Treatment plan using Anti-NKG2a antibody

Abstract

To provide an antibody that specifically binds to and inhibits human NKG2A.SOLUTION: Provided is an anti-NKG2A antibody that binds to and neutralizes the inhibitory activity of NKG2A for use in treating an individual having a cancer that includes cancer cells expressing HLA-E on the cell surface, the anti-NKG2A antibody comprising a heavy chain that includes a specific amino acid sequence and a light chain that includes a specific amino acid sequence, the treatment comprising administering the anti-NKG2A antibody to the individual for at least one administration cycle, and the anti-NKG2A antibody being administered at least twice throughout the treatment cycle in an amount effective to maintain a sustained blood concentration of the anti-NKG2A antibody of at least 100 μg / mL between two successive administrations of the anti-NKG2A antibody.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 / 050,948, filed September 16, 2014, U.S. Provisional Patent Application No. 62 / 067,642, filed October 23, 2014, U.S. Provisional Patent Application No. 62 / 083,929, filed November 25, 2014, U.S. Provisional Patent Application No. 62 / 093,141, filed December 17, 2014, and U.S. Provisional Patent Application No. 62 / 093,124, filed December 17, 2014, all of which are incorporated by reference in their entirety, including any drawings.

[0002] Sequence Listing Reference This application is filed with an electronic sequence listing, which is provided as a file entitled "NKG2A-ST25", created on September 15, 2015, and 26 KB in size. The information in the electronic format of this sequence listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to the use of anti-NKG2A antibodies for therapy, particularly for the treatment of cancer. [Background technology]

[0004] NK cell activity is regulated by a complex mechanism involving both activating and inhibitory signals. Several different NK-specific receptors have been identified that play important roles in NK cell-mediated recognition and killing of HLA class I-deficient target cells. Natural cytotoxicity receptors (NCRs) refer to a class of activating receptor proteins specifically expressed in NK cells and the genes that express them. Examples of NCRs include NKp30, NKp44, and NKp46 (see, e.g., (1), (2), (3), (4), (5), (6), the entire disclosures of which are incorporated herein by reference). These receptors are members of the Ig superfamily, and their cross-linking induced by specific mAbs increases the intracellular Ca2+ levels. ++ It induces potent NK cell activation leading to increased levels of NK cells, induction of cytotoxicity, and release of lymphokines, and activation of NK cytotoxicity against many types of target cells.

[0005] CD94 / NKG2A is a cytotoxic receptor found on natural killer cells (NK cells), natural killer T cells (NKT cells), and subsets of T cells (α / β and γ / δ). CD94 / NKG2A limits the cytokine release and cytotoxic response of lymphocytes toward cells expressing HLA-E, the ligand of CD94 / NKG2A (see, for example, Patent Document 1). HLA-E has also been found to be secreted in a soluble form by some types of tumor cells (Non-Patent Document 7) and by activated endothelial cells (Non-Patent Document 8). Antibodies that block CD94 / NKG2A signaling (such as the induction of CD94 / NKG2A-positive NK cell responses to virus-infected cells) can increase the cytokine release and cytolytic activity of lymphocytes toward HLA-E-positive target cells. Thus, therapeutic antibodies that inhibit CD94 / NKG2A but do not cause the death of CD94 / NKG2A expressing cells (i.e., non-depleting antibodies) can induce tumor growth control in cancer patients. In addition, anti-NKG2A antibodies for the treatment of autoimmune or inflammatory diseases have also been proposed (see, for example, Patent Document 2 and Patent Document 3).

[0006] Various antibodies against NKG2A have been disclosed in the art. Patent Document 3 and Patent Document 4 (see also Patent Document 5) describe anti-NKG2A antibody Z270, while Patent Document 6 describes humanized anti-NKG2A antibody Z199. Non-Patent Document 9 refers to rat anti-mouse NKG2 antibody 20D5 (currently marketed by BD Biosciences Pharmingen, Catalog No. 550518, USA), and Patent Document 2 describes mouse antibody 3S9. Antibody Z270 binds to and neutralizes the cytotoxic receptor NKG2A without neutralizing the activating receptors NKG2C and NKG2E. Antibodies Z199, 20D5, and 3S9 all bind to the activating NKG2 family members NKG2C and NKG2E in addition to NKG2A. Antibody Z270 blocks the binding of HLA-E to NKG2A, whereas antibody Z199 neutralizes NKG2A without interfering with the binding of NKG2A to HLA-E. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 99 / 28748 Brochure [Patent Document 2] US Patent Publication No. 20030095965 [Patent Document 3] International Publication No. 2006070286 Brochure [Patent Document 4] U.S. Pat. No. 8,206,709 [Patent Document 5] International Publication No. 2008 / 009545 Brochure [Patent Document 6] International Publication No. 2009 / 092805 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Lanier (2001) Nat Immunol 2:23~27 [Non-Patent Document 2] Pende et al. (1999) J Exp Med. 190:1505~1516 [Non-Patent Document 3] Cantoni et al. (1999) J Exp Med. 189:787~796 [Non-Patent Document 4] Sivori et al. (1997) J.Exp.Med.186:1129~1136 [Non-Patent Document 5] Pessino et al. (1998) J Exp Med. 188(5):953~60 [Non-Patent Document 6] Mandelboim et al. (2001) Nature 409:1055~1060 [Non-Patent Document 7] Derre et al., J Immunol 2006;177:3100~7) [Non-Patent Document 8] Coupel et al.,Blood 2007;109:2806~14 [Non-Patent Document 9] Vance et al., J Exp Med 1999;190:1801~12) Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have discovered that doses and concentrations of anti-NKG2A antibodies that fully occupy the NKG2A receptor on lymphocytes do not result in maximal neutralization of the NKG2A receptor in the presence of HLA-E-expressing target cells in vivo. +The concentration of anti-NKG2A antibodies required for complete neutralization of the NKG2A receptor on NK cells in the presence of HLA-E-expressing target cells is 100-fold higher than that observed in lymphocyte-based binding assays. At the same time, the anti-tumor effect of anti-NKG2A is found to increase with higher expression of HLA-E on tumor cells.

[0010] As shown herein, NKG2A+ NK and CD8+ T lymphocytes are present not only in the circulating blood but also in the tumor environment, and furthermore, NKG2A+ CD8 T cells can be found at significantly higher frequencies in tumor-infiltrating subsets compared to CD8+ T cells in tumor draining lymph nodes and the spleen. Thus, the therapeutic regimen of the present invention further provides an advantageous method for treating solid tumors by modulating lymphocytes in the tumor environment using anti-NKG2A antibodies. [Means for solving the problem]

[0011] In one embodiment, a method of treating or preventing a disease (e.g., cancer, solid tumor, hematological tumor) in an individual is provided, the method comprising administering to an individual having the disease (e.g., cancer, solid tumor) an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide, the anti-NKG2A antibody inhibiting at least an EC50 of an NKG2A+ NK cell response. 50 , in some cases E.C. 100 In one embodiment, the amount is administered in an amount effective to achieve a blood (serum) concentration of anti-NKG2A antibody equivalent to at least the EC 50 , in some cases E.C. 100 In one embodiment, the NKG2A+ NK cell response is assessed using an assay for the cytotoxic activity of NKG2A-expressing NK cells against HLA-E-expressing target cells. The present disclosure provides, for example: [Section 1] An anti-NKG2A antibody that binds to and neutralizes the inhibitory activity of NKG2A, for use in treating an individual having a cancer comprising cancer cells that express HLA-E on the cell surface, said anti-NKG2A antibody comprising a heavy chain comprising the amino acid sequence of any of SEQ ID NOs: 2, 4 to 6 and a light chain comprising the amino acid sequence of SEQ ID NO: 7, said treatment comprising administering said anti-NKG2A antibody to said individual for at least one administration cycle, wherein the anti-NKG2A antibody is administered at least twice in an amount effective to maintain a sustained blood concentration of the anti-NKG2A antibody of at least 100 μg / mL between two successive administrations of the anti-NKG2A antibody throughout the treatment cycle. [Section 2] The anti-NKG2A antibody of item 1, wherein the anti-NKG2A antibody is administered in an amount effective to provide a sustained blood concentration of the anti-NKG2A antibody of at least about 100 μg / mL for at least one week after administration of the anti-NKG2A antibody. [Section 3] The anti-NKG2A antibody of item 1, wherein the anti-NKG2A antibody is administered twice per month. [Section 4] Item 2. The anti-NKG2A antibody according to Item 1, wherein the anti-NKG2A antibody is administered intravenously twice a month in an amount of 6 to 10 mg per kg of body weight. [Section 5] The anti-NKG2A antibody of item 1, wherein the treatment comprises a loading period followed by a maintenance period, wherein during the loading period the anti-NKG2A antibody is administered at least once at an initial dose effective to maintain a blood concentration of at least 100 μg / mL until the next successive administration of the anti-NKG2A antibody, and during the maintenance period the anti-NKG2A antibody is administered at least twice at a second dose and frequency effective to maintain a sustained blood concentration of the anti-NKG2A antibody of at least 100 μg / mL between successive administrations of the anti-NKG2A antibody. [Section 6] Item 6. The anti-NKG2A antibody according to Item 5, wherein the anti-NKG2A antibody is administered intravenously, the loading period comprises a single administration of the anti-NKG2A antibody at a dose of 8 to 10 mg per kg of body weight, and the maintenance period comprises at least two administrations of the anti-NKG2A antibody at a dose of 2 to 6 mg per kg of body weight at intervals of about two weeks. [Section 7] The anti-NKG2A antibody of clause 1, wherein the individual has a blood cancer selected from the group consisting of leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, promyelocytic leukemia, and myelodysplastic syndrome. [Section 8] The anti-NKG2A antibody of paragraph 1, wherein the individual has a solid tumor. [Section 9] The antibody of paragraph 8, wherein the individual has head and neck squamous cell carcinoma (HNSCC). [Section 10] The antibody of paragraph 1, wherein the anti-NKG2A antibody comprises a human IgG4 constant region or comprises an engineered Fc constant region that contains amino acid modifications that reduce binding to human Fcγ receptors.

[0012] In one embodiment, a method of treating or preventing a disease in an individual (e.g., cancer, solid tumor, hematological tumor) is provided, the method comprising administering to an individual having the disease (e.g., cancer, solid tumor) an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide, wherein the anti-NKG2A antibody is in an amount effective to achieve (and / or maintain for a specified period of time or between two successive administrations) a blood (serum) concentration of the anti-NKG2A antibody of at least 10 μg / mL (or optionally at least 20, 30, 40, 50, 80, or 100 μg / mL).

[0013] Thus, in one embodiment, there is provided a method of treating or preventing a disease (e.g., cancer, solid tumor, hematological tumor) in an individual, the method comprising administering to the individual an antibody that binds to NKG2A and neutralizes the inhibitory activity of a human NKG2A polypeptide for at least one administration cycle, the administration cycle comprising at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or more) administrations of an anti-NKG2A antibody, the anti-NKG2A antibody being administered to achieve a blood concentration of the anti-NKG2A antibody and / or two successive (e.g., the first administration described above) administrations. and a second, and optionally more) dose, that is at least 10-fold (e.g., 10-20-fold, 10-50-fold, 10-100-fold, 20-50-fold, 20-100-fold, 30-100-fold, 50-100-fold), and optionally at least 50-fold, 60-fold, 80-fold, or 100-fold, the minimum concentration required to substantially completely (e.g., 90%, 95%) occupy (saturate) the NKG2A receptors on the surface of NKG2A+ cells (e.g., as determined by titrating the anti-NKG2A antibody on NKG2A-expressing cells in PBMCs). In one embodiment, the anti-NKG2A antibody competes with HLA-E for binding to human NKG2A.

[0014] In one embodiment, a method is provided for treating or preventing a disease (e.g., cancer, solid tumor, hematological tumor) in an individual, the method comprising administering to an individual having the disease (e.g., cancer, solid tumor) an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide for at least one administration cycle, the administration cycle comprising at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or more) administration of an anti-NKG2A antibody, the anti-NKG2A antibody being administered in an amount effective to achieve or maintain between two successive administrations a blood (serum) concentration of the anti-NKG2A antibody of at least 10 μg / mL (or optionally at least 20, 30, 40, or 50 μg / mL). In one embodiment, a specified sustained blood concentration is maintained, the blood concentration not substantially falling below the specified blood concentration for the duration of the specified period (e.g., between two administrations of the antibody, i.e., for a period of multiple weeks). That is, blood concentrations can fluctuate over a specified period of time, but the specified sustained blood concentration represents a minimum or "trough" concentration. In one embodiment, a therapeutically effective amount of an anti-NKG2A antibody is one that suppresses (at least) the EC50 of an NKG2A+ NK cell response in the blood and / or tissues for a period of at least about one week, about two weeks, or about one month after administration of the antibody. 50 Concentration, in some cases practically EC 100 In one embodiment, the anti-NKG2A antibody is administered in an amount effective to achieve a blood (serum) concentration of the anti-NKG2A antibody of at least 10 μg / mL (or optionally at least 20, 30, 40, or 50 μg / mL) and that allows for significant "desaturation" between two successive administrations (the successive administrations may be, for example, one month, two months, or more apart), for a period of at least about one week, about two weeks. In one embodiment, a therapeutically effective amount of an anti-NKG2A antibody is an amount that is effective to achieve a blood (serum) concentration of the anti-NKG2A antibody of at least 10 μg / mL (or optionally at least 20, 30, 40, or 50 μg / mL) and that allows for significant "desaturation" between two successive administrations (the successive administrations may be, for example, one month, two months, or more apart), for a period of at least about one week, about two weeks, in a tissue with respect to the NKG2A+ NK cell response. 50 Concentration, in some cases practically EC 100The amount of such antibody that can provide a concentration and allow significant "desaturation" between two successive doses thereafter (optionally the antibody is administered at a dosing frequency of about once every month, or about once every two months). During the desaturation period, the blood concentration of the anti-NKG2A antibody is below a specified concentration that should be achieved during an earlier period (e.g., 1 week, 2 weeks, etc.). For example, the blood and / or tissue anti-NKG2A antibody concentration during the desaturation period may be indicative of the EC of the NKG2A+ NK cell response. 100 below EC 50 can be specified to be less than

[0015] In one embodiment, particularly where it is intended to modulate NKG2A+ T or NK cells in extravascular tissues, for example for the treatment of solid tumors, the anti-NKG2A antibody is administered in an amount effective to achieve a peak concentration or to maintain a blood concentration of at least about 40, 50, 60, 70, or 80 μg / mL, and optionally at least about 100 μg / mL, at the time of administration (e.g., within 1 or 2 days of administration). In one embodiment, the NKG2A antibody is administered in an amount effective to maintain a specified blood concentration for at least 1 week, and optionally at least 2 weeks, following administration of the anti-NKG2A antibody. In one embodiment, particularly when it is intended to modulate NKG2A+T or NK cells in extravascular tissues, for example for the treatment of solid tumors, the anti-NKG2A antibody is administered in an amount effective to achieve or maintain a sustained (minimum) blood concentration of about or at least about 50, 60, 70, or 80 μg / mL, and optionally at least about 100 μg / mL, of the anti-NKG2A antibody between the first and second (and optionally more) administrations. In one embodiment, the successive administrations (e.g., the first and second administrations) are separated in time by at least one week, and optionally by about two weeks. The anti-NKG2A antibody can optionally be administered according to an amount and frequency effective to achieve or maintain a specified blood concentration for the entire duration of the administration cycle.

