1, intraperitoneal (ip), on days 13, 17 and 20). Tumor volume was measured twice a week, and mice were sacrificed when the tumor grew larger (volume > 2000 mm 3 ), ulcerated or necrosed, and NK and CD8 T cells were characterized by flow cytometry.

[0187] The progression of individual tumor volumes over time is shown in Figure 6. Neither anti-PD-L1 nor anti-PD-1 antibody produced a significant anti-tumor effect. Among 9 mice treated with isotype control (IC), no partial regression, temporary complete regression or complete regression was observed. Among 9 mice treated with anti-PD-1 antibody treatment, none of the mice had partial regression or temporary complete regression observed, and 2 mice had complete regression. Among 10 mice treated with anti-PD-L1 antibody, no partial regression or temporary complete regression was observed, and 2 mice had complete regression.

[0188] Despite the high expression of PD-1 in many immune infiltrating cells and the high expression of PD-L1 in tumor cells, monotherapy with anti-PD-1 or -PD-L1 mAb only produced a slight reduction in tumor growth. Interestingly, more than 50% of A20 tumor infiltrating NK cells and approximately 10% of CD8 T cells expressed CD94 / NKG2A. NKG2A + The CD8 T cell population also co-expressed PD-1. Qa-1 b expression was induced not only on the surface of tumor cells but also in infiltrating immune cells in vivo.

[0189] Example 7 - Anti-NKG2A antibody and anti-PD1 antibody result in complete tumor regression of PD-1 / -L1 resistant A20 tumors A20 tumor cells are PD-L1 and Qa-1 bBecause both antibodies were simultaneously expressed, mice carrying A20 tumors and administered a neutralizing anti-PD-1 antibody (see Example 6) were further treated with a neutralizing anti-NKG2A antibody.

[0190] Balb / c mice (female, 8-10 weeks old) were given A20 tumor cells (1 × 10⁶). 6 Engrafted (individual cells) subcutaneously (sc) and treated with isotype control (IC), neutralizing anti-mouse PD-1 antibody, or anti-PD-1 and neutralizing anti-NKG2A antibody (200 μg, intraperitoneal (ip), days 10, 13, and 17). Tumor volume was measured twice weekly using calipers. Tumor growth (volume > 2000 mm) was assessed. 3 ), or when ulceration or necrosis occurred, the animals were euthanized. The data represents the individual tumor volume per experiment.

[0191] Figure 7 shows the progression of individual tumor volume over time. Similar to what was observed in Example 6, the tumors were resistant to either the anti-PD-1 antibody or the isotype control (IC). Among 10 mice treated with the isotype control (IC), no partial regression was observed, and one transient complete regression and one complete regression were observed, while with anti-NKG2A alone, one partial regression was observed, no transient complete regression was observed, and one complete regression was observed. Among 9 mice treated with anti-PD-1 antibody, none of the mice showed partial regression, one transient complete regression was observed, and three complete regressions were observed. However, when anti-NKG2A antibody was added (indicated as "combination" in Figure 7), among 10 mice treated with the added anti-NKG2A antibody, seven complete regressions were observed (no partial or transient complete regressions). Therefore, anti-NKG2A antibodies have promising therapeutic potential in treating cancers that are resistant to treatment with anti-PD-1 or anti-PD-L1 antibodies.

[0192] Example 8 - Anti-NKG2A antibody and anti-PD-L1 antibody result in complete tumor regression of anti-PD1 / -L1 resistant A20 tumors. A20 tumor cells (5 × 10) subcutaneously were found in Balb / c mice (female, 8 weeks old, n=11 / group). 6 (Individual cells) were engrafted, and the mice were raised to day 11 (tumor volume approximately 50 mm). 3 Animals were randomized to receive treatment with an isotype control (IC), neutralizing anti-PD-L1 antibody (50 μg, intraperitoneal (ip), days 11, 14, 18, 21, 25, 28), blocking anti-NKG2A mAb (200 μg, intravenous (iv), days 11, 14, and 18), or a combination of both mAbs. Tumor volume was measured twice weekly using calipers. Animals were euthanized when tumor volume exceeded 2000 mm3, or when ulceration or necrosis occurred. Data represent individual tumor curves. PR: partial regression, CR: complete regression.

