EA047799B1 - HEAD AND NECK CANCER TREATMENT - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the use of NKG2A-targeted agents for the treatment of cancers, in particular head and neck cancers. The present invention also provides advantageous combination regimens with NKG2A-targeted agents for the treatment of cancers.

Background of the invention

Natural killer (NK) cell activity is regulated by a complex mechanism that involves both activating and inhibitory signals. Several different NK-specific receptors have been identified as playing important roles in NK cell-mediated recognition and killing of HLA class I-deficient target cells. Natural cytotoxicity receptors (NCRs) refer to a class of activating receptor proteins and the genes expressing them that are specifically expressed on NK cells. 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 target cell lysis is NKG2D. NKG2D recognizes major histocompatibility complex (MHC) class I-related antigens from the MICA/B and ULBP protein families; the latter are stress-associated proteins that can act as tumor-specific antigens, allowing NK cells to recognize and eliminate tumor cells.

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 limits cytokine release and cytotoxic responses of these 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 some tumor cells (Derre et al., J Immunol 2006; 177:3100-7) and activated by endothelial cells (Coupel et al., Blood 2007; 109:2806-14). Antibodies that inhibit CD94/NKG2A signaling can enhance cytokine release and lymphocyte cytolytic activity against HLA-E-positive target cells, such as CD94/NKG2A-positive NK cell responses against virus-infected cells. Therefore, therapeutic antibodies that inhibit CD94/NKG2A but do not induce killing of CD94/NKG2A-expressing cells (i.e., non-depleting antibodies) may induce tumor growth control in cancer patients.

Various antibodies to NKG2A have been described in the art. WO 2008/009545 describes a humanized anti-NKG2A antibody Z270, while WO 2009/092805 describes a humanized anti-NKG2A antibody Z199. Vance et al. (J Exp Med 1999; 190: 1801-12) refer to a rat anti-mouse NKG2 antibody 20D5 (now commercially available through BD Biosciences Pharmingen, catalog number 550518, USA); and US Patent Application Publication 20030095965 describes a mouse antibody 3S9, which is purported to bind to NKG2A, NKG2C and NKG2E.

Head and neck squamous cell carcinoma (HNSCC) has an incidence of approximately 600,000 cases per year and a mortality rate of approximately 50%. The major risk factors for HNSCC are tobacco smoking, alcohol consumption, and human papillomavirus (HPV) infection. Despite advances in understanding the epidemiology and pathogenesis, survival rates for the many types of HNSCC have improved little over the past four decades. The overall 5-year survival rate for patients with HNSCC is only approximately 50%. Tobacco smoking, alcohol consumption, and viral pathogens are the major risk factors for the development of HNSCC. Such risk factors, together with genetic predisposition, lead to the accumulation of multiple genetic and epigenetic alterations during the multistep process of cancer development, and understanding this molecular carcinogenesis of HNSCC is being applied to the development of targeted agents for the treatment of HNSCC.

The concept of using immunotherapy as a treatment for HNSCC has been around for decades, and attempts to treat HNSCC involve targeting tumor-specific antigens. Although improvements have been made in the application of such immunostimulatory treatment strategies to various solid cancers, the application of such strategies to patients suffering from head and neck squamous cell carcinoma (HNSCC) has lagged. Immunotherapeutic approaches to HNSCC are particularly complicated by the significant immunosuppression induced by HNSCC, which potentially reduces the effectiveness of immunostimulatory activity. A review of the mechanisms by which HNSCC evades the antitumor immune response, such as suppressive modulation of HLA class I, is presented in Duray et al. (2010) Clin. Dev. Immunol. Article ID 701657; 2010: 1-15.

Standard of care for HNSCC includes cisplatin-based chemotherapies, including combinations with cetuximab for the treatment of metastatic HNSCC. For the treatment of unresectable non-metastatic HNSCC, treatments include cisplatin-based chemotherapies, as well as a combination of cetuximab and radiation therapy. c225 (cetuximab, ERBITUX®) is an anti-EGFR antibody that has been shown to inhibit EGF-mediated growth

- 1,047,799 tumor cells in vitro, which was approved by the FDA for the treatment of head and neck cancer in 2011. Cetuximab is thought to act by blocking oncogenic EGF receptor pathway signaling and by inducing Fcy receptor-mediated antibody-dependent cellular cytotoxicity (ADCC). However, in HNSCC, ADCC may act by induced significant immunosuppression. Meanwhile, Vantourout et al., Sci. Transl. Med. 6: 231ra49 (2014) report that blocking the oncogenic EGF receptor pathway signaling results in post-transcriptional upregulation of major histocompatibility complex (MHC) class I antigens of the MICA/B and ULBP protein family in tumor cells, which are recognized by the activating receptor NKG2D on NK cells and T cell subsets. Specifically, tumor cell expression of these stress-related antigens, which are natural ligands of NKG2D, is reduced by clinical EGFR inhibitors, thereby potentially reducing tumor cell visibility to NK and T cells.

However, because many patients with HNSCC experience variable treatment outcomes despite currently approved therapies, there is a need in the art to identify patient populations that could benefit more from treatment with immunotherapeutic agents.

Brief description of the present invention

The present invention arises, inter alia, from the observation that blockade of the inhibitory receptor NKG2A using an antibody to NKG2A in combination with cetuximab provides a clinical response in patients suffering from cancer, in particular head and neck cancer, and especially HNSCC, including patients previously treated with a platinum-based drug and whose cancer is progressive (e.g., has failed to respond, has relapsed, or has otherwise progressed). In addition, the combination of an antibody to NKG2A in combination with cetuximab provides clinical responses even in patients with progressive cancer during or after prior treatment with cetuximab. In some embodiments, the patient may have been previously treated with cetuximab and/or a chemotherapeutic agent (e.g., therapy using a platinum-based drug) or cetuximab and radiation therapy. In one embodiment, the combination therapies disclosed herein may be particularly useful for use in treating a patient population suffering from incurable, unresectable and/or metastatic HNSCC, wherein the patient has optionally additionally been treated with a platinum-based drug, radiation therapy and/or cetuximab treatment.

Accordingly, in one aspect, a neutralizing antibody to NKG2A in combination with cetuximab can provide a significant reduction in cancer intensity in a population of individuals characterized by inoperable (e.g., incurable, unresectable and/or metastatic) head and neck cancer, in particular HNSCC, where the cancer is considered resistant to cetuximab. This combination treatment can provide an opportunity for a significant portion of the population of individuals suffering from head and neck cancer, in particular HNSCC, whose cancer progresses despite treatment with cetuximab. In particular, the combination treatment can be useful in preventing further progression, in particular in delaying or preventing metastatic cancer (e.g., in individuals suffering from non-metastatic cancer).

The methods provided herein are for treating head and neck cancer in individuals suffering from head and neck cancer. In one embodiment, the cancer is inoperable (head and neck cancer that cannot be completely removed by surgery). In one embodiment, a method is provided for reducing tumor burden (e.g., reducing the sum of diameters of target cell lesions compared to a baseline sum of diameters) in an individual suffering from inoperable and/or metastatic head and neck cancer.

In one embodiment, the method comprises treating head and neck cancer in an individual, wherein the method comprises administering to the individual a therapeutically active amount of an agent that neutralizes the inhibitory activity of a human NKG2A polypeptide, in combination with cetuximab. In one embodiment, the individual has a cancer that has progressed during or after a prior course of treatment with cetuximab (a prior course of treatment with cetuximab does not include administration of a compound that neutralizes NKG2A activity, but may be administered in combination with other treatments, particularly radiation therapy and/or chemotherapy). In one embodiment, in addition to the prior course of therapy with cetuximab (optionally combined with a chemotherapeutic agent or radiation therapy), the individual has received an additional prior course of therapy with a chemotherapeutic agent; for example, the individual has received a first prior course of therapy with a platinum-based agent, followed by a second prior course of therapy with cetuximab. In one embodiment, the cancer is inoperable (e.g., incurable, unresectable) and/or metastatic head and neck cancer.

In one embodiment, a method of treating head and neck cancer in an individual suffering from inoperable, optionally non-metastatic head and neck cancer is provided, in particular

- 2 047799

HNSCC, wherein the method comprises administering to the subject: (A) a therapeutically active amount of an agent that neutralizes the activity of a human NKG2A polypeptide, (b) a therapeutically active amount of cetuximab. In one embodiment, the subject has previously received treatment with a chemotherapeutic agent (e.g., therapy using a platinum-based agent), radiation therapy, and/or cetuximab (e.g., a prior treatment regimen that includes administration of the chemotherapeutic agent, radiation therapy, and/or cetuximab) and whose head and neck cancer has progressed during or after such prior treatment. In one embodiment, the subject has a head and neck cancer that has failed to respond or has responded insufficiently to treatment with such chemotherapeutic agent, radiation therapy, and/or cetuximab. In one embodiment, the subject has a head and neck cancer that has recurred after treatment with such chemotherapeutic agent, radiation therapy, and/or cetuximab. In one example, the individual has received a prior course of treatment that included administration of a chemotherapeutic agent (e.g., platinum-based therapy), radiation therapy, and/or cetuximab treatment, and has experienced cancer progression or recurrence after completion of the course of treatment, such as within 3 years or less after completion of the course of treatment. In one embodiment, the individual has received cetuximab in combination with radiation therapy. In one embodiment, the individual has received platinum-based therapy followed by or in combination with cetuximab therapy. In another embodiment, the individual has previously received cetuximab treatment.

In one embodiment, a method of treating head and neck cancer in an individual suffering from head and neck cancer that has previously been treated with cetuximab (e.g., a prior treatment that includes cetuximab) and whose cancer is progressing is provided, wherein the method includes administering to the individual: (A) a therapeutically active amount of an agent that neutralizes the activity of a human NKG2A polypeptide, (b) a therapeutically active amount of cetuximab. In one example, the individual suffering from head and neck cancer has received a prior treatment that included cetuximab and has not responded or has not responded sufficiently to such treatment. In one example, the individual has received a prior treatment that included cetuximab and has experienced cancer progression or recurrence during or after said treatment that included cetuximab. In one example, the individual has received a prior course of treatment that included cetuximab administration and has experienced cancer progression or relapse after completion of said prior course of treatment that included cetuximab administration, such as within 3 years after completion of said prior course of treatment. The prior course of treatment that included cetuximab administration may, for example, include cetuximab administration in combination with radiation therapy. In one embodiment, the prior course of treatment that included cetuximab administration includes cetuximab administration in combination with radiation and/or chemotherapeutic agent, such as a platinum-based agent.