[0016] In one embodiment, a method of treating or preventing a solid tumor in an individual is provided, the method comprising administering to an individual having a solid tumor an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide for at least one administration cycle, the administration cycle comprising at least two administrations of the anti-NKG2A antibody, the anti-NKG2A antibody being administered in an amount effective to achieve or maintain a (minimum) concentration in extravascular tissue (e.g., in the tumor environment) of at least 4 μg / mL, and optionally at least 10 μg / mL, between two successive administrations. Optionally, the anti-NKG2A antibody is administered in an amount effective to achieve or maintain a (minimum) concentration in extravascular tissue (e.g., in the tumor environment) of at least 4 μg / mL, and optionally at least 10 μg / mL, for the entire duration of the administration cycle. In one embodiment, the anti-NKG2A antibody is administered in an amount effective to maintain a sustained blood concentration of the anti-NKG2A antibody of at least 40 μg / mL, and optionally at least 100 μg / mL, between two successive administrations or for the duration of the administration cycle. In one embodiment, the anti-NKG2A antibody is administered in an amount effective to achieve a blood concentration of at least 40 μg / mL, and optionally at least 100 μg / mL, of the anti-NKG2A antibody at the time of administration (e.g., for at least 1 week, at least 2 weeks after administration), followed by a period allowing for significant "desaturation" of circulating NKG2A-expressing cells between two successive administrations (wherein the blood concentration of the anti-NKG2A antibody is less than the above-mentioned at least 40 μg / mL or at least 100 μg / mL during the period of desaturation).

[0017] In one embodiment, a method of treating or preventing a hematological tumor in an individual is provided, the method comprising administering to an individual having a hematological tumor an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide for at least one administration cycle, the administration cycle comprising at least two administrations of the anti-NKG2A antibody, the anti-NKG2A antibody being administered in an amount effective to achieve or maintain a sustained (minimum) blood concentration of the anti-NKG2A antibody of at least 10 μg / mL between two successive administrations. Optionally, the anti-NKG2A antibody is administered in an amount effective to achieve or maintain a sustained (minimum) blood concentration of the anti-NKG2A antibody of at least 10 μg / mL for the entire duration of the administration cycle.

[0018] In one embodiment, the antibody is administered to achieve a peak blood concentration of at least about 40 μg / mL, and optionally about or at least about 100 μg / mL, at the time of administration (e.g., on the day of administration, within 24 or 48 hours of administration).

[0019] In one embodiment, a blood concentration of 40 μg / mL is the EC 50 In one embodiment, a blood concentration of 100 μg / mL can result in an EC20 concentration in tissues (outside the vasculature, e.g., in the tumor environment) of NKG2A+ NK cell responses. 100 Concentration can be achieved.

[0020] In one embodiment, the method of treatment involves administering a treatment cycle having (a) an induction (i.e., loading) period during which a first dose of an anti-NKG2A antibody is administered to achieve or maintain a sustained (minimum) blood concentration of at least about 40 μg / mL, and optionally at least about 100 μg / mL, between the first and subsequent administrations of the anti-NKG2A antibody, and (b) a maintenance period during which a second, lower dose of the anti-NKG2A antibody is administered in an amount sufficient to achieve or maintain a sustained (minimum) blood concentration of at least about 40 μg / mL, and optionally at least about 100 μg / mL, of the anti-NKG2A antibody until the next administration of the anti-NKG2A antibody (e.g., for the entire treatment cycle). This maintenance period follows the induction period. In one embodiment, the maintenance period includes at least two administrations of the anti-NKG2A antibody. In one embodiment, the maintenance period includes at least two administrations at a dose and frequency that results in a sustained blood concentration of at least about 40 μg / mL, and optionally at least about 100 μg / mL, of the anti-NKG2A antibody between the two administrations.

[0021] In any embodiment it may be specified that the blood concentration is a serum concentration.

[0022] In one embodiment, the anti-NKG2A antibody substantially completely neutralizes the inhibitory activity of human CD94 / NKG2A in a human patient (in vivo) on NKG2A-positive lymphocytes in the circulating blood or in extravascular tissues for about one week or for about two weeks.

[0023] In one embodiment, a method of treating an individual having cancer (e.g., a solid tumor) is provided, the method comprising administering to the individual an antibody that binds to and neutralizes the inhibitory activity of NKG2A for at least one administration cycle, where the anti-NKG2A antibody is administered intravenously at a dose of between 4-10 mg / kg body weight, optionally between 4-6 mg / kg, optionally between 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 twice per month.

[0024] In one embodiment, a method of treating an individual having cancer (e.g., a solid tumor) is provided, the method comprising administering to the individual an antibody that binds to and neutralizes the inhibitory activity of NKG2A for at least one administration cycle, the method comprising: a. a loading period in which the antibody is administered intravenously at least once at an initial dose of between 8-10 mg / kg body weight, and in some cases approximately 10 mg / kg body weight; and b. A maintenance period in which the antibody is administered intravenously at a dose of 2-6 mg / kg, optionally between 2-5 mg / kg, optionally between 2-4 mg / kg, optionally about 2 mg / kg, optionally about 3 mg / kg, optionally about 4 mg / kg, or optionally about 6 mg / kg, at least twice every two weeks, optionally with the first administration in the maintenance period occurring within two weeks of the initial dose.

[0025] In one embodiment, the treatment regimens described herein are administered to an individual with cancer before the individual undergoes surgery to remove cancer cells, i.e., the anti-NKG2A antibody regimen is used as a pre-operative treatment.

[0026] In one embodiment, at least the EC 50 , in some cases E.C. 100 A treatment regimen or course of treatment designed to achieve a concentration of anti-NKG2A antibodies corresponding to is administered to an individual with cancer prior to surgery to remove cancer cells, ie, as a pre-operative treatment.

[0027] In one embodiment, anti-NKG2A antibodies are administered to individuals with cancer cells that express HLA-E on their cell surface. In some cases, the HLA-E status of the cancer can be assessed prior to treatment with anti-NKG2A antibodies. In one embodiment, a method is provided that combines a step of HLA-E detection to identify patients with HLA-E+ tumors. Those patients can then be treated with anti-NKG2A antibodies according to the treatment methods described herein.

[0028] In one embodiment of any of the therapeutic uses or methods of treating or preventing cancer herein, the treatment and prevention of cancer in an individual comprises: a) determining the HLA-E polypeptide status of malignant cells in an individual with cancer; and b) based on a determination that the patient has an HLA-E polypeptide prominently expressed on the surface of malignant cells, administering to the individual an anti-NKG2A antibody that binds to and neutralizes the inhibitory activity of NKG2A, e.g., according to any of the therapeutic methods described herein. Optionally, the antibody prevents binding of NKG2A by HLA-E.

[0029] In one embodiment of any of the therapeutic uses or methods of treating or preventing cancer herein, the treatment and prevention of cancer in an individual comprises: a) determining the level of expression of HLA-E nucleic acid or polypeptide of malignant cells in an individual with cancer; and b) based on a determination that the malignant cells express HLA-E nucleic acid or polypeptide at a high, and optionally a high level (e.g., high numerical value, intense surface staining, etc.) relative to baseline levels, administering to the individual an anti-NKG2A antibody that binds to and neutralizes the inhibitory activity of NKG2A, e.g., according to any of the therapeutic methods described herein; Optionally, the antibody prevents binding of NKG2A by HLA-E.

[0030] In one embodiment of any of these methods, determining the status of HLA-E polypeptide or determining the level of expression in step a) comprises determining the level of expression of HLA-E nucleic acid or polypeptide of malignant cells in the biological sample and comparing the level to a reference level (e.g., numerical value, weak cell surface staining, etc.). The reference level can correspond, for example, to a healthy individual, to an individual who will get no / low clinical benefit from treatment with an anti-NKG2A antibody, or to an individual who will get a significant clinical benefit from treatment with an anti-NKG2A antibody. A determination that the biological sample expresses HLA-E nucleic acid or polypeptide at a high level (e.g., high numerical value, strong surface staining, a level higher than that corresponding to an individual who will get no / low clinical benefit from treatment with an anti-NKG2A antibody, etc.) indicates that the individual has a cancer that can be treated with an anti-NKG2A antibody, for example according to the treatment methods described herein.

[0031] In one embodiment, the anti-NKG2A antibody is administered as a monotherapy. In one embodiment, the anti-NKG2A antibody is administered in combination therapy with one or more other anti-cancer agents.

[0032] In one embodiment of any of the therapeutic uses or treatment or prophylactic methods herein, the method further comprises administering to the individual a therapeutically effective amount of a second anti-cancer agent. In one embodiment, the cancer is a solid tumor. In one embodiment, the second anti-cancer agent is an antibody capable of mediating ADCC (e.g., binds to a human Fcγ receptor, such as CD16, via its Fc domain). In one embodiment, the antibody mediating ADCC is administered in an amount effective to induce antibody-dependent cellular cytotoxicity against human tumor cells (in vivo) in a human patient expressing a polypeptide against which the second anti-cancer agent is directed.

[0033] In one embodiment, the second anti-cancer agent is an anti-EGFR antibody. In one embodiment, patients treated with an anti-NKG2A antibody according to the treatment regimen of the present invention in combination with an anti-EGFR antibody have responded inadequately to prior treatment with an anti-EGFR antibody (e.g., are non-responders or have progressive disease) or have an unfavorable prognosis for response to treatment with an anti-EGFR antibody (in the absence of treatment with an anti-NKG2A).

[0034] In one embodiment, the combination is administered (or is intended to be administered) according to a particular clinical regimen, particularly according to a particular dosage amount or according to a particular administration schedule (e.g., according to the dosage amounts and / or particular administration schedules provided herein).

[0035] Antibodies that neutralize the inhibitory activity of NKG2A polypeptides (anti-NKG2A agents) are those that enhance the ability of NKG2A-expressing NK and / or T cells to cause the death of HLA-E expressing cells.

[0036] In one embodiment, the anti-NKG2A substance reduces the inhibitory activity of NKG2A by blocking the binding of its ligand, HLA-E. That is, the anti-NKG2A substance prevents the binding of NKG2A by HLA-E. Examples of such antibodies include antibodies having a heavy chain of any one of SEQ ID NOs: 2 to 6 and a light chain of SEQ ID NO: 7.

[0037] In one embodiment, the anti-NKG2A antibody is an antibody that binds with much higher affinity to NKG2A than to one or more activating NKG2 receptors. For example, in one embodiment, the antibody binds with much higher affinity to NKG2A than to NKG2C. In further or alternative embodiments, the antibody binds with much higher affinity to NKG2A than to NKG2E. In further or alternative embodiments, the antibody binds with much higher affinity to NKG2A than to NKG2H. An antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 binds to NKG2A without substantially binding to NKG2C, NKG2E, or NKG2H.

[0038] In further or alternative embodiments, the anti-NKG2A antibody binds to the same epitope on NKG2A and / or competes for binding to CD94 / NKG2A with an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7. The antibody can be, for example, a human or humanized anti-NKG2A antibody.

[0039] In one embodiment, the anti-NKG2A antibody is a humanized antibody having a heavy chain CDR of any one of the heavy chain sequences of SEQ ID NO: 2-6 and a light chain CDR of the light chain sequence of SEQ ID NO: 7. Examples are provided of residues or sequences in the complementarity determining regions (CDRs) and / or sites of amino acid substitution in the framework regions (FRs) of such humanized antibodies that have improved properties, e.g., lower antigenicity, improved properties, e.g., antigen binding or other functional properties, and / or improved physicochemical properties, e.g., better stability.

[0040] In other embodiments, pharmaceutical compositions and kits and methods for their use are provided.

[0041] These aspects are more fully described in the Detailed Description of the Invention provided herein, and additional aspects, features, and advantages will become apparent from the Detailed Description of the Invention provided herein. [Brief description of the drawings]

