Treatment of Head and Neck Cancer

Abstract

The present invention relates to the use of NKG2A-targeting agents for the treatment of cancer, particularly head and neck cancer, in patients who have been previously treated with cetuximab. The present invention also provides advantageous combination regimens for the use of NKG2A-targeting agents for cancer treatment.

Description

TECHNICAL FIELD

[0001] The present invention relates to the use of NKG2A-targeting agents for the treatment of cancer, particularly head and neck cancer. The present invention also provides advantageous combination regimens for using NKG2A-targeting agents for cancer treatment. BACKGROUND OF THE INVENTION

[0002] Natural killer (NK) cell activity is regulated by a complex mechanism involving activating and inhibitory signals. A variety of different NK-specific receptors have been identified, which play important roles in NK cell-mediated recognition and killing of HLA class I-deficient target cells. Natural cytotoxic receptors (NCRs) refer to a class of activating receptor proteins specifically expressed in NK cells and the genes encoding them. Examples of NCRs include NKp30, NKp44, and NKp46 (see, e.g., Lanier (2001) Nat Immunol 2:23-27). Another important activating receptor involved in NK cell lysis of target cells is NKG2D. NKG2D recognizes class I major histocompatibility complex (MHC)-associated antigens of the MICA / B and ULBP protein families; the latter are stress-related proteins that can serve as tumor-specific antigens, enabling NK cells to recognize and eliminate tumor cells.

[0003] CD94 / NKG2A is an inhibitory receptor found on subsets of natural killer (NK) cells, natural killer T (NKT) cells, and T cells (α / β and γ / δ). CD94 / NKG2A restricts cytokine release and the cytotoxic response of the above lymphocytes to cells expressing the CD94 / NKG2A-ligand HLA-E (see, e.g., WO99 / 28748). HLA-E has also been found to be secreted in soluble form by certain tumor cells (Derre et al., J Immunol 2006; 177:3100-7) and activated endothelial cells (Coupel et al., Blood 2007; 109:2806-14). Antibodies that inhibit CD94 / NKG2A signaling can increase cytokine release and the cytolytic activity of lymphocytes against HLA-E-positive target cells, such as the response of CD94 / NKG2A-positive NK cells to virus-infected cells. Thus, therapeutic antibodies that inhibit CD94 / NKG2A but do not cause killing of CD94 / NKG2A-expressing cells (i.e., non-depleting antibodies) may induce control of tumor growth in cancer patients.

[0004] A variety of antibodies against NKG2A have been described in the art. WO 2008 / 009545 describes the humanized anti-NKG2A antibody Z270, while WO 2009 / 092805 describes the humanized anti-NKG2A antibody Z199. Vance et al. (J Exp Med [Journal of Experimental Medicine] 1999; 190: 1801-12) refers to the rat anti-mouse NKG2-antibody 20D5 (now commercially available from BD Biosciences Pharmingen, catalog number 550518, USA); and U.S. Patent Application Publication 20030095965 describes the murine antibody 3S9, which is alleged to bind to NKG2A, NKG2C, and NKG2E.

[0005] The incidence of head and neck squamous cell carcinoma (HNSCC) is approximately 600,000 cases per year, and the mortality rate is approximately 50%. The main risk factors for HNSCC are smoking, alcohol consumption, and infection with human papillomavirus (HPV). Despite advances in knowledge regarding epidemiology and pathogenesis, the survival rates for many types of HNSCC have shown little improvement over the past four decades. The 5-year overall survival rate for patients with HNSCC is only approximately 50%. Smoking, alcohol consumption, and viral agents are the main risk factors for the development of HNSCC. These risk factors, combined with genetic susceptibility, lead to the accumulation of multiple genetic and epigenetic alterations during the multi-step process of cancer development, and the understanding of the molecular carcinogenesis of such HNSCC is being used to develop targeted agents for the treatment of HNSCC.

[0006] The idea of immunotherapy as a treatment for HNSCC has been around for decades, and attempts to treat HNSCC have involved the targeting of tumor-specific antigens. Although progress has been made regarding such immunostimulatory treatment strategies for use in various solid cancers, the use of these strategies in patients with head and neck squamous cell carcinoma (HNSCC) has been relatively lagged. The immunotherapy approach for HNSCC is particularly complex because of the severe immunosuppression induced by HNSCC, which may reduce the effectiveness of immunostimulation. A review of the mechanisms by which HNSCC evades the anti-tumor immune response (such as downregulation of HLA class I) is provided in: Duray et al. (2010) Clin. Dev. Immunol. [Clinical and Developmental Immunology] Article ID 701657; 2010: 1-15.

[0007] The standard of care for HNSCC includes cisplatin-based chemotherapy, including in combination with cetuximab for the treatment of metastatic HNSCC. For the treatment of unresectable non-metastatic HNSCC, the treatment includes chemotherapy with cisplatin as well as a combination of cetuximab and radiotherapy. The c225 antibody (cetuximab, ) is an anti-EGFR antibody that has been shown to inhibit EGF-mediated tumor cell growth in vitro and was approved by the FDA in 2011 for the treatment of head and neck cancer. Cetuximab is thought to act by blocking the oncogenic signaling of the EGF receptor pathway and by inducing Fcγ receptor-mediated antibody-dependent cellular cytotoxicity (ADCC). However, in HNSCC, ADCC may be affected by the profound immunosuppression induced. Meanwhile, Vantourout et al., Sci. Transl. Med. [Science Translational Medicine] 6:231ra49 (2014) reported that blocking the oncogenic signaling of the EGF receptor pathway results in the post-transcriptional regulation of major histocompatibility complex (MHC) class I-related antigens of the MICA / B and ULBP protein families in tumor cells, which are recognized by the activating receptor NKG2D on NK cells and T cell subsets. In particular, the expression of these stress-related antigens, which are natural ligands for NKG2D, on tumor cells is reduced by clinical EGFR inhibitors, thus potentially reducing the visibility of tumor cells to NK and T cells.

[0008] However, since many HNSCC patients receive different treatment regimens of currently approved therapies, there remains a need in the art to identify the patient population that would most benefit from treatment with immunotherapy agents. SUMMARY OF THE INVENTION

[0009] The present invention particularly arises from the observation that blockade of the inhibitory receptor NKG2A using an anti-NKG2A antibody in combination with cetuximab provides a clinical response in patients with cancer, specifically patients with head and neck cancer (particularly HNSCC), including patients who have previously received platinum-based therapy and patients in whom the cancer has progressed (e.g., non-responding, relapsed, or progressive). In addition, the combination of an anti-NKG2A antibody and cetuximab results in a clinical response even in patients in whom the cancer has progressed during or after treatment with cetuximab. In certain embodiments, the patient may have been previously treated with cetuximab and / or chemotherapy agents (e.g., platinum-based therapy) or cetuximab and radiotherapy. In one embodiment, the combination therapy disclosed herein is particularly suitable for treating the patient population with incurable, unresectable, and / or metastatic HNSCC, optionally further treating the patient population that has previously received platinum-based therapy, radiotherapy, and / or cetuximab.

[0010] Accordingly, in one aspect, a neutralizing anti-NKG2A antibody in combination with cetuximab can significantly improve the cancer of a population of individuals with unresectable (e.g., incurable unresectable) and / or metastatic head and neck cancer, particularly cancer HNSCC that has been considered resistant to cetuximab. Such combination therapy can provide an opportunity for a large population of individuals with head and neck cancer, particularly patients with HNSCC whose cancer is progressing despite treatment with cetuximab. In particular, the combination therapy may be valuable in preventing further progression, particularly in delaying or preventing metastatic cancer (e.g., in individuals with non-metastatic cancer).

[0011] Provided herein are methods of treating head and neck cancer in an individual having head and neck cancer. In one embodiment, the cancer is unresectable (head and neck cancer that cannot be completely removed by surgery). In one embodiment, a method is provided for reducing the tumor burden (e.g., a reduction in the total diameter of target cell foci compared to the baseline total diameter) in an individual having unresectable and / or metastatic head and neck cancer.

[0012] In one embodiment, the method comprises treating the head and neck cancer of the individual, the method comprising administering to the individual a therapeutically effective amount of an agent that, in combination with cetuximab, neutralizes the inhibitory activity of the human NKG2A polypeptide. In one embodiment, an individual having cancer in which the cancer has progressed during or after treatment with cetuximab (previous courses of treatment with cetuximab do not include a compound that neutralizes NKG2A activity, but may be administered in combination with other treatments, particularly radiotherapy and / or chemotherapy). In one embodiment, in addition to a previous course of treatment with cetuximab (optionally in combination with a chemotherapy agent or radiotherapy), the individual has also received a further previous course of treatment with a chemotherapy agent; for example, the individual has received a first previous course of treatment with a platinum-based agent and then a second previous course of treatment with cetuximab. In one embodiment, the cancer is unresectable (e.g., incurable unresectable) and / or metastatic head and neck cancer.

[0013] In one embodiment, provided is a method for treating head and neck cancer in an individual having unresectable, optionally non-metastatic head and neck cancer, particularly HNSCC, the method comprising administering to the individual: (a) a therapeutically effective amount of an agent that neutralizes human NKG2A polypeptide activity, (b) a therapeutically effective amount of cetuximab. In one embodiment, the individual has received prior treatment with a chemotherapeutic agent (e.g., platinum-based therapy), radiotherapy, and / or cetuximab (e.g., including a prior course of administration of such chemotherapeutic agent, radiotherapy, and / or cetuximab) and their head and neck cancer has progressed during or after such prior treatment. In one embodiment, the individual has head and neck cancer that has not responded or has not adequately responded to treatment with such chemotherapeutic agent, radiotherapy, and / or cetuximab. In one embodiment, the individual has head and neck cancer that has recurred after treatment with a chemotherapeutic agent, radiotherapy, and / or cetuximab. In one example, the individual has received a prior course of administration of a chemotherapeutic agent (e.g., platinum-based therapy), radiotherapy, and / or cetuximab and has experienced cancer progression or recurrence after completion of the treatment course (e.g., within 3 years or less after completion of the treatment course). In one embodiment, the individual has received cetuximab in combination with radiotherapy. In one embodiment, the individual has received platinum-based therapy followed by or in combination with cetuximab therapy. In another embodiment, the individual has received prior cetuximab treatment.

[0014] In one embodiment, provided is a method for treating head and neck cancer in an individual having head and neck cancer who has received prior treatment with cetuximab (e.g., including a prior course of administration of cetuximab) and whose cancer has progressed, the method comprising administering to the individual: (a) a therapeutically effective amount of an agent that neutralizes human NKG2A polypeptide activity, (b) a therapeutically effective amount of cetuximab. In one example, the individual having head and neck cancer has received a prior course of administration of cetuximab and has not responded or has not adequately responded to such treatment. In one example, the individual has received a prior course of administration of cetuximab and has experienced cancer progression or recurrence during or after the treatment course including administration of cetuximab. In one example, the individual has received a prior course of administration of cetuximab and has experienced cancer progression or recurrence after completion of the prior treatment course including cetuximab (e.g., within 3 years after completion of the prior treatment course). The prior course of administration of cetuximab can, for example, include cetuximab in combination with radiotherapy. In one embodiment, the prior course of administration of cetuximab includes cetuximab in combination with a radiotherapy agent and / or a chemotherapeutic agent (e.g., a platinum-based agent).