In one embodiment, a method for treating or preventing the progression of HNSCC in an individual is provided, comprising: (i) identifying an individual who has HNSCC that is resistant to treatment with cetuximab (e.g., progressing despite treatment with cetuximab, optionally cetuximab in combination with radiation therapy and/or chemotherapy), and (ii) administering to the individual an effective amount of an agent that neutralizes an inhibitory receptor NKG2A, in combination with cetuximab. In one embodiment, the individual of step (i) has an inoperable, non-metastatic head and neck cancer. In one embodiment, the individual of step (i) has an inoperable and metastatic head and neck cancer.

In another embodiment, a method is provided for determining whether an individual (or, e.g., a population of individuals) suffering from HNSCC, optionally inoperable head and neck cancer, optionally non-metastatic head and neck cancer, optionally metastatic head and neck cancer, may benefit from, be responsive to, and/or be suitable for treatment with an agent that neutralizes an inhibitory receptor NKG2A, in combination with cetuximab, wherein the method comprises determining whether the individual(s) suffer from head and neck cancer that is resistant to cetuximab (e.g., cetuximab alone, cetuximab in combination with radiation therapy, and/or cetuximab in combination with chemotherapy), wherein determining that the individual(s) suffer from head and neck cancer that is resistant to cetuximab indicates that the individual(s) may benefit from, be susceptible 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 an agent that neutralizes the inhibitory receptor NKG2A, in combination with cetuximab, to the individual(s) determined to benefit from, be susceptible to, and/or be suitable for such treatment.

Also provided herein are compositions for use in the treatment of a disease, such as cancer, that is resistant to cetuximab. In some embodiments, an agent is provided,

- 3 047799 that neutralizes the inhibitory activity of human NKG2A, and/or cetuximab for use in the treatment of HNSCC in an individual whose HNSCC cancer is resistant to treatment (e.g., prior treatment) with cetuximab (e.g., alone with cetuximab or in combination with another agent such as chemotherapy or radiation therapy). In one embodiment, an agent that neutralizes the inhibitory activity of human NKG2A is provided for use in the treatment of cancer in an individual who has received prior treatment with cetuximab. In one embodiment, an agent that neutralizes the inhibitory activity of human NKG2A is administered in combination with cetuximab. In one embodiment, there is provided an agent that neutralizes the inhibitory activity of human NKG2A for use in treating HNSCC in a human subject who has received prior treatment with cetuximab, wherein the treatment comprises administering to the subject an effective amount of each of: (a) an agent, optionally an antibody, that neutralizes the inhibitory activity of human NKG2A, and (b) cetuximab. In one embodiment, the cancer or carcinoma is HNSCC. In one embodiment, there is provided an agent that neutralizes the inhibitory activity of human NKG2A for use in treating cancer in a human having an inoperable, optionally non-metastatic carcinoma, wherein the treatment comprises administering to the subject an effective amount of each of: (a) an agent, optionally an antibody, that neutralizes the inhibitory activity of human NKG2A, and (b) cetuximab. In one embodiment, the cancer or carcinoma is HNSCC. In one embodiment, cetuximab is provided for use in the treatment of HNSCC in an individual who has received prior treatment (e.g., a first course of treatment with cetuximab), wherein said treatment comprises administering to the individual an effective amount of each of: (a) an agent, optionally an antibody, that neutralizes human NKG2A inhibitory activity, and (b) cetuximab (e.g., a second course of treatment with cetuximab).

In any embodiment herein, the subject is characterized as having an HNSCC cancer that is progressing despite treatment with cetuximab. Optionally, the subject has an HNSCC cancer that is progressing despite prior treatment with cetuximab combined with radiation therapy.

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

In one aspect of any of the embodiments herein, the treatment of the present invention causes a decrease in tumor burden, optionally a decrease in the sum of the diameters of the lesions of target cells compared to the initial sum of the diameters. In one embodiment, the treatment delays the progression of cancer. In one embodiment, the treatment delays or prevents metastasis in cancer. In one embodiment, the treatment by reducing the growth or delaying the growth of cancer improves the patient's symptom scores or well-being.

In any embodiment herein, the individual may be characterized as having received prior therapy with a platinum-based agent (and may possibly have a cancer that is progressing despite such therapy). The therapy with a platinum-based agent may include, for example, administration of a regimen that includes cisplatin or carboplatin, for example, in a regimen that includes a platinum-based agent and additionally paclitaxel, docetaxel, gemcitabine, or 5FU (5-fluorouracil).

In one embodiment, a method of treating HNSCC in an individual is provided, comprising:

(a) administering to the individual cetuximab (e.g., in a cycle or course of therapy comprising cetuximab, optionally in a course of therapy comprising cetuximab and radiation therapy or comprising cetuximab and chemotherapy); and (b) if the individual's stage (a) cancer is resistant to cetuximab (e.g., to a course of therapy for stage (a)), optionally where the patient has cancer that is progressive, has spread to other organs, or is unresponsive), administering to the individual a therapeutically active amount of

- 4 047799 an agent that neutralizes the activity of the human NKG2A polypeptide, in combination with a therapeutically active amount of cetuximab.

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

In one embodiment, the individual has received prior therapy with a platinum-based drug (e.g., and the cancer has progressed despite such therapy).

In one embodiment, treatment with cetuximab in step (a) comprises combination treatment with radiation therapy and/or chemotherapy.

In one embodiment of any aspect herein, an agent that neutralizes the inhibitory activity of a human NKG2A polypeptide is an antibody capable of binding to NKG2A. In one aspect, an agent that neutralizes the inhibitory activity of a human NKG2A polypeptide is a non-depleting antibody (e.g., an antibody lacking an Fc domain or having an Fc domain characterized by minimal or no binding to one or more FcY receptors).

In one embodiment, the cancer is a tumor of the oropharynx, a tumor of the larynx, a tumor of the oral cavity, a tumor of the nasopharynx, or a tumor of the hypopharynx. In one embodiment, the FINSCC is an oral SCC (OCSCC). OCSCC includes squamous cell carcinoma of the lip, anterior 2/3 of the tongue, floor of the mouth, buccal mucosa, gingiva, hard palate, and retromolar triangle.

In one embodiment, the FINSCC is a non-metastatic cancer.

In one embodiment, the individual is human papillomavirus (FLPV) positive (e.g., characterized by the presence of human papillomavirus, positive for an HPV genotype associated with high cancer risk, positive for the FLPV16 genotype, and/or positive for P16 ^INKh expression).

In one embodiment, the individual is negative for human papillomavirus (FLPV) (e.g., characterized by being negative for human papillomavirus, negative for an HPV genotype associated with high cancer risk, negative for the FLPV16 genotype, and/or negative for P16 ^INKh expression).

In one embodiment, the individual has head and neck cancer characterized by the presence of lymphocytes in the tumor environment (e.g., within the tumor tissue and/or within tissue adjacent to the tumor).

In one embodiment, the anti-NKG2A antibody is administered in an amount that neutralizes the inhibitory activity of human CD94/NKG2A in a human patient (in vivo), optionally the anti-NKG2A antibody is administered at a dose that saturates NKG2A polypeptides 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 of 1 mg/kg to 10 mg/kg, optionally about 4 mg/kg, optionally about 10 mg/kg. In one embodiment, the anti-NKG2A antibody is administered at a fixed dose in the range of 100-1000 mg, optionally in the range of 200-1200 mg, such as 750 mg.

Optionally, the HLA-E status of the cancer can be assessed prior to treatment with an agent directed against NKGA. In one embodiment, a method is provided that combines the step of detecting HLA-E to identify patients suffering from HLA-E+ HNSCC; these patients can then be treated with an agent that neutralizes the inhibitory activity of the NKG2A polypeptide.

In one embodiment, the agent that neutralizes the activity of a human NKG2A polypeptide is an anti-NKG2A antibody that 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 the anti-NKG2A antibody is administered at a dose that results in neutralization of NKG2A in peripheral blood NK and T lymphocytes for at least two weeks, optionally at least four weeks. In one aspect, the combination is administered (or is intended to be administered) according to a specific clinical administration regimen, in particular in a specific dose amount and in a specific dosing regimen (e.g., in a dose amount and/or in a specific dosing regimen as provided herein).

In one embodiment, the agent that neutralizes the activity of a 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 antibody interferes with the binding of NKG2A via HLA-E. An antibody having the heavy chains of any of SEQ ID NOs: 2-6 and the light chain of SEQ ID NO: 7 is an example of such an antibody. 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 agent directed against NKG2A is a non-competitive antagonist and does not interfere with the binding of NKG2A via HLA-E. An antibody having the light and heavy chain variable regions of SEQ ID NOs: 16 and 17, respectively, is an example of such an antibody.

In one embodiment, the anti-NKG2A agent is an

- 5,047,799 a titer that binds with significantly higher affinity to NKG2A than to one or more activating NKG2 receptors. For example, in one embodiment, the agent is an antibody that binds with significantly higher affinity to NKG2A than to NKG2C. In a further or alternative embodiment, the agent is an antibody that binds with significantly higher affinity to NKG2A than to NKG2E. In a further or alternative embodiment, the agent is an antibody that binds with significantly higher affinity to NKG2A than to NKG2H. An antibody having the heavy chains of any of SEQ ID NOs: 2-6 and the light chain of SEQ ID NO: 7, respectively, binds NKG2A without directly binding to NKG2C, NKG2E, or NKG2H.

In a further or alternative embodiment, the agent directed against NKG2A competes with an antibody having the heavy chains of any of SEQ ID NOs: 2-6 and the light chain of SEQ ID NO: 7, and/or an antibody having the light and heavy chain variable regions of SEQ ID NOs: 16 and 17, respectively, in binding to CD94/NKG2A. The agent can be, for example, a human or humanized antibody to NKG2A.

In one embodiment, the anti-NKG2A antibody is a humanized antibody having heavy chains of any 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 framework region (FR) amino acid substitution sites of such humanized antibodies having improved properties, such as, for example, lower immunogenicity, improved antigen binding or other functional properties, and/or improved physicochemical properties, such as, for example, better stability.

In other embodiments, pharmaceutical compositions and kits, as well as methods of using them, are provided.

These aspects are more precisely described in, and additional aspects, features and advantages will be apparent from, the description of the present invention provided herein.

Definitions

When the term containing is used, it may optionally be replaced by the terms essentially consisting of or consisting of.