[0042] [Figure 1]Figure 1 shows receptor occupancy in human blood for anti-NKG2A antibody (humZ270, also called IPH2201) at various concentrations (ng / mL) listed on the x-axis. The MESF signal of receptor binding is shown on the y-axis. The antibody had a binding affinity (EC50) of approximately 4 ng / mL for NKG2A+ cells (KD approximately 4 ng / mL). This KD is consistent with KD values ​​observed in other assays, notably the affinity for binding to PBMC in the Biacore assay and for recombinant NKG2A. The KD for full receptor occupancy (EC100) was approximately 100 ng / mL. [Diagram 2] FIG. 1 shows the expected receptor occupancy of anti-NKG2A antibodies based on human patients treated with a single dose of humZ270 in a 92-patient Phase I clinical trial. Substantially complete (at least 90%) receptor saturation of NKG2A+ cells can be achieved by dosing at 0.03 mg / kg every 2 weeks. Receptor saturation increases with increasing dose, with the plotted line in the figure having the lowest receptor saturation corresponding to the lowest dose (0.01 mg / kg). Each progressively higher dose level corresponds to the plotted line with the next higher receptor saturation, with the line corresponding to the highest dose (10 mg / kg) having the highest receptor saturation. A dose of 0.1 mg / kg can be administered every 4 weeks to maintain substantially complete saturation of NKG2A. [Figure 3A] Figure 1 shows results from an autologous cell assay demonstrating that the concentration of anti-NKG2A required for efficacy (blocking NKG2A in the presence of cells expressing its HLA-E ligand) is 100-fold higher than the concentration that results in full receptor saturation. The EC50 concentration (amount achieving 50% of maximum) in the CD107 mobilization assay was determined to be approximately 400 ng / mL, and the EC100 was approximately 10,000 ng / mL (10 μg / mL). [Figure 3B]FIG. 1 shows predicted IPH2201 blood concentrations after iv injections every 2 weeks based on preliminary PK data and NCA analysis from a Phase I clinical trial of IPH2201 (anti-NKG2A). Different doses administered by iv injection every 2 weeks achieved different blood concentrations. IPH2201 concentrations increased with increasing dose, with the plotted line in the figure having the lowest blood concentration corresponding to the lowest dose (0.01 mg / kg). Each progressively higher dose level corresponds to the plotted line showing the next higher blood concentration, with the line corresponding to the highest dose (10 mg / kg) representing the highest blood concentration. The 4 mg / kg dose showed initial (peak) blood concentrations of about 100 μg / mL, i.e., near the EC100 of efficacy in tissues, and sustained (minimum) blood concentrations of greater than 30 μg / mL out to the 2-week time point, or about 100 μg / mL at the 4th dose. A dose of 10 mg / kg produced sustained (minimum) blood concentrations of approximately 100 μg / mL. [Figure 3C] FIG. 1 shows predicted IPH2201 blood levels (shown as the upper line) after iv injections once every two weeks with a loading dose of 10 mg / kg body weight followed by a maintenance dose of 4 mg / kg body weight. This results in initial and sustained blood levels of approximately 100 μg / mL. For comparison, a flat dose of 4 mg / kg is shown (lower line in the figure) that results in sustained (or minimum) blood levels of at least 30 μg / mL at the two week time point. [Figure 4A]FIG. 4 shows the ability of anti-NKG2A to improve recognition of HNSCC cell lines by NK cells. FACS readouts of CD107 (top) and CD137 (bottom) on NKG2A-NK (left) or NKG2A+NK cells (right) in the presence of the indicated target HNSCC cell lines and in the presence or absence of anti-NKG2A at a concentration of 10 μg / mL are shown. The cell lines are arranged from left to right according to the level of surface expression of HLA-E. Each dot represents PBMC from a healthy volunteer. FIG. 4A shows control and K562 targets with low or high levels of surface HLA-E, and FIG. 4B demonstrates that anti-NKG2A can restore lysis of NHSCC due to expression of endogenous HLA-E. This effect is seen only on NKG2A-positive NK cells and is dependent on the level of expression of HLA-E. [Figure 4B] FIG. 4 shows the ability of anti-NKG2A to improve recognition of HNSCC cell lines by NK cells. FACS readouts of CD107 (top) and CD137 (bottom) on NKG2A-NK (left) or NKG2A+NK cells (right) in the presence of the indicated target HNSCC cell lines and in the presence or absence of anti-NKG2A at a concentration of 10 μg / mL are shown. The cell lines are arranged from left to right according to the level of surface expression of HLA-E. Each dot represents PBMC from a healthy volunteer. FIG. 4A shows control and K562 targets with low or high levels of surface HLA-E, and FIG. 4B demonstrates that anti-NKG2A can restore lysis of NHSCC due to expression of endogenous HLA-E. This effect is seen only on NKG2A-positive NK cells and is dependent on the level of expression of HLA-E. [Diagram 5] FIG. 1 shows that NK cells directed towards HNSCC FaDu cells were induced by a suboptimal dose of the anti-EGFR cetuximab (ctx), whereby an optimal dose of anti-NKG2A enhanced ADCC. [Figure 6A]Figure 3A shows the effect of increasing doses of anti-NKG2A and increasing doses of anti-EGFR (cetuximab). Figure 3A shows the readout of CD107 on untargeted and K562-HLA-E transfectant controls. Healthy volunteers are represented by different symbols, squares and circles. Cross-open symbols correspond to the condition where anti-NKG2A was replaced by 10 μg / mL of hIgG4 isotype control co-incubated with 0.1 μg / mL of cetuximab. Figure 6B shows the readout of CD107 on HNSCC cell lines. Symbols (circles) for each concentration of anti-NKG2A for each concentration of cetuximab correspond from left to right to 0 μg / mL, 0.1 μg / mL, 1 μg / mL, and 10 μg / mL. [Figure 6B] Figure 3A shows the effect of increasing doses of anti-NKG2A and increasing doses of anti-EGFR (cetuximab). Figure 3A shows the readout of CD107 on untargeted and K562-HLA-E transfectant controls. Healthy volunteers are represented by different symbols, squares and circles. Cross-open symbols correspond to the condition where anti-NKG2A was replaced by 10 μg / mL of hIgG4 isotype control co-incubated with 0.1 μg / mL of cetuximab. Figure 6B shows the readout of CD107 on HNSCC cell lines. Symbols (circles) for each concentration of anti-NKG2A for each concentration of cetuximab correspond from left to right to 0 μg / mL, 0.1 μg / mL, 1 μg / mL, and 10 μg / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] definition As used herein, "a" or "an" can mean one or more than one. When used in the claims, when used with the word "comprising", the word "a" or "an" can mean one or more than one. As used herein, "another" can mean at least a second, or more.

[0044] When "comprising (in its entirety)" is used, it may be replaced in some cases by "consisting essentially of" or by "consisting of".

[0045] NKG2A (OMIM 161555, the entire disclosure of which 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 that show several alternative splices. Together with CD94, NKG2A forms the heterodimeric cytotoxic receptor CD94 / NKG2A, which is found on the surface of NK cells, α / β T cells, γ / δ T cells, and a subset of NKT cells. Like the cytotoxic KIR receptors, it possesses an ITIM in its cytoplasmic domain. As used herein, "NKG2A" refers to any variant, derivative, or isoform of the NKG2A gene or encoded protein. Also encompassed are any nucleic acid or protein sequences that share one or more biological properties or functions with wild-type full-length NKG2A and share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide or amino acid identity therewith. Human NKG2A contains 233 amino acids of the following sequence in three domains: the cytoplasmic domain includes residues 1-70, the transmembrane region includes residues 71-93, and the extracellular region includes residues 94-233. [ka]

[0046] NKG2C (OMIM 602891, the entire disclosure of which is incorporated herein by reference) and NKG2E (OMIM 602892, the entire disclosure of which is incorporated herein by reference) are two other members of the NKG2 group of transcripts (Gilenke, et al. (1998), Immunogenetics 48:163-173). The CD94 / NKG2C and CD94 / NKG2E receptors are activating receptors found on the surface of subsets of lymphocytes, such as NK cells and T cells.

[0047] HLA-E (OMIM 143010, the entire disclosure of which is incorporated herein by reference) is a non-classical MHC molecule that is expressed on the cell surface and regulated by peptide binding, such as fragments derived from the signal sequences of other MHC class I molecules. Soluble versions of HLA-E have 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, e.g., 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 encoded protein. Also encompassed is any nucleic acid or protein sequence that shares one or more biological properties or functions with wild-type full-length HLA-E and shares at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide or amino acid identity therewith.

[0048] In the context of the present invention, "CD94 / NKG2A positive lymphocytes" refers to cells of the lymphoid lineage (e.g., NK cells, NKT cells, and T cells) that express CD94 / NKG2A on the cell surface. This can be detected, for example, by flow cytometry using an antibody that specifically recognizes a combined epitope on CD94 and NKG2A and / or an epitope on NKG2A alone. "CD94 / NKG2A positive lymphocytes" also include immortal cell lines derived from lymphocytes (e.g., NKL, NK-92).

[0049] In the context of the present invention, "reducing the inhibitory activity of NKG2A", "neutralizing NKG2A" or "neutralizing the inhibitory activity of NKG2A" refers to a process in which CD94 / NKG2A is inhibited with respect to its ability to adversely affect intracellular processes leading to lymphocyte responses, such as cytokine release and cytotoxic responses. This can be measured, for example, in a cytotoxicity assay using NK cells or T cells. In this assay, the ability of a therapeutic compound to promote the killing of HLA-E positive cells by CD94 / NKG2A positive lymphocytes is measured. In one embodiment, the antibody preparation causes at least a 10% increase in lymphocyte cytotoxicity that is CD94 / NKG2A restricted, preferably at least a 40% or 50% increase in lymphocyte cytotoxicity, or more preferably at least a 70% increase in NK cytotoxicity. This also refers to the cytotoxicity assay described above. If an anti-NKG2A antibody reduces or blocks the interaction of CD94 / NKG2A with HLA-E, it can increase the cytotoxicity of lymphocytes that are CD94 / NKG2A-restricted. This can be assessed, 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 kill targets expressing HLA-E efficiently because CD94 / NKG2A recognizes HLA-E, which triggers the initiation and propagation of inhibitory signals that prevent lymphocyte-mediated cytolysis. Such in vitro cytotoxicity assays can be performed by standard methods well known in the art, for example as described in Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY (1992, 1993).Chromium release and / or other parameters to assess the ability of the antibodies to stimulate lymphocytes to kill target cells, such as P815 cells, K562 cells, or appropriate tumor cells, are also 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 entire disclosures of each of which are incorporated herein by reference. The target cells are added prior to the addition of the NK cells. 51 Cr, and then the lethality is determined as a result of 51 The release of Cr from the cells into the medium is estimated as being proportional to the release of Cr from the cells into the medium. Addition of antibodies that prevent CD94 / NKG2A from binding to HLA-E causes a block in the initiation and propagation of inhibitory signaling through CD94 / NKG2A. Addition of such agents therefore causes an increase in lymphocyte-mediated killing of target cells. This step thereby identifies agents that block CD94 / NKG2A-induced negative signaling, for example by blocking ligand binding. 51 In Cr release cytotoxicity assays, CD94 / NKG2A-expressing NK effector cells were able to kill HLA-E-negative LCL 721.221 target cells, but poorly killed HLA-E-expressing LCL 721.221-Cw3 control cells. In contrast, YTS effector cells lacking CD94 / NKG2A efficiently killed both cell lines. Thus, NK effector cells are able to inhibit HLA-E expression due to the transmission of inhibitory signals via CD94 / NKG2A induced by HLA-E. + NK cells do not kill LCL 721.221-Cw3 cells very efficiently. 51When preincubated with blocking anti-CD94 / NKG2A antibodies according to the invention in a Cr release cytotoxicity assay, HLA-E expressing LCL 721.221-Cw3 cells are killed more efficiently in an antibody concentration dependent manner. The inhibitory activity (i.e. potential to enhance cytotoxicity) of anti-NKG2A antibodies can also be assessed in any of several other ways, for example by their effect on intracellular free calcium, as described in, for example, Sivori et al., J. Exp. Med. 1997; 186: 1129-1136, the disclosure of which is incorporated herein by reference. NK cell cytotoxic activation can be assessed, for example, by measuring an increase in cytokine production (e.g., production of IFN-γ) or cytotoxicity markers (e.g., CD107 or CD137 mobilization). In a typical protocol, IFN-γ production from PBMCs is assessed by cell surface and cytoplasmic staining and flow cytometric analysis after 4 days in culture. Briefly, Brefeldin A (Sigma Aldrich) is added to a final concentration of 5 μg / mL for the last 4 hours of culture. The cells are then incubated with anti-CD3 and anti-CD56 mAbs before being permeabilized (IntraPrep TM , Beckman Coulter), PE-anti-IFN-γ or PE-IgG1 (Pharmingen). GM-CSF and IFN-γ production from polyclonally activated NK cells is measured in supernatants using ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, MN, IFN-γ: OptEIA set, Pharmingen).

[0050] Throughout this specification, whenever "treatment of cancer" or the like is mentioned in connection with an anti-NKG2A binding agent (e.g. an antibody), it means: (a) a method of treating cancer comprising the step of administering (for at least one treatment) an anti-NKG2A binding agent (preferably in a pharma- ceutically acceptable carrier material) to an individual, mammal, especially a human in need of such treatment, in a dose (therapeutically effective amount) allowing such treatment, preferably in the doses (amounts) specified herein; (b) a method of use of an anti-NKG2A binding agent for the treatment of cancer, or for use in said treatment (especially in humans); (c) a method of use of an anti-NKG2A binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, comprising combining the anti-NKG2A binding agent with a pharma- ceutical acceptable carrier, or a pharmaceutical preparation comprising an effective dose of an anti-NKG2A binding agent suitable for the treatment of cancer; or (d) any combination of a), b), and c), depending on the subject matter permitted for the grant of a patent in the country in which this application is filed.

[0051] The term "biopsy" as used herein is defined as the removal of tissue for the purpose of examination, for example to establish a diagnosis. Examples of types of biopsy include application of suction, e.g., via a needle attached to a syringe, by excision of a piece of tissue with a tool, by excision via an endoscope with a suitable tool, by surgical excision of, e.g., the entire lesion, etc.

[0052] 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 divided into subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, etc. A typical example of an immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The variable light chain (VLC) is a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. L ) and variable heavy chain (V H The terms "antibody", "antibody specific", "antibody specific antibody ...

[0053] The term "specifically binds to" means that the antibody can bind to a binding partner, e.g., NKG2A, assessed using either a recombinant form of the protein, an epitope therein, or a native protein present on the surface of isolated target cells, preferably in a competitive binding test. Competitive binding tests and other methods for measuring specific binding are well known in the art. For example, binding can be detected via physical methods such as radiolabeling, mass spectrometry, or direct or indirect fluorescent labeling, e.g., detected using cytofluorometry (e.g., FACScan). Non-specific substances indicate that they bind to the target if they bind above the amount seen in the control. Substances that specifically bind to NKG2A can be bound to NKG2A alone or to NKG2A as a dimer together with CD94.

[0054] When an antibody is said to "compete" with a particular monoclonal antibody, it means that the antibody competes with the monoclonal antibody in binding assays using either a recombinant molecule (e.g., NKG2A) or a surface-expressed molecule (e.g., NKG2A). For example, if a test antibody reduces binding of an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 to an NKG2A polypeptide or an NKG2A-expressing cell in a binding assay, then the antibody is said to "compete" with such antibody, respectively.

[0055] The term "affinity" as used herein means the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molecular concentration of the antibody-antigen complex, [Ab] is the molecular concentration of unbound antibody, and [Ag] is the molecular concentration of unbound antigen. The affinity constant K ais 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), which are incorporated herein by reference in their entireties. One standard method well known in the art for determining the affinity of mAbs is surface plasmon resonance (SPR) screening (e.g., BIAcore TM The use of SPR analysis equipment.

[0056] By "determinant" in the context of this specification is meant a site of interaction or binding on a polypeptide.

[0057] The term "epitope" refers to an antigenic determinant and is the site or region on an antigen to which an antibody binds. A protein epitope can include amino acid residues that are directly involved in binding and those that are effectively blocked by the binding of a specific antigen to an antibody or peptide, i.e., amino acid residues within the "footprint" of an antibody. It is the simplest form or smallest structural site of a complex antigen molecule that can bind, for example, to an antibody or receptor. Epitopes can be linear or conformational / structural. The term "linear epitope" is defined as an epitope composed of amino acid residues that are contiguous (primary structure) on a linear sequence of amino acids. The term "conformational or structural epitope" is defined as an epitope composed of amino acid residues that represent discrete portions of a linear sequence of amino acids that are not all contiguous and thus brought into close proximity to each other by folding of the molecule (secondary, tertiary, and / or quaternary structure). A conformational epitope is determined by its three-dimensional structure. Thus, the term "conformational" is often used interchangeably with "structural."

[0058] The term "substance" is used herein to refer to a chemical compound, a mixture of compounds, a biopolymer, or an extract made from biological materials. The term "therapeutic agent" refers to a substance that has biological activity.

[0059] For purposes herein, a "humanized" or "human" antibody refers to an antibody in which the constant and variable framework regions of one or more human immunoglobulins are fused to the binding regions, e.g., CDRs, of an animal's immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune response against the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "genetically engineered" to produce specific human antibodies in response to antigenic challenge (see, e.g., 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 teachings of which are incorporated herein by reference). Fully human antibodies can also be constructed by genetic or chromosomal transfection methods and phage display technology, all of which are known in the art (see, e.g., McCafferty, et al. (1990), Nature 348:552-553). Human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Patent Nos. 5,567,610 and 5,229,275, which are incorporated by reference in their entireties).