[0015] In one embodiment, provided is a method for treating HNSCC in an individual or preventing the progression of HNSCC in an individual, the method comprising: (i) identifying an individual with HNSCC who is resistant to cetuximab treatment (e.g., has progression despite treatment with cetuximab, optionally in combination with radiotherapy and / or chemotherapy), and (ii) administering to the individual an effective dose of an agent in combination with cetuximab, the agent neutralizing the inhibitory receptor NKG2A. In one embodiment, the individual of step (i) has unresectable, non-metastatic head and neck cancer. In one embodiment, the individual of step (i) has unresectable and metastatic head and neck cancer.

[0016] In another embodiment, provided is a method for determining whether an individual (or, for example, a population of individuals) with HNSCC, optionally unresectable head and neck cancer, optionally non-metastatic head and neck cancer, optionally metastatic head and neck cancer, can specifically benefit from, respond to, and / or be suitable for treatment with an agent that neutralizes the inhibitory receptor NKG2A in combination with cetuximab, the method comprising determining whether one or more individuals have head and neck cancer that is resistant to cetuximab (e.g., cetuximab as monotherapy, cetuximab in combination with radiotherapy, and / or cetuximab in combination with chemotherapy), wherein determining that an individual has head and neck cancer that is resistant to cetuximab indicates that the one or more individuals may specifically benefit from, respond to, and / or be suitable for treatment with an agent that neutralizes the inhibitory receptor NKG2A in combination with cetuximab. Optionally, the method further comprises the step of administering to the individual determined to specifically benefit from, respond to, and / or be suitable for such treatment an agent that neutralizes the inhibitory receptor NKG2A in combination with cetuximab.

[0017] The present disclosure also provides compositions for treating diseases (such as cetuximab-resistant cancers). In some embodiments, provided are agents and / or cetuximab for treating HNSCC in an individual having HNSCC that is resistant to treatment (such as prior treatment) with cetuximab (such as cetuximab alone or in combination with another agent such as chemotherapy or radiotherapy), wherein the agent neutralizes the inhibitory activity of human NKG2A. In one embodiment, provided is an agent that neutralizes the inhibitory activity of human NKG2A for treating cancer in an individual who has received prior treatment with cetuximab. In one embodiment, the agent that neutralizes the inhibitory activity of human NKG2A is administered in combination with cetuximab. In one embodiment, provided is an agent that neutralizes the inhibitory activity of human NKG2A for treating HNSCC in a human individual who has received prior treatment with cetuximab, the treatment comprising administering to the individual an effective amount of each of the following: (a) an agent that neutralizes the inhibitory activity of human NKG2A, optionally an antibody, and (b) cetuximab. In one embodiment, the cancer is HNSCC. In one embodiment, provided is an agent that neutralizes the inhibitory activity of human NKG2A for treating cancer in a human individual having an inoperable, optionally non-metastatic cancer, the treatment comprising administering to the individual an effective amount of each of the following: (a) an agent that neutralizes the inhibitory activity of human NKG2A, optionally an antibody, and (b) cetuximab. In one embodiment, the cancer is HNSCC. In one embodiment, provided is cetuximab for treating HNSCC in an individual who has received prior treatment (such as a first course of treatment with cetuximab), wherein the treatment comprises administering to the individual an effective amount of each of the following: (a) an agent that neutralizes the inhibitory activity of human NKG2A, optionally an antibody, and (b) cetuximab (such as a second course of treatment with cetuximab).

[0018] In any of the embodiments herein, the individual is characterized as having HNSCC cancer that has progressed despite treatment with cetuximab. Optionally, the individual has HNSCC cancer that has progressed despite prior treatment with cetuximab in combination with radiotherapy.

[0019] In any embodiment herein, head and neck cancer resistant to treatment with cetuximab refers to cancer that has progressed or recurred during or after a prior course of treatment that included cetuximab. In one example, cancer resistant to treatment with cetuximab is cancer that has progressed or recurred after the end of a prior course of treatment that included cetuximab (e.g., within 3 years or less). For example, the cancer may progress within 2 years or within 12 months, 6 months, 4 months, or 3 months after the end of a prior course of treatment that included cetuximab. Optionally, the recurrent cancer is not a new primary cancer but represents a recurrence of the original HNSCC. Optionally, the recurrent cancer is a cancer having the same region and / or laterality (e.g., right or left). A prior course of treatment that includes cetuximab may, for example, include a combination of cetuximab and radiotherapy. A prior course of treatment that includes cetuximab may, for example, include a combination of cetuximab and chemotherapy (e.g., platinum-based therapy).

[0020] In one aspect of any embodiment herein, the treatment of the invention results in a reduction in tumor burden, optionally a reduction in the sum of the diameters of the target cell lesions compared to the sum of the baseline diameters. In one embodiment, the treatment delays the progression of the cancer. In one embodiment, the treatment delays or prevents the metastasis of the cancer. In one embodiment, treatment by shrinking or delaying the growth of the cancer may improve the symptoms or well-being of the patient.

[0021] In any embodiment herein, the individual may be characterized as having received a prior platinum-based therapy (and may have cancer that has progressed despite such therapy). Platinum-based therapy may include, for example, administration of a treatment regimen that includes cisplatin or carboplatin, for example, a regimen that includes a platinum agent and further paclitaxel, docetaxel, gemcitabine, or 5FU (5-fluorouracil).

[0022] In one embodiment, provided is a method for treating HNSCC in an individual, the method comprising:

[0023] (a) administering cetuximab to the individual (e.g., administering a cycle or course that includes cetuximab, optionally a course that includes cetuximab and radiotherapy or a course that includes cetuximab and chemotherapy); and

[0024] (b) if the cancer of the individual in step (a) is resistant to cetuximab (e.g., to the course of step (a)), optionally wherein the cancer the patient has is progressing, spreading to other organs, or non-responsive, administering to the individual a therapeutically active amount of an agent that neutralizes the activity of a human NKG2A polypeptide in combination with a therapeutically active amount of cetuximab.

[0025] In one embodiment, the individual has non-metastatic HNSCC.

[0026] In one embodiment, the individual has received prior platinum-based therapy (e.g., and has progressed despite such treatment).

[0027] In one embodiment, the cetuximab treatment of step (a) comprises combination therapy with radiotherapy and / or chemotherapy.

[0028] In any aspect of the embodiments herein, an agent that neutralizes the inhibitory activity of the human NKG2A polypeptide is an antibody capable of binding NKG2A. In one aspect, the agent that neutralizes the inhibitory activity of the human NKG2A polypeptide is a non-consumptive antibody (e.g., an antibody lacking an Fc domain or having an Fc domain with minimal or no binding to one or more Fcγ receptors).

[0029] In one embodiment, the cancer is an oropharyngeal tumor, laryngeal tumor, oral tumor, nasopharyngeal tumor, or hypopharyngeal tumor. In one embodiment, the HNSCC is oral SCC (OCSCC). OCSCC includes squamous cell carcinoma of the lip, anterior two-thirds of the tongue, floor of the mouth, buccal mucosa, gingiva, hard palate, and retromolar trigone.

[0030] In one embodiment, the HNSCC is non-metastatic cancer.

[0031] In one embodiment, the individual is positive for human papillomavirus (HPV) (e.g., characterized by the presence of human papillomavirus, positive for HPV genotypes associated with high cancer risk, positive for the HPV16 genotype, and / or positive for P16 INKa expression).

[0032] In one embodiment, the individual is negative for human papillomavirus (HPV) (e.g., characterized by the absence of human papillomavirus, absence of HPV genotypes associated with high cancer risk, negative for the HPV16 genotype, and / or negative for P16 INKa expression).

[0033] In one embodiment, the individual has a head and neck cancer characterized by the presence of lymphocytes in the tumor microenvironment (e.g., within the tumor tissue and / or within the tumor-adjacent tissue).

[0034] In one embodiment, the anti-NKG2A antibody is administered in an amount that results in neutralization of the inhibitory activity of human CD94 / NKG2A in a human patient (in vivo), optionally wherein the anti-NKG2A antibody is administered at a dose that results in saturation of NKG2A polypeptide on peripheral blood NK and T lymphocytes for at least two weeks, optionally at least four weeks. In one embodiment, the anti-NKG2A antibody is administered at a dose between 1 mg / kg and 10 mg / kg (optionally at about 4 mg / kg, optionally at about 10 mg / kg). In one embodiment, the fixed dose of the anti-NKG2A antibody administered is in the range of 100 - 1000 mg, optionally in the range of 200 - 1200 mg, such as 750 mg.

[0035] Optionally, the HLA-E status of the cancer can be evaluated prior to treatment with an anti-NKGA agent. In one embodiment, a method is provided for combining HLA-E detection steps to identify patients with HLA-E+HNSCC; these patients can subsequently be treated with an agent that neutralizes the inhibitory activity of the NKG2A polypeptide.

[0036] In one embodiment, the agent that neutralizes the activity of the human NKG2A polypeptide is an anti-NKG2A antibody, which is administered in an effective amount that results in neutralization of the inhibitory activity of human CD94 / NKG2A in a human patient (in vivo), optionally wherein the anti-NKG2A antibody is administered at a dose that results in NKG2A neutralization in peripheral blood NK and T lymphocytes for at least two weeks, optionally at least four weeks. In one aspect, the combination (or for administration) is administered according to a specific clinical dosing regimen, in particular at a specific dose amount and according to a specific dosing schedule (such as the dose amount and / or according to the specific dosing schedules provided herein).

[0037] In one embodiment, the agent that neutralizes the activity of the human NKG2A polypeptide is an antibody that reduces the inhibitory activity of NKG2A by blocking the binding of its ligand HLA-E (i.e., the anti-NKG2A agent interferes with the binding of NKG2A to HLA-E). Antibodies having a heavy chain of any one of SEQ ID NO:2 - 6 and a light chain of SEQ ID NO:7 are examples of such antibodies. In one embodiment, the anti-NKG2A antibody reduces the inhibitory activity of NKG2A without blocking the binding of its ligand HLA-E, i.e., the anti-NKG2A agent is a non-competitive antagonist and does not interfere with the binding of NKG2A to HLA-E. Antibodies having the heavy and light chain variable regions of SEQ ID NO:16 and SEQ ID NO:17 respectively are examples of such antibodies.

[0038] In one embodiment, the anti-NKG2A agent is an antibody that binds NKG2A with a significantly higher affinity than one or more activating NKG2 receptors. For example, in one embodiment, the agent is an antibody that binds NKG2A with a significantly higher affinity than NKG2C. In additional or alternative embodiments, the agent is an antibody that binds NKG2A with a significantly higher affinity than NKG2E. In additional or alternative embodiments, the agent is an antibody that binds NKG2A with a significantly higher affinity than NKG2H. Antibodies having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 bind NKG2A and substantially do not bind NKG2C, NKG2E, or NKG2H.

[0039] In additional or alternative embodiments, the anti-NKG2A agent competes 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, and / or an antibody having heavy and light chain variable regions of SEQ ID NOs: 16 and 17, respectively, for binding to CD94 / NKG2A. The agent can be, for example, a human or humanized anti-NKG2A antibody.

[0040] In one embodiment, the anti-NKG2A antibody is a humanized antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7. Exemplary complementarity-determining region (CDR) residues or sequences and / or sites of amino acid substitutions in the framework region (FR) of such a humanized antibody are provided, the humanized antibody having improved properties such as lower immunogenicity, improved antigen binding or other functional characteristics, and / or improved physicochemical properties such as better stability.

[0041] In other embodiments, pharmaceutical compositions and kits, and methods of using them are provided.

[0042] These aspects are disclosed in more detail in the specification of the invention provided herein, and additional aspects, features, and advantages will be apparent from the specification.