[0193] Figure 8 shows the progression of individual tumor volume over time. Similar to what was observed in Example 6, the tumors were resistant to either the anti-PD-L1 antibody or the isotype control (IC). Among the 11 mice treated with the isotype control (IC), no partial regression was observed, and two complete regressions (18%) were observed, compared to four complete regressions with anti-NKG2A alone. Among the 11 mice treated with anti-PD-L1 antibody alone, none of the mice showed partial regression, and six complete regressions were observed. However, when anti-NKG2A antibody was added, nine complete regressions were observed among the 11 mice treated with the addition of anti-NKG2A antibody. Therefore, the results indicate that anti-NKG2A antibody has promising therapeutic potential in treating cancers resistant to anti-PD-L1 antibody treatment.

[0194] All references, including publications, patent applications, and patents, that are invoked herein by reference are invoked in their entirety, and each reference is individually and specifically indicated as being invoked by reference, notwithstanding any other use of separately provided references elsewhere in this specification, and is invoked in its entirety to the same extent as described herein (to the maximum extent permitted by law).

[0195] The terms “a,” “an,” and “the,” as well as the use of similar referents, should be interpreted as encompassing both singular and plural unless otherwise stated herein or clearly negated by the context, in relation to the description of the present invention.

[0196] Unless otherwise specified, all exact values ​​provided herein represent the corresponding approximate values ​​(for example, all exact exemplary values ​​provided for a particular factor or measurement can be considered to also provide the corresponding approximate values, which may be modified as necessary). Where "approximately" is used in relation to a number, it may be specified to include values ​​corresponding to + / - 10% of the given number.

[0197] Any description of any aspect or embodiment of the Invention herein using terms such as “contains,” “has,” “includes,” or “contains” with respect to one or more elements is intended to provide support for similar aspects or embodiments of the Invention that “consist of,” “essentially consist of,” or “substantially contain” those one or more specific elements, unless otherwise specified or clearly refuted by the context (for example, a composition described herein as containing a particular element should be understood to also describe a composition consisting of that element, unless otherwise specified or clearly refuted by the context).