[0060] A "chimeric antibody" is an antibody molecule in which (a) the antigen binding site (variable region) has been modified, substituted or exchanged so that the constant region or portion thereof is linked to a constant region of a different or altered class (effector function) and / or species, or to an entirely different molecule that confers new properties on the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or portion thereof has been modified, substituted or exchanged with a variable region having a different or altered antibody specificity.

[0061] The terms "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of the human gamma (gamma) heavy chain, or its counterpart sequence in other types of antibody heavy chains (e.g., alpha, delta, epsilon, and mu of human antibodies), or its naturally occurring allotypes. Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (Kabat, et al. (1991), Sequences of Proteins of Immunological Interest, 5 th ed, United States Public Health Service, National Institute of Health, Bethesda, MD).

[0062] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its natural state. Purity and homogeneity are typically measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0063] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acids.

[0064] The term "recombinant" when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the modification of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, such recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or that express naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0065] Within the context of this specification, the term antibody that "binds" to a polypeptide or epitope refers to an antibody that binds to a determinant with the above-mentioned specificity and / or affinity.

[0066] The term "identity" or "identical" when used in the context of two or more polypeptide sequences refers to the degree of sequence relatedness between the polypeptides as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of exact matches between two or more sequences, the smaller of which has gap alignment (if any) processed by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk, AM, ed., Computational Molecular Biology, Oxford University Press, New York, 1988; Smith, DW, ed., Biocomputing: Informatics and Genome Projects, Academic Press New York, 1993; Griffin AM and Griffin, HG, eds., Computer Analysis of Sequence Data, Part 1, Humana Press, New Jersey, 1994; von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, 1987; Gribskov, M. and Devereux, J., eds., Sequence Analysis Primer, M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0067] Methods for determining identity are designed to give the greatest match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG program package, including 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 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., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0068] Antibody production The anti-NKG2A agent binds to the extracellular portion of the human CD94 / NKG2A receptor and reduces 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 in binding to CD94 / NKG2A. That is, the agent interferes with and reduces the interaction between CD94 / NKG2A and its ligand, HLA-E. The antibody can bind to a composite epitope on CD94 and NKG2A, or to an epitope on NKG2A alone. In one embodiment, the antibody binds to an epitope on NKG2A that at least partially overlaps with the HLA-E binding site.

[0069] In one embodiment, the anti-NKG2A agent is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody. In one embodiment, the agent comprises a constant domain from a human IgG1, IgG2, IgG3, or IgG4 antibody. In one embodiment, the agent is a fragment of an antibody selected from an IgA, IgD, IgG, IgE, and IgM antibody. In one embodiment, the agent is a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab) 2 fragment, F(ab')2 fragment, Fv fragment, heavy chain Ig (llama or camel Ig), V HH The antibody fragment is selected from a fragment, a single domain FV, and a single chain antibody fragment. In one embodiment, the agent is a synthetic or semi-synthetic antibody-derived molecule selected from a scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR, and a multispecific antibody.

[0070] Preferably, the anti-NKG2A antibody does not exhibit substantial specific binding to Fcγ receptors, such as CD16. Such antibodies may comprise constant regions of various heavy chains known not to bind to Fc receptors. One such example is the constant region of human IgG4. In one embodiment, the IgG4 antibody comprises a modification that prevents the formation of half antibodies (fab arm exchange) in vivo. For example, the antibody may be one that is 5'-fold lower than the EU index (Kabat et al., "Sequences of Proteins of Immunological Interest", ... th The modified IgG4 antibodies include an IgG4 heavy chain containing a serine to proline mutation at residue 241, corresponding to residue 228, according to the method of the present invention (Ed., NIH, Bethesda, ML, 1991). Such modified IgG4 antibodies will remain intact in vivo and maintain bivalent (high affinity) binding to NKG2A, in contrast to native IgG4s, which will undergo fab arm exchange in vivo such that they bind NKG2A monovalently, which can alter the binding affinity. Alternatively, antibody fragments that do not contain one or more constant regions, such as Fab or F(ab') 2Fragments can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing the antibody for binding to Fc receptor proteins in a Biacore assay. Any human antibody type (e.g., IgG1, IgG2, IgG3, or IgG4) whose Fc portion has been modified to minimize or eliminate Fc receptor binding can also be used (see, for example, WO 03101485, the disclosure of which is incorporated herein by reference). Assays, such as, for example, cell-based assays, for assessing Fc receptor binding are well known in the art and are described, for example, in WO 03101485.

[0071] The anti-NKG2A antibody can advantageously bind to the extracellular portion of NKG2A with a KD at least 100-fold lower than that of binding to NKG2C. In one embodiment, the antibody binds to the extracellular portion of NKG2A with a KD at least 150-fold lower, 200-fold lower, 300-fold lower, 400-fold lower, or 10,000-fold lower than that of binding to NKG2C. In another embodiment, the antibody binds to the extracellular portion of NKG2A with a KD at least 100-fold lower than that of binding to NKG2C, NKG2E, and / or NKG2H molecules. In a further embodiment, the antibody binds to the extracellular portion of NKG2A with a KD at least 150-fold lower, 200-fold lower, 300-fold lower, 400-fold lower, or 10,000-fold lower than that of binding to NKG2C, NKG2C, and / or NKG2H molecules. This is measured, for example, in a Biacore experiment. In this experiment, the ability of the substances 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 the substances are compared to CD94 / NKG2C and / or other CD941 / NKG2 variants similarly produced in the same assay. Alternatively, the binding of the antibody to cells that either naturally express or overexpress (e.g., after transient or stable transfection) can be measured and compared to the binding of cells expressing CD94 / NKG2C and / or other NKG2 variants. Anti-NKG2A antibodies can optionally bind to NKG2B, an NKG2A splice variant that forms a cytotoxic receptor with CD94. In one embodiment, affinity can be measured using the methods disclosed in U.S. Pat. No. 8,206,709, for example by assessing binding to a covalently immobilized NKG2A-CD94-Fc fusion protein by Biacore as shown in Example 8 of U.S. Pat. No. 8,206,709, the disclosure of which is incorporated herein by reference.

[0072] For example, the antibody has an EC of 0.5-10 ng / mL, optionally 1-5 ng / mL, optionally 1-10 ng / mL, optionally 1-20 ng / mL, for example about 4 ng / mL, for binding to NKG2A-expressing cells (high affinity). 50 The NKG2A-expressing cells can be, for example, NKG2A-expressing cells in human PBMCs. In one embodiment, the NKG2A-expressing cells are cells engineered to express CD94 / NKG2A, for example Ba / F3 cells stably overexpressing CD94 / NKG2A as shown in Example 13 of U.S. Pat. No. 8,206,709, the disclosure of which is incorporated by reference. In one embodiment, the antibody is -9 Less than M, sometimes 10 -10 Less than M, or in some cases 10 -11 Less than M, sometimes 10 -10 M~10 -12 Between M and 10 -10 M~10 -11 Binding affinity (K) for human NKG2A polypeptide between M D ) (in some cases the binding affinity is bivalent). Affinity can be assessed for binding to a single chain NKG2A-CD94-mFc construct, for example as described in U.S. Pat. No. 7,932,055, the disclosure of which is incorporated by reference.

[0073] The anti-NKG2A antibody can be, for example, a human or humanized antibody that includes a VH human receptor framework derived from a human receptor 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 frameworks known in the art. The VL region human receptor sequence can be, for example, VLI_O2 / JK4.

[0074] In one embodiment, the antibody is a humanized antibody based on antibody Z270. Various humanized Z270VH chains are shown in SEQ ID NOs: 2-6 (amino acids of variable region domains underlined). Humanized Z270VH light chain is shown in SEQ ID NO: 7. HumZ270 antibodies are also disclosed in U.S. Pat. No. 8,206,709, the disclosure of which is incorporated herein by reference. humZ270VH6 (SEQ ID NO: 2) is based on VH5_51, humZ270VH1 (SEQ ID NO: 3) is based on VH1_18, humZ270VH5 (SEQ ID NO: 4) is based on VH5_a, humZ270VH7 (SEQ ID NO: 5) is based on VH1_f, and humZ270VH8 (SEQ ID NO: 6) is based on VH1_46. All of them have a JH6 J segment. Each of these antibodies retains high affinity binding to NKG2C. This reduces the likelihood of a host immune response to the antibody, since the six C-terminal amino acid residues of the Kabat CDR-H2 of each of the humanized constructs are identical to the human receptor framework. Using the alignment program VectorNTI, we obtained sequence identities of 78.2% (VH1 vs. VH5), 79.0% (VH1 vs. VH6), 88.7% (VH1 vs. VH7), and 96.0% (VH1 vs. VH8) between humZ270VH1 and humZ270VH5, humZ270VH6, humZ270VH7, and humZ270VH8.

[0075] In one aspect, the substance comprises (i) a heavy chain variable region of any of SEQ ID NOs: 2-6, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical thereto, and (ii) a light chain variable region of SEQ ID NO: 7, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical thereto. In one embodiment, the substance comprises (i) a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 2-6, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical thereto, and (ii) a light chain variable region of SEQ ID NO: 7, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical thereto, and (ii) a light chain comprising an amino acid sequence of SEQ ID NO: 7, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical thereto. An antibody having a heavy chain comprising a sequence of any one of SEQ ID NOs: 2-6 and a light chain comprising a sequence of SEQ ID NO: 7 neutralizes the inhibitory activity of NKG2A, but does not substantially bind to the activating receptors NKG2C, NKGE, or NKG2H. The antibody further competes with HLA-E for binding to NKG2A on the cell surface. In one embodiment, the substance comprises the sequence of HCDR1, HCDR2, and / or HCDR3 from a heavy chain having the amino acid sequence of any of SEQ ID NOs: 2 to 6. In one embodiment of the invention, the substance comprises the sequence of HCDR1, HCDR2, and / or HCDR3 from a light chain having the amino acid sequence of SEQ ID NO: 7.

[0076] Heavy chain (variable regions are underlined) [ka] [ka]

[0077] Light chain [ka]

[0078] In one aspect, the anti-NKG2A antibody is an antibody comprising a CDR-H1 (amino acid sequence SYWMN (SEQ ID NO: 8)) corresponding to residues 31-35 of any of SEQ ID NOs: 2-6, a CDR-H2 (amino acid sequence RIDPYDSETHY (SEQ ID NO: 9)) corresponding to residues 50-60 of any of SEQ ID NOs: 2-6 (optionally 50-66 when including the six terminal amino acids of human origin, i.e., VH6 heavy chain sequence RIDPYDSETHYSPSFQG (SEQ ID NO: 10), VH1 heavy chain sequence RIDPYDSETHYAQKLQG (SEQ ID NO: 11), etc.), and a CDR-H3 (amino acid sequence GGYDFDVGTLYWFFDV (SEQ ID NO: 12)) corresponding to residues 99-114 (95-102 according to Kabat) of any of SEQ ID NOs: 2-6. In one embodiment, the CDR-H2 corresponds to residues 50-66 of any of SEQ ID NOs: 2-6. In some cases, the CDRs may contain one, two, three, four or more amino acid substitutions.

[0079] In one embodiment, the anti-NKG2A antibody is an antibody that comprises a CDR-L1 (amino acid sequence RASENIYSYLA (SEQ ID NO: 13)) corresponding to residues 24-34 of SEQ ID NO: 7, a CDR-L2 (amino acid sequence NAKTLAE (SEQ ID NO: 14)) corresponding to residues 50-56 of SEQ ID NO: 7, and a CDR-L3 (amino acid sequence QHHYGTPRT (SEQ ID NO: 15)) corresponding to residues 89-97 of SEQ ID NO: 7. Optionally, the CDRs can contain one, two, three, four or more amino acid substitutions.

[0080] In one aspect, the anti-NKG2A antibody is an antibody comprising a CDR-H1 corresponding to residues 31 to 35 of any of SEQ ID NOs: 2 to 6, a CDR-H2 corresponding to residues 50 to 60 (and optionally 50 to 66) of any of SEQ ID NOs: 2 to 6, a CDR-H3 corresponding to residues 99 to 114 (and possibly 95 to 102 according to Kabat) of any of SEQ ID NOs: 2 to 6, a CDR-L2 corresponding to residues 50 to 56 of SEQ ID NO: 7, and a CDR-L3 corresponding to residues 89 to 97 of SEQ ID NO: 7.

[0081] In one embodiment, the agent is a fully human antibody raised against the CD94 / NKG2A epitope bound by any of the aforementioned antibodies.

[0082] Although the aforementioned antibodies can be used, it should be understood that other antibodies can also be prepared. For example, any fragment of NKG2A (preferably, but not limited to, human NKG2A) or any combination of NKG2A fragments can be used as an immunogen to produce antibodies, and they can do so anywhere within the NKG2A polypeptide, so long as they can recognize an epitope on the NKG2A-expressing NK cells described herein. Most preferably, this epitope is an epitope that is specifically recognized by an antibody having a heavy chain of any of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7.

[0083] In one embodiment, the substance competes with the humZ270 antibody disclosed in U.S. Pat. No. 8,206,709 (the disclosure of which is incorporated herein by reference) for binding to the extracellular portion of human CD94 / NKG2A. Competitive binding can be measured, for example, in a Biacore experiment, by measuring the ability of the substance to bind to the extracellular portion of immobilized CD94 / NKG2A receptor (e.g., purified from CD94 / NKG2-expressing cells or produced in a biological system) saturated with humZ270. Alternatively, the binding of the substance is measured to cells that naturally express or overexpress (e.g., after transient or stable transfection) CD94 / NKG2A receptor and are pre-incubated with a saturating dose of Z270. In one embodiment, competitive binding can be measured using the methods disclosed in U.S. Pat. No. 8,206,709, for example, by assessing binding to Ba / F3-CD94 / NKG2A cells by flow cytometry as shown in Example 15 of U.S. Pat. No. 8,206,709, the disclosure of which is incorporated herein by reference.

[0084] The anti-NKG2A antibody can be incorporated into a pharmaceutical formulation, and includes a concentration of 1 mg / mL to 500 mg / mL (the formulation has a pH of 2.0 to 10.0). The formulation can further include a buffer system, a preservative, an isotonicity agent, a chelating agent, a stabilizer, and a surfactant. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation that includes water. Such formulations are generally solutions or suspensions. In a further embodiment, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation that includes at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution that includes at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension that includes at least 50% w / w water.

[0085] In another embodiment, the pharmaceutical formulation is a lyophilized formulation, whereby the physician or the patient adds solvents and / or diluents prior to use.

[0086] In another embodiment, the pharmaceutical formulation is a dry formulation (eg, freeze-dried or spray-dried) ready for use without prior dissolution.

[0087] In a further embodiment, the pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present at a concentration of 1 mg / mL or greater, and wherein the formulation has a pH of about 2.0 to about 10.0.

[0088] In another embodiment, the pH of the formulation is in the range selected from the list consisting of about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.

[0089] In further embodiments, the buffering agent 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 one of these specific buffering agents constitutes an alternative embodiment of the invention.