[0043] Definitions

[0044] When used, "comprising" can optionally be replaced 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 transcriptome (Houchins et al. (1991) J. Exp. Med. 173:1017-1020). NKG2A is encoded by 7 exons spanning 25 kb and shows some differential splicing. NKG2A forms a heterodimeric inhibitory receptor CD94 / NKG2A found on the surface of subsets of NK cells, α / β T cells, γ / δ T cells, and NKT cells together with CD94. Similar to inhibitory KIR receptors, it has an ITIM in its cytoplasmic domain. As used herein, "NKG2A" refers to the NKG2A gene or any variant, derivative, or isoform of the encoded protein. Also covered 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. Human NKG2A contains 233 amino acids in 3 domains, including a cytoplasmic domain of residues 1-70, a transmembrane region of residues 71-93, and an extracellular region of residues 94-233, having the following sequence:

[0046] MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO:1).

[0047] 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 transcriptome (Gilenke et al. (1998) Immunogenetics 48:163-173). The CD94 / NKG2C and CD94 / NKG2E receptors are activating receptors found on the surface of lymphocyte subsets, such as NK cells and T cells.

[0048] 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 the binding of peptides, such as fragments of signal sequences derived from 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 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 cells, T cells, 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 covered are any nucleic acid or protein sequences that share one or more biological properties or functions with wild-type, full-length HLA-E and share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide or amino acid identity.

[0049] In the context of this disclosure, "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, which can be detected, for example, by flow cytometry using antibodies that specifically recognize the combined epitope on CD94 and NKG2A or the epitope on NKG2A alone. "CD94 / NKG2A-positive lymphocytes" also includes lymphoid-derived immortalized cell lines (e.g., NKL, NK-92).

[0050] In the context of the present disclosure, "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 in its ability to negatively impact intracellular processes that result in lymphocyte responses such as cytokine release and cytotoxic responses. This can be measured, for example, in an NK cell- or T cell-based cytotoxicity assay, where the ability of a therapeutic compound to stimulate the killing of HLA-E positive cells by CD94 / NKG2A positive lymphocytes is measured. In one embodiment, the antibody preparation results in at least a 10% increase in the cytotoxicity of CD94 / NKG2A-restricted lymphocytes, preferably at least a 40% or 50% increase in lymphocyte cytotoxicity, or more preferably at least a 70% increase in NK cell cytotoxicity, and involves the cytotoxicity assay described. If an anti-NKG2A antibody reduces or blocks the interaction of CD94 / NKG2A with HLA-E, it can increase the cytotoxicity of CD94 / NKG2A-restricted lymphocytes. This can be evaluated, for example, in a standard 4-hour in vitro cytotoxicity assay using, for example, NK cells expressing CD94 / NKG2A and target cells expressing HLA-E. Such NK cells cannot effectively kill HLA-E-expressing targets because CD94 / NKG2A recognizes HLA-E, leading to the initiation and propagation of inhibitory signaling that prevents lymphocyte-mediated cytolysis. This in vitro cytotoxicity assay can be performed by standard methods well known in the art, such as those described in the examples of Coligan et al., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992, 1993). The chromium release and / or other parameters for assessing the ability of an antibody to stimulate lymphocytes to kill target cells (such as P815, K562 cells or suitable 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. Immunol. 2001; 8: 1131-1135; Pende et al., J Exp Med. 1999; 190: 1505-1516, each of which is incorporated herein by reference in its entirety. Before adding the NK cells, with 51Label the target cells with Cr, and then kill those estimated to be proportional to the 51 Cr release from the cells to the culture medium as a result of killing. Addition of an antibody that prevents CD94 / NKG2A from binding to HLA-E results in prevention of the initiation and propagation of inhibitory signaling via CD94 / NKG2A. Thus, addition of such an agent results in an increase in lymphocyte-mediated target cell killing. This step thereby identifies agents that prevent CD94 / NKG2A-induced negative signaling by, for example, blocking ligand binding. In a particular 51 Cr release cytotoxicity assay, NK effector cells expressing CD94 / NKG2A can kill HLA-E-negative LCL 721.221 target cells, but are less able to kill HLA-E-expressing LCL721.221-Cw3 control cells. In contrast, YTS effector cells lacking CD94 / NKG2A efficiently kill both cell lines. Thus, due to HLA-E-induced inhibitory signaling via CD94 / NKG2A, NK effector cells kill HLA-E+LCL 721.221-Cw3 cells less efficiently. When in this type of 51 Cr release cytotoxicity assay, NK cells are pre-incubated with a blocking anti-CD94 / NKG2A antibody according to the present invention, they more efficiently kill HLA-E-expressing LCL 721.221-Cw3 cells in an antibody concentration-dependent manner. The inhibitory activity (i.e., cytotoxicity enhancing potential) of the anti-NKG2A antibody can also be evaluated by any one of many other means, for example, by its effect on intracellular free calcium, as described, for example, in Sivori et al., J Exp Med. [Journal of Experimental Medicine] 1997; 186:1129-1136, the disclosure of which is incorporated herein by reference.. Activation of NK cell cytotoxicity can be evaluated, for example, by measuring an increase in cytokine production (such as IFN-γ production) or cytotoxicity markers (such as CD107 or CD137 mobilization). In an exemplary protocol, IFN-y production from PBMC is evaluated by cell surface and intracellular staining and by flow cytometry analysis after 4 days of culture. Briefly, brefeldin A (Sigma Aldrich) is added at a final concentration of 5 μg / ml during the last 4 hours of the culture. Then, after permeabilization (IntraPrep TM; Cells were incubated with anti-CD3 and anti-CD56 mAbs before staining with PE-anti-IFN-γ or PE-IgG1 (Pharmingen), Beckman Coulter). GM-CSF and IFN-γ production from polyclonally activated NK cells was measured in the supernatant using ELISA (GM-CSF: DuoSet ELISA, R&D Systems, Minneapolis, MN, IFN-γ: OptEIA set, Pharmingen).

[0051] Whenever the entire specification refers to "treating HNSCC" etc. when referring to an NKG2A neutralizing agent (such as an antibody), it means: (a) a method of treating HNSCC, the method comprising administering (for at least one treatment) an NKG2A neutralizing agent (preferably in a pharmaceutically acceptable carrier material) to an individual, mammal, especially a human, in need of such treatment in a dose (therapeutically effective amount) that permits treating HNSCC, preferably in a dose (amount) as specified herein; (b) using an NKG2A neutralizing agent for treating HNSCC, or using an NKG2A neutralizing agent for said treatment (especially in humans); (c) using an NKG2A neutralizing agent for manufacturing a pharmaceutical preparation for treating HNSCC, (d) using an NKG2A neutralizing agent for manufacturing a pharmaceutical preparation for treating HNSCC (including mixing an NKG2A neutralizing agent with a pharmaceutically acceptable carrier) or a method of a pharmaceutical preparation comprising an effective dose of an NKG2A neutralizing agent suitable for treating HNSCC; or (e) any combination of (a), (b), (c) and (d), which is the subject matter of the present invention that permits patenting in the country where this application is filed.

[0052] As used herein, the term "biopsy sample" is defined as tissue removed for examination, for example for the purpose of establishing a diagnosis. Examples of types of biopsies include by aspiration, for example with a needle attached to a syringe; by removal of a tissue fragment with an instrument; by removal through an endoscope with a suitable instrument; by surgical excision, for example of an entire lesion; and so on.

[0053] As used herein, the term "antibody" refers to polyclonal and monoclonal antibodies. Antibodies are assigned to one of five main classes based on the type of constant region in the heavy chain: IgA, IgD, IgE, IgG, and IgM. Some of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 kDa - 70 kDa). The N-terminus of each chain defines a variable region having about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. The terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively. The constant regions of the heavy chains corresponding to the different classes of immunoglobulins are correspondingly referred to as "alpha", "delta", "epsilon", "gamma", and "mu". The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. IgG is the exemplary antibody class used herein because they are the most common antibodies under physiological conditions and are the easiest to prepare under laboratory conditions. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human, or other suitable human antibodies. "Antibody" also includes any fragment or derivative of any one of the antibodies.

[0054] The term "specifically binds to" means that preferably in a competitive binding assay, the antibody can bind to a binding partner (e.g., NKG2A), as evaluated using a recombinant form of the protein, an epitope therein, or the native protein present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected via radiolabeling, physical methods such as mass spectrometry, or direct or indirect fluorescent labeling detected using, for example, cell fluorescence analysis (e.g., FACScan). Binding above the amount seen with a control indicates that the agent binds to the target. An agent that specifically binds NKG2A can bind NKG2A alone or as a dimer with CD94.

[0055] When an antibody is said to compete with a specific monoclonal antibody, it means that in a binding assay using a recombinant molecule (e.g., NKG2A) or a surface-expressed molecule (e.g., NKG2A), the antibody competes with the monoclonal antibody. For example, if a test antibody reduces the binding of an antibody having any one of the heavy chains of SEQ ID NO:2 and the light chain of SEQ ID NO:7 to an NKG2A polypeptide or cells expressing NKG2A in a binding assay, then the antibody is said to "compete" with such an antibody, respectively.

[0056] As used herein, the term "affinity" refers to the strength of binding of an antibody to an epitope. The avidity of an antibody is given by the dissociation constant Kd (defined as [Ab] x [Ag] / [Ab-Ag]), where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The avidity constant K a is defined as 1 / Kd. Methods for determining the avidity of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983), which are hereby incorporated by reference in their entirety. A standard method well known in the art for determining the avidity of mAbs is to use surface plasmon resonance (SPR) screening (e.g., by analyzing with a BIAcore TM SPR analysis apparatus).

[0057] In the context of the present invention, "determinant" refers to a site on a polypeptide that interacts or binds.

[0058] The term "epitope" refers to an antigenic determinant and is the area or region on an antigen that binds to an antibody. A protein epitope can include the amino acid residues directly involved in the binding together with those amino acid residues effectively blocked by an antibody or peptide that specifically binds the antigen, i.e., the amino acid residues within the antibody "footprint". It is the simplest form or the smallest structural region on a complex antigen molecule that can combine with, for example, an antibody or a receptor. Epitopes can be linear or conformational / structural. The term "linear epitope" is defined as an epitope consisting of multiple amino acid residues that are contiguous in the linear sequence of amino acids (primary structure). The term "conformational epitope or structural epitope" is defined as an epitope consisting of multiple amino acid residues that are not all contiguous and thus represents separated portions of the linear sequences of amino acids that are brought into proximity to each other by molecular folding (secondary, tertiary, and / or quaternary structure). Conformational epitopes depend on the three-dimensional structure. Thus, the term "conformational" is often used interchangeably with "structural".

[0059] The term "agent" is used herein to denote a compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The term "therapeutic agent" refers to an agent having biological activity.

[0060] For the purposes of this disclosure, a "humanized" or "human" antibody refers to an antibody in which one or more constant and variable framework regions of human immunoglobulins are fused to the binding regions (e.g., CDRs) of an animal immunoglobulin. Such antibodies are designed to retain the binding specificity of the non-human antibody from which these 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 "engineered" to produce specific human antibodies in response to antigen stimulation (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 disclosures of which are incorporated herein by reference). Fully human antibodies can also be constructed by gene or chromosome transfection methods in conjunction with phage display techniques, which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies can also be produced from in vitro activated B cells (see, e.g., U.S. Patent Nos. 5,567,610 and 5,229,275, which are incorporated herein by reference in their entireties).

[0061] "Chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a different or altered constant region of a different class, effector function, and / or species, or to a completely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is altered, replaced, or exchanged with a variable region having a different or altered antigen specificity.