NKG2A (OMIM 161555, the entire disclosure of which is incorporated herein by reference) is a member of the NKG2 group of transcripts (Houchins, et al. (1991) J. Exp. Med. 173:1017-1020). NKG2A is encoded by 7 exons spanning 25 kb, showing some differential splicing. Together with CD94, NKG2A forms the heterodimeric inhibitory receptor CD94/NKG2A found on the surface of NK cell subsets, α/β T cells, γ/δ T cells, and NKT cells. Like the inhibitory receptors KIR, it has an ITIM in its cytoplasmic domain. As used herein, the term NKG2A refers to any variant, derivative, or isoform of the NKG2A gene or the encoded protein. Also encompassed are any nucleic acid or protein sequences with one or more biological properties or functions with wild-type, full-length NKG2A and with 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, with the cytoplasmic domain containing residues 1-70, the transmembrane region containing residues 71-93 and the extracellular region containing residues 94-233 of the following sequence:

MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTY HCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCPE EWITYSNSCYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSH HPWVTMNGLAFKHEI KDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID

NO:1).

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

HLA-E (OMIM 143010, the disclosure of which is incorporated herein by reference) is a non-classical MHC molecule that is expressed on the cell surface and is regulated by binding peptides, such as fragments derived from the signal sequence of other MHC class I molecules. Soluble variants of HLA-E have also been identified. In addition to its T cell receptor binding properties, HLA-E binds a subset of natural killer (NK) cells, natural killer T cells (NKT) and T cells (α/β and γ/δ) by

- 6 047 799 specific binding to CD94/NKG2A, CD94/NKG2B and CD94/NKG2C (see, e.g., Braud et al. (1998) Nature 391:795-799, the entire disclosure of which is incorporated herein by reference). Surface expression of HLA-E protects target cells from lysis by CD94/NKG2A+ NK, T or NKT cell clones. As used herein, the term HLA-E refers to any variant, derivative or isoform of the HLA-E gene or the encoded protein. Also included are any nucleic acid or protein sequences with one or more biological properties or functions of wild-type, full-length HLA-E and with at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher nucleotide or amino acid identity.

In the context of the present invention, a CD94/NKG2A-positive lymphocyte refers to cells of the lymphoid lineage (e.g., NK, NKT and T cells) expressing CD94/NKG2A on the cell surface, which can be detected by, for example, flow cytometry using antibodies that specifically recognize a combined epitope on CD94 and NKG2A or an epitope on NKG2A alone. A CD94/NKG2A-positive lymphocyte also includes an immortal cell line of lymphoid origin (e.g., NKL, NK-92).

In the context of the present invention, the term reduces the inhibitory activity of NKG2A, neutralizes NKG2A or neutralizes the inhibitory activity of NKG2A refers to a process in which CD94/NKG2A is inhibited with respect to its ability to negatively affect intracellular processes leading to lymphocyte responses, such as cytokine release and cytotoxic responses. This can be measured, for example, using an NK or T cell-based cytotoxicity assay, which measures the ability of a therapeutic compound to stimulate the killing of HLA-E positive cells by CD94/NKG2A positive lymphocytes. In one embodiment, the production of the antibody causes at least a 10% enhancement of CD94/NKG2A-restricted lymphocyte cytotoxicity, preferably at least a 40% or 50% enhancement of lymphocyte cytotoxicity, or more preferably at least a 70% enhancement of NK cytotoxicity, referring to the described cytotoxicity assays. If an anti-NKG2A antibody reduces or blocks the interactions of CD94/NKG2A with HLA-E, this may increase the cytotoxicity of CD94/NKG2A-restricted lymphocytes. This can be assessed, for example, using a standard 4-hour in vitro cytotoxicity assay using, for example, NK cells that express CD94/NKG2A and target cells that express HLA-E. These NK cells are unable to effectively kill targets that express HLA-E because CD94/NKG2A recognizes HLA-E, resulting in the generation and propagation of inhibitory signaling that prevents lymphocyte-mediated cytolysis. Such an in vitro cytotoxicity assay can be performed using standard methods that are widely known in the art and are described, for example, in Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993). Chromium release and/or other parameters for assessing the ability of an antibody to stimulate lymphocytes to kill target cells such as P815, K562, 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. Immun. 2001;8:11311135; Pende et al. J. Exp. Med. 1999;190:1505-1516, the entire disclosures of each of which are incorporated herein by reference. Target cells were labeled with ^51Cr prior to addition of NK cells, and killing was then assessed in proportion to the release of ^51Cr from the cells into the medium as a result of killing. Addition of an antibody that prevents CD94/NKG2A binding to HLA-E results in the prevention of the onset and propagation of inhibitory signaling via CD94/NKG2A. Therefore, addition of these agents results in increased lymphocyte-mediated killing of target cells. This step thus identifies agents that prevent CD94/NKG2A-induced negative signaling by, for example, blocking ligand binding. In a specific ^51Cr release cytotoxicity assay, NK effector cells expressing CD94/NKG2A could kill HLA-E-negative target 721.221 LCL cells but less effectively killed HLA-E-expressing control 721.221-Cw3 LCL cells. In contrast, YTS effector cells lacking CD94/NKG2A efficiently killed both cell lines. Thus, NK effector cells were less efficient at killing HLA-E-negative target 721.221 LCL cells due to HLA-E-induced inhibitory signaling through CD94/NKG2A. When NK cells are pre-incubated with blocking anti-CD94/NKG2A antibodies according to the present invention in such a ⁵¹ Cr release cytotoxicity assay, HLA-E expressing 721.221-Cw3 LCL cells are killed more efficiently in an antibody concentration-dependent manner. The inhibitory activity (i.e., cytotoxicity enhancing potential) of an anti-NKG2A antibody can also be assessed by any of a variety of other methods, such as by its effect on intracellular free calcium, as described, for example, in Sivori et al., J. Exp. Med. 1997; 186:1129-1136, the disclosure of which is incorporated herein by reference. Activation of NK cell cytotoxicity can be assessed, for example, by measuring an increase in cytokine production (e.g., IFN-γ production) or cytotoxicity markers (e.g., molecular

- 7 047 799 CD107 or CD137 bilization). In an exemplary protocol, IFN-γ production from PBMCs is assessed by cell surface and intracytoplasmic staining and flow cytometric analysis after 4 days in culture medium. Briefly, brefeldin A (Sigma Aldrich) is added at a final concentration of 5 μg/ml during the last 4 hours of culture. Cells are then incubated with anti-CD3 and anti-CD56 mAbs prior to permeabilization (IntraPrep™; Beckman Coulter) and staining with PE anti-IFN-γ antibody or PE-IgG1 (Pharmingen). GM-CSF and IFN-y production from polyclonal activated NK cells was measured in supernatants using ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, MN, IFN-y: OptElA set, Pharmingen).

Whenever throughout this specification, treatment of HNSCC or the like is mentioned with reference to an NKG2A neutralizing agent (e.g., an antibody), what is meant is: (a) a method of treating HNSCC, said method comprising the step of 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 that provides treatment for HNSCC (a therapeutically effective amount), preferably in the dose (amount) specified herein; (b) the use of an NKG2A neutralizing agent for treating HNSCC or an NKG2A neutralizing agent for use in said treatment (especially a human); (c) the use of an NKG2A neutralizing agent for the manufacture of a pharmaceutical preparation for the treatment of HNSCC, (d) a method of using an NKG2A neutralizing agent for the manufacture of a pharmaceutical preparation for the treatment of HNSCC comprising a mixed NKG2A neutralizing agent with a pharmaceutically acceptable carrier or a pharmaceutical preparation comprising an effective dose of an NKG2A neutralizing agent that is suitable for the treatment of HNSCC; or (e) any combination of (a), (b), (c) and (d) in accordance with the subject matter eligible for patenting in the country where the application is filed.

The term biopsy as used in this document is defined as the removal of tissue for the purpose of examination, such as to establish a diagnosis. Examples of types of biopsy include the use of suction, such as through a needle attached to a syringe; instrumental removal of a piece of tissue using ; removal using suitable instruments through an endoscope; surgical removal, such as of an entire lesion; etc.

The term antibody as used herein refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are classified into one of five major classes: IgA, IgD, IgE, IgG, and IgM. Some are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. An exemplary structural unit of an immunoglobulin (antibody) comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, where each pair has one light (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The N-terminus of each chain is characterized by a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms light chain variable region (V _L ) and heavy chain variable region (V _H ) refer to these light and heavy chains, respectively. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins, are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. IgG is an illustrative class of antibodies used herein because they are the most common antibodies in the physiological case and because they are the most easily produced in the laboratory. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human, or other human-appropriate antibodies. The term antibodies also includes any fragment or derivative of any antibody.

The term "specifically binds" means that the antibody can bind preferentially in a competitive binding assay to a binding partner, such as NKG2A, as assessed using either recombinant forms of the proteins, epitopes thereof, or native proteins present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected using radioactive labels, physical methods such as mass spectrometry, or direct or indirect fluorescent labels detected using, for example, a flow cytometry assay (e.g., FAC Scan). Binding in excess of the amount observed with a control, non-specific agent indicates that the agent binds to the target. An agent that specifically binds NKG2A can bind NKG2A alone or NKG2A as a dimer to CD94.

When an antibody is stated to compete with a particular monoclonal antibody, it means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant molecules (e.g., NKG2A) or surface-expressed molecules (e.g., NKG2A). For example, if the antibody being tested reduces the binding of an antibody having

- 8 047799 heavy chain according to any of SEQ ID NO: 2 and light chain under SEQ ID NO: 7 with an NKG2A polypeptide or a cell expressing NKG2A in a binding assay, then, accordingly, the antibody is called competing with this antibody accordingly.

The term affinity as used herein refers to the strength of binding of an antibody to an epitope. Antibody affinity is represented 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 affinity constant K _a is defined as 1/Kd. Methods for determining the affinity of mAbs are given in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), the references of which are incorporated herein by reference. One standard method, well known in the art, for determining the affinity of a mAb is the use of surface plasmon resonance (SPR) screening (e.g., by analysis with a BIAcore™ SPR assay device).

In the context of this document, a determinant refers to a region of interaction or binding on a polypeptide.

The term epitope refers to an antigenic determinant and is a region or site on an antigen to which an antibody binds. A protein epitope may comprise amino acid residues directly involved in binding as well as amino acid residues that are effectively blocked by the specific antigen-binding antibody or peptide, i.e. amino acid residues within the recognition region of the antibody. It is the simplest form or smallest structural region on a complex antigen molecule that can associate with, for example, an antibody or a receptor. Epitopes may be linear or conformational/structural. The term linear epitope is defined as an epitope consisting of amino acid residues that are contiguous in a linear sequence of amino acids (primary structure). The term conformational or structural epitope is defined as an epitope consisting of amino acid residues that are not all contiguous and thus represent distinct portions of a linear sequence of amino acids that are brought into close proximity to each other as a result of the folding of the molecule (secondary, tertiary and/or quaternary structure). A conformational epitope depends on its 3-dimensional structure. Therefore, the term conformational is often used interchangeably with the term structural.