[0090] In a further embodiment, the formulation further comprises a pharma- ceutically acceptable preservative. In a further embodiment, the formulation further comprises an isotonicity agent. In a further embodiment, the formulation also comprises a chelating agent. In a further embodiment of the invention, the formulation further comprises a stabilizer. In a further embodiment, the formulation further comprises a surfactant. Conveniently, the formulation is described in Remington: The Science and Practice of Pharmacy, 1999. th edition, 1995 is referred to.

[0091] Other ingredients can also be present in the peptide pharmaceutical formulation of the present invention. Such additional ingredients can include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity adjusting agents, chelating agents, metal ions, oil 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). Of course, such additional ingredients must not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

[0092] Pharmaceutical compositions containing antibodies can be administered to a patient in need of such treatment at several sites, including local sites (e.g., skin and mucosal sites), sites that avoid absorption (e.g., administration into arteries, veins, the heart), and sites that are relevant for absorption (e.g., administration into the skin, subcutaneously, muscle, or abdomen). Administration of the pharmaceutical composition can be by several routes of administration, including subcutaneous, intramuscular, intraperitoneal, intravenous, lingual, sublingual, buccal, oral, oral, gastric and intestinal, nasal, pulmonary (e.g., via the bronchial and alveolar tubes, or a combination thereof), epidermal, dermal, transdermal, vaginal, rectal, ocular (e.g., via the conjunctiva), urinary, and parenteral administration to a patient in need of such treatment.

[0093] Diagnosis and treatment of malignant tumors Methods are described that are useful for the diagnosis, prognosis, monitoring, treatment, and prevention of cancer in individuals. The methods described herein are particularly useful for the treatment of solid tumors, but the treatment regimens described herein can also be used for a variety of hematological cancers as well as infectious, inflammatory, and autoimmune diseases. The methods and compositions of the present invention can be used to treat, treat, and diagnose, and treat a variety of diseases, including, but not limited to, carcinomas (including carcinomas of the bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid, and skin, including squamous cell carcinomas), hematopoietic tumors of the lymphoid lineage (including leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, and multiple myeloma), hematopoietic tumors of the myeloid lineage (including acute and chronic myeloid leukemia, pre-osteoblastic leukemia, myeloma, and leukemia), and other cancers. It is used in the treatment of a variety of cancers and other proliferative disorders, including myeloid leukemia, myelodysplastic syndromes, tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma), other tumors (including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma), tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma, and schwannoma), tumors of mesenchymal origin (including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma), and other tumors (including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma).

[0094] Examples of hematopoietic tumors of lymphoid lineage include, but are not limited to, tumors of T and B cells, including disorders of T cells such as, for example, T-prolymphocytic leukemia (T-PLL) (including those of small cell and gyrocyte-like cell type), large granular lymphocytic leukemia (LGL), preferably of the T-cell type, Sézary syndrome (SS), adult T-cell leukemia-lymphoma (ATLL), T-NHL splenic hepatic lymphoma, peripheral / mature T-cell lymphoma (pleomorphic and immunoblastic subtypes), angioimmunoblastic T-cell lymphoma, angiocentric (nasal) T-cell lymphoma, anaplastic (Ki 1+) large cell lymphoma, intestinal T-cell lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), multiple myeloma.

[0095] In one embodiment, the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, the HNSCC is oropharyngeal cancer, pharyngeal cancer, tumor of the oral cavity, or tumor of the hypopharynx. In one embodiment, the HNSCC is oral cavity SCC (OCSCC). OCSCC includes squamous cell carcinoma of the lip, anterior two-thirds of the tongue, floor of the mouth, buccal mucosa, gums, hard palate, and retromolar triangle. In one embodiment, the HNSCC is a metastatic cancer.

[0096] When treating an individual with a solid tumor, a compound (e.g., an antibody) that neutralizes the inhibitory activity of a human NKG2A polypeptide can be advantageously administered according to the treatment regimen described herein to an individual with cancer who has not undergone or is not currently undergoing surgery to remove cancer cells. However, it should be understood that the compound can also be administered to a patient who has undergone or is about to undergo surgery to remove cancer cells. When an anti-NKG2A compound is administered to an individual who has not undergone a surgical intervention to remove cancer cells (e.g., to remove HNSCC cells), the NKG2A binding compound can be administered, for example, about 1 to 8 weeks prior to surgery. In one embodiment, at least one (e.g., 1, 2, 3, or more) complete cycle of treatment with the anti-NKG2A compound is administered prior to surgery. In one embodiment, the administration is for a period of 2 to 8 weeks.

[0097] In one embodiment, the cancer treated with the methods disclosed herein is an HLA-E expressing cancer.

[0098] Patients with cancer can be treated with anti-NKG2A agents with or without a prior detection step of evaluating the expression of HLA-E on the surface of tumor cells. Advantageously, the treatment method can include detecting HLA-E nucleic acid or polypeptide (e.g., on tumor cells) in a biological sample of a tumor from an individual. Examples of biological samples include any suitable biological fluid (e.g., serum, lymph, blood), cell sample, or tissue sample. For example, the tissue sample can be tumor tissue or tissue adjacent to the tumor. In some cases, HLA-E polypeptide is detected on the surface of malignant cells. Determining that a biological sample expresses HLA-E (e.g., significantly expresses anti-HLA-E antibodies, expresses at a high level, stains highly intensely with HLA-E compared to a reference) indicates that the individual has a cancer that can benefit strongly from treatment with an agent that inhibits NKG2A. In one embodiment, the method includes determining the level of expression of HLA-E nucleic acid or polypeptide in the biological sample and comparing the level to a reference level (e.g., numerical value, weak staining on the cell surface, etc.) corresponding to a healthy individual. A determination that the biological sample expresses an HLA-E nucleic acid or polypeptide at an elevated level relative to a reference level indicates that the individual has a cancer that can be treated with an agent that inhibits NKG2C.

[0099] In one embodiment, the determination that a biological sample (e.g., a sample containing tumor cells, tumor tissue, and / or tissue adjacent to a tumor) significantly expresses an HLA-E nucleic acid or polypeptide indicates that the individual has a cancer that can be treated with an agent that inhibits NKG2A. When referring to an HLA-E polypeptide, "significantly expressed" means that the HLA-E polypeptide is expressed in a substantial number of tumor cells taken from a given individual. The definition of the term "significantly expressed" is not bound to an exact percentage value, but in some instances, the receptor said to be "significantly expressed" will be present in at least 30%, 40%, 50%, 60%, 70%, 80% or more of tumor cells taken from a patient.

[0100] Determining whether an individual has cancer cells that express HLA-E polypeptides includes, for example, obtaining a biological sample from an individual that contains cancer cells (e.g., by performing a biopsy), contacting the cells with an antibody that binds to HLA-E polypeptides, and detecting whether the cells express HLA-E on their surface. In some cases, determining whether an individual has cancer cells that express HLA-E includes performing an immunohistochemistry test. In some cases, determining whether an individual has cancer cells that express HLA-E includes performing a flow cytometry test.

[0101] In one exemplary embodiment, a method of alleviating the progression of cancer in a mammalian host (e.g., a human patient) having detectable levels of cancer cells is provided, the method comprising administering an anti-NKG2A antibody at a dosage and frequency (according to the present disclosure) sufficient to detectably alleviate the progression of cancer in the host.

[0102] In one embodiment, a method is provided for treating or preventing a disease in an individual (e.g., hematological cancer, inflammatory or autoimmune disease, infectious disease), the method comprising administering to an individual having the disease an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide in an amount that achieves a concentration in the circulating blood that is at least 10, 20, 30, or 50 times the concentration required for substantially complete (e.g., 90%, 95%) receptor saturation (e.g., as assessed by, e.g., titrating the anti-NKG2A antibody on NKG2A-expressing cells in PBMCs). The antibody has, e.g., an EC of binding to NKG2A-expressing cells of between 0.5-10 ng / mL, optionally 1-10 ng / mL, optionally 1-20 ng / mL, e.g., about 4 ng / mL, in human PBMCs. 50 may have the following structure:

[0103] In one embodiment, a method is provided for treating or preventing a disease in an individual (e.g., solid tumor, inflammatory or autoimmune disease, invention), the method comprising administering to an individual having the disease an antibody that neutralizes the inhibitory activity of a human NKG2A polypeptide in an amount that achieves a concentration in the extravascular tissue of interest (e.g., tumor or tumor milieu) that is at least 10, 20, 30, or 50 times the concentration required for substantially complete (e.g., 90%, 95%) receptor saturation (e.g., as assessed by, e.g., titrating anti-NKG2A antibodies on NKG2A-expressing cells in PBMCs).

[0104] EC of NKG2A+ NK cell responses of the blocking anti-NKG2A antibody humZ270 50 is about 4 μg / mL, and thus the amount of anti-NKG2A antibody is administered to achieve and / or maintain a blood concentration of at least about 4 μg / mL. Advantageously, the amount of anti-NKG2A antibody is at least about 10 μg / mL (EC of NKG2A+ NK cell response). 50) in the individual. For example, the blood concentration achieved and / or maintained can be between 10-12 μg / mL, between 10-15 μg / mL, between 10-20 μg / mL, between 10-30 μg / mL, between 10-40 μg / mL, between 10-50 μg / mL, between 10-70 μg / mL, between 10-100 μg / mL, between 10-150 μg / mL, or between 10-200 μg / mL. In one embodiment, the amount of anti-NKG2A antibody is at least about 4 μg / mL (EC of NKG2A+ NK cell response). 50 ), or in some cases at least about 10 μg / mL (EC 100 The amount of anti-NKG2A antibody is administered to an individual to achieve and / or maintain a tissue concentration in the individual of at least about 10 μg / mL. When tissue outside the vasculature is targeted (e.g., in the treatment of solid tumors), the amount of anti-NKG2A antibody is administered to achieve and / or maintain a tissue concentration of at least about 10 μg / mL. For example, by administering an amount of anti-NKG2A antibody that achieves a blood concentration of at least about 100 μg / mL, it is expected that an extravascular tissue (e.g., tumor tissue) concentration of at least 10 μg / mL will be achieved. For example, the blood concentration to be achieved and / or maintained to achieve / maintain 10 μg / mL in the tissue can be between 100-110 μg / mL, between 100-120 μg / mL, between 100-130 μg / mL, between 100-140 μg / mL, between 100-150 μg / mL, between 100-200 μg / mL, between 100-250 μg / mL, or between 100-300 μg / mL.

[0105] In some embodiments, the amount of anti-NKG antibody is at least an EC 50 , in some cases about or at least about EC 100The NKG2A+ NK cell response can be assessed using a suitable assay for 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 as shown in the Examples herein. "EC 50 " refers to the effective concentration of an anti-NKG2A antibody that produces 50% of its maximum response or effect on such NKG2A+ NK cell response. 100 " refers to the effective concentration of an anti-NKG2A antibody that produces substantially its maximal response or effect with respect to such NKG2A+ NK cell response. In some embodiments, particularly for the treatment of solid tumors, the concentration achieved is at least the EC 50 , in some cases about or at least about EC 100 The antibody is designed to produce a concentration in tissue (outside the vasculature, e.g., in a tumor or tumor environment) equivalent to

[0106] A suitable treatment protocol for treating a human, for example, includes administering to the patient an effective amount of an anti-NKG2A antibody, the method including at least one administration cycle in which at least one dose of the anti-NKG2A antibody is administered at a dose of 2-10 mg / kg body weight, optionally 4-10 mg / kg body weight, optionally 6-10 mg / kg body weight, optionally 8-10 mg / kg body weight, optionally 2-4 mg / kg body weight, optionally 4-6 mg / kg body weight, optionally 4-8 mg / kg body weight, optionally 6-8 mg / kg body weight. Optionally at least 2, 3, 4, 5, 6, 7, or 8 doses of the anti-NKG2A antibody are administered. In one embodiment, the administration cycle is between 2 weeks and 8 weeks. In one embodiment, the administration cycle is 8 weeks. In one embodiment, the administration cycle is 8 weeks and includes administering one dose of the anti-NKG2A antibody every two weeks (for a total of 4 doses).

[0107] In one aspect of any of the embodiments herein, the anti-NKG2A antibody is administered approximately once every two weeks.

[0108] A suitable treatment protocol for treating a human may, for example, include administering to the patient an effective amount of an anti-NKG2A antibody, where the antibody is administered twice per month, and the amount effective to maintain a sustained blood concentration of at least 10 μg / mL of the anti-NKG2A antibody between at least two successive administrations of the anti-NKG2A antibody is between 2-10 mg / kg, optionally between 2-6 mg / kg, optionally between 2-8 mg / kg, optionally between 2-4 mg / kg, optionally between 2-3 mg / kg, or optionally about 2, 3, or 4 mg / kg of body weight. These doses may optionally be selected to provide a sustained blood concentration of at least 10 μg / mL of the anti-NKG2A antibody throughout the entire treatment cycle. Achieving a blood concentration of 10 μg / mL of the anti-NKG2A antibody may be indicative of the EC of an antibody, such as humanized Z270. 100 is equivalent to.

[0109] Further suitable treatment protocols for treating humans include, for example, administering to the patient an effective amount of an anti-NKG2A antibody, where the antibody is administered twice per month, and the amount effective to maintain a sustained blood concentration of at least 40 μg / mL of the anti-NKG2A antibody between at least two successive administrations of the anti-NKG2A antibody is between 2-10 mg / kg, optionally between 2-8 mg / kg, optionally between 2-6 mg / kg, optionally between 2-4 mg / kg, optionally between 2-3 mg / kg, or optionally about 2, 3, or 4 mg / kg of body weight. These doses can optionally be administered to provide a sustained blood concentration of at least 40 μg / mL of the anti-NKG2A antibody throughout the entire treatment cycle. Achieving a blood concentration of the anti-NKG2A antibody of 40 μg / mL is equivalent to an EC of about 4 μg / mL, and also an EC of the NKG2A+ NK cell response to an antibody such as humanized Z270. 50 is expected to result in tissue (e.g., extravascular tissue, tumor environment of solid tumors) concentrations equivalent to

[0110] Further advantageous suitable treatment protocols for treating humans include, for example, administering to the patient an effective amount of an anti-NKG2A antibody, the antibody being administered twice per month, and an amount effective to maintain a sustained blood concentration of at least 100 μg / mL of the anti-NKG2A antibody between at least two successive administrations of the anti-NKG2A antibody is between 4-10 mg / kg, optionally between 4-6 mg / kg, optionally between 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 can optionally be administered to provide a sustained blood concentration of at least 100 μg / mL of the anti-NKG2A antibody throughout the entire treatment cycle. Achieving a blood concentration of 100 μg / mL of the anti-NKG2A antibody can be achieved by administering to the patient an effective amount of an anti-NKG2A antibody, the EC of the antibody being about 10 μg / mL, and also of an antibody such as humanized Z270. 100 is expected to result in tissue (e.g., extravascular tumor environment) concentrations equivalent to

[0111] A further advantageous suitable treatment protocol for treating a human with cancer includes a dosing regimen using a loading period with a higher dose followed by a maintenance period. For example, the loading period can include administering to the patient an effective amount of an anti-NKG2A antibody, the antibody being administered one or more times in an amount effective to maintain a sustained blood concentration of at least 100 μg / mL of the anti-NKG2A antibody until the first administration of the anti-NKG2A antibody in the maintenance regimen. For example, when administered once, a loading dose of 10 mg / kg of the anti-NKG2A antibody can be administered, with the first administration of the anti-NKG2A antibody in the maintenance regimen occurring about 2 weeks (or less) after the loading dose. The maintenance regimen can then use a lower dose and / or less frequent administration to maintain a sustained blood concentration of at least 100 μg / mL of the anti-NKG2A antibody between successive administrations in the maintenance regimen. For example, a maintenance dosing regimen may include administering an anti-NKG2A antibody every two weeks at a dose of between 2-10 mg / kg, optionally between 4-10 mg / kg, optionally between 2-4 mg / kg, optionally between 4-6 mg / kg, optionally between 4-8 mg / kg, optionally about 4 mg / kg, optionally about 6 mg / kg, or optionally about 8 mg / kg.