[0198] The use of any and all examples or exemplary words (e.g., "etc.") provided herein is intended solely to better illustrate the invention and, unless otherwise asserted, does not impose any limitation on the scope of the invention. The words herein should not be construed as indicating that any unclaimed element is essential for the practice of the invention. This disclosure provides, for example, the following embodiments. [Section 1] A drug that neutralizes the inhibitory activity of human NKG2A, for use in treating individuals with cancer that is less responsive to treatment with antibodies that neutralize the inhibitory activity of human PD-1. [Section 2] The compound for use as described in item 1, wherein the individual has cancer that has recurred or progressed during or after treatment with an antibody that neutralizes the inhibitory activity of human PD-1. [Section 3] The compound for use according to item 1, wherein the individual has cancer that is predicted to progress or recur during or after treatment with an antibody that neutralizes the inhibitory activity of human PD-1, based on one or more predictors. [Section 4] The aforementioned individual has not produced a complete response during or after treatment with an antibody that neutralizes the inhibitory activity of human PD-1, and the compound for use as described in any one of claims 1 to 3. [Section 5] The individual having an increased number of NKG2A-expressing NK and / or CD8 T cells is a compound for use according to any one of items 1 to 4. [Section 6] The individual having increased levels of expression of NKG2A polypeptide in NK and / or CD8 T cells, the compound for use according to any one of claims 1 to 5. [Section 7] The agent for neutralizing the inhibitory activity of human NKG2A is an antibody that binds to NKG2A, and is a compound for use according to any one of claims 1 to 6. [Section 8] The antibody that neutralizes the inhibitory activity of human NKG2A is administered in combination with an antibody that neutralizes the inhibitory activity of PD-1, as described in item 7. [Section 9] The antibody for neutralizing the inhibitory activity of human NKG2A and the antibody for neutralizing the inhibitory activity of PD-1 are compounds for use as described in item 8, formulated for separate administrations and administered simultaneously or sequentially. [Section 10] The antibody that neutralizes the inhibitory activity of human NKG2A and the antibody that neutralizes the inhibitory activity of PD-1 are compounds for use as described in item 8, formulated for simultaneous administration in a single formulation. [Section 11] The method according to any one of claims 7 to 10, wherein the antibody that neutralizes the inhibitory activity of human NKG2A is optionally administered following at least one administration of at least one treatment cycle with the antibody that neutralizes the inhibitory activity of PD-1. [Section 12] The individual is optionally determined to have an increased number of NKG2A-expressing NK and / or CD8 T cells after at least one administration of an antibody that neutralizes the inhibitory activity of PD-1, as described in any one of the compounds for use according to any one of claims 1 to 11. [Section 13] The aforementioned cancer is a solid tumor, and the compound for use according to any one of the claims 1 to 12. [Section 14] The aforementioned cancer is selected from the group consisting of lung cancer, renal cell carcinoma (RCC), melanoma, colorectal cancer, and ovarian cancer, and is a compound for use according to any one of claims 1 to 13. [Section 15] The aforementioned cancer is head and neck squamous cell carcinoma, and the compound for use according to any one of claims 1 to 14. [Section 16] An antibody that neutralizes the inhibitory activity of NKG2A is a compound for use as described in any one of items 1 to 15, comprising heavy chain CDR1, CDR2 and CDR3 domains having the sequence described in any one of SEQ ID NOs: 4 to 8, and light chain CDR1, CDR2 and CDR3 domains having the sequence described in SEQ ID NO: 9. [Section 17] An antibody that neutralizes the inhibitory activity of PD-1 is an antibody that binds to the PD-1 polypeptide, and is a compound for use as described in any one of items 1 to 16. [Section 18] The antibody that neutralizes the inhibitory activity of PD-1 is an antibody that binds to the PD-L1 polypeptide, and is a compound for use as described in any one of items 1 to 17. [Section 19] The antibody is a chimeric, human, or humanized antibody, and is a compound for use as described in any one of claims 1 to 18. [Section 20] The antibody that neutralizes the inhibitory activity of NKG2A is a non-depleted antibody, and is a compound for use as described in any one of items 1 to 19. [Section 21] The antibody that neutralizes the inhibitory activity of PD-1 is a non-depleted antibody, and is a compound for use as described in any one of items 1 to 20. [Section 22] The antibody is an IgG4 antibody, the compound for use as described in any one of claims 1 to 21. [Section 23] The antibody is a compound for use according to any one of claims 1 to 22, comprising an Fc domain that lacks an Fc domain or an Fc domain that is modified to reduce the binding of the Fc domain to the Fcγ receptor. [Section 24] The antibody is an antibody fragment, a compound for use as described in any one of claims 1 to 23. [Section 25] The antibody fragment is selected from Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, single-chain antibody fragment, or a polyspecific antibody comprising multiple different antibody fragments, as described in Section 24. [Section 26] The antibody that neutralizes the inhibitory activity of NKG2A is administered to the individual in an amount effective to neutralize the inhibitory activity of NKG2A in NK and / or CD8 T cells, as described in any one of the compounds for use in any one of claims 1 to 25. [Section 27] A method for identifying individuals with cancer who are poor responders to treatment with agents that neutralize the inhibitory activity of human PD-1, a) A step of determining the level of NKG2A expression in NK and / or CD8 T cells and / or the number of NKG2A-expressing NK and / or CD8 T cells in an individual treated with a drug that neutralizes the inhibitory activity of human PD-1, b) Once it is determined that the individual has increased levels of NKG2A expression in NK and / or CD8 T cells and / or an increased number of NKG2A-expressing NK and / or CD8 T cells, the individual is identified as a low responder to treatment with a drug that neutralizes the inhibitory activity of human PD-1. A method that includes this. [Section 28] An antibody that neutralizes the inhibitory activity of NKG2A for use in the treatment or prevention of cancer in an individual treated with a drug that neutralizes the inhibitory activity of PD-1, wherein the treatment is a) A step of determining the level of NKG2A expression in NK and / or CD8 T cells and / or the number of NKG2A-expressing NK and / or CD8 T cells in the individual, b) If it is determined that the individual has increased levels of NKG2A expression in NK and / or CD8 T cells and / or an increased number of NKG2A-expressing NK and / or CD8 T cells, the individual is administered a compound that neutralizes the inhibitory activity of human NKG2A polypeptide. Antibodies containing antibodies.