The term agent is used in this document to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from biological materials. The term therapeutic agent refers to an agent that is characterized by biological activity.

In the context of the present invention, a humanized or human antibody refers to an antibody in which the constant and variable framework regions of one or more human immunoglobulins are fused to a binding region, e.g., a CDR, of an animal immunoglobulin. These antibodies are designed to retain the binding specificity of the non-human antibody from which the binding regions are derived, but to prevent an immune reaction against the non-human antibody. Such an antibody may 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, all of which teachings are incorporated herein by reference). A fully human antibody can also be constructed using genetic or chromosomal transfection techniques, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies can also be produced by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated by reference in their entireties).

A chimeric antibody is an antibody molecule in which (a) the constant region or portions thereof are altered, replaced or substituted such that the antigen-binding portion (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule that provides new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or portions thereof are altered, replaced or substituted by a variable region having a different or altered specificity for the antigen.

The terms Fc domain, Fc portion and Fc region refer to the C-terminal portion of an antibody heavy chain, e.g., from about 230 (aa) amino acids to about 450 aa of the human γ (gamma) heavy chain or its analogous sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ for human antibody) or naturally occurring allotypes thereof. Unless otherwise specified

- 9 047799 otherwise, throughout the present invention, the generally accepted Kabat amino acid numbering for immunoglobulins is used (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

The terms isolated, purified, or biologically pure refer to a material that is substantially or essentially free of the components that would normally accompany it as it is found in its natural state. Purity and homogeneity are typically determined using analytical chemistry procedures such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, which is the predominant species present in the preparation, is substantially purified.

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

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

In the context of this document, the term "antibody that binds a polypeptide or epitope" means an antibody that binds the said determinant with specificity and/or affinity.

The terms identity or identical, when used in the context of 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 interstrand matches of two or more amino acid residues. Identity measures the percentage of identical matches between the smaller of the two or more sequences, with gap alignment (if any), considered by a particular mathematical model or computer program (i.e., algorithms). 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, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Methods for determining identity are designed to ensure the greatest possible match between the sequences under study. Methods for determining identity are described in publicly available computer programs. Methods using a computer program to determine identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and elsewhere (BLAST Manual, Altschul et al. NCB/NLM/NTH Bethesda, Md. 20894; Altschul et al. supra). The well-known Smith-Waterman algorithm can also be used to determine identity.

Obtaining NKG2A-neutralizing agents

An agent that neutralizes an inhibitory NKG2A receptor may, for example, include an agent (e.g., a protein) that binds 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 a CD94/NKG2A-positive lymphocyte. 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 an inhibitory NKG2A receptor is a protein (e.g., an antibody) that binds to a 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 for binding to CD94/NKG2A; i.e., the agent binds NKG2A and is capable of binding CD94/NKG2A simultaneously with HLA-E. The antibody may bind a combined epitope on CD94 and NKG2A or an epitope on NKG2A separately. In one embodiment, the antibody binds an epitope on NKG2A that is

- 10 047799 at least partially overlaps with the HLA-E binding site.

In one aspect, the agent directed against NKG2A is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody. In one aspect, the agent comprises a constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 antibody. In one aspect, the agent is an antibody fragment selected from an IgA, IgD, IgG, IgE, and IgM antibody. In one aspect, the agent is an antibody fragment selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(Ab) ₂ fragment, an F(Ab') ₂ fragment, an Fv fragment, an Ig heavy chain (llama or camel Ig), a VHH fragment, a single domain FV, and a single chain antibody fragment. In one aspect, the agent is a synthetic or semi-synthetic molecule derived from an antibody selected from scFV, dsFV, minibody, diabody, tribody, kappa body, IgNAR; and a polyspecific antibody.

In one embodiment, an anti-NKG2A antibody does not exhibit significant specific binding to FcY receptors, such as one or more (or all) of human CD16, CD32a, CD32b, and CD64. Such an antibody may include constant regions of various heavy chains that are not known to bind to Fc receptors. One such example is the constant region of IgG4. IgG4 Alternatively, antibody fragments that do not include constant regions, such as Fab or F(Ab')2 fragments, can be used to prevent Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing the binding of the antibody to the Fc receptor protein using the BIACORE assay. Any type of human antibody (e.g., IgG1, IgG2, IgG3, or IgG4) in which the Fc portion is modified to minimize or eliminate binding to Fc receptors may also be used (see, e.g., WO 03101485, the disclosure of which is incorporated herein by reference). Assays, such as, e.g., cell-based assays, for assessing Fc receptor binding are widely known in the art and are described in, e.g., WO 03101485.

Thus, the present invention relates to an antibody or other means of binding to NKG2A. In one aspect, the antibody binds to NKG2A with a KD at least 100-fold lower than to human NKG2C and/or NKG2E.

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

In one aspect of the present invention, the agent binds to the extracellular portion of NKG2A with a KD that is at least 100 times lower than that of NKG2C. In a further 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 times lower than that of NKG2C. In another aspect of the disclosure, the agent binds to the extracellular portion of NKG2A with a KD that is at least 100 times lower than that of NKG2C, NKG2E, and/or NKG2H molecules. In a further 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 times lower than that of NKG2C, NKG2C, and/or NKG2H molecules. This can be measured, for example, in BiaCore experiments, in which the ability of agents to bind the extracellular portion of immobilized CD94/NKG2A (e.g., isolated from cells expressing CD94/NKG2 or produced in a biosystem) is measured and compared to the binding of agents to similarly produced CD94/NKG2C and/or other CD94/NKG2 variants in the same assay. Alternatively, the binding of agents to cells that either naturally express or overexpress (e.g., after transient or stable transfection) CD94/NKG2A can be measured and compared to the binding of cells expressing CD94/NKG2C and/or other CD94/NKG2 variants. Anti-NKG2A antibodies may optionally bind NKG2B, which is a splice variant of NKG2A that forms an inhibitory receptor together with CD94. In one embodiment, affinity can be measured using the methods disclosed in U.S. Patent No. 8,206,709, for example, by assessing binding to a covalently immobilized NKG2A-CD94-Fc fusion protein using Biacore, as shown in Example 8 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference.

The antibody may, for example, have an EC ₅₀ for binding (high affinity) to cells expressing NKG2A of 0.5-10 ng/ml, optionally 1-5 ng/ml, optionally 1-10 ng/ml, optionally 1-20 ng/ml, such as about 4 ng/ml. The cells expressing NKG2A may be, for example, cells expressing NKG2A in human PBMC. In one embodiment, the cells expressing NKG2A are cells engineered to express CD94/NKG2A, such as 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 by reference. In one embodiment, the antibody has a binding affinity (KD), optionally the binding affinity is bivalent, for a human NKG2A polypeptide of less than 10 ^-9 M, optionally less than 10 ^-10 M, or optionally less than 10 ^-11 M, optionally from 10 ^-10 M to 10 ^-12 M, optionally from

- 11 047799

10 ^-10 M to 10 ^-11 M. Affinity can be assessed, for example, for binding to the single chain NKG2A-CD94-mFc construct described in U.S. Patent No. 7,932,055, the disclosure of which is incorporated by reference).

The NKG2A antibody may be a human or humanized antibody, such as one comprising the corresponding VH and VL regions of the antibodies shown in the table below.

Antibody VH VL VH6 SEQ GO NO: 2 SEQ GO NO: 7 VH1 SEQ GO NO: 3 SEQ GO NO: 7 VH5 SEQ GO NO: 4 SEQ GO NO: 7 VH7 SEQ GO NO: 5 SEQ GO NO: 7 VH8 SEQ GO NO: 6 SEQ GO NO: 7 Z199 SEQIDNO: 16 SEQIDNO: 17

The NKG2A antibody may be a human or humanized antibody, for example, comprising a human VH acceptor framework region of a human acceptor sequence selected from, for example, VH1_18, VH5_a, VH5_51, VH_1f and VH1_46, and the JH6 J segment, or other human germline VH framework region sequences known in the art. The human VL region acceptor sequence may be, for example, VKI_O2/JK4.

In one embodiment, the antibody is a humanized antibody or antibody fragment based on the Z270 antibody. Various humanized chains of Z270VH are shown as SEQ ID NOs: 2-6 (variable region domain amino acids are underlined). The humanized light chain of Z270VH is shown as 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). HumZ270VH6 (SEQ ID NO: 3) is based on VH5_51; HumZ270VH1 (SEQ ID NO: 2) is based on VH1_18; humZ270VH5 (SEQ ID NO: 4) is based on VH5_a; humZ270VH7 (SEQ ID NO: 5) is based on VH1_f; and humZ270VH8 (SEQ ID NO: 6) is based on VH146; all with the JH6 J segment. Each of these antibodies retains high binding affinity for NKG2A, with a low probability of eliciting a host immune response against the antibody, since the 6 C-terminal amino acid residues of the Kabat CDR-H2 of each humanized construct are identical to the human acceptor framework. Using the VectorNTI alignment program, the following sequence identities were obtained between humZ270VH1 and humZ270VH5, -6, -7, and -8: 78.2% (VH1 versus VH5), 79.0% (VH1 versus VH6), 88.7% (VH1 versus VH7), and 96.0% (VH1 versus VH8).

In one aspect, the agent comprises (i) a heavy chain variable region of any of SEQ ID NOs: 2-6, or an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity thereto, and (ii) a light chain variable region of SEQ ID NO: 7, or an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity thereto. In one aspect, the agent comprises (i) a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 2-6 or an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity thereto and (ii) a light chain comprising an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity thereto. An antibody having a heavy chain comprising a sequence of any one of SEQ ID NOs: 2-6 and a light chain comprising a sequence of SEQ ID NO: 7 neutralizes the inhibitory activity of NKG2A, but does not directly bind activating receptors NKG2C, NKGE or NKG2H. This antibody further 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 characterized by the amino acid sequence under any 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 characterized by the amino acid sequence under any of SEQ ID NOs: 7.

- 12 047799

Heavy chains (variable regions underlined).

VH1.

OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGOGLEWMGRIDPYDS ETHYAOKLOGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYDFDVGTLYWFFD VWGOGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFP AVLQS SGLYSLS VVTVPS S SLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDW

LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF

YPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLS LSLGK (SEQ ID NO: 2)

VH6.