[0112] In one embodiment, the anti-NKG2A antibody inhibits at least an EC50 of an NKG2A+ NK cell response over a period of at least about one week, at least about two weeks, without significant "desaturation" of NKG2A on cells in the circulating blood between at least two successive doses of the anti-NKG2A antibody. 50 , in some cases approximately or at least approximately EC 100 In another embodiment, the antibody is administered in an amount to obtain a concentration in the blood (serum) and / or tissue (e.g., tumor tissue) equivalent to at least the EC of an NKG2A+ NK cell response over a period of at least about one week, at least about two weeks. 50 , in some cases approximately or at least approximately EC 100and that allows significant "desaturation" of NKG2A on cells in the circulation between (or between) two successive administrations of the anti-NKG2A antibody. For example, the anti-NKG2A antibody can be administered in an amount and frequency that can cause at least 50% desaturation of NKG2A on cells in the circulation during the treatment period, for example, between at least two successive administrations of the anti-NKG2A antibody. In one example, an amount that allows significant "desaturation" of NKG2A on cells in the circulation is the EC of an NKG2A+ NK cell response. 50 Less than or equal to the EC 20 This is an amount that results in a blood concentration of less than

[0113] An advantageous suitable therapeutic regimen for treating humans includes, for example, administering to the patient an effective amount of an anti-NKG2A antibody, the antibody being administered in an amount effective to achieve a blood concentration of at least 10 μg / mL, 40 μg / mL, or 100 μg / mL of the anti-NKG2A antibody over a period of at least about one week, or at least two weeks, and allowing significant "desaturation" (e.g., subsequent desaturation) of NKG2A on cells in the circulating blood between two successive administrations (or between each administration) of the anti-NKG2A antibody.

[0114] For example, in that case the antibody is administered no more than once per month (or no more than once every two months), and an amount effective to achieve a blood concentration of at least 10 μg / mL, 40 μg / mL, or 100 μg / mL of anti-NKG2A antibody for a period of at least about one week, or at least about two weeks, is between 2-10 mg / kg, optionally between 2-8 mg / kg, optionally between 2-6 mg / kg, optionally between 2-4 mg / kg, optionally between 2-3 mg / kg, or optionally about 2, 3, or 4 mg / kg of body weight.

[0115] Saturation (and desaturation) of NKG2A on cells in the circulating blood can be assessed by obtaining peripheral blood samples and using standard methods to assess receptor occupancy.

[0116] In another aspect, a method is provided for reducing the risk of cancer progression, reducing the risk of further cancer progression in a cell population that has undergone initiation, and / or providing a treatment regimen for ameliorating cancer progression in a human patient, the method comprising administering to the patient one or more initial treatments (e.g., induction therapy such as a chemotherapeutic agent) in an amount and dosing regimen sufficient to achieve a response (partial or complete response), and then administering to the patient an amount of an anti-NKG2A antibody at a dosage and frequency according to the present disclosure.

[0117] In a further aspect, a method is provided for promoting remission of cancer in an individual, such as a human patient, comprising administering to the individual a composition comprising an anti-NKG2A antibody at a dose and frequency according to the present disclosure to promote remission of cancer in the individual. In a further aspect, a method is provided for preventing recurrence of cancer in an individual, such as a human patient, during remission following a prior anti-cancer treatment, comprising administering to the individual a composition comprising an anti-NKG2A antibody at a dose and frequency according to the present disclosure to promote remission of cancer in the individual.

[0118] In a further aspect, a method is provided for reducing the risk of developing cancer, reducing the risk of shortening the time to onset of cancer symptoms, and / or reducing the risk of cancer being diagnosed at an earlier stage of severity, comprising administering to an individual a prophylactically effective amount of an anti-NKG2A antibody at a dosage and frequency according to the present disclosure to achieve a desired physiological effect.

[0119] In a further aspect, a method is provided for increasing the likelihood that a human patient diagnosed with cancer will survive beyond a reasonable period of time. In another aspect, a method is provided for improving the quality of life of a cancer patient, comprising administering to the patient a composition in an amount effective to improve the quality of life. In a further aspect, the method described herein can be applied to significantly reduce the number of cancer cells, e.g., the total number of cancer cells, in a vertebrate host. In a related sense, a method is provided for killing (e.g., causing the death of, either directly or indirectly) cancer cells in a vertebrate, e.g., human, cancer patient.

[0120] The anti-NKG2A antibody can be administered as a monotherapy or by adjunctive or combined administration (co-administration) with a second therapeutic agent, such as an anti-EGFR antibody. Adjunctive or combined administration includes simultaneous administration of the compounds in the same or different dosage forms, or separate administration (e.g., sequential administration) of the compounds. Thus, the anti-NKG2A antibody and the second therapeutic agent can be administered simultaneously as a single formulation. Alternatively, the anti-NKG2A antibody and the second therapeutic agent can be formulated for separate administration, which are administered simultaneously or sequentially. The second therapeutic agent will usually be administered in an amount and treatment regimen generally used for that agent in monotherapy for the particular disease or condition being treated. EXAMPLES

[0121] Example 1: Biacore analysis of humanized Z270 Humanized Z270 (humZ270) is described in U.S. Pat. No. 8,206,709 (Novo Nordisk), the disclosure of which is incorporated herein by reference. As described in U.S. Pat. No. 8,206,709, the hum270 VKI_O2 / JK4 light chain and various heavy chain receptor frameworks were produced as human IgG4 antibodies. The heavy chain frameworks include "VH1" based on VH1_18 / JH6, "VH5" based on VH5_a, "VH6" based on VH5_51, "VH7" based on VH1_f, and "VH8" based on VH1_46, all with a JH6 J segment. Antigen binding properties were analyzed with Biacore T100 (Biacore AB, Uppsala, Sweeden). The antigen was in the form of a single-chain NKG2A-CD94-mFc construct covalently immobilized on a sensor CM5 chip (Biacore AB, Uppsala, Sweeden) via amine groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The immobilization level was targeted at 300 RU. The Z270 antibody variants were diluted in a concentration series (0.157, 0.313, 0.625, 1.25, 2.5 nM) in running buffer HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) Tween-20). All samples were then injected over the immobilized antigen for 2 min at a flow rate of 40 μL / min. Subsequently, running buffer was injected at 40 μL / min for 3 min for antibody dissociation analysis. After each run, regeneration buffer (10 mM NaOH, 500 mM NaCl) was injected (30 s, 10 μL / min) to completely remove the remaining antibodies from the antigen. Data were evaluated with Biacore T100 evaluation software.

[0122] The affinity of humanized Z270 VH1 was determined to be 67 pM. The affinities of VH5, VH6, VH7, and VH8 were similar.

[0123] [Table 1]

[0124] Example 2: humZ270 is a competitive CD94 / NKG2A antagonist To test whether humZ270 blocks ligand (i.e., HLA-E) binding to CD94 / NKG2A as described in US Pat. No. 8,206,709, humZ270 was tested for its ability to block HLA-E tetramer binding to CD94 / NKG2A-overexpressing Ba / F3 cells (Ba / F3-CD94 / NKG2A). humZ270VL1 / NH1, with heavy and light chains of SEQ ID NOs: 3 and 7, respectively, was used in this example, and unless otherwise stated, this antibody was also used as humZ270 for all other examples below. In this case, Ba / F3-CD94 / NKG2A was 1) incubated with various concentrations of humZ270 or 2) first incubated with a saturating concentration of HLA-E tetramer (4.7 μg / mL) and then incubated with various concentrations of humZ270. All incubations were performed in tissue culture medium containing 2% FCS on ice. Cells were then incubated with APC-conjugated secondary antibodies specific for mouse Ab's and analyzed by flow cytometry using a BD BioSciences FACSarray.

[0125] humZ270 binds efficiently to Ba / F3-CD94 / NKG2A cells in a concentration-dependent manner (diamonds). However, when cells were preincubated with HLA-E tetramers, humZ270 was prevented from binding to Ba / F3-CD94 / NKG2A cells. Thus, humZ270 and HLA-E bind overlapping epitopes on CD94 / NKG2A. The CD94 / NKG2A inhibitory effect of humZ270 in the NK cytotoxicity assay is therefore likely a result of blocking the ability of HLA-E to induce negative signals on cytotoxic lymphocytes via CD94 / NKG2A. Thus, humZ270 can be considered as a competitive CD94 / NKG2A antagonist.

[0126] Example 3: Distribution of NK and T cell subsets in rma-rae1 tumor-bearing C57 / bI6 mice based on NKG2A expression To further examine NKG2A expression in the tumor setting, the distribution of NKG2A was examined on NK and T cell subsets in mice. Lymphocytes were harvested from the spleen, from tumor-draining lymph nodes, and from within solid tumors.

[0127] C57 / BL6 mice were implanted (sc) with RMA-Rae clone 6 (2 million cells). These tumor cells express the CD94 / NKG2A ligand Qa-1. Mice were sacrificed on day 12, with a mean tumor volume of 723 mm. 3 , SD:161mm 3 , n = 4. After preparing cell suspensions from spleen, LN, and tumor, cells were stained: CD3e: PerCP CY5.5, NKP46: Alexa 647, NKG2A / C / E: FITC, CD8: Pacific Blue.

[0128] The results shown in Tables 1-3 reveal that a significant percentage of NKG2A-expressing NK and CD8+ T lymphocytes were found within the tumor environment, and that a higher percentage of NKG2A+CD8+ T cells were present in the tumor environment but not in the spleen or tumor-draining lymph nodes. In this NK cell subset, approximately half of the cells in both the draining lymph nodes and the spleen were NKG2A positive and half were NKG2A negative. In this T cell subset, the majority of cells were NKG2A negative (only 1.1% of the lymph nodes and 4.7% of the spleen were NKG2A negative). + In this CD8 T cell subset, the majority of cells were NKG2A negative (only 1.6% of lymph nodes and 3.9% of spleen cells were NKG2A negative). + However, CD8 T cells outside the tumor barely express NKG2A, whereas the tumor-infiltrating CD8 T cell subset had a mean of 26.3% NKG2A+ positive cells. -Among T cell subsets, little difference in NKG2A expression was observed between TILs and splenic or lymph node cells, whereas CD8 - Only 5.1% of T cells expressed NKG2A.

[0129] [Table 2]

[0130] [Table 3]

[0131] [Table 4]

[0132] Example 4: In vitro receptor saturation, i.e., binding of humanized anti-NKG2A antibody IPH2201 to NKG2A+ NK cells in whole blood Receptor occupancy of anti-NKG2A was evaluated in vitro in human blood. This provides a prediction of anti-NKG2A pharmacokinetics in human patients. This study aimed to estimate ex vivo the cellular affinity of humZ270 in whole blood and the absolute number of available NKG2A receptors per μL of whole blood and also per human cell. Receptor affinity and total number can affect both the pharmacokinetics (PK) and pharmacodynamics (PD) of the mAb in humans. These data will be used as input to a PK / PD model that will be used to design the first human dose study. Human whole blood was collected from each donor and immediately processed. Titrations of humZ270 were performed in whole blood from eight volunteers (mAb was incubated for 30 min at room temperature (RT)). Fifteen mAb concentrations were tested to span the full range of binding between 0 and 100% of maximum binding, which corresponds to 90 μg / mL down to 0.000019 μg / mL (1 / 3 serial dilutions, 15 points and 0). Samples were processed to remove red blood cells, fix cells, and acquired on a hemocytometer. Calibration beads were used to convert MFI results to MESF (Molecules of Equivalent Soluble Fluorochrome). Gating was performed on CD45+ lymphocytes expressing NKG2A.

[0133] Three tubes were dedicated to the absolute enumeration of lymphocyte subsets using flow counting beads added to the blood. First, titrations were performed with humZ270 and bound humZ270 was detected using a PE-conjugated anti-hIgG4 secondary antibody. Titrations were also performed with PE-conjugated humZ270 to compare it with titrations performed with bound humZ270 to examine the effect of conjugation with PE on the pharmacological properties of humZ270 and to evaluate the total number of receptors relative to saturating concentrations of humZ270.

[0134] The analysis focused on NKG2A+ cell subsets, namely NK cells and T cells. These subsets of lymphocytes do not all express NKG2A, therefore the NKG2A- subset of each population in question was examined and compared to the NKG2A+ subset. These cell populations are defined as follows: Lymphocytes: defined according to their granularity and size, and according to the expression of CD45, on a CD45 / SSC plot. - Human NK lymphocytes: CD3-CD56+ lymphocytes. - CD8 T cells: CD3+CD8+ lymphocytes. - NKG2A+ lymphocytes: NKG2A+ cells among lymphocytes.

[0135] Overall mean fluorescence intensity (MFI) was recorded for each population. Calibration beads were used as MESFs to express fluorescence in the PE channel. The primary population of interest in this study is NKG2A+ lymphocytes, which primarily comprise NK cells.

[0136] The results showed that the human anti-NKG2A mAb (humZ270) bound to two subsets of peripheral blood lymphocytes, NK cells and CD8+ T cells.

[0137] The results are summarized in Figure 1. humZ270 (also called IPH2201) is shown at various concentrations (ng / mL) plotted on the x-axis and the MESF signal of receptor binding is shown on the y-axis. The antibody had a binding affinity (EC50) of approximately 4 ng / mL for NKG2A+ cells (K D ~4ng / mL). This K D The K values ​​observed in other assays, particularly for affinity for binding to PBMC in the Biacore assay and for affinity for recombinant NKG2A, were D The K value for full receptor occupancy (EC100) D was approximately 100ng / mL.