[0062] The terms "Fc domain", "Fc portion", and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, such as the sequence from amino acid (aa) approximately 230 to approximately aa 450 of the human γ (gamma) heavy chain or its corresponding sequence in other types of antibody heavy chains (such as α, δ, ε, and μ for human antibodies), or its naturally occurring allotypes. Unless otherwise specified, the Kabat amino acid numbering commonly accepted for immunoglobulins is used throughout this disclosure (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0063] The terms "isolated", "purified", or "biologically pure" refer to a substance that is substantially or essentially free of the components that are normally associated with it in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The major species of protein present in a preparation is substantially purified.

[0064] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and 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 mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0065] The term "recombinant", when used in reference to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of heterologous nucleic acid or protein or by the alteration of native nucleic acid or protein, or that the cell is derived from such a modified cell. Thus, for example, such recombinant cells express genes that are not found in the native form (non-recombinant) of the cell or express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0066] In this context, an antibody that "binds" to a polypeptide or epitope designates an antibody that binds to the polypeptide or epitope with specificity and / or avidity determinants.

[0067] The terms "identity" or "identical," when used in reference to the relationship between the sequences of two or more polypeptides, refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matching residues between two or more chains of amino acid residues. "Identity" is measured using gap alignment (if any) solved by a specific mathematical model or computer program (i.e., "algorithm") to determine the percentage of identical matches between the shorter of two or more sequences. The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, Totowa, NJ, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0068] The method for determining identity is designed to give the maximum match between the sequences being tested. The method for determining identity is described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. [Nucleic Acid Research] 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215, 403 - 410 (1990)). The BLASTX program can be publicly obtained 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., ibid). The well-known Smith Waterman algorithm can also be used to determine identity.

[0069] Generation of NKG2A neutralizing agents

[0070] For example, an agent that neutralizes the inhibitory receptor NKG2A can include an agent (such as a protein) that binds to the extracellular portion of the human CD94 / NKG2A receptor or its natural ligand HLA-E 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 for binding to CD94 / NKG2A, i.e., the agent blocks the interaction between CD94 / NKG2A and its ligand HLA-E. In one embodiment, the agent (e.g., an antibody) binds to CD94 / NKG2A and blocks the interaction between CD94 / NKG2A and its ligand HLA-E. In another embodiment, an agent that neutralizes the inhibitory receptor NKG2A is a protein (such as an antibody) that binds to the human HLA-E polypeptide and inhibits the interaction between the human HLA-E protein and the human CD94 / NKG2A protein. In another embodiment, the agent does not compete with HLA-E in binding to CD94 / NKG2A; i.e., the agent binds to NKG2A and is capable of binding to CD94 / NKG2A simultaneously with HLA-E. The antibody can bind to a combined epitope on CD94 and NKG2A or 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.

[0071] In one aspect, the anti-NKG2A agent is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody. In one aspect, the agent comprises a constant region derived from a human IgG1, IgG2, IgG3, or IgG4 antibody. In one aspect, the agent is a fragment of an antibody selected from: IgA, IgD, IgG, IgE, and IgM antibodies. In one aspect, the agent is an antibody fragment selected from: Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab)2 fragment, F(ab')2 fragment, Fv fragment, heavy chain Ig (camelid or camel Ig), V HH fragment, single domain FV, and single chain antibody fragment. In one aspect, the agent is a synthetic or semi-synthetic antibody-derived molecule selected from: scFV, dsFV, minibody, diabody, triabody, kappa body, IgNAR, and multispecific antibody.

[0072] In one embodiment, the anti-NKG2A antibody does not exhibit substantial specific binding to human Fcγ receptors (such as one or more (or all) of human CD16, CD32a, CD32b, and CD64). Such antibodies can comprise constant regions of various heavy chains known not to bind Fc receptors. One such example is the IgG4 constant region. Alternatively, IgG4 antibody fragments that do not contain a constant region (such as Fab or F(ab')2 fragments) can be used to avoid Fc receptor binding. Fc receptor binding can be evaluated according to methods known in the art, including, for example, testing the binding of the antibody to Fc receptor proteins in a BIACORE assay. Additionally, any human antibody type (such as IgG1, IgG2, IgG3, or IgG4) can be used, where the Fc portion is modified to minimize or eliminate binding to Fc receptors (see, for example, WO 03101485, the disclosure of which is incorporated herein by reference). Assays for evaluating Fc receptor binding (such as cell-based assays) are well known in the art and are described, for example, in WO 03101485.

[0073] The present disclosure thus relates to an antibody or other agent that binds NKG2A. In one aspect, the antibody binds NKG2A with a KD that is at least 100-fold lower than that for human NKG2C and / or NKG2E.

[0074] In one aspect of the present disclosure, the agent reduces CD94 / NKG2A-mediated inhibition of lymphocytes expressing CD94 / NKG2A by interfering with CD94 / NKG2A signal transduction, such as by interfering with the binding of NKG2A to HLA-E, preventing or inducing a conformational change in the CD94 / NKG2A receptor, and / or affecting the dimerization and / or aggregation of the CD94 / NKG2A receptor.

[0075] In one aspect of the disclosure, an agent binds to the extracellular portion of NKG2A with a KD that is at least 100-fold lower than that of NKG2C. In another preferred aspect, the agent binds to the extracellular portion of NKG2A with a KD that is at least 150, 200, 300, 400, or 10,000-fold lower than that of NKG2C. In another aspect of the disclosure, an agent binds to the extracellular portion of NKG2A with a KD that is at least 100-fold lower than that of NKG2C, NKG2E, and / or NKG2H molecules. In another preferred aspect, the agent binds to the extracellular portion of NKG2A with a KD that is at least 150, 200, 300, 400, or 10,000-fold lower than that of NKG2C, NKG2C, and / or NKG2H molecules. This can be measured, for example, in a BiaCore experiment, where the ability of the agent to bind to the extracellular portion of immobilized CD94 / NKG2A (e.g., purified from cells expressing CD94 / NKG2 or produced in a biological system) is measured and compared to the binding of the agent to similarly produced CD94 / NKG2C and / or other CD94 / NKG2 variants in the same assay. Alternatively, the binding of the agent to cells that naturally express or overexpress (e.g., after transient or stable transfection) CD94 / NKG2A can be measured and compared to the binding to cells expressing CD94 / NKG2C and / or other CD94 / NKG2 variants. An anti-NKG2A antibody can optionally bind to NKG2B, which is an NKG2A splice variant that forms an inhibitory receptor together with CD94. In one embodiment, the affinity can be measured using the method disclosed in U.S. Patent No. 8,206,709, for example, by Biacore evaluation of binding to a covalently immobilized NKG2A-CD94-Fc fusion protein as shown in Example 8 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference.

[0076] The antibody can, for example, have an EC for binding to cells expressing NKG2A (high affinity) between 0.5 - 10 ng / ml, optionally 1 - 5 ng / ml, optionally 1 - 10 ng / ml, optionally 1 - 20 ng / ml, such as approximately 4 ng / ml 50 . The cells expressing NKG2A can be, for example, cells expressing NKG2A in human PBMCs. In one embodiment, the cells expressing NKG2A are cells expressing CD94 / NKG2A, such as the Ba / F3 cells stably overexpressing CD94 / NKG2A as shown in Example 13 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference. In one embodiment, the binding affinity (K D ) of the antibody for the human NKG2A polypeptide, optionally where the binding affinity is bivalent, is less than 10 -9 M, optionally less than 10-10 M, or optionally less than 10 -11 M, optionally between 10 -10 M and 10 -12 M, optionally between 10 -10 M and 10 -11 M. Affinity can be evaluated, for example, as described in U.S. Patent No. 7,932,055, by binding to a single-chain NKG2A-CD94-mFc construct, the disclosure of which is incorporated herein by reference.

[0077] The anti-NKG2A antibody is a human or humanized antibody, such as comprising the corresponding VH and VL regions of the antibodies shown in the following table.

[0078] Antibody VH VL VH6 SEQ ID NO:2 SEQ ID NO:7 VH1 SEQ ID NO:3 SEQ ID NO:7 VH5 SEQ ID NO:4 SEQ ID NO:7 VH7 SEQ ID NO:5 SEQ ID NO:7 VH8 SEQ ID NO:6 SEQ ID NO:7 Z199 SEQ ID NO:16 SEQ ID NO:17

[0079] The anti-NKG2A antibody can be a human or humanized antibody, such as including a VH human receptor framework 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 framework sequences known in the art. The VL region human receptor sequence can be, for example, VKI_O2 / JK4.

[0080] In one embodiment, the antibody is a humanized antibody or antibody fragment based on antibody Z270. Different humanized Z270 VH chains are shown in SEQ ID NO: 2-6 (the amino acids of the variable domain are underlined). The humanized Z270 VL chain is shown in SEQ ID NO: 7. The HumZ270 antibody is also disclosed in U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference. HumZ270 VH6 (SEQ ID NO: 3) is based on VH5_51; HumZ270 VH1 (SEQ ID NO: 2) is based on VH1_18; humZ270 VH5 (SEQ ID NO: 4) is based on VH5_a; humZ270 VH7 (SEQ ID NO: 5) is based on VH1_f; and humZ270 VH8 (SEQ ID NO: 6) is based on VH1_46; all having the JH6 J segment. Each of these antibodies maintains high affinity binding to NKG2A and has a low likelihood of a host immune response to the antibody because the 6 C-terminal amino acid residues of the Kabat CDR-H2 of each humanized construct are identical to the human receptor framework. Using the alignment program VectorNTI, the following sequence identities were obtained between humZ270 VH1 and humZ270 VH5, -6, -7, and -8: 78.2% (VH1 vs. VH5), 79.0% (VH1 vs. VH6), 88.7% (VH1 vs. VH7), and 96.0% (VH1 vs. VH8).

[0081] In one aspect, the agent comprises (i) a heavy chain variable region having any one of SEQ ID NOs: 2-6, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity thereto, and (ii) a light chain variable region having SEQ ID NO: 7, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity thereto. In one aspect, the agent comprises (i) a heavy chain having the amino acid sequence of any one of SEQ ID NOs: 2-6, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity thereto, and (ii) a light chain having the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity thereto. An antibody having a heavy chain comprising any one of the sequences of SEQ ID NOs: 2-6 and a light chain comprising the sequence of SEQ ID NO: 7 neutralizes the inhibitory activity of NKG2A, but binds substantially not to the activating receptors NKG2C, NKGE or NKG2H. This antibody also competes with HLA-E for binding to NKG2A on the cell surface. In one aspect, the agent comprises HCDR1, HCDR2 and / or HCDR3 sequences derived from a heavy chain having the amino acid sequence of any one of SEQ ID NOs: 2-6. In one aspect of the present invention, the agent comprises LCDR1, LCDR2 and / or LCDR3 sequences derived from a light chain having the amino acid sequence of SEQ ID NO: 7.

[0082] Heavy chain (variable region underlined)

[0083] VH1:

[0084]

[0085] VH6:

[0086]

[0087] VH5:

[0088] VH7:

[0089] VH8:

[0090] Light chain

[0091]

[0092] In one aspect, the anti-NKG2A antibody is an antibody or antibody fragment comprising a CDR-H1 corresponding to residues 31-35 (amino acid sequence SYWMN (SEQ ID NO:8)) of any one of SEQ ID NOs: 2-6, a CDR-H2 corresponding to residues 50-60 (amino acid sequence RIDPYDSETHY (SEQ ID NO:9)) (optionally 50-66 when including 6 terminal amino acids of human origin, i.e., the sequence RIDPYDSETHYSPSFQG (SEQ ID NO:10) of VH6 heavy chain, the sequence RIDPYDSETHYAQKLQG (SEQ ID NO:11) of VH1 heavy chain, etc.) of any one of SEQ ID NOs: 2-6, and a CDR-H3 corresponding to residues 99-114 (95-102 according to Kabat) of any one of SEQ ID NOs: 2-6 (amino acid sequence GGYDFDVGTLYWFFDV (SEQ ID NO:12)). In one embodiment, CDR-H2 corresponds to residues 50-66 of any one of SEQ ID NOs: 2-6. Optionally, the CDR may contain one, two, three, four or more amino acid substitutions.