EVOLVOSGAEVKKPGESLKISCKGSGYSFTSYWMNWVRQMPGKGLEWMGRIDPYDSE THYSPSFOGOVTISADKSISTAYLQWSSLKASDTAMYYCARGGYDFDVGTLYWFFDVW GO

GTTVTySSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAV

LQ S SGLYSLS S VVTVP S S SLGTKT YTCNVDHKP SNTKVDKRVESK YGPPCPPCP APEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQ

VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCS VMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3)

VH5.

EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWMNWVRQMPGKGLEWMGRIDPYDSE THYSPSFOGHVTISADKSISTAYLOWSSLKASDTAMYYCARGGYDFDVGTLYWFFDVW GOGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APE

FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR

- 13 047799

EEQFN

STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE

EMTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHE ALHNHYTQKSLSLSLGK (SEQ Ш NO: 4)

VH7.

EVOLVOSGAEVKKPGATVKISCKVSGYTFTSYWMNWVOQAPGKGLEWMGRIDPYDSE THY

AEKFOGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYDFDVGTLYWFFDVWGQ GTTVTVS

SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLY

SLS S VVTVPS S SLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPS VFLFP

PKPKDT

LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDW

LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF

YPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLS LSLGK (SEQ ID NO: 5)

VH8.

OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGOGLEWMGRIDPYDS

ETHY

AQKFOGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDFDVGTLYWFFDVWGQ GTTVTVS

SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYS

LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP

KPKDTL

MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWL

NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP

- 14 047799

SDIAVEW

ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK

SLSLSLGK (SEQ ID NO: 6)

Light chain

DIQMTQSPSSLSSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPS

RFSGS

GSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK

SGTASVVC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQG LSSPVTKSFNRGEC (SEQ Ш NO: 7)

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 (the amino acid sequence of SYWMN (SEQ ID NO: 8)), a CDR-H2 corresponding to residues 50-60 (the amino acid sequence of RIDPYDSETHY (SEQ ID NO: 9)) (optionally 50-66, wherein including the 6 terminal amino acids of human origin, i.e., the sequence RIDPYDSETHYSPSFQG (SEQ ID NO: 10) for the VH6 heavy chain, the sequence RIDPYDSETHYAQKLQG (SEQ ID NO: 11) for the 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 with Kabat), any 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 of SEQ ID NOs: 2-6. Optionally, the CDR may contain one, two, three, four or more amino acid substitutions.

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

In one aspect, the anti-NKG2A antibody is an antibody or antibody fragment comprising a CDR-H1 corresponding to residues 31-35 of any of SEQ ID NOs: 2-6, a CDR-H2 corresponding to residues 50-60 (optionally 50-66) of any of SEQ ID NOs: 2-6, and a CDR-H3 corresponding to residues 99-114 (95-102 according to Kabat) of any 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.

In one aspect, the agent is a fully human antibody that is directed against an epitope of CD94/NKG2A to which any of the above antibodies binds.

It will be understood that while the above 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 fragments of NKG2A, can be used as immunogens for the production of antibodies, and the antibodies can recognize epitopes at any position in the NKG2A polypeptide, if they can do so on NKG2A expressing NK cells as described herein. Most preferably, the epitope is an epitope specifically recognized by an antibody having a heavy chain of any of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7.

In one aspect, an anti-NKG2A antibody binds directly to the same epitope as an antibody having a heavy chain of any of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7, such as monalizumab. An antibody having a heavy chain of any of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7 is characterized by loss of binding to a mutant NKG2A having the following amino acid substitutions: K199A/D202A/V213S/R215A/K217A (GenBank Accession No. AAL65234.1). In one embodiment, an anti-NKG2A antibody used in accordance with the present invention binds to an epitope of NKG2A that at least partially overlaps with or includes at least one residue in an epitope bound by an antibody having a heavy chain of any 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 determined to be present on the surface of an NKG2A polypeptide, e.g., in an NKG2A polypeptide expressed on the surface of a cell. The amino acid residues on the NKG2A bound by the antibody can be selected, for example, from the group of residues consisting of K199, D202,

V213, R215 and K217 (GenBank accession no. AAL65234.1 or NKG2A amino acid sequence

- 15 047799 sequence under SEQ ID NO: 1).

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

In some embodiments, a significant decrease in binding means that the binding affinity and/or binding capacity between the anti-NKG2A antibody and the mutant NKG2A polypeptide is reduced 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 some embodiments, the binding is reduced to values that are below a detectable limit. In some embodiments, a significant reduction 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.

In some embodiments, NKG2A antibodies are provided that exhibit significantly reduced binding to a mutant NKG2A polypeptide in which a residue in the segment containing the amino acid residue bound by an antibody having a heavy chain of any of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7, such as monalizumab, is substituted with a different amino acid compared to binding to a wild-type NKG2A polypeptide (e.g., the polypeptide of SEQ ID NO: 1). In one embodiment, the mutant is characterized by the substitutions K199A, D202A, V213S, R215A, and K217A, with reference to the wild-type NKG2A of SEQ ID NO: 1.

In one aspect, the agent comprises HCDR1, HCDR2 and/or HCDR3 sequences derived from a VH characterized by the amino acid sequence of any of SEQ ID NO: 16. In one aspect of the present invention, the agent comprises LCDR1, LCDR2 and/or LCDR3 sequences derived from a VL characterized by the amino acid sequence of any of SEQ ID NO: 17. In one aspect, the agent comprises HCDR1, HCDR2 and/or HCDR3 sequences derived from a VH characterized by the amino acid sequence of any of SEQ ID NO: 16 and an LCDR1, LCDR2 and/or LCDR3 sequence derived from a VL characterized by the amino acid sequence of SEQ ID NO: 17. An antibody having a heavy chain of SEQ ID NO: 16 and a light chain of SEQ ID NO: 17 neutralizes the inhibitory activity of NKG2A and also binds 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 NKG2A antagonist).

EVQLVESGGGLVKPGGSLKLSCAASGFFSSYAMSWVRQSPEKRLEWVAEISSGGSYTY

Y

PDT VTGRFTISRDNAKNTLYLEIS SLRSEDTAMYYCTRHGDYPRFFD VWGAGTT VTVS S (SEQIDNO: 16)

QIVLTQ SP ALMS ASPGEK VTMTC SASS S VS YIYW YQQKPRS SPKPWIYLT SNL ASGVP AR

FSGSGSGTSYSLTISSMEAEDAATYYCQQWSGNPYTFGGGTKLEIKR (SEQ Ш NO: 17)

In one aspect, the agent comprises amino acid residues 31-35, 50-60, 62, 64, 66 and 99-108 of the variable domain of the heavy chain (Vh) (SEQ ID NO: 16) and amino acid residues 24-33, 49-55 and 88-96 of the variable domain of the light chain (Vl) (SEQ ID NO: 17) optionally with one, two, three, four or more amino acid substitutions.

In one aspect, the agent is a fully human antibody that is directed against an epitope of CD94/NKG2A to which any of the above antibodies binds.

It will be appreciated that while the above antibodies may be used, other antibodies may recognize and be raised against a portion of the NKG2A polypeptide as long as the antibody causes neutralization of the inhibitory activity of NKG2A. For example, any fragment of NKG2A pred

- 16 047799 respectfully, but not exclusively, human NKG2A or any combination of NKG2A fragments can be used as immunogens to generate antibodies, and the antibodies can recognize epitopes at any position in the NKG2A polypeptide if they can do so on NKG2A-expressing NK cells as described herein. In one embodiment, the epitope is an epitope specifically recognized by an antibody having a heavy chain of any of SEQ ID NOs: 2-6 and a light chain of SEQ ID NO: 7.

In one aspect, the agent competes with the antibody humZ270 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 antibody Z199 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, using BiaCore experiments, which measure the ability of agents to bind the extracellular portion of an immobilized CD94/NKG2A receptor (e.g., isolated from cells expressing CD94/NKG2 or produced in a biosystem) loaded with humZ270. Alternatively, binding of the agents to cells that either naturally express or overexpress (e.g., after transient or stable transfection) the CD94/NKG2A receptor and that have been pre-incubated with saturating doses of Z270 is measured. In one embodiment, competitive binding can be measured using the methods disclosed in U.S. Patent No. 8,206,709, for example, by assessing binding to Ba/F3-CD94-NKG2A cells using flow cytometry, as shown in Example 15 of U.S. Patent No. 8,206,709, the disclosure of which is incorporated herein by reference.

An agent directed against NKG2A, such as an antibody, can be included in a pharmaceutical formulation comprising the antibody at a concentration of 1 mg/mL to 500 mg/mL, wherein said formulation is characterized by a pH of 2.0 to 10.0. The formulation can further comprise a buffer system, a preservative(s), a tonicity adjusting agent(s), a chelating agent(s), stabilizers and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e. a formulation containing water. Such a formulation is typically a solution or a suspension. In a further embodiment, the pharmaceutical formulation is an aqueous solution. The term aqueous formulation is defined as a formulation comprising at least 50% w/w water. Similarly, the term aqueous solution is defined as a solution comprising at least 50% w/w water, and the term aqueous suspension is defined as a suspension comprising at least 50% w/w water.

In another embodiment, the pharmaceutical composition is a freeze-dried composition to which the physician or patient adds solvents and/or diluents prior to use.

In another embodiment, the pharmaceutical formulation is a dried formulation (e.g., freeze-dried or spray-dried) ready for use without any prior reconstitution.

In a further aspect, the pharmaceutical composition comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present at a concentration of 1 mg/mL or higher, and wherein said composition is characterized by a pH of from about 2.0 to about 10.0.

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

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

In a further embodiment, the composition further comprises a pharmaceutically acceptable preservative. In a further embodiment, the composition further comprises an isotonic agent. In a further embodiment, the composition further comprises a chelating agent. In a further embodiment, the composition further comprises a stabilizer. In a further embodiment, the composition further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, ^19th edition, 1995.

It is possible that other ingredients may be present in the pharmaceutical formulation of the peptide. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oil-containing carriers, proteins (e.g., human serum albumin, gelatin or proteins) and zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and hist

- 17 047799 din). These additional ingredients, of course, should not negatively affect the overall stability of the pharmaceutical composition.

Pharmaceutical compositions containing an antibody can be administered to a patient in need of such treatment in several areas, such as a local area, such as the skin and mucous membrane, areas bypassing absorption, such as administration into an artery, into a vein, into the heart, and areas that involve absorption, such as administration into the skin, under the skin, into a muscle, or into the abdominal cavity. The administration of pharmaceutical compositions can be carried out by several routes of administration, for example, subcutaneously, intramuscularly, intraperitoneally, intravenously, lingually, sublingually, transbuccally, orally, orally, into the stomach and intestines, nasally, by inhalation, for example, through the bronchioles and alveoli or a combination thereof, epidermally, dermally, transdermally, vaginally, rectally, by the ocular route, for example, through the conjunctiva, urethrally and parenterally to patients in need of such treatment.