[0138] Example 5: Receptor Saturation of IPH2201 in a Human Phase I Clinical Trial for Rheumatoid Arthritis humZ270, a human IgG4 antibody, was generated with a heavy chain containing a serine to proline mutation at residue 241 (S241P mutation), corresponding to EU index number 228, to maintain high affinity bivalent binding in vivo. The safety profile of humZ270 (IPH2201) was investigated in a double-blind, placebo-controlled, dose-escalating Phase I clinical trial in 92 patients with stable and controlled rheumatoid arthritis. In this triple-arm Phase I clinical trial, Z270 or placebo was administered as a single dose (iv) of up to 10 mg / kg, or as single (sc) or repeated (sc) doses (4 doses given at 2-week intervals) of up to 4 mg / kg. Doses tested as a single dose iv were 0.0002 mg / kg, 0.001 mg / kg, 0.005 mg / kg, 0.025 mg / kg, 0.1 mg / kg, 0.4 mg / kg, 1.1 mg / kg, 3.5 mg / kg, and 10 mg / kg. All patients were followed for a minimum of 12 weeks. The safety profile was extremely favorable. The MTD was not reached. There were no SUSARs, no drug-related serious adverse events, no infusion-related reactions, and no immune-related adverse events. Nasopharyngitis and headache were the two most frequently reported adverse events.

[0139] Receptor saturation was assessed using a conventional sandwich ELISA format with a mouse Fc-human NKG2A fusion protein used to capture the humZ270 antibody. After incubation with serum samples containing humZ270 antibody, bound antibodies (analytes in this study) are detected using a biotinylated mouse anti-human IgG4. Avidin labeled with HRP is added to tag the solid-phase-bound biotinylated anti-human IgG4, and a chromogenic HRP substrate (TMB) is used for endpoint detection. The optimal dose and dosing frequency for the anti-NKG2A (IPH2201) dose level used in clinical trials that would result in virtually complete receptor saturation was predicted using a PK / PD model developed using a pharmacokinetic software package (WinNonLin 6.3.0395, Pharsight Corporation), a model based on clinical data from a Phase I clinical trial of IPH2201 in RA patients. The dosing frequency for clinical treatment with IPH2201 will depend on the steady-state plasma concentration required for saturation, as well as the clearance and amount of IPH2201 distribution.

[0140] Preliminary PK data from the IPH2201 Phase I clinical trial (iv injection) and NCA analysis allowed us to estimate the PK parameters of the PK model. Nominal times were used. A standard two-compartment model with first-order elimination and non-linear target mediated drug disposition modeled by Michaelis-Menten kinetics was selected. Dose-dependent clearance was observed in the NCA analysis and applied to this model. Clearance decreases with increasing dose below 0.4 mg / kg and then remains constant at higher doses. This preliminary PK model is consistent with known PK properties for IgG, where dose dependence reflects saturation of target receptors (at low doses) and saturation of FcRn receptors (at higher doses) (Brambell).

[0141] Population PD modeling was performed based on preliminary NKG2A occupancy data available from an ongoing Phase I clinical trial.

[0142] A sigmoidal Emax model was used to fit the plasma concentration-effect relationship. This model is max EC is defined here as the serum drug concentration that achieves 50% of the NKG2A occupancy rate. 50 was characterized by.

[0143] However, when the PD data were plotted against the PK data, it was observed that with increasing time post-dose, the maximum NKG2A occupancy (Emax) achieved for the highest IPH2201 concentration decreased linearly in a time-dependent manner, without any dose effect. Similarly, the EC50 appeared to increase linearly with time. To model this observation on the NKG2A occupancy profile, a decrease in Emax and an increase in EC50 over time were incorporated to describe the NKG2A occupancy data.

[0144] NKG2A occupancy rate% = (E max (E max F all ×TSLD))×C γ / ((EC 50 +EC 50 inc×TSLD) γ +C γ ) Where TSLD = Time Since Last Dose, E max =100%, E max F all = rate of decline in Emax over time, EC 50 inc=EC 50 The rate of increase.

[0145] The modeling results are shown in Figure 2. Virtually complete (at least 90%) receptor saturation of NKG2A+ cells can be achieved by administering 0.03 mg / kg every 2 weeks. A dose of 0.1 mg / kg can be administered every 4 weeks to maintain virtually complete saturation of NKG2A. If saturation in extravascular tissues (e.g. solid tumors) is desired, approximately 10 times the dose would be required, which translates to doses of approximately 0.4 mg / kg every 2 weeks and 1 mg / kg every 4 weeks.

[0146] The NKG2A receptor saturation results from this human clinical trial are consistent with the concentration of Anti-NKG2A required for full receptor occupancy (EC100) determined in vitro (see Example 4).

[0147] Example 6: CD107 response to NKG2A blockade using IPH2201 To evaluate killing mediated by purified cytokine-activated NK cells (effector cells), autologous in vitro experiments using effector and target cells were performed in 12 human individuals. CD107 mobilization and occupancy were evaluated in parallel in three donors. The target cells were autologous SEB blasts. The killing was thought to be mediated by NK cells, and could be specifically increased by anti-NKG2A antibodies, as it would be expected to counteract the inhibitory signal from HLA-E. A concentration range response for the antibody humZ270 was performed in a 4-h cytotoxicity test. The choice of using purified NK cells and the CD107 mobilization assay was based on the experimental results generated. For the first time, this FACS-based CD107 assay allows the cytotoxic response of NK cells expressing NKG2A to be specifically examined and compared with NK cells not expressing NKG2A. Alternative protocols such as chromium release assays do not allow one to distinguish effects dependent on NKG2A expression, which may be problematic in donors with a low percentage of NKG2A+ cells.

[0148] Autologous SEB blasts were generated by incubating frozen PBMCs with 200IU / mL recombinant human IL-2 (Proleukin) and 100ng / mL SEB (Staphylococcal Enterotoxin B, Sigma) in complete medium (RPMI, 10% FCS, penicillin / streptomycin) for 4 days. CD4 T cells were then negatively selected by using the "Stemsep negative selection human CD4 T cell enrichment kit" (product no. #14052A). Purity was assessed by FACS and HLA-E expression was measured with PE-conjugated anti-HLA-E clone 3D12 (E-Biosciences). T CD4 SEB blasts are considered an excellent model to mimic autoreactive T CD4+ cells to generate autologous cells.

[0149] Purified NK cells were prepared from frozen PBMCs: human NKs were purified by the Stem Sep system and cultured O / N in complete medium supplemented with 10 IU / mL recombinant IL-2. PBMCs were prepared from collected blood. The blood was eligible for transfusion, so the donor was anonymous and presumably healthy. PBMCs were prepared, aliquoted, and stored in a nitrogen bath prior to study.

[0150] Kd of receptor saturation and EC of CD107 mobilization 50 Calculations were performed with GraphPad Prism software using a four-parameter nonlinear regression fit. To estimate the affinity constant Kd from titration curves of anti-NKG2A (IPH2201) binding to purified NK cells, values ​​for 30 and 90 μg / mL were excluded because some artifacts were observed at those concentrations.

[0151] The results are summarized in Figure 3A. Surprisingly, the concentration of anti-NKG2A required for efficacy (blocking) is 100-fold higher than that resulting in complete receptor saturation. The EC50 concentration (amount giving 50% of maximum) in the CD107 mobilization assay was determined to be about 400 ng / mL, with an EC100 of about 10,000 ng / mL (10 μg / mL). In contrast, as shown in Example 4, the EC50 concentration for receptor saturation is about 4 ng / mL, with an EC100 of about 100 ng / mL, concentrations also supported by data from a Phase I clinical trial (see Example 5).

[0152] For three donors, parallel assessment of CD107 mobilization and occupancy was performed on individual ECs of CD107. 50 Values ​​were approximately 100-fold the individual Kd for receptor saturation. The same ratios were observed when all data were evaluated (median Kd for receptor saturation 0.005 μg / mL, n=5; median EC50 for CD107 mobilization 0.40 μg / mL, n=9), suggesting that the donors collectively have similar receptor affinities for IPH2201 and that the relationship between receptor saturation and biological activity is similar.

[0153] Example 7: Effect of HLA-E expression levels on the response of CD107 to NKG2A blockade This 100-fold difference between the Kd obtained for receptor saturation and the EC for CD107 mobilization could potentially be explained by membrane-associated HLA-E on target cells in the CD107 assay, which binds with high avidity to NKG2A on NK cells, requiring higher amounts of IPH2201 to reach functional saturation of the receptor compared to receptor saturation experiments not involving HLA-E.

[0154] To investigate this possibility, the EC observed for the biological effect of autologous SEB blasts in Example 6 was compared with the EC of cells with various levels of HLA-E expression. Human K562 cells transfected with HLA-E were selected as high HLA-E expressing cells. CD107 assays were performed using FACS with purified NK cells as described in Example 6.

[0155] In each experiment, autologous CD4+ SEB blasts and selected K562-HLA-E clones were tested for expression of HLA-E using a PE-conjugated anti-HLA-E mAb, 3D12. HLA-E expression on K562-HLA-E transfectants was at least 20-fold higher than the constitutive HLA-E expression observed on autologous SEB blasts. The purity of the SEB blasts was 96% ± 0.818 (SD, n = 12, range 94.6%-97.1%). Expression of HLA-E on T CD4 SEB blasts and K562-HLA-E was similar among all donors.

[0156] Corroborating this observation of a 100-fold difference between the Kd obtained for receptor saturation and the EC50 for CD107 mobilization, this lower EC observed for the biological effect of SEB blasts (median 0.40 μg / mL) compared to the EC50 of K562-HLA-E transfectants (median 4.1 μg / mL) can be explained by at least a 20-fold difference in HLA-E expression at the cell surface, where HLA-E is expressed at a higher density on K562-HLA-E transfectants and therefore a higher amount of IPH2201 is required to compete with HLA-E when compared to autologous SEB blasts.

[0157] Example 8: Prediction of pharmacokinetic model for repeated injections of IPH2201 Based on the ECs for CD107 mobilization observed in autologous (SEB) cells in Example 7, and incorporating blood concentration results from the Phase I clinical trial described in Example 5, modeling was performed to determine the optimal dosing frequency for the dose levels of anti-NKG2A (IPH2201) used in this clinical trial that would confer efficacy.

[0158] The optimal dose and dosing frequency for the dose levels of anti-NKG2A (IPH2201) used in this clinical trial was predicted using a PK / PD model developed using a pharmacokinetic software package (WinNonLin 6.3.0.395, Pharsight Corporation), a model based on clinical data from a Phase I clinical trial of IPH2201 in RA patients. The dosing frequency in clinical treatment with IPH2201 will depend on the required steady-state plasma concentration and the clearance and amount of distribution of IPH2201.

[0159] Preliminary PK data for IPH2201 Phase I clinical trial (iv injection) and NCA analysis allowed us to estimate PK parameters for the PK model. Nominal times were used. A standard two-compartment model with first-order elimination and nonlinear target-mediated drug elimination modeled by Michaelis-Menten kinetics was selected. Dose-dependent clearance was observed in the NCA analysis and applied to this model. Clearance decreases with increasing dose below 0.4 mg / kg and then remains constant for higher doses. This preliminary PK model is consistent with known PK properties for IgG, where dose dependence reflects saturation of target receptors (at low doses) and saturation of FcRn receptors (at higher doses) (Brambell).

[0160] Based on preliminary PK and PD data from a Phase I clinical trial, the following PK and PD parameters best fit the observed data.

[0161] [Table 5]

[0162] FIG. 3B shows the different blood concentrations reached at different doses administered by iv every 2 weeks. The dose levels were 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.2 mg, 0.4 mg, 2 mg, 4 mg, and 10 mg per kg of body weight. The lines in FIG. 3B correspond to increasing doses of IPH2201 from bottom to top, with the lowest dose corresponding to the lowest line at the bottom of the graph and the highest dose corresponding to the highest line at the top of the graph. A dose of 0.01 mg / kg resulted in an initial (peak) blood concentration of about 0.2 μg / mL. A dose of 0.04 mg / kg resulted in an EC of about 1 μg / mL, i.e., an EC of efficacy of 400 ng / mL. 50 The initial (peak) blood concentration exceeded the EC 50 The 0.4 mg / kg dose resulted in an approximately EC of 10 μg / mL, or approximately the EC of efficacy in the circulation. 100 and sustained blood concentrations of greater than 3 μg / mL out to the 2-week time point. The 4 mg / kg dose produced an initial (peak) blood concentration of approximately 100 μg / mL, i.e., approximately the EC 100 and sustained blood levels of >30 μg / mL out to 2 weeks. However, when a 4 mg / kg dose is administered in repeated doses, the fourth dose results in a sustained blood level of approximately 100 μg / mL. A 10 mg / kg dose results in a sustained blood level of approximately 100 μg / mL.

[0163] Figure 3C shows the various blood concentrations reached with various doses administered by iv every 2 weeks when a loading dose and a maintenance dose are used. A loading dose of 10 mg / kg body weight followed by a maintenance dose of 4 mg / kg body weight at a 2-week dosing frequency (upper line in Figure 3C) resulted in initial and sustained blood concentrations of approximately 100 μg / mL. For comparison, a flat dose of 4 mg / kg is shown as the lower line in Figure 3C. This flat dose results in sustained (or minimum) blood concentrations of at least 30 μg / mL between the first and subsequent injections for 2 weeks, sustained (or minimum or residual) blood concentrations of at least 50 μg / mL for 2 weeks after the second injection, and sustained (or minimum or residual) blood concentrations of at least 60-70 μg / mL for 2 weeks after the third injection.

[0164] Example 9: Dose-response effect of anti-NKG2A on NK cell activation Immunotherapeutic approaches to HNSCC are particularly complicated by the profound immunosuppression induced by HNSCC, which may reduce the effectiveness of immunostimulatory effects (see, e.g., Duray et al., (2010), Clin. Dev. Immunol. 2010:1-15). The aim of this experiment was to examine whether anti-NKG2A antibodies targeting NKG2A could eliminate HNSCC cells.

[0165] The effect of the dose response of anti-NKG2A on NK cell activation was examined by analysis of CD107 and CD137 expression. CD107 mobilization at 4 hours is a marker of lytic granule release by NK cells (Alter et al., (2004), J Immunol Methods 294(1-2):15-22). Increased expression of CD137 at 24 hours correlates with activation of several types of lymphocytes, including NK cells (Kohrt et al., (2011), Blood 117(8):2423-2432). Analysis of CD107 and CD137 expression was performed on NK cells expressing or not expressing NKG2A (NKG2A+NK cells or NKG2A-NK cells, respectively). Since the antibody targets NKG2A, its effects are expected to be seen only on NKG2A+ NK cells, and therefore NKG2A- NK cells can be considered as an internal control in these experiments.

[0166] The effector cells used were freshly isolated PBMCs from healthy volunteers and the target cells were either HLA-E transfected HNSCC cell lines or clones of the K562 cell line. Cells were counted and subcultured every 2 days in complete medium. Viability was measured and should have been greater than 90%. They were maintained in medium for up to 12 passages. The day before the experiment, cells were counted and adjusted to 100,000 cells / well. Viability was measured and should have been greater than 90%.

[0167] The K562 cell lines were K562 clone E6 (HLA-E positive, CD32 low) and K562 clone F7 (HLA-E negative / low, CD32 low). Human head and neck cancer cell lines were screened by flow cytometry for HLA-E expression (see Table C below). Three cell lines were selected for functional studies: FaDu (ATCC #HTB-43), HN (DSMZ #ACC 417), and CAL-27 (DSMZ #ACC 446).