[0093] In one aspect, the anti-NKG2A antibody is an antibody or antibody fragment comprising a CDR-L1 corresponding to residues 24-34 (amino acid sequence RASENIYSYLA (SEQ ID NO:13)) of SEQ ID NO:7, a CDR-L2 corresponding to residues 50-56 (amino acid sequence NAKTLAE (SEQ ID NO:14)) of SEQ ID NO:7, and a CDR-L3 corresponding to residues 89-97 (amino acid sequence QHHYGTPRT (SEQ ID NO:15)) of SEQ ID NO:7. Optionally, the CDR may contain one, two, three, four or more amino acid substitutions.

[0094] In one aspect, the anti-NKG2A antibody is an antibody or antibody fragment comprising a CDR-H1 corresponding to residues 31-35 of any one of SEQ ID NOs: 2-6, a CDR-H2 corresponding to residues 50-60 (optionally 50-66) of SEQ ID NOs: 2-6, a CDR-H3 corresponding to residues 99-114 (95-102 according to Kabat) of any one of SEQ ID NOs: 2-6, a CDR-L1 corresponding to residues 24-34 of SEQ ID NO:7, a CDR-L2 corresponding to residues 50-56 of SEQ ID NO:7, and a CDR-L3 corresponding to residues 89-97 of SEQ ID NO:7.

[0095] In one aspect, the agent is a fully human antibody raised against a CD94 / NKG2A epitope bound by any of the foregoing antibodies.

[0096] It should be understood that while the foregoing antibodies can be used, other antibodies can be prepared. For example, any fragment of NKG2A (preferably but not exclusively human NKG2A) or any combination of NKG2A fragments can be used as an immunogen to generate antibodies, and these antibodies can recognize epitopes at any position within the NKG2A polypeptide, provided that they can do so on NK cells expressing NKG2A as described herein. Most preferably, the epitope is the epitope specifically recognized by an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7.

[0097] In one aspect, the anti-NKG2A antibody binds substantially the same epitope as an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 (e.g., monalizumab). Antibodies having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 lose binding to an NKG2A mutant having the following amino acid substitutions: K199A / D202A / V213S / R215A / K217A (reference GenBank accession number AAL65234.1). In one embodiment, the anti-NKG2A antibody used according to the present disclosure binds an epitope of NKG2A that is at least partially overlapping with, or includes at least one residue of, the epitope bound by an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 (e.g., monalizumab). The residues bound by the antibody can be designated as being on the surface of the NKG2A polypeptide (e.g., the NKG2A polypeptide expressed on the cell surface). For example, the amino acid residues on NKG2A bound by the antibody can be selected, for example, from the group consisting of the residues K199, D202, V213, R215, and K217 (reference GenBank accession number AAL65234.1 or the NKG2A amino acid sequence of SEQ ID NO: 1).

[0098] The binding of an anti-NKG2A antibody to cells transfected with an NKG2A mutant can be measured and compared to the ability of the anti-NKG2A antibody to bind to the wild-type NKG2A polypeptide (SEQ ID NO:1). A decrease in the binding between the anti-NKG2A antibody and the mutant NKG2A polypeptide (e.g., mutant NKG2A having the substitutions K199A / D202A / V213S / R215A / K217A) implies a decrease in binding affinity (e.g., as measured by known methods such as FACS testing of cells expressing a particular mutant or binding to the mutant polypeptide by Biacore testing), and / or a decrease in the total binding capacity of the anti-NKG2A antibody (e.g., as evidenced by a decrease in Bmax in a graph of anti-NKG2A antibody concentration versus polypeptide concentration). A significant decrease in binding indicates that the mutated residue(s) are directly involved in the binding to the anti-NKG2A antibody when the anti-NKG2A antibody binds to NKG2A, or are in close proximity to the binding protein.

[0099] In some embodiments, a significant decrease in binding means that the binding affinity and / or capacity between the anti-NKG2A antibody and the mutant NKG2A polypeptide is decreased by more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95% relative to the binding between the antibody and the wild-type NKG2A polypeptide. In certain embodiments, the binding drops below the limit of detection. In some embodiments, a significant decrease in binding is confirmed when the binding of the anti-NKG2A antibody to the mutant NKG2A polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%) of the binding observed between the anti-NKG2A antibody and the wild-type NKG2A polypeptide.

[0100] In some embodiments, there is provided an anti-NKG2A antibody that exhibits significantly lower binding to a mutant NKG2A polypeptide (wherein residues in the segment of the amino acid residues bound by an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 (e.g., monalizumab) are substituted with different amino acids) compared to its binding to the wild-type NKG2A polypeptide (e.g., the polypeptide of SEQ ID NO:1). In one embodiment, the mutant has the substitutions K199A, D202A, V213S, R215A and K217A relative to the wild-type NKG2A of SEQ ID NO:1.

[0101] In one aspect, the agent comprises HCDR1, HCDR2 and / or HCDR3 sequences derived from a VH having an amino acid sequence of SEQ ID NO: 16. In one aspect of the disclosure, the agent comprises LCDR1, LCDR2 and / or LCDR3 sequences derived from a VL having an amino acid sequence of SEQ ID NO: 17. In one aspect, the agent comprises HCDR1, HCDR2 and / or HCDR3 sequences derived from a VH having an amino acid sequence of SEQ ID NO: 16, and LCDR1, LCDR2 and / or LCDR3 sequences derived from a VL having an amino acid sequence of SEQ ID NO: 17. An antibody having a heavy chain of SEQ ID NO: 16 and a light chain comprising SEQ ID NO: 17 neutralizes the inhibitory activity of NKG2A and also binds to activating receptors NKG2C, NKGE or NKG2H. The antibody does not compete with HLA-E for binding to NKG2A on the cell surface (i.e., it is a non-competitive antagonist of NKG2A).

[0102]

[0103] In one aspect, the agent comprises a variable heavy chain (V H ) domain (SEQ ID NO: 16), and amino acid residues 31-35, 50-60, 62, 64, 66, and 99-108 of the variable light chain (V L ) domain (SEQ ID NO: 17), optionally with one, two, three, four or more amino acid substitutions.

[0104] In one aspect, the agent is a fully human antibody raised against the CD94 / NKG2A epitope bound by any of the foregoing antibodies.

[0105] It should be understood that, although the above antibodies can be used, other antibodies can recognize and be raised against any portion of the NKG2A polypeptide, as long as the antibody results in neutralization of the inhibitory activity of NKG2A. For example, any fragment of NKG2A (preferably but not exclusively human NKG2A) or any combination of NKG2A fragments can be used as an immunogen to generate antibodies, and these antibodies can recognize epitopes at any position within the NKG2A polypeptide, as long as they can do so on NK cells expressing NKG2A as described herein. In one embodiment, the epitope is an epitope specifically recognized by an antibody having a heavy chain of any one of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7.

[0106] In one aspect, the agent competes with the humZ270 antibody disclosed in U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference, for binding to the extracellular portion of the human CD94 / NKG2A receptor. In one aspect, the agent competes with the humanized Z199 antibody disclosed in U.S. Patent No. 8,796,427, the disclosure of which is incorporated herein by reference, for binding to the extracellular portion of the human CD94 / NKG2A receptor. Competitive binding can be measured, for example, in a BiaCore experiment, where the ability of the agent to bind to the extracellular portion of immobilized CD94 / NKG2A receptor (e.g., purified from cells expressing CD94 / NKG2 or produced in a biological system) saturated with humZ270 is measured. Alternatively, the binding of the agent to cells that naturally express or overexpress (e.g., after transient or stable transfection) the CD94 / NKG2A receptor and have been pre-incubated with a saturating dose of Z270 is measured. In one embodiment, competitive binding can be measured using the method disclosed in U.S. Patent No. 8,206,709, for example, by evaluating binding to Ba / F3-CD94-NKG2A cells by flow cytometry as shown in Example 15 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference.

[0107] An anti-NKG2A agent, such as an antibody, can be incorporated into a pharmaceutical formulation containing the agent at a concentration from 1 mg / ml to 500 mg / ml, wherein the formulation has a pH from 2.0 to 10.0. The formulation can further contain a buffer system, one or more preservatives, one or more tonicity agents, one or more chelating agents, stabilizers, and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous solution, i.e., a formulation containing water. Such formulations are typically solutions or suspensions. In additional embodiments, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0108] In another embodiment, the pharmaceutical formulation is a lyophilized formulation, to which a solvent and / or diluent is added by a physician or patient prior to use.

[0109] In another embodiment, the pharmaceutical formulation is a dry formulation (e.g., lyophilized or spray-dried) that is ready for use without any prior dissolution.

[0110] In additional aspects, 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 more, and wherein the pH of the formulation ranges from about 2.0 to about 10.0.

[0111] In another embodiment, the pH of the formulation is in a range selected from the list consisting of: from 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.

[0112] In additional embodiments, the buffer is selected from the group consisting of: sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, diglycine, tricine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment.

[0113] In additional embodiments, the formulation further comprises a pharmaceutically acceptable preservative. In additional embodiments, the formulation further comprises an isotonic agent. In additional embodiments, the formulation further comprises a chelating agent. In additional embodiments, the formulation further comprises a stabilizer. In additional embodiments, the formulation further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th Edition, 1995.

[0114] It is possible to have other components present in the peptide drug formulation. These additional components can include wetting agents, emulsifying agents, antioxidants, swelling agents, tonicity modifiers, chelating agents, metal ions, oily carriers, proteins (such as human serum albumin, gelatin, or proteins), and zwitterions (such as amino acids like betaine, taurine, arginine, glycine, lysine, and histidine). Of course, these additional components should not adversely affect the overall stability of the drug formulation.

[0115] A pharmaceutical composition comprising an antibody can be administered to a patient in need of such treatment at several sites: for example, at local sites (such as the skin) and mucosal sites, at sites that avoid absorption (such as administration to an artery, blood vessel, or heart), and at sites involving absorption (such as administration to the skin, subcutaneous, muscle, or abdomen). The pharmaceutical composition can be administered to a patient in need of such treatment by the following administration routes: for example, subcutaneous, intramuscular, intraperitoneal, intravenous, lingual, sublingual, buccal, intraoral, oral, gastric, and enteral, nasal, pulmonary (such as via bronchioles and alveoli or a combination thereof), epidermal, dermal, transdermal, vaginal, rectal, ocular (such as via the conjunctiva), ureteral, and parenteral.

[0116] Suitable antibody formulations can also be determined by examining the experience of other already developed therapeutic monoclonal antibodies. Clinically, several monoclonal antibodies have been shown to be effective, such as (rituximab), (trastuzumab), (obinutuzumab), (tositumomab) and (alemtuzumab), and similar formulations can be used with the disclosed compounds. For example, the monoclonal antibody can be provided at a concentration of 10 mg / mL in single-use vials of 100 mg (10 mL) or 500 mg (50 mL), and the monoclonal antibody is formulated for IV administration with 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. In another embodiment, the antibody is provided in a formulation containing approximately 20 mM sodium citrate and approximately 150 mM NaCl at a pH of approximately 6.0.