Claims

1. A composition for use in the treatment of lung cancer in human subjects, An antibody that neutralizes the inhibitory activity of human NKG2A by binding to NKG2A and competes with HLA-E for binding to NKG2A, comprising a heavy chain CDR1, CDR2, and CDR3 domain having the sequence shown in any one of SEQ ID NOs: 4 to 8, and a light chain CDR1, CDR2, and CDR3 domain having the sequence shown in SEQ ID NO: 9, This antibody neutralizes the inhibitory activity of PD-1 and is used in combination with the antibody MEDI-4736. composition.

2. The composition according to claim 1, wherein the antibody that neutralizes the inhibitory activity of NKG2A comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequence shown in SEQ ID NO: 5, and light chain CDR1, CDR2, and CDR3 domains having the sequence shown in SEQ ID NO:

9.

3. The composition according to claim 1 or 2, wherein the antibody that neutralizes the inhibitory activity of NKG2A comprises a heavy chain having the sequence shown in SEQ ID NO: 5 and a light chain having the sequence shown in SEQ ID NO:

9.

4. The composition according to any one of claims 1 to 3, wherein the lung cancer is non-small cell lung cancer.

5. A composition according to any one of claims 1 to 4, wherein the lung cancer is advanced and / or refractory.

6. The composition according to any one of claims 1 to 5, wherein a human individual has an increased number of NK cells and / or CD8 T cells expressing NKG2A.

7. The composition according to any one of claims 1 to 6, wherein a human individual is determined to have an increased number of NK cells and / or CD8 T cells expressing NKG2A after at least one administration of an antibody that neutralizes the inhibitory activity of PD-1, and optionally after at least one treatment cycle with the antibody.

8. The composition according to any one of claims 1 to 7, wherein the antibody that neutralizes the inhibitory activity of NKG2A is an IgG4 antibody, an antibody fragment, an antibody lacking an Fc domain, or an antibody containing an Fc domain, which is modified to reduce the binding between the Fc domain and the Fcγ receptor.

9. The composition according to any one of claims 1 to 8, wherein an antibody that neutralizes the inhibitory activity of NKG2A is administered to the human individual in an effective amount that neutralizes the inhibitory activity of NKG2A against NK cells and / or CD8 T cells.

10. A composition for use in the treatment of non-small cell lung cancer in human subjects, An antibody that neutralizes the inhibitory activity of human NKG2A by binding to NKG2A and competes with HLA-E for binding to NKG2A, comprising an antibody having a heavy chain having the sequence shown in SEQ ID NO: 5 and a light chain having the sequence shown in SEQ ID NO: 9, This antibody neutralizes the inhibitory activity of PD-1 and is used in combination with the antibody MEDI-4736. composition.

11. The composition according to claim 10, wherein a human individual develops a progressive disease during (or after) treatment with an antibody that neutralizes the inhibitory activity of human PD-1.

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