[0168] [Table 6]

[0169] Effector cells used were freshly isolated PBMCs from healthy volunteers. Target cells were HNSCC cell lines (FaDu, HN, and CAL-27) at an E:T ratio of 2.5 / 1 and a clone of the K562 cell line transfected with HLA-E (clone E6 = HLA-E + , Clone F7=HLA-E - ). Readout was CD107 at 4 hours versus CD137 at 24 hours. Seven donors (FaDu and HN) and two donors (CAL-27) were tested. Anti-NKG2A antibody humZ270 VH1, whose heavy chain amino acid sequence is shown in SEQ ID NO:3 and whose light chain amino acid sequence is shown in SEQ ID NO:7, was used at a final concentration of 10 μg / mL.

[0170] Figures 4A and 4B show FACS readouts of CD107 (top) and CD137 (bottom) on NKG2A-NK (left) or NKG2A+NK cells (right) in the presence of control or indicated target cell lines, and in the presence or absence of anti-NKG2A at a concentration of 10 μg / mL. Cell lines are arranged from left to right according to the level of surface expression of HLA-E. Each dot represents a PBMC from a healthy volunteer. Figure 4B shows HNSCC cell lines and demonstrates that anti-NKG2A can restore lysis of HNSCC with endogenous HLA-E expression, or lysis of K562 transfected with HLA-E. This effect is only seen on NKG2A-positive NK cells and depends on the level of HLA-E expression. Indeed, the effect of anti-NKG2A is seen on cell lines with medium to high levels of HLA-E expression.

[0171] Anti-NKG2A can induce NK-mediated lysis of HLA-E expressing cell lines by blocking the interaction of the cytotoxic receptor NKG2A with HLA-E. This effect is observed on HLA-E transfected K562 cell lines, but more importantly on HNSCC cell lines with endogenous expression of HLA-E, such as FaDu and CAL-27 cell lines. The extent of the effect of anti-NKG2A depends on the level of HLA-E expression on the cell surface of the target cells.

[0172] Example 10: Effect of the combination of an optimal dose of anti-NKG2A with a suboptimal dose of cetuximab Epidermal growth factor receptor (EGFR) (also ErbB-1, HER1 in humans) is a ubiquitously expressed transmembrane glycoprotein in the ErbB / HER family of receptor tyrosine kinases. High expression of EGFR occurs in the majority of epithelial malignancies, including HNSCC, and is associated with poor prognosis. Its activation by natural ligands leads to the initiation of intracellular signaling pathways that regulate activation of cell proliferation, invasion, angiogenesis, and metastasis that drive tumor growth.

[0173] The anti-EGFR monoclonal antibody cetuximab is thought to act through blockade of tumor signaling of the EGF receptor pathway and by inducing antibody-dependent cellular cytotoxicity (ADCC) mediated by Fcγ receptors. However, in HNSCC ADCC may be affected by the profound immunosuppression induced. At the same time, blockade of tumor signaling of the EGF receptor pathway leads to post-transcriptional regulation in tumor cells of major histocompatibility complex (MHC) class I-associated antigens of the MICA / B and ULBP protein families that are recognized by the activating receptor NKG2D on NK cells and on a subset of T cells. Specifically, tumor cell expression of these stress-associated antigens, which are natural ligands for NKG2A, may be reduced by clinical EGFR inhibitors, thus decreasing the degree to which tumor cells are recognized by NK and T cells (Vantourout et al., Sci. Transl. Med. 6:231ra49 (2014)).

[0174] This experiment was designed to examine the effect of EGFR blocking antibodies on the ability of anti-NKG2A antibodies to activate NK cells in HNSCC. The dose-response impact of cetuximab on NK cell activation was determined by CD107 and CD137 analysis using freshly isolated PBMCs from healthy volunteers as effector cells and HNSCC cell lines (FaDu and HN) as target cells at an E:T ratio of 2.5 / 1. Readouts were CD107 at 4 hours versus CD137 at 24 hours using three donors (CD107 readout at 4 hours) and four donors (CD107 readout at 24 hours). Cetuximab was tested at 1 / 10 serial dilutions starting at 10 μg / mL for the dose response. For one healthy volunteer, NK cells were further divided into NKG2A+ and NKG2A- subsets. A suboptimal dose of cetuximab was chosen for further testing to examine the effect of EGFR-blocking antibodies on the ability of anti-NKG2A antibodies to activate NK cells in HNSCC. A dose of 0.001 μg / mL (1 ng / mL) is the starting point for cetuximab effects observed on both CD107 and CD137 readouts. A dose of 0.001 μg / mL (10 ng / mL) is approximately the EC50 for cetuximab effects.

[0175] The combined effects of anti-NKG2A and a suboptimal dose of an EGFR inhibitor were examined using freshly isolated PBMCs from healthy volunteers as effector cells and HNSCC cell lines (FaDu, HN, and CAL-27) and a clone of the K562 cell line transfected with HLA-E (clone E6 = HLA-E) as target cells. + , Clone F7=HLA-E - ) and CD137 expression at an E:T ratio of 2.5 / 1. The readout was CD107 at 4 hours versus CD137 at 24 hours using seven donors (FaDu and HN) and two donors (CAL-27).

[0176] The anti-NKG2A antibody, whose heavy chain amino acid sequence is shown in SEQ ID NO:3 and whose light chain amino acid sequence is shown in SEQ ID NO:7, was used at a final concentration of 10 μg / mL, corresponding to full functional activity, and the EGFR inhibitor cetuximab was used at two suboptimal doses of 0.001 μg / mL or 0.01 μg / mL.

[0177] Fully activating concentrations of anti-NKG2A enhanced ADCC against HNSCC cell lines induced by suboptimal doses of cetuximab. No cetuximab-dependent ADCC was observed on K562-transfected cell lines because they do not express EGF-R. Anti-NKG2A effects are only seen on NKG2A-positive NK cells and depend on the level of HLA-E expression. In fact, anti-NKG2A effects are seen on cell lines with medium to high levels of expression (FaDu, CAL-27, and K562-HLA-E clone E6). Figure 5 shows a representative example seen with FaDu cells. It can be seen in Figure 5 that a fully activating dose of anti-NKG2A enhances ADCC of HNSCC cell lines induced by suboptimal doses of cetuximab (ctx).

[0178] Example 11: Combined effect of increasing doses of anti-NKG2A with increasing doses of cetuximab The combined effect of increasing doses of an EGFR-blocking antibody and increasing doses of anti-NKG2A was evaluated for their ability to activate NK cells directed against HNSCC target cells. These experiments aimed to assess whether anti-NKG2A treatment could still enhance ADCC when cetuximab was used at saturating doses, and whether the anti-NKG2A effect was dose-dependent.

[0179] Briefly, the effector cells used were freshly isolated PBMCs from healthy volunteers, and the target cells were HNSCC cell lines (FaDu, HN, and CAL-27) and a clone of the K562 cell line transfected with HLA-E (clone E6 = HLA-E + , Clone F7=HLA-E -) with an E:T ratio of 2.5 / 1. The readout was CD107 at 4 hours versus CD137 at 24 hours using two donors. Anti-NKG2A antibodies with heavy chain amino acid sequence shown in SEQ ID NO:3 and light chain amino acid sequence shown in SEQ ID NO:7 were used at two suboptimal doses of 0.1 and 1 μg / mL and at a saturating dose of 10 μg / mL. In these experimental settings, cetuximab was used at two suboptimal doses (approximately EC50) of 0.001 μg / mL and 0.01 μg / mL, and at a saturating dose of 0.1 μg / mL.

[0180] The results are shown in Figures 6A and 6B. Figure 6A shows the readout of CD107 on a control without target and on a control with K562-HLA-E transfectants. Each healthy volunteer is represented by a different symbol, i.e., a square or a circle. The cross-open symbol corresponds to the condition where anti-NKG2A was replaced by 10 μg / mL of hIgG4 isotype control co-incubated with 0.1 μg / mL of cetuximab. It can be seen that the effect of anti-NKG2A is dose-dependent, depends on the expression level of HLA-E, and is still observed at a saturating dose of 0.1 μg / mL of cetuximab in clone E6, which expresses HLA-E at high levels. In clone F7 (low HLA-E expression level), the effect of anti-NKG2A was limited, and high doses (e.g., full activation doses) of anti-NKG2A did not further increase the effect.

[0181] FIG. 6B shows the readout of CD107 on HNSCC cell lines. Each healthy volunteer is represented by a different symbol, i.e., a square or a circle. The cross-open symbol corresponds to the condition where anti-NKG2A was replaced by 10 μg / mL of hIgG4 isotype control co-incubated with 0.1 μg / mL of cetuximab. It can be seen that the effect of anti-NKG2A is dose-dependent and depends on the expression level of HLA-E, and is still observed at a saturating dose of 0.1 μg / mL of cetuximab on FaDu or CAL-27, which express / strongly stain HLA-E at high levels.

[0182] The effect of anti-NKG2A was dose-dependent, dependent on the expression level of HLA-E, and was still observed at a saturating dose of cetuximab of 0.1 μg / mL.

[0183] Example 12: Human clinical trial of humanized Z270 as a single agent with repeated injections to treat cancer The primary objective of this clinical trial is to evaluate the antitumor activity of preoperative IPH2201 (humanized Z270 containing the S241P mutation) in patients with operable squamous cell carcinoma of the oral cavity. The secondary objectives are to evaluate the safety, pharmacokinetics, antigenicity, and pharmacodynamics of IPH2201, including intratumoral biomarkers.

[0184] Clinical trial design This clinical trial is a single-center, open-label, single-arm, Phase Ib-II study, including partial trials. Previously untreated patients with measurable clinically intermediate or high-risk stage III or IVa squamous cell carcinoma of the oral cavity will be treated with single-agent IPH2201 iv every 2 weeks (q2w) for four doses by intravenous route (iv) over 1 hour. The first six patients will receive IPH2201 at a dose of 4 mg / kg q2w x 4. A minimum interval of 1 week will be observed between each first dose of IPH2201 for the first three patients treated with 4 mg / kg. Subsequent patients will be treated with a dose of 10 mg / kg q2w x 4, with dose escalation approved by the safety committee after a minimum follow-up of 4 weeks after the first dose of the last patient treated with 4 mg / kg. Standard locoregional treatment with surgery followed by adjuvant therapy (radiotherapy (RT) or radiochemotherapy (RCT)) depending on histopathological risk factors will be initiated after the last dose of IPH2201. If the tumor progresses, locoregional treatment will be initiated immediately.

[0185] Antitumor activity will be evaluated clinically and radiologically before the third dose of IPH2201 prior to surgery and 2 weeks after the last dose of IPH2201. Tumor measurements will be obtained at target lesions according to RECIST 1.1 criteria. Similar imaging techniques will be used for evaluation of response at baseline and during the preoperative period, for the primary endpoint and / or postoperative evaluation, and for monitoring possible recurrence. Imaging techniques appropriate at the investigator's discretion (computed tomography (CT) and / or magnetic resonance imaging (MRI)) and photographic evaluation of accessible tumor lesions will be performed in all patients.

[0186] Fresh tumor samples will be obtained by baseline biopsy and resected specimens will be collected at the time of surgery. Pharmacology (PK / PD) and biomarker studies will be performed pre- and post-operatively.

[0187] Patients will be followed up until one year after the first cycle of treatment. After study visits are completed, recurrence and survival will be recorded according to local practice for an additional two years after the study.

[0188] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference in their entirety (to the maximum extent permitted by law) to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein, regardless of any separate provided incorporation of a specific document made elsewhere in this specification.

[0189] The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention should be construed to encompass both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0190] Unless otherwise noted, the exact numerical values ​​provided herein represent the corresponding approximate numerical values ​​(e.g., all exemplary numerical values ​​provided with respect to a particular coefficient or measurement value can also be considered to provide the corresponding approximate measurement value, where appropriate modified by "about." When "about" is used in conjunction with a number, it can be specified to include the numerical value corresponding to + / - 10% of the specified number.

[0191] Description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" in reference to an element or elements is intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" that particular element or elements, unless otherwise noted herein or clearly contradicted by context (e.g., a composition described herein as comprising a particular element or elements will be understood to also describe a composition consisting of that element, unless otherwise noted herein or clearly contradicted by context).

[0192] Any or all of the examples provided herein, or exemplary language (e.g., "etc."), are intended merely to shed more light on the invention and do not pose a limitation on the scope of the invention unless otherwise required. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Claims

1. An anti-NKG2A antibody that binds to and neutralizes the inhibitory activity of NKG2A for use in treating an individual having a cancer comprising cancer cells that express HLA-E on their cell surface, said anti-NKG2A antibody comprising a heavy chain comprising an amino acid sequence of any of SEQ ID NOs: 2, 4-6 and a light chain comprising the amino acid sequence of SEQ ID NO: 7, said treatment comprising administering said anti-NKG2A antibody to said individual for at least one dosing cycle, said anti-NKG2A antibody being administered at least twice in an amount effective to maintain a sustained blood concentration of the anti-NKG2A antibody of at least 100 μg / mL between two successive administrations of said anti-NKG2A antibody throughout the treatment cycle.

2. The anti-NKG2A antibody of claim 1, wherein the anti-NKG2A antibody is administered in an amount effective to provide a sustained blood concentration of the anti-NKG2A antibody of at least about 100 μg / mL for at least one week after administration of the anti-NKG2A antibody.

3. The anti-NKG2A antibody of claim 1, wherein the anti-NKG2A antibody is administered twice per month.

4. The anti-NKG2A antibody of claim 1, wherein the anti-NKG2A antibody is administered intravenously twice a month, and the amount of the anti-NKG2A antibody administered is 6-10 mg per kg of body weight.

5. The anti-NKG2A antibody of claim 1, wherein the treatment comprises a loading period followed by a maintenance period, during which the anti-NKG2A antibody is administered at least once at an initial dose effective to maintain a blood concentration of at least 100 μg / mL until the next successive administration of the anti-NKG2A antibody, and during which the anti-NKG2A antibody is administered at least twice at a second dose and frequency effective to maintain a sustained blood concentration of the anti-NKG2A antibody of at least 100 μg / mL between successive administrations of the anti-NKG2A antibody.

6. 6. The anti-NKG2A antibody of claim 5, wherein the anti-NKG2A antibody is administered intravenously, and the loading period comprises a single administration of the anti-NKG2A antibody at a dose of 8-10 mg / kg body weight, and the maintenance period comprises at least two administrations of the anti-NKG2A antibody at a dose of 2-6 mg / kg body weight, spaced apart by about two weeks.

7. 2. The anti-NKG2A antibody of claim 1, wherein the individual has a hematological cancer selected from the group consisting of leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, promyelocytic leukemia, and myelodysplastic syndrome.

8. The anti-NKG2A antibody of claim 1 , wherein the individual has a solid tumor.

9. The antibody of claim 8, wherein the individual has head and neck squamous cell carcinoma (HNSCC).

10. The antibody of claim 1, wherein the anti-NKG2A antibody comprises a human IgG4 constant region or comprises an Fc engineered constant region that contains amino acid modifications that reduce binding to human Fcγ receptors.

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