Suitable antibody formulations can also be identified by examining experience with other therapeutic monoclonal antibodies that have already been developed. Several monoclonal antibodies have been shown to be effective in clinical conditions, for example, Rituxan® (rituximab), Herceptin® (trastuzumab), Xolair® (omalizumab), Bexxa®g (tositumomab), and Campath® (alemtuzumab) and similar formulations can be used with the antibodies of the present invention. For example, a monoclonal antibody can be supplied at a concentration of 10 mg/mL in single-use vials of either 100 mg (10 mL) or 500 mg (50 mL) formulated for IV administration in 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 supplied in a formulation containing about 20 mM Na-citrate, about 150 mM NaCl, at a pH of about 6.0.

Treatment of HNSCC

Methods are described for the treatment of carcinomas, in particular HNSCC cancer, in particular unresectable carcinoma or HNSCC that is resistant to cetuximab. Cetuximab (ERBITUX®) is an antibody to EGFR that was approved by the regulatory authorities (FDA) in 2011 for HNSCC.

Cetuximab-resistant carcinoma may progress (e.g., fail to respond, recur, spread to other organs) despite prior treatment with cetuximab (and optionally radiation therapy or other treatments). In some embodiments, the individual whose cancer has progressed may have received prior treatment with cetuximab alone. In some embodiments, the individual whose cancer has progressed may have received prior treatment with cetuximab combined with a chemotherapeutic agent (e.g., platinum-based therapy) or cetuximab combined with radiation therapy. In some embodiments, in addition to a prior course of therapy with cetuximab (optionally further in combination with radiation therapy, chemotherapy and/or other agents), the individual has received an additional prior course of therapy with a platinum-based chemotherapeutic agent, wherein the additional prior course of therapy with the platinum-based agent is administered prior to the course of therapy with cetuximab.

In one embodiment, the carcinoma is HNSCC. HNSCC is a squamous cell or basaloid tumor that occurs in the head or neck region and includes tumors of the nasal cavity, nasopharynx, sinuses, lips, mouth and oral cavity, salivary glands, pharynx, hypopharynx or larynx. The treatments disclosed herein may be particularly useful, for example, in the treatment of oropharyngeal tumors, laryngeal tumors, oral cavity tumors and hypopharyngeal tumors. These tumors are routinely detected by oncology practitioners, such as physicians, medical oncologists, pathologists and otolaryngologists, and head and neck surgeons. Optionally, the HNSCC is non-metastatic HNSCC.

In any embodiments herein, unless the context indicates otherwise, head and neck cancer (e.g., HNSCC) may optionally be defined as locally recurrent, distantly metastatic, and/or may be considered incurable (and any combination thereof, such as, for example, locally recurrent, locally recurrent and distantly metastatic, locally recurrent and non-distantly metastatic, may further be considered incurable in any of the above cases.

In one exemplary aspect, a method is provided for halting, reversing, or reducing the progression of HNSCC in a mammalian host (e.g., a human patient) suffering from inoperable HNSCC cancer and in which the disease or cancer has progressed despite prior treatment with cetuximab alone or cetuximab combined with chemotherapy or with radiation therapy, the method comprising administering to the patient an anti-NKG2A agent (e.g., an anti-NKG2A antibody), an anti-NKG2A antibody composition, or a corresponding composition (e.g., an anti-NKG2A antibody composition), or a corresponding composition (e.g., an anti-NKG2A antibody composition), to the patient.

- 18 047799 example, nucleic acid encoding an antibody to NKG2A) in an amount sufficient to detectably reduce the progression of HNSCC in the host.

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

In any embodiment herein, a disease, cancer, or HNSCC that progresses despite prior treatment with cetuximab may refer to a disease, cancer, or HNSCC that is resistant to cetuximab. An individual who has a cancer or HNSCC that progresses despite prior treatment with cetuximab may be referred to as an individual who is (or whose disease, cancer, or HNSCC is) resistant to cetuximab.

In one embodiment, an agent directed against NKG2A is administered in combination with cetuximab. A cancer that is resistant or, for example, has progressed may optionally be characterized as a tumor resistant to treatment with cetuximab, rather than, for example, a new primary tumor. A tumor resistant to cetuximab may, for example, arise in the same area and during the course of treatment with cetuximab or in a limited period after the end of treatment with cetuximab (e.g., 1, 2 or 3 years or less, such as 3, 4, 6, 9 months).

In one embodiment, compositions are provided for use in treating a disease, such as cancer, that is resistant to cetuximab. In some embodiments, an agent that neutralizes human NKG2A inhibitory activity and/or cetuximab is provided for use in treating HNSCC in an individual whose HNSCC cancer is resistant to treatment (e.g., prior treatment) with cetuximab (e.g., alone with cetuximab or in combination with another agent such as chemotherapy or radiation therapy). In one embodiment, an agent that neutralizes human NKG2A inhibitory activity is provided for use in treating HNSCC in a human individual who has received prior treatment with cetuximab, wherein the treatment comprises administering to the individual an effective amount of each of: (a) an agent, optionally an antibody, that neutralizes human NKG2A inhibitory activity, and (b) cetuximab. In one embodiment, the cancer or carcinoma is HNSCC.

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

In one aspect, the use of cetuximab and/or an agent that neutralizes the inhibitory activity of NKG2A causes a decrease in tumor burden, optionally causes a decrease in the sum of the diameters of the lesions of the target cell compared to the initial sum of the diameters. In one embodiment, the use of cetuximab and/or an agent that neutralizes the inhibitory activity of NKG2A is for slowing the progression of cancer. In one embodiment, the use of cetuximab and/or an agent that neutralizes the inhibitory activity of NKG2A is for slowing or preventing metastasis in cancer.

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

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

In one embodiment, the anti-NKG2A agent is administered in an amount effective to saturate NKG2A receptors on lymphocytes for at least one week, two weeks, three weeks, or four weeks. In some embodiments, the dose (e.g.,

- 19 047799 each dose) antibodies to NKG2A are administered in an amount of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg.

In one aspect of any embodiment herein, the anti-NKG2A antibody is administered once about every two or four weeks.

Delivery of an anti-NKG2A antibody to an individual (either by direct administration or expression from a nucleic acid therein, such as from a poxvirus gene transfer vector containing a nucleic acid sequence(s) encoding an anti-NKG2A antibody) and the use of other methods herein may be used to reduce, treat, prevent or otherwise ameliorate any suitable aspect of cancer progression (particularly the progression of HNSCC). The methods disclosed herein may be particularly useful in reducing and/or decreasing tumor growth, tumor cell count and any parameter or symptom associated therewith (e.g. biomarkers). The methods disclosed herein may be particularly useful in preventing tumor recurrence, increasing progression-free survival, preventing metastasis. Methods that reduce, prevent or otherwise ameliorate the severity of such aspects of cancer progression, independently and collectively, are advantageous elements.

In another aspect, there is provided a method for reducing the risk of cancer progression, reducing the risk of further cancer progression in a cell population that has been stimulated, and/or providing a therapeutic regimen for reducing cancer progression in a human patient. In a further aspect, there is provided a method for increasing the likelihood of survival for an appropriate period in a human patient diagnosed with HNSCC. In another aspect, there is provided a method for improving the quality of life of patients suffering from HNSCC, comprising administering to the patient a composition in an amount effective to improve their quality of life. In a further aspect, the methods described herein can be used to significantly reduce tumor size or tumor burden. In a further aspect, the methods described herein can be used to significantly reduce the number of HNSCC cells in a vertebrate host animal, such that, for example, the total number of HNSCC cells is reduced. In this regard, there is provided a method for killing (e.g., directly or indirectly causing death) of HNSCC cells in a vertebrate, such as a human cancer patient.

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

The NKG2A neutralizing agent (e.g., an anti-NKG2A antibody) disclosed herein can be used to treat an individual in combination with cetuximab. Thus, if HNSCC has progressed during or after a prior cetuximab-based regimen (the prior cetuximab regimen does not include combination therapy with an NKG2A neutralizing agent), the individual (e.g., with inoperable, optionally non-metastatic cancer) can be treated with a combination of an NKG2A neutralizing agent and cetuximab. The prior cetuximab regimen can include one or more additional agents or therapies, in particular, radiation therapy and/or chemotherapy.

In some cases, prior to a prior course of cetuximab (either as monotherapy or optionally in combination with radiation therapy and/or chemotherapy), an individual will have received an even earlier course of treatment with a platinum-based chemotherapeutic agent; for example, an individual received a course of treatment with a platinum-based agent followed by a course of treatment with cetuximab. In these circumstances, the individual may have experienced cancer progression despite treatment with a platinum-based agent and then received subsequent treatment with cetuximab (a prior course of cetuximab), with the cetuximab optionally administered in combination with radiation therapy and/or a chemotherapeutic agent. If the individual then experiences cancer progression despite treatment with a prior course of cetuximab, the individual may be treated with a combination of an NKG2A neutralizing agent and cetuximab.

The combined administration of an NKG2A neutralizing agent and cetuximab includes the simultaneous administration of the compound in identical or different dosage forms or separate administration of the compounds (e.g., sequential administration). Thus, the NKG2A neutralizing agent and cetuximab may be defined as being formulated for separate administration and administered simultaneously or sequentially. The treatment may optionally be combined with the administration of one or more additional therapeutic agents. In one embodiment, the treatment course with an NKG2A neutralizing agent comprises an NKG2A neutralizing agent and cetuximab without other therapeutic (e.g., anticancer) agents. In another embodiment, the treatment course with an NKG2A neutralizing agent comprises an NKG2A neutralizing agent, cetuximab, and another additional therapeutic (e.g., anticancer) agent.

- 20 047799 remedy.

Additional or combined administration of an anti-NKG2A agent and cetuximab, as used herein, includes simultaneous administration of the compounds in identical or different dosage forms, or separate administration of the compounds (e.g., sequential administration). Thus, the anti-NKG2A agent and cetuximab can be administered simultaneously in a single formulation. Alternatively, the anti-NKG2A agent and cetuximab can be formulated for separate administration and administered simultaneously or sequentially.

In the treatment methods, 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 is administered. For example, the anti-NKG2A antibody can be administered from about 0 to 30 days before cetuximab is administered. 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 is administered. In some embodiments, the anti-NKG2A antibody is administered simultaneously with cetuximab. In some embodiments, the anti-NKG2A antibody is administered after cetuximab is administered. For example, the anti-NKG2A antibody can be administered from about 0 to 30 days after cetuximab is administered. 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 is administered.