[0117] Treatment of HNSCC

[0118] Described is a method for treating cancer, particularly HNSCC cancer, particularly unresectable cancer or HNSCC resistant to cetuximab. Cetuximab is an anti-EGFR antibody that received regulatory (FDA) approval for HNSCC in 2011.

[0119] Despite prior treatment with cetuximab (and optionally radiotherapy or other treatments), cetuximab-resistant cancers may have progressed (such as no response, recurrence, spread to other organs). In some embodiments, individuals in whom the cancer has progressed may have received prior treatment with cetuximab as a single agent. In some embodiments, individuals in whom the cancer has progressed may have received prior treatment with a combination of cetuximab and a chemotherapy agent (such as a platinum-based therapy) or a combination of cetuximab and radiotherapy. In some embodiments, in addition to a prior course of treatment with cetuximab (optionally additionally in combination with radiotherapy, chemotherapy, and / or other agents), the individual has further received a prior course of a platinum-based chemotherapy agent, wherein the prior course of the platinum-based agent is administered prior to the course of cetuximab.

[0120] In one embodiment, the cancer is HNSCC. HNSCC is a squamous or basaloid tumor that arises in the head or neck region and includes tumors of the nasal cavity, nasopharynx, paranasal sinuses, lips, mouth and oral cavity, salivary glands, pharynx, hypopharynx, or larynx. The treatments disclosed herein can be particularly useful for the treatment of, for example, oropharyngeal tumors, laryngeal tumors, oral tumors, and hypopharyngeal tumors. These tumors are routinely identified by practitioners in the field of oncology, such as physicians, medical oncologists, histopathologists, and otolaryngologists, as well as head and neck surgeons. Optionally, the HNSCC is non-metastatic HNSCC.

[0121] In any embodiment herein, unless the context otherwise indicates, a head and neck cancer (e.g., HNSCC) can optionally be designated as locally recurrent, distantly metastatic, and / or deemed incurable (and any combination thereof, such as locally recurrent, locally recurrent and distantly metastatic, locally recurrent without distant metastasis, optionally additionally in any of the above cases, deemed incurable).

[0122] In one exemplary aspect, provided is a method of arresting, reversing, or reducing the progression of HNSCC in a mammalian host (e.g., a human patient) having inoperable HNSCC cancer that has progressed despite prior treatment with cetuximab alone or in combination with chemotherapy or radiation, the method comprising administering to the patient a sufficient amount of an anti-NKG2A agent (e.g., an anti-NKG2A antibody), an anti-NKG2A antibody composition, or a related composition (e.g., a nucleic acid encoding an anti-NKG2A antibody), the amount being sufficient to detectably reduce the progression of HNSCC in the host.

[0123] In another aspect, provided is a method of preventing the progression of HNSCC to metastatic cancer in a mammalian host (e.g., a human patient) having inoperable, non-metastatic cancer that has optionally further progressed despite prior treatment with cetuximab, the method comprising administering an anti-NKG2A agent to the patient.

[0124] In any embodiment herein, a disease, cancer, or HNSCC that has progressed despite prior treatment with cetuximab can be referred to as a cetuximab-resistant disease, cancer, or HNSCC. An individual in whom cancer or HNSCC has progressed despite prior treatment with cetuximab can be referred to as (or their disease, cancer, or HNSCC is) a cetuximab-resistant individual.

[0125] In one embodiment, the anti-NKG2A agent is administered in combination with cetuximab. For example, a cancer that is resistant or has progressed may optionally be characterized as a tumor that is resistant to cetuximab treatment and is not, for example, a new primary tumor. For example, cetuximab-resistant tumors may occur in the same area and within a limited time during or after the course of cetuximab treatment (e.g., 1, 2, 3 years or less, e.g., 3, 4, 6, 9 months).

[0126] In one embodiment, provided is a composition for treating a disease (such as a cetuximab-resistant cancer). In some embodiments, provided is an agent and / or cetuximab for treating HNSCC in an individual with HNSCC that is resistant to treatment (such as prior treatment) with cetuximab (e.g., cetuximab alone or in combination with another agent such as chemotherapy or radiotherapy), wherein the agent neutralizes the inhibitory activity of human NKG2A. In one embodiment, provided is an agent that neutralizes the inhibitory activity of human NKG2A, which is used to treat HNSCC in a human individual previously treated with cetuximab, the treatment comprising administering to the individual an effective amount of each of the following: (a) an agent that neutralizes the inhibitory activity of human NKG2A, optionally an antibody, and (b) cetuximab. In one embodiment, the cancer or carcinoma is HNSCC.

[0127] In one embodiment, provided is cetuximab for treating HNSCC in an individual who has received prior treatment (such as a first course of treatment with cetuximab), wherein the treatment comprises administering to the individual an effective amount of each of the following: (a) an agent that neutralizes the inhibitory activity of human NKG2A, optionally an antibody, and (b) cetuximab (e.g., a second course of treatment with cetuximab). Thus, an individual who has received a first prior course of cetuximab and whose disease has progressed during or after the first course of treatment with cetuximab can be treated with a second course of treatment with cetuximab, wherein cetuximab is used in combination with an agent (optionally an antibody) that neutralizes the inhibitory activity of human NKG2A. Thus, an individual who has received a first prior course of cetuximab can be treated with an effective amount of each of the following: (a) an agent that neutralizes the inhibitory activity of human NKG2A, optionally an antibody, and (b) cetuximab (e.g., a second course of treatment with cetuximab).

[0128] In one aspect, the use of cetuximab and / or an agent that neutralizes NKG2A inhibitory activity is for reducing the tumor burden, optionally reducing the total diameter of target cell foci compared to the sum of baseline diameters. In one embodiment, the use of cetuximab and / or an agent that neutralizes NKG2A inhibitory activity is for delaying the progression of cancer. In one embodiment, the use of cetuximab and / or an agent that neutralizes NKG2A inhibitory activity is for delaying or preventing cancer metastasis.

[0129] Suitable treatment regimens for treating an individual include, for example, administering to the individual an effective amount of an antibody that neutralizes the inhibitory activity of human NKG2A, wherein the method comprises at least one administration cycle, and wherein at least one dose of the anti-NKG2A antibody is administered at a dose of 1 - 10 mg / kg body weight. In one embodiment, the administration cycle is between 2 and 8 weeks.

[0130] In one embodiment, the method comprises at least one administration cycle, wherein the cycle is a time of eight weeks or less, and wherein for each of the at least one cycle, two, three, or four doses of the anti-NKG2A antibody are administered at a dose of 1 - 10 mg / kg body weight.

[0131] In one embodiment, the anti-NKG2A is administered in an amount effective to saturate the NKG2A receptor on lymphocytes for at least one, two, three, or four weeks. In certain embodiments, the anti-NKG2A antibody is administered at a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg (e.g., per dose).

[0132] In one aspect of any of the embodiments herein, the anti-NKG2A antibody is administered approximately every 2 or 4 weeks.

[0133] Delivery of the anti-NKG2A antibody to an individual (by direct administration, or expression from nucleic acid therein, such as from a poxvirus gene transfer vector comprising one or more anti-NKG2A antibody-encoding nucleic acid sequences) and practicing the other methods herein can be used to reduce, treat, prevent, or otherwise improve any suitable aspect of cancer progression (particularly HNSCC progression). The methods disclosed herein can be particularly useful for reducing and / or alleviating tumor growth, tumor cell number, and any associated parameters or symptoms (such as biomarkers). The methods disclosed herein are particularly useful in preventing tumor recurrence, prolonging progression-free survival, and preventing metastasis. The methods of independently and comprehensively reducing, preventing, or otherwise improving these aspects of cancer progression are advantageous features.

[0134] In another aspect, there is provided a method that reduces the risk of cancer progression in a human patient, reduces the risk of further cancer progression in a cell population that has undergone priming, and / or provides a treatment regimen for reducing cancer progression. In a further aspect, there is provided a method of increasing the likelihood of survival beyond a relevant period in a human patient diagnosed with HNSCC. In another aspect, there is provided a method for improving the quality of life of a patient with HNSCC, the method comprising administering to the patient an amount of a composition effective to improve their quality of life. In further aspects, the methods described herein can also be used to significantly reduce tumor size or tumor burden. In a further aspect, the methods described herein can be administered to significantly reduce the number of HNSCC cells in a vertebrate host such that, for example, the total number of HNSCC cells is reduced. In a related sense, there is provided a method for killing (e.g., directly or indirectly causing death) HNSCC cells in a vertebrate (such as a human cancer patient).

[0135] Typically, cancer progression and response can be determined by an investigator according to standard tumor response criteria practices, such as according to the "Response Evaluation Criteria in Solid Tumors" (RECIST) v1.1 detailed by Eisenhauer, EA, et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1), Eur J Cancer 2009:45:228-247, the disclosure of which is incorporated herein by reference.

[0136] As disclosed herein, an NKG2A neutralizing agent (such as an anti-NKG2A antibody) can be used in combination with cetuximab to treat an individual. Thus, after progression of HNSCC during or after prior cetuximab (prior cetuximab does not include combination therapy with an NKG2A neutralizing agent), an individual (such as having unresectable, optionally non-metastatic cancer) can be treated with an NKG2A neutralizing agent and cetuximab in combination. The prior course of cetuximab can include one or more additional agents or therapies, particularly radiation therapy and / or chemotherapy.

[0137] In some cases, prior to prior courses of cetuximab (used as a single agent or optionally in combination with radiotherapy and / or chemotherapy), patients will have been treated earlier with prior courses of platinum-based chemotherapy agents; for example, an individual received a prior course of treatment with a platinum-based agent and then received a prior course of treatment with cetuximab. In such cases, despite treatment with a platinum-based agent followed by subsequent treatment with cetuximab (prior course of cetuximab), optionally wherein cetuximab is administered in combination with radiotherapy and / or chemotherapy agents, the patient may have experienced cancer progression. If an individual experiences cancer progression despite a prior course of cetuximab, the individual can be treated with a combination of an NKG2A neutralizing agent and cetuximab.

[0138] Combined administration of an NKG2A neutralizing agent and cetuximab includes simultaneous administration of the compounds in the same or different dosage forms, or separate administration (e.g., sequential administration) of these compounds. Thus, an NKG2A neutralizing agent and cetuximab can be designated for separate administration and administered simultaneously or sequentially. The treatment can optionally be administered in combination with one or more additional therapeutic agents. In one embodiment, a course of treatment with an NKG2A neutralizing agent includes an NKG2A neutralizing agent and cetuximab and does not include other therapeutic (such as anti-cancer) agents. In another embodiment, a course of treatment with an NKG2A neutralizing agent includes an NKG2A neutralizing agent, cetuximab, and one or more additional therapeutic (such as anti-cancer) agents.

[0139] As used herein, co-administration or combination administration of an anti-NKG2A agent and cetuximab includes simultaneous administration of the compounds in the same or different dosage forms, or separate administration (e.g., sequential administration) of these compounds. Thus, an anti-NKG2A and cetuximab can be administered simultaneously in a single formulation. Alternatively, an anti-NKG2A and cetuximab can be formulated for separate administration and administered simultaneously or sequentially.