Suitable treatment protocols for treating a human patient with 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 administration cycle in which at least one dose of the NKG2A antibody is administered at a dose of 1-10 mg/kg body weight (e.g. every two weeks) and at least one dose, optionally at least two doses of cetuximab are administered, optionally wherein cetuximab is administered at a dose of 250 mg/ ^m2 weekly, optionally wherein cetuximab is administered at a dose of 400 mg/ ^m2 as an initial dose, followed by at least one dose of 250 mg/m2 weekly. In any embodiment herein, each dose of the NKG2A antibody may be administered in a fixed dose, such as a fixed dose of 100 to 1000 mg, optionally 200 to 1200 mg, such as 750 mg.

In one embodiment, the method comprises at least one period of administration (e.g., a cycle of administration), wherein the cycle or period is eight weeks (or less), wherein for each of at least one period, two, three, or four doses of the anti-NKG2A antibody are administered at a dose of 1-10 mg/kg body weight (e.g., a fixed dose of 100-1000 mg, optionally 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 at a dose of 250 mg/ ^m2 . Optionally, the cycle comprises a loading dose of cetuximab; i.e., The first dose in the cycle consists of one initial dose of cetuximab at a dose of 400 mg/ ^m2 and a subsequent dose of cetuximab at a dose of 250 mg/ ^m2 .

The NKG2A antibody may advantageously be administered in an amount that achieves a concentration in the bloodstream that is at least 10, 20, or 30 times higher than the concentration required for substantially complete (e.g., 90%, 95%) saturation of the receptor (e.g., as assessed by titration of NKG2A antibodies on NKG2A-expressing cells, such as in PBMCs), or optionally in an amount that achieves a concentration in extravascular tissue (e.g., tumor tissue or the environment) that is at least 10, 20, or 30 times higher than the concentration required for substantially complete saturation of the receptor (e.g., as assessed by titration of NKG2A antibodies on NKG2A-expressing cells, such as in PBMCs).

The NKG2A+ NK cell response can be assessed using a suitable assay for the cytotoxic activity of NK cells expressing NKG2A against HLA-E expressing target cells. Examples include assays based on markers of NK cell activation, such as CD107 or CD137 expression. Advantageously, the amount of anti-NKG2A antibody can be administered in such a way as to achieve and/or maintain a constant (trough) tissue concentration of at least 10 μg/mL. For example, the blood concentration that must be achieved and/or maintained to achieve/maintain 10 mcg/mL in tissue may be 100-110 mcg/mL, 100-120 mcg/mL, 100-130 mcg/mL, 100-140 mcg/mL, 100-150 mcg/mL, 100-200 mcg/mL, 100-250 mcg/mL, or 100-300 mcg/mL.

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Illustrative treatment protocols for an anti-NKG2A antibody, such as humZ270 (e.g., monalizumab), as used in the examples herein, characterized by an EC ₁₀₀ for an NKG2A+ NK cell response of 1-10 μg/mL, such as about 10 μg/mL, comprise at least one administration cycle in which at least one dose of the anti-NKG2A antibody is administered at a dose of about 10 mg/kg, optionally 2-10 mg/kg, optionally 4-10 mg/kg, optionally 6-10 mg/kg, optionally 2-6 mg/kg, optionally 2-8 mg/kg, or optionally 2-4 mg/kg body weight, optionally a fixed dose of 100 to 1000 mg, optionally 200 to 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 administration cycle is from 2 weeks to 8 weeks. In one embodiment, the administration cycle is 8 weeks. In one embodiment, the administration cycle is 8 weeks and includes administering one dose of the anti-NKG2A antibody every two weeks (i.e., a total of four doses).

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

Exemplary treatment protocols for use with an anti-NKG2A antibody include, for example, administering an anti-NKG2A antibody to a patient twice a month and an amount effective to maintain a constant blood concentration of at least 40 μg/mL of the anti-NKG2A antibody between at least two subsequent administrations of the anti-NKG2A antibody of 2 to 10 mg/kg, optionally 2-6 mg/kg, optionally 2-4 mg/kg, optionally about 4 mg/kg body weight, or optionally a fixed dose in the range of 100-1000 mg, optionally in the range of 200-1200 mg, such as 750 mg. Such doses can optionally be administered in a manner that provides a constant blood concentration of at least 40 μg/mL of the anti-NKG2A antibody throughout the treatment cycle. Achieving a blood concentration of 40 μg/mL of an anti-NKG2A antibody is expected to provide a tissue concentration (e.g., extravascular tissue, tumor environment) of approximately 4 μg/mL, providing at least an EC ₅₀ for an antibody such as humanized Z270 (e.g., monalizumab).

Illustrative treatment protocols for use with an anti-NKG2A antibody include, for example, administering to a patient an effective amount of an anti-NKG2A antibody, wherein the antibody is administered twice a month, and an amount effective to maintain a constant blood concentration of the anti-NKG2A antibody of at least 100 μg/mL between at least two subsequent administrations of the anti-NKG2A antibody, is from 4-10 mg/kg, optionally 4-6 mg/kg, optionally 4-8 mg/kg, optionally about 4 mg/kg, optionally about 6 mg/kg, optionally about 8 mg/kg, optionally about 10 mg/kg body weight, or optionally a fixed dose in the range of 100-1000 mg, optionally in the range of 200-1200 mg, such as 750 mg. Such doses may optionally be administered in a manner that provides a constant blood concentration of the NKG2A antibody of at least 100 μg/mL throughout the treatment cycle. Achieving a blood concentration of 100 μg/mL of the NKG2A antibody is expected to provide a tissue concentration (e.g., extravascular tissue, tumor environment) of approximately 10 μg/mL, which in turn corresponds to at least an EC ₁₀₀ for an antibody such as humanized Z270.

A patient suffering from head and neck cancer that is progressive or has not responded sufficiently to prior or subsequent treatment with cetuximab may be treated with an NKG2A neutralizing agent without a prior detection step to assess HLA-E expression on the surface of tumor cells. Thus, the treatment methods may optionally include a step of detecting an HLA-E nucleic acid or polypeptide in a tumor biological sample (e.g., on a tumor cell) from the individual. Determining that a biological sample expresses HLA-E (e.g., expresses HLA-E at a detectable level, expresses HLA-E at least at a predetermined level, expresses HLA-E significantly, expresses HLA-E at a high level or with high intensity of staining with an antibody to HLA-E, in each case optionally compared to a reference) can be used to designate a patient as suffering from head and neck cancer who may receive a particularly strong benefit from treatment with an agent that neutralizes NKG2A activity. In one embodiment, the method includes determining the level of expression of an HLA-E nucleic acid or polypeptide in a biological sample and comparing the level with a reference level (e.g., a value, intense cell surface staining, etc.) corresponding to an individual who benefits from treatment with an agent that inhibits, neutralizes NKG2A activity. Determining that a biological sample expresses an HLA-E nucleic acid or polypeptide at a level that meets and/or increases the reference level indicates that an individual who may benefit particularly from treatment with an agent that inhibits, neutralizes NKG2A activity has head and neck cancer. Optionally, detecting an HLA-E polypeptide in a biological sample includes detecting an HLA-E polypeptide expressed on the surface of a malignant HNSCC cell. In one embodiment, determining that a biological sample significantly expresses an HLA-E nucleic acid or polypeptide indicates

- 22 047799 that an individual who may benefit particularly from treatment with an NKG2A activity-neutralizing agent suffers from head and neck cancer. Significantly expressed with respect to an HLA-E polypeptide means that the HLA-E polypeptide is expressed in a significant number of tumor cells taken from the patient. Although the definition of the term expressed is not limited to a precise percentage, in some examples, a receptor that is considered to be significantly expressed will be present on at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the HNSCC cells taken from the patient.

Determining whether an individual has head and neck cancer cells that express an HLA-E polypeptide may, for example, include obtaining a biological sample (e.g., by performing a biopsy) from an individual who has head and neck cancer cells, contacting the cells with an antibody that binds an HLA-E polypeptide, and determining 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 immunohistochemistry assay. Optionally, determining whether an individual has head and neck cancer cells that express HLA-E includes performing an assay using flow cytometry.

A patient suffering from head and neck cancer, in particular HNSCC, can further be treated with agents directed against NKG2A, with or without a prior detection step to assess whether the patient (or the patient's tumor) is HPV-positive. In some embodiments, the individual treated with the methods of the present invention is HPV-positive. In some embodiments, the individual treated with the methods of the present invention is HPV-negative.

Examples

Example 1. A case of response treatment of HNSCC with repeated injections of monalizumab in combination with cetuximab in a patient resistant to cetuximab.

A phase A 1b/2 study of IPH2201 and cetuximab was conducted in patients with human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma. Although cetuximab is approved for the treatment of HNSCC following platinum-based therapy, it has limited activity in this setting (response rate of 12%). The clinical trial evaluated the efficacy of monalizumab in combination with cetuximab in HNSCC. Monalizumab (see WHO Drug Information Vol. 30, No. 1, 2016), also referred to as IPH2201, is a neutralizing antibody to NKG2A having the heavy chain amino acid sequence shown as SEQ ID NO: 2 and the light chain amino acid sequence shown as SEQ ID NO: 7.

The inclusion criteria were as follows.

Age >18 years.

1. Histologically or cytologically confirmed HPV (+) or HPV (-) status of squamous cell carcinoma of the nasopharynx (type 1 according to the WHO classification), oropharynx, hypopharynx, larynx (epiglottis, glottis, subglottis) or oral cavity.

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

3. Progression after chemotherapy with a platinum-based drug.

4. For Phase Ib only: Pretreated patients who are not eligible for further treatment with curative intent. This part is open to pretreated patients, regardless of the number of prior lines of treatment.

For Phase II only: patients who have received no more than two prior systemic regimens for relapsed and/or metastatic disease and are refractory to further therapy with curative intent.

5. No previous treatment with cetuximab, except when it is indicated for primary treatment (locally advanced disease) in the absence of disease progression for at least 4 months after completion of previous treatment with cetuximab.

6. Recovery from previous surgery and recovery from adverse effects to grade 1 or less (except alopecia) due to previous radiation therapy and any systemic therapy.

7. The performance status index according to the Eastern Cooperative Oncology Group (ECOG) is 0-1.

8. Life expectancy >3 months.

9. Patients with treated brain metastases are eligible if >4 weeks have passed since completion of therapy (including radiotherapy and/or surgery), if they are clinically stable at study entry and not receiving corticosteroid therapy at study entry.

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10. Adequate hematological, immunological, liver and renal function defined as hemoglobin >9.0 g/dL;

absolute neutrophil count >1500/mm3;

platelets >100000/mm3;

total bilirubin <1.5 X established upper limit of normal (UNL);

aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <2.5 X;

established UNL, serum creatinine level <1.5 X established UNL or estimated (using Cockcroft-Gault formula) or measured creatinine clearance >50 ml/min.

11. A negative serum pregnancy test within 72 hours prior to study treatment for women of childbearing potential. Women of childbearing potential and all men must agree to use appropriate contraception (hormonal or barrier method of contraception; abstinence) prior to study entry, while receiving monalizumab, and for 5 months after the last dose of monalizumab.

12. Ability to understand a written informed consent document.

13. Signed informed consent prior to any procedures related to the protocol.

The exclusion criteria were as follows.

1. For Phase II only: patients who have previously received more than 2 systemic treatment regimens for recurrent and/or metastatic disease (without restrictions in the Phase Ib portion of the study).

2. For Phase II only: Patients receiving cetuximab or another epidermal growth factor receptor inhibitor are excluded from the Phase II study unless cetuximab was administered as part of the primary treatment approach in the absence of disease progression for at least 4 months after completion of prior cetuximab treatment.

3. History of allergic reactions associated with compounds similar in chemical or biological composition to cetuximab.

4. Patients with known, untreated, and uncontrolled brain metastases are excluded. However, brain imaging is not required for selection if the patient has no neurological signs or symptoms.

5. Serious concomitant uncontrolled medical disorder.

6. An autoimmune disease that:

1) currently or previously required systemic immunosuppressive or immunomodulatory therapy (including systemically administered corticosteroids), and/or

2) has a significant potential to cause irreversible damage to any tissue, and/or

3) was diagnosed less than 3 months prior to study inclusion, and/or

4) clinically unstable, and/or

5) is characterized by a significant risk of progression and causing serious complications.

7. Abnormal cardiac status with any of the following.

1) Unstable angina.

2) Arrhythmia requiring treatment that is not stabilized by treatment.

3) QTc> 450 ms (M) or 470 ms (F) (Bazett formula - QT/f interval (RR interval), where RR interval = 60/HR).

8. History of cardiac dysfunction, including any of the following.

1) Myocardial infarction within the last 6 months.

2) Documented history of congestive heart failure (stages III-IV according to the New York Heart Association Functional Classification).

9. Known interstitial lung disease.

10. Pregnant women are excluded from this study; breastfeeding should be discontinued.

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

12. Treatment with other investigational agents less than 14 days prior to study inclusion.

13. Systemic treatment with steroids or other immunosuppressants within 30 days prior to study inclusion. Physiological replacement with hydrocortisone or its equivalent is allowed.

14. Current active infectious disease.

15. Positive serology for HIV.

16. Positive HBs-Ag or positive HBV viremia, Positive HCV viremia.

17. Psychological, family, sociological or geographical conditions that do not allow for medical supervision and compliance with the research protocol.

The trial included a dose escalation portion in which patients received escalating dose levels of monalizumab of 0.4, 1, 2, 4, or 10 mg/kg every two weeks in combination with

- 24,047,799 fixed-dose cetuximab (400 mg/m2 loading dose, then 250 mg/m2 weekly) in a 3+3 design. The expansion cohort used monalizumab at the highest dose tested, 10 mg/kg, and included a futility analysis after the first 11 patients. Response rates were assessed by RECIST every 8 weeks. Patients were treated until disease progression or unacceptable toxicity. The trial is ongoing to enroll additional patients and evaluate duration of response, progression-free survival, and overall survival.

Among the observed initial partial responses was a patient with a history of squamous cell carcinoma of the right alveolar process of the mandible, stage T4aN2bM0/IVA, who underwent surgical resection with right posterior lateral mandibulectomy with fibular free flap reconstruction followed by adjuvant irradiation. The patient had a recurrent squamous cell carcinoma of the oral cavity (which is not a new primary cancer), p16-negative, that was unresectable. The patient had previously received 3 cycles of cisplatin-taxotere-5-FU (TPF) induction (replacing cisplatin with carboplatin) followed by concurrent chemoradiation with weekly cetuximab. Approximately 4 months after completing the cetuximab course, the patient's cancer recurred with PET/CT. The patient was considered resistant to cetuximab because the recurrence occurred just over 4 months after completion of cetuximab plus definitive radiotherapy, the oral cancer recurrence occurred within three years (and was thus not considered a new primary cancer) and was in the same site, in this case all on the right side. The patient was recommended for the phase 1b/2 study of IPH2201 and cetuximab. Treatment with monalizumab and cetuximab according to protocol resulted in an objective response (PR), with a best response being a 50% reduction in target lesions on treatment compared with baseline (before treatment). The lesion score is the sum of the longest diameters or the shortest axes in the case of lymph nodes.

Also among the observed initial partial responses was a patient with stage TxN2bMMVA, right-sided laterality, oropharyngeal squamous cell carcinoma. The patient’s prior treatments included chemotherapy (cisplatin/docetaxel/5FU) for initial disease followed by radiation. With progressive disease, the patient was treated for recurrent/metastatic disease with surgery to the initial site and lymph nodes followed by chemotherapy (cisplatin/carboplatin/SFU) then cetuximab. The patient was found to have progressive disease (as best response) after treatment with cetuximab, after which the patient was treated with paclitaxel and carboplatin. As the patient’s disease progressed, the patient was enrolled in a phase 1b/2 study of IPH2201 and cetuximab. Treatment with monalizumab and cetuximab according to protocol resulted in response (PR).

The finding that a neutralizing antibody to NKG2A in combination with cetuximab can provide significant reduction in the intensity of HNSCC in patients who have been found to be resistant to cetuximab provides an opportunity for the large population of patients with HNSCC who have unresectable cancer and whose cancer progresses despite treatment with cetuximab, particularly in combination with radiation therapy. The treatment may be useful in preventing further progression, particularly in delaying or preventing metastatic cancer.

All references, including publications, patent applications and patents, cited in this document are herein incorporated by reference in their entirety and to the same extent as if each document was specifically and individually indicated to be incorporated by reference and was provided in its entirety herein (to the maximum extent permitted by law), notwithstanding any special incorporation of specific documents made elsewhere herein.

The use of the terms "a" and "an" and similar definitions in the context of describing the present invention shall be construed as including both the singular and the plural unless otherwise indicated herein or otherwise clearly contradicted by context.

Unless otherwise stated, all exact values presented in this document are illustrative of the corresponding approximate values (e.g., all exact illustrative values provided with respect to a particular factor or measurement may also be considered to provide the corresponding approximate measurement, modified to approximately where appropriate). When approximately is used in connection with a number, it may be stated as including values corresponding to ±10% of the stated number.

A description herein of any aspect or embodiment of the invention using terms such as comprising, having, including, or containing with reference to an element or elements is intended to support a similar aspect or embodiment of the invention that consists of, consists essentially of, or essentially includes that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should also be understood as describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

- 25 047799 context).

The use of all possible examples or illustrative phrases (e.g., such as) provided herein is intended solely to better illuminate the present invention and not to formulate a limitation on the scope of the present invention unless otherwise stated. No phrase in this specification should be construed as indicating that any unreported element is essential to the practice of the present invention.

Claims (16)

1. The use of an antibody that neutralizes the inhibitory activity of human NKG2A in the treatment of cancer in an individual who is a human with inoperable HNSCC, wherein the antibody is an anti-NKG2A antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain having the sequence set forth in SEQ ID NO: 2 and the CDR1, CDR2, and CDR3 domains of a light chain having the sequence set forth in SEQ ID NO: 7, wherein the treatment comprises administering to the individual an effective amount of each of: (a) an antibody that neutralizes the inhibitory activity of human NKG2A, and (b) cetuximab, wherein the individual has HNSCC cancer that has progressed despite prior treatment with cetuximab in combination with radiation therapy. 2. The use according to claim 1, wherein the treatment of HNSCC in an individual comprises a) determining whether an 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 any one of claims 1, 2, wherein the treatment of HNSCC in an individual comprises a) determining whether the HLA-E polypeptide is expressed by malignant cells of 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 from an individual a biological sample that contains HNSCC cells, contacting said cells with an antibody that binds the HLA-E polypeptide, and identifying cells that express HLA-E. 5. The use according to any one of claims 1 to 4, wherein the antibody to NKG2A is characterized by a decrease in binding to a mutant NKG2A polypeptide having the substitutions K199A/D202A/V213S/R215A/K217A (with reference to SEQ ID NO: 1), compared to the wild-type NKG2A polypeptide (SEQ ID NO: 1). 6. The use according to any one of claims 1 to 5, wherein the antibody to NKG2A comprises a constant region of human IgG4. 7. The use according to any one of claims 1 to 6, wherein the antibody to NKG2A comprises an Fc-engineered constant region comprising an amino acid modification that reduces binding to the human Fcy receptor. 8. The use according to any one of claims 1 to 7, wherein the antibody to NKG2A competes with antibodies having heavy and light chains comprising SEQ ID NO: 2 and 7, respectively, for binding to human NKG2A. 9. The use according to any one of claims 1 to 8, wherein the antibody to NKG2A comprises a heavy chain having the sequence presented under SEQ ID NO: 2 and a light chain having the sequence presented under SEQ ID NO: 7. 10. The use according to any one of claims 1 to 9, wherein the antibody to NKG2A is administered in the form of a pharmaceutically acceptable composition containing a therapeutically effective amount of the antibody to NKG2A. 11. Use according to claim 10, wherein the composition does not contain any other pharmaceutically active agents. 12. The use according to any one of claims 1 to 11, wherein the antibody to NKG2A is administered several times with a frequency of administration of doses from once every week to once every month. 13. Use according to any one of claims 1 to 12, wherein cetuximab is administered weekly. 14. The use according to any one of claims 1 to 13, wherein the treatment comprises at least one cycle of administration, wherein the agent that neutralizes the inhibitory activity of human NKG2A is administered every two weeks, and cetuximab is administered weekly. 15. The use according to any one of claims 1 to 14, wherein the treatment comprises at least one period of two weeks, wherein for each of at least one period the antibody that neutralizes human NKG2A inhibitory activity is administered at a dose of 1-10 mg/kg body weight, and cetuximab is administered at an initial dose of 400 mg/ ^m2 and then weekly at 250 mg/ ^m2 16. The use according to any one of claims 1 to 15, wherein the treatment comprises at least one period of two weeks, wherein for each of the at least one period the antibody that - 26 047799 neutralizes the inhibitory activity of human NKG2A, is administered at a fixed dose of 100 to 1000 mg, optionally 750 mg, and cetuximab is administered at an initial dose of 400 mg/ ^m2 and then weekly at 250 mg/ ^m2 .