[0140] In a treatment method, the anti-NKG2A antibody and cetuximab can be administered separately, together, or sequentially, or in a mixture. In some embodiments, cetuximab is administered before the anti-NKG2A antibody. For example, the anti-NKG2A antibody can be administered about 0 to 30 days before cetuximab. In some embodiments, the anti-NKG2A antibody is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days before cetuximab. In some embodiments, the anti-NKG2A antibody is administered simultaneously with cetuximab. In some embodiments, the anti-NKG2A antibody is administered after cetuximab. For example, the anti-NKG2A antibody can be administered about 0 to 30 days after cetuximab. In some embodiments, the anti-NKG2A antibody is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days after cetuximab.

[0141] Suitable treatment regimens for treating a human having HNSCC include, for example, administering to the patient an effective amount of each antibody that neutralizes NKG2A activity and cetuximab, wherein the method comprises at least one dosing cycle, wherein at least one dose of the anti-NKG2A antibody is administered at a dose of 1-10 mg / kg body weight (e.g., once every two weeks), and administering at least one dose, optionally at least two doses, of cetuximab, optionally wherein cetuximab is administered at a dose of 250 mg / m 2 per week, optionally wherein cetuximab is administered as an initial dose at a dose of 400 mg / m 2 and subsequently at a dose of at least 250 mg / m 2 per week. In any of the embodiments herein, each dose of the anti-NKG2A antibody can be administered at a fixed dose, such as a fixed dose between 100-1000 mg, optionally between 200-1200 mg, for example 750 mg.

[0142] In one embodiment, the method comprises at least one dosing period (e.g., dosing cycle), wherein the cycle or period is a time of eight weeks (or less), and wherein for each of the at least one period, two, three, or four doses of an anti-NKG2A antibody are administered at a dose of 1-10 mg / kg body weight (e.g., a fixed dose between 100-1000 mg, optionally between 200-1200 mg, such as 750 mg). In one embodiment, each cycle further comprises administering two, three, four, five, six, seven, or eight doses of cetuximab, each dose being 250 mg / m 2 . Optionally, the cycle comprises a loading dose of cetuximab; i.e., the first dose in the cycle comprises administering an initial dose of 400 mg / m 2 of cetuximab, and subsequent doses of 250 mg / m 2 of cetuximab.

[0143] The anti-NKG2A antibody can advantageously be administered in an amount that achieves a concentration in the circulation at least 10-fold, 20-fold, or 30-fold higher than the concentration required for substantially complete (e.g., 90%, 95%) receptor saturation (as evaluated by titrating the anti-NKG2A antibody on cells expressing NKG2A (e.g., in PBMCs)), or optionally in an amount that achieves a concentration in extravascular tissues (e.g., tumor tissue or milieu) at least 10-fold, 20-fold, or 30-fold higher than the concentration required for substantially complete receptor saturation (as evaluated by titrating the anti-NKG2A antibody on cells expressing NKG2A (e.g., in PBMCs)).

[0144] A suitable assay of the cytotoxic activity of NKG2A-expressing NK cells against HLA-E-expressing target cells can be used to evaluate the NKG2A+ NK cell response. Examples include assays based on markers of NK cell activation, such as CD107 or CD137 expression. Advantageously, an amount of the anti-NKG2A antibody can be administered to achieve and / or maintain a continuous (minimum) tissue concentration of at least 10 μg / ml. For example, to achieve / maintain 10 μg / ml in tissue, the blood concentration to be achieved and / or maintained can be between 100 μg / ml - 110 μg / ml, 100 μg / ml - 120 μg / ml, 100 μg / ml - 130 μg / ml, 100 μg / ml - 140 μg / ml, 100 μg / ml - 150 μg / ml, 100 μg / ml - 200 μg / ml, 100 μg / ml - 250 μg / ml, or 100 μg / ml - 300 μg / ml.

[0145] For the EC of the NKG2A+ NK cell response used in the examples herein having approximately 1-10 μg / ml (e.g., approximately 10 μg / ml) 100Exemplary treatment regimens for anti-NKG2A antibodies (such as humZ270, such as monalizumab) include at least one cycle of administration, wherein at least one dose of the anti-NKG2A antibody is administered at the following doses: about 10 mg / kg body weight, optionally 2-10 mg / kg body weight, optionally 4-10 mg / kg body weight, optionally 6-10 mg / kg body weight, optionally 2-6 mg / kg body weight, optionally 2-8 mg / kg body weight, or optionally 2-4 mg / kg body weight, optionally a fixed dose between 100-1000 mg, optionally between 200-1200 mg, such as 750 mg. Optionally, at least 2, 3, 4, 5, 6, 7 or 8 doses of the anti-NKG2A antibody are administered. In one embodiment, the cycle of administration is between 2 weeks and 8 weeks. In one embodiment, the cycle of administration is 8 weeks. In one embodiment, the cycle of administration is 8 weeks and includes administering one dose of the anti-NKG2A antibody every two weeks (i.e., a total of four doses).

[0146] In one aspect of any of the embodiments herein, the anti-NKG2A antibody is administered approximately every two weeks.

[0147] Exemplary treatment regimens for use with anti-NKG2A antibodies include, for example, administering the anti-NKG2A antibody to a patient twice a month and maintaining an effective amount of at least 40 μg / ml of the anti-NKG2A antibody in the continuous blood concentration between at least two consecutive administrations of the anti-NKG2A antibody at a dose of 2-10 mg / kg body weight, optionally 2-6 mg / kg body weight, optionally 2-4 mg / kg body weight, optionally about 4 mg / kg body weight, or optionally in the range of 100-1000 mg, optionally in the range of 200-1200 mg, such as 750 mg. These doses can be optionally administered so as to provide a continuous blood concentration of the anti-NKG2A antibody of at least 40 μg / ml throughout the treatment cycle. A blood concentration of the anti-NKG2A antibody reaching 40 μg / ml is expected to provide a tissue (e.g., extravascular tissue, tumor microenvironment) concentration of about 4 μg / ml, providing at least the EC for the antibody (such as, humanized Z270, such as monalizumab) 50 .

[0148] Exemplary treatment regimens for use with anti-NKG2A antibodies include, for example, administering to a patient an effective amount of an anti-NKG2A antibody, wherein the antibody is administered 2 times per month, and maintaining a continuous blood concentration of the anti-NKG2A antibody between at least two consecutive anti-NKG2A antibody administrations at an effective amount of at least 100 μg / ml is in the range of 4 - 10 mg / kg body weight, optionally 4 - 6 mg / kg body weight, optionally 4 - 8 mg / kg body weight, optionally 4 mg / kg body weight, optionally about 6 mg / kg body weight, optionally about 8 mg / kg body weight, or optionally 10 mg / kg body weight, or optionally in the range of 100 - 1000 mg, optionally in the range of 200 - 1200 mg, for example 750 mg. These doses can be optionally administered in order to provide a continuous blood concentration of the anti-NKG2A antibody of at least 100 μg / ml throughout the treatment cycle. A blood concentration of the anti-NKG2A antibody of 100 μg / ml is expected to provide a tissue (e.g., extravascular, tumor microenvironment) concentration of about 10 μg / ml, which in turn corresponds to at least an EC of an antibody such as humanized Z270 100 .

[0149] With or without a pre-detection step to evaluate the expression of HLA-E on the surface of tumor cells, patients with head and neck cancer can be treated with an NKG2A neutralizing agent, where the head and neck cancer has progressed or failed to respond effectively during or after prior treatment with cetuximab. Thus, optionally, the method of treatment can include the step of detecting HLA-E nucleic acid or polypeptide in a biological sample from the individual's tumor (e.g., on tumor cells). The determination of the expression of HLA-E in the biological sample (e.g., expressing HLA-E at a detectable level, expressing at least a predetermined level of HLA-E, significantly expressing HLA-E, expressing HLA-E at a high level, or expressing HLA-E in the case of intense staining with an anti-HLA-E antibody, optionally compared to a reference in each case) can be used to designate whether the patient is a head and neck cancer patient who particularly strongly benefits from treatment with an agent that neutralizes the activity of NKG2A. In one embodiment, the method includes determining the expression level of HLA-E nucleic acid or polypeptide in the biological sample and comparing that level to a reference level (e.g., value, strong cell surface staining, etc.) corresponding to an individual who benefits from treatment with an agent that inhibits the activity of NKG2A. The determination that the biological sample expresses HLA-E nucleic acid or polypeptide at a level corresponding to and / or increased compared to the reference level indicates that the individual has a head and neck cancer that particularly strongly benefits from treatment with an agent that inhibits the activity of NKG2A. Optionally, detecting HLA-E polypeptide in a biological sample includes detecting HLA-E polypeptide expressed on the surface of malignant HNSCC cells. In one embodiment, the determination that the biological sample significantly expresses HLA-E nucleic acid or polypeptide indicates that the individual has a head and neck cancer that particularly strongly benefits from treatment with an agent that neutralizes the activity of NKG2A. When referring to HLA-E polypeptide, "significantly expresses" means that the HLA-E polypeptide is expressed in a substantial number of tumor cells taken from a given patient. However, the definition of the term "significantly expresses" is not limited to an exact percentage value, and in some instances, the receptor referred to as "significantly expresses" will be present on at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the HNSCC cells taken from the patient.

[0150] Determining whether an individual has head and neck cancer cells that express HLA-E polypeptide can for example include obtaining a biological sample from the individual containing head and neck cancer cells (e.g., by performing a biopsy), contacting the cells with an antibody that binds to the HLA-E polypeptide, and detecting whether the cells express HLA-E on their surface. Optionally, determining whether an individual has head and neck cancer cells that express HLA-E includes performing an immunohistochemical assay. Optionally, determining whether an individual has head and neck cancer cells that express HLA-E includes performing a flow cytometry assay.

[0151] Patients with head and neck cancer, particularly those with HNSCC, can be further treated with an anti-NKG2A agent, with or without a pre-detection step to evaluate whether the patient (or the patient's tumor) is HPV-positive. In some embodiments, the individuals treated using the methods of the invention are HPV-positive. In some embodiments, the individuals treated using the methods of the invention are HPV-negative.

[0152] Example

[0153] Example 1 - Response case of a patient resistant to cetuximab treated with repeated injections of the combination of monalizumab and cetuximab for the treatment of HNSCC

[0154] A phase 1b / 2 trial of IPH2201 and cetuximab was conducted in patients with human papillomavirus (HPV)(+) and HPV(-) squamous cell carcinoma of the head and neck. Although cetuximab after platinum-based therapy is approved for HNSCC, its activity in this setting is limited (response rate of 12%). A clinical trial evaluated the efficacy of the combination of monalizumab and cetuximab for the treatment of HNSCC. Monalizumab (see WHO Drug Information, Volume 30, No. 1, 2016), also known as IPH2201, is a neutralizing anti-NKG2A antibody that has the heavy chain amino acid sequence shown in SEQ ID NO:2 and the light chain amino acid sequence shown in SEQ ID NO:7.

[0155] The inclusion criteria were as follows:

[0156] Age ≥ 18 years

[0157] 1. Histologically or cytologically confirmed HPV(+) or HPV(-) squamous cell carcinoma of the nasopharynx (WHO type 1), oropharynx, hypopharynx, larynx (supraglottis, glottis, subglottis) or oral cavity.

[0158] 2. Recurrent or metastatic disease, confirmed by imaging (CT scan, MRI, X-ray) and / or physical examination. In phase II, measurable disease according to Response Evaluation Criteria in Solid Tumors [RECIST] 1.1 was mandatory. In phase Ib, patients with or without measurable disease were eligible.

[0159] 3. Progression after platinum-based chemotherapy.

[0160] 4. Only applicable to phase Ib: patients before treatment, not compliant with further treatment with curative intent. This part was open to patients before treatment, regardless of the number of previous treatment lines.

[0161] Indicated only for Phase II: Patients who have received a maximum of two prior systemic treatment regimens due to recurrent and / or metastatic disease and are not compliant with further curative-intent treatment.

[0162] 5. Excluded if not previously treated with cetuximab, but with primary treatment (locally advanced disease) at least 4 months after the end of cetuximab treatment and without progressive disease.

[0163] 6. Recovery from prior surgery and from adverse events to Grade 1 or less (except alopecia) due to prior radiotherapy and systemic treatment.

[0164] 7. Eastern Cooperative Oncology Group (ECOG) performance score of 0 or 1.

[0165] 8. Life expectancy ≥ 3 months.

[0166] 9. Patients receiving treatment for brain metastases are eligible if > 4 weeks after the end of treatment (including radiotherapy and / or surgery), clinically stable at the start of the study, and not receiving corticosteroids at the start of the study.

[0167] 10. Adequate blood, immune, hepatic, and renal function, defined as:

[0168] o Hemoglobin ≥ 9.0 g / dL,

[0169] o Absolute neutrophil count ≥ 1,500 / mm3,

[0170] o Platelets ≥ 100,000 / mm3,

[0171] o Total bilirubin ≤ 1.5 X the institutional upper limit of normal (UNL),

[0172] o Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 X the institutional UNL,

[0173] o Serum creatinine ≤ 1.5 X the institutional UNL or estimated (Cockcroft-Gault formula) or measured creatinine clearance ≥ 50 mL / min.

[0174] 11. Serum pregnancy test negative in women of childbearing potential within 72 hours before starting study treatment. Women of childbearing potential and all men must agree to use appropriate contraceptive measures (hormonal or barrier contraception; abstinence) before the start of the study, during monalizumab administration, and for 5 months after the last monalizumab dose.

[0175] 12. Be able to understand the written informed consent form.

[0176] 13. Sign the informed consent form before any protocol - specific procedure.

[0177] The exclusion criteria are as follows:

[0178] 1. Only applicable to Phase II: Patients who have received more than 2 systemic regimens for recurrent and / or metastatic disease (no restriction in the Ib - phase part of the trial).

[0179] 2. Only applicable to Phase II: Patients who have received cetuximab or another epidermal growth factor receptor inhibitor are excluded from the Phase II of the trial, unless cetuximab was part of the primary treatment method and there has been no progressive disease for at least 4 months after the end of the previous cetuximab treatment.

[0180] 3. History of allergic reactions caused by compounds with chemical or biological components similar to cetuximab.

[0181] 4. Patients with known untreated and uncontrolled brain metastases are excluded. However, if the patient has no neurological signs or symptoms, brain imaging studies are not required for eligibility.

[0182] 5. Severe concurrent and uncontrolled medical diseases.

[0183] 6. Autoimmune diseases, which:

[0184] 1. Currently or previously required systemic immunosuppressive or immunomodulatory treatment (including administration of corticosteroids via the systemic route) and / or

[0185] 2. Have a high likelihood of causing irreversible damage to any tissue and / or

[0186] 3. Were diagnosed less than 3 months before the start of the study and / or

[0187] 4. Are clinically unstable and / or

[0188] 5. Have a high risk of progression and cause severe complications.

[0189] 7. Have any of the following abnormal cardiac conditions:

[0190] 1. Unstable angina

[0191] 2. Arrhythmia requiring treatment and not stabilized by treatment

[0192] 3. QTc > 450 ms (M) or 470 ms (F) (Bazett formula - QT interval / √(RR interval) where RR interval = 60 / HR).

[0193] 8. History of cardiac dysfunction including any of the following:

[0194] 1. Myocardial infarction within the last 6 months

[0195] 2. Documented history of congestive heart failure (New York Heart Association functional classification III-IV).

[0196] 9. Known interstitial lung disease.

[0197] 10. Pregnant women are excluded from this study; breastfeeding must be discontinued.

[0198] 11. Other active invasive malignancies (except treated basal or squamous cell skin cancer, or carcinoma in situ of the cervix).

[0199] 12. Treatment with other study agents within less than 14 days before the start of the study.

[0200] 13. Systemic treatment with steroids or other immunosuppressants within 30 days before the start of the study. Physiological replacement with hydrocortisone or equivalent is acceptable.

[0201] 14. Current active infectious diseases.

[0202] 15. HIV seropositive.

[0203] 16. HBs Ag positive or HBV viremia positive, HCV viremia positive.

[0204] 17. Psychological, family, social, or geographical conditions that do not allow medical follow-up and compliance with the study protocol.

[0205] The trial includes a dose-escalation part in which patients receive escalating dose levels of monalizumab at 0.4, 1, 2, 4, or 10 mg / kg every two weeks, in combination with a fixed dose of cetuximab (400 mg / m2 loading dose then 250 mg / m2 weekly), using a 3+3 design. The cohort expansion part uses the highest tested dose of monalizumab, 10 mg / kg, and a futility analysis is performed after the first 11 patients. Response rates are evaluated according to RECIST, every 8 weeks. Patients are treated until cancer progression or unacceptable toxicity. The trial is still ongoing to recruit more patients and to evaluate response duration, progression-free, and overall survival.

[0206] In the initial part of the observed responses, one patient had a history of squamous cell carcinoma of the right mandibular alveolar ridge, stage T4aN2bM0 / IVA, treated surgically by right posterior mandibulectomy with free fibula flap reconstruction, followed by radiotherapy. The patient had recurrent oral squamous cell carcinoma (not a new primary cancer), p16 negative, and was unresectable. The patient had received 3 cycles of cisplatin-paclitaxel-5FU (TPF) induction (with carboplatin substituted for cisplatin), followed by concurrent chemoradiotherapy with cetuximab weekly. At approximately 4 months after completion of cetuximab, the patient's PET / CT showed tumor recurrence. The patient was considered resistant to cetuximab, with minor recurrence observed more than 4 months after completion of cetuximab plus definitive radiotherapy, and recurrent oral cancer occurred within three years (and thus was not considered a new primary cancer) and in the same area, all on the right side in this case. The patient was recommended for a phase 1b / 2 trial of IPH2201 and cetuximab. Treatment with monalizumab and cetuximab according to the protocol resulted in an objective response (PR), with the best response of target lesions during treatment being a 50% reduction compared to baseline (before treatment). Lesions were measured as the sum of the longest diameter or the shortest axis (if lymph nodes).

[0207] In the initial part of the observed responses, there was also one patient with squamous cell carcinoma of the oropharynx, right-sided predominance, stage TxN2bM0 / IVA. The patient's previous treatment regimen included chemotherapy (cisplatin / docetaxel / 5FU) in the initial disease stage, followed by radiotherapy. At disease progression, the patient was treated for recurrent / metastatic disease, first with surgery at the initial site and lymph nodes, then chemotherapy (cisplatin / carboplatin / 5FU), and then cetuximab. The patient had disease progression (as the best response) after cetuximab treatment, and subsequently the patient was treated with paclitaxel and carboplatin. After disease progression, the patient was enrolled in a phase 1b / 2 trial of IPH2201 and cetuximab. Treatment with monalizumab and cetuximab according to the protocol resulted in a response (PR).

[0208] The finding that the combination of a neutralizing anti-NKG2A antibody and cetuximab can significantly improve patients with HNSCC who are considered resistant to cetuximab provides an opportunity for a large patient population with HNSCC who have unresectable cancer and whose cancer has progressed despite treatment with cetuximab, particularly in combination with radiotherapy. This treatment may be valuable in preventing further progression, particularly in delaying or preventing metastatic cancer.

[0209] All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference in their entirety, to the extent permitted by law, as if each reference were individually and specifically indicated to be incorporated by reference and set forth in full herein, without regard to any separate incorporation of any specific document made elsewhere in this text.

[0210] Unless otherwise indicated herein or clearly contradicted by context, in the context of describing the present invention, the use of the terms "a" and "the" and similar referents shall be construed to include both the singular and the plural.

[0211] Unless otherwise noted, all exact values provided herein represent corresponding approximations (e.g., all exact example values are provided relative to a specific factor, or a measurement can be considered to also include providing the corresponding approximate measurement, when appropriate, modified by "about"). Where "about" is used in conjunction with a number, this may be designated to include values corresponding to + / - 10% of the specified number.

[0212] Unless otherwise stated or clearly contradicted by context, any description herein of any aspect or embodiment of the present invention using terms such as "comprising", "having", "including", or "containing" in reference to one or more elements is intended to provide support for a similar aspect or embodiment of the present invention "consisting of" that one or more specific elements, "consisting essentially of" that one or more specific elements, or "essentially containing" that one or more specific elements (e.g., unless otherwise stated or clearly contradicted by context, a composition described herein as containing a specific element should be understood to also describe a composition consisting of that element).

[0213] The use of any and all examples or exemplary language (such as "for example") provided herein is intended only to better illustrate the present invention and does not limit the scope of the present invention as otherwise claimed. The language in the specification should not be construed as indicating that any non-claimed element is essential for the practice of the present invention.

Claims

1. Use of an antibody that neutralizes the inhibitory activity of human NKG2A and cetuximab in the manufacture of a medicament for treating cancer in a human individual suffering from unresectable HNSCC, the treatment comprising administering to the individual an effective amount of each of the following: (a) an antibody that neutralizes the inhibitory activity of human NKG2A, and (b) cetuximab, wherein the individual has HNSCC cancer that has progressed despite previous treatment with cetuximab in combination with radiotherapy, and wherein the anti-NKG2A antibody comprises CDR1, CDR2, and CDR3 domains of a heavy chain having the sequence shown in SEQ ID NO:2, and CDR1, CDR2, and CDR3 domains of a light chain having the sequence shown in SEQ ID NO:

7.

2. The use according to claim 1, wherein the treatment of HNSCC in the individual comprises: a) determining whether the individual has HNSCC that is resistant to cetuximab, and b) after determining that the individual has HNSCC that is resistant to cetuximab, administering to the individual: (i) an antibody that neutralizes the inhibitory activity of human NKG2A, and (ii) cetuximab.

3. The use according to claim 1 or 2, wherein the treatment of HNSCC in the individual comprises: a) determining whether an HLA-E polypeptide is expressed by malignant cells from an individual suffering from HNSCC, and b) after determining that the malignant cells express the HLA-E polypeptide, administering to the individual (i) an antibody that neutralizes the inhibitory activity of human NKG2A, and (ii) cetuximab.

4. The use according to claim 3, wherein determining whether the HLA-E polypeptide is expressed by malignant cells comprises obtaining a biological sample from the individual that contains HNSCC cells, contacting the cells with an antibody that binds to the HLA-E polypeptide, and detecting the cells that express HLA-E.

5. The use according to claim 1 or 2, wherein the anti-NKG2A antibody is characterized by reduced binding to a mutant NKG2A polypeptide having substitutions K199A / D202A / V213S / R215A / K217A as compared to the wild-type NKG2A polypeptide, the amino acid sequence of the wild-type NKG2A polypeptide being as shown in SEQ ID NO:

1.

6. The use according to claim 1 or 2, wherein the anti-NKG2A antibody comprises a human IgG4 constant region.

7. The use according to claim 1 or 2, wherein the anti-NKG2A antibody comprises an Fc-engineered constant region that comprises amino acid modifications that reduce binding to human Fcγ receptors.

8. The use according to claim 1 or 2, wherein the anti-NKG2A antibody is administered as a pharmaceutically acceptable composition that comprises a therapeutically effective amount of the anti-NKG2A antibody.

9. The use according to claim 8, wherein the composition does not contain any other pharmaceutically active agents.

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