WO2021096970A1 - Natural killer cell therapies - Google Patents

Abstract

The instant technology generally relates to methods and compositions for treating cancer in a patient by treating the patient with natural killer (NK) cells. For example, the NK cell treatment may restore or induce responsiveness of a cancer/tumor to a checkpoint inhibitor.

Description

NATURAL KILLER CELL THERAPIES

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 62/934,394, filed November 12, 2019, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The field of this technology is immunotherapy for treating cancer, in particular treatment with natural killer (NK) cells, which may restore or induce responsiveness of a cancer/tumor to a checkpoint inhibitor.

BACKGROUND

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] Immune checkpoint inhibitors have been a major advance in cancer treatment. Although treatment with immune checkpoint inhibitors has been demonstrated to generate significant clinical benefit for some cancers in some subjects, many subjects do not clinically respond to such inhibition. For example, metastatic Merkel cell carcinoma (MCC) is associated with high response rates to PD-1/PD-L1 blockade, yet about 50% of patients are refractory to treatment.

[0006] There remains a need to improve responsiveness to immunotherapies, including immune checkpoint inhibitors, for treatment of cancer. SUMMARY

[0007] The instant technology generally relates to methods and compositions for treating cancer in a patient.

[0008] In an aspect, a method of preparing a subject having cancer for treatment with a checkpoint inhibitor is provided.

[0009] In an aspect, a method of treating cancer in a subject is provided.

[0010] In embodiments, the method may include administering to the subject an effective amount of activated natural killer (aNK) cells, wherein the cancer is refractory to checkpoint inhibitor treatment prior to administration of the aNK cells.

[0011] In embodiments, the method may include (a) providing or identifying a subject having a cancer that is refractory to checkpoint inhibitor treatment; and (b) administering to the subject an effective amount of activated natural killer (aNK) cells to potentiate the activity of the checkpoint inhibitor.

[0012] In embodiments, the method may include administering N-803 to the subject. In embodiments, the N-803 can be administered concurrently with the aNK cells.

[0013] In embodiments, a checkpoint inhibitor may be administered to the subject. In embodiments, the checkpoint inhibitor may be administered after administration of the aNK cells.

[0014] In embodiments, the checkpoint inhibitor may not be administered concurrently with the aNK cells. For example, the checkpoint inhibitor may be administered some time after the aNK treatment (e.g., aNK treatment cycle) ends. In embodiments, the checkpoint inhibitor may be administered between 1 week and 5 years after the aNK treatment (e.g., aNK treatment cycle) ends.

[0015] In embodiments, the cancer may be a skin cancer. In embodiments, the skin cancer may be Merkel cell carcinoma.

[0016] In embodiments, the checkpoint inhibitor may be a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In embodiments, the checkpoint inhibitor may be a therapeutic antibody. In embodiments, the checkpoint inhibitor may be ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, or spartalizumab. In embodiments, the checkpoint inhibitor is pembrolizumab. In embodiments, the checkpoint inhibitor is avelumab.

[0017] In embodiments, the aNK cells can be allogeneic to the subject. In embodiments, the aNK cells can be autologous to the subject.

[0018] In embodiments, the subject may have been treated with a checkpoint inhibitor before administration of the aNK cells. The checkpoint inhibitor can be the same checkpoint inhibitor that is administered to (or intended to be administered to) the subject after administration of the aNK cell. Alternatively, the checkpoint inhibitor can be a different checkpoint inhibitor that is administered to (or intended to be administered to) the subject after administration of the aNK cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 illustrates 2 week treatment cycles from a Phase 2 study of aNK (Activated NK-92, natural killer cells) infusions in combination with ALT-803 (IL-15) in patients with Stage III (IIIB) or Stage IV Merkel Cell Carcinoma (MCC).

[0020] FIG. 2 shows a patient (Patient 02-02) during the first consultation on October, 2014.

[0021] FIG. 3 shows Patient 02-02 in January 2015 (left) and April 2015 (right). As shown in the figure, the patient showed no response to pembrolizumab despite high PDL-1 expression.

[0022] FIG. 4 shows Patient 02-02 in July 2015 (top) and September 2015 (bottom). As shown in the figure, the patient showed robust response to salvage neutron radiation therapy.

[0023] FIG. 5 shows Patient 02-02 in March 2016 (left) and September 2016 (right). As shown in the figure, the patient showed radiologic complete response (CR) in response to aNK monotherapy.

[0024] FIG. 6 shows Patient 02-02 in September 2016 (left) and July 2019 (right). As shown in the figure, pembrolizumab re-challenge has resulted in an ongoing CR. [0025] FIG. 7 shows immune cell infiltration in the TME is increased after aNK monotherapy.

DETAILED DESCRIPTION

[0026] After reading this description it will become apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure as set forth herein.

[0027] Before the present technology is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0028] The detailed description divided into various sections only for the reader’s convenience and disclosure found in any section may be combined with that in another section. Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present disclosure.

Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. [0031] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0032] The term “about” when used before a numerical designation, e.g ., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” when used with regard to a dose amount means that the dose may vary by +/- 10%.

[0033] “Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0034] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody plays a significant role in determining the specificity and affinity of binding. In some embodiments, antibodies or fragments of antibodies may be derived from different organisms, including humans, mice, rats, hamsters, camels, etc. Antibodies may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (e.g. glycosylation, expression, antigen recognition, effector functions, antigen binding, specificity, etc.). [0035] Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable ("V") region involved in binding the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3 -dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Rabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

[0036] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0037] As used herein, the terms “Fc domain” or “fragment crystallizable domain” are used in accordance with their plain and ordinary meanings and refer to any of the recombinant or naturally-occurring forms of the “base” or tail-end region (C-terminal) of an antibody. The Fc domain is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. The Fc region is comprised of two heavy chain constant Ig domains in the antibodies IgG, IgA, and IgD, and of three heavy chain constant Ig domains in the antibodies IgE and IgM.

[0038] The term "Fc" refers to a non-antigen-binding fragment of an antibody. Such an "Fc" can be in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins. In embodiments, the Fc is an IgGl or IgG2 Fc. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0039] In embodiments, the term "Fc" refers to a molecule or sequence that is modified from a native Fc, but still comprises a binding site for a receptor. As with modified Fc and native Fc's, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by recombinant gene expression or by other means.

[0040] Antibodies exist, for example, as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially the antigen binding portion with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0041] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. [0042] A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0043] A “protein complex” or “complex” as used herein refers to two or more polypeptides that assoicate simultaneously. The complexes may be constructed through binding between proteins and/or binding between receptors and ligands. The proteins may be associated through non-covalent protein-protein interactions, though certain polypeptides in the complex may also be covalently linked directly or indirectly through, for example, a chemical linker, a bond or another protein.

[0044] As used herein, the term "cancer" refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and Non- Hodgkin's Lymphomas. Exemplary cancers that may be treated with a compound or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0045] The term "leukemia" refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease- acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0046] As used herein, the term “lymphoma” refers to a group of cancers affecting hematopoietic and lymphoid tissues. It begins in lymphocytes, the blood cells that are found primarily in lymph nodes, spleen, thymus, and bone marrow. Two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin’s disease. Hodgkin’s disease represents approximately 15% of all diagnosed lymphomas. This is a cancer associated with Reed-

Stemberg malignant B lymphocytes. Non-Hodgkin’s lymphomas (NHL) can be classified based on the rate at which cancer grows and the type of cells involved. There are aggressive

(high grade) and indolent (low grade) types of NHL. Based on the type of cells involved, there are B-cell and T-cell NHLs. Exemplary B-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, small lymphocytic lymphoma, Mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large cell B-lymphoma, Burkitt’s lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B-lymphoblastic lymphoma. Exemplary T- cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, cunateous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.

[0047] The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

[0048] The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding- Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0049] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky- cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.

[0050] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. “Metastatic cancer” is also called “Stage IV cancer.” Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0051] The terms “treating”, or “treatment” refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. The term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing.

[0052] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or a combination thereof.

[0053] Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, the treating or treatment is no prophylactic treatment.

[0054] As used herein, the term “modulator” refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule. The term “modulate” is used in accordance with its plain ordinary meaning and refers to the act of changing or varying one or more properties. “Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule.

[0055] The terms “immune response” and the like refer, in the usual and customary sense, to a response by an organism that protects against disease. The response can be mounted by the innate immune system or by the adaptive immune system, as well known in the art.

[0056] The terms “modulating immune response” and the like refer to a change in the immune response of a subject as a consequence of administration of an agent, e.g., a compound or composition as disclosed herein, including embodiments thereof. Accordingly, an immune response can be activated or deactivated as a consequence of administration of an agent, e.g., a compound or composition as disclosed herein, including embodiments thereof. [0057] The terms “agonist”, “activator”, “upregulator”, etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.

[0058] The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist.

[0059] Likewise, an “immune checkpoint inhibitor” or “checkpoint inhibitor” as provided herein refers to a substance (e.g., an antibody or fragment thereof, a small molecule) that is capable of inhibiting, negatively affecting (e.g., decreasing) the activity or function of a checkpoint protein (e.g., decreasing expression or decreasing the activity of a checkpoint protein) relative to the activity or function of the checkpoint protein in the absence of the inhibitor. The checkpoint inhibitor may at least in part, partially or totally block stimulation, decrease, prevent, or delay activation, or inactivate, desensitize, or down-regulate signal transduction or enzymatic activity or the amount of a checkpoint protein. A "checkpoint inhibitor" may inhibit a checkpoint protein, e.g.,, by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity of the checkpoint protein. The inhibited expression or activity of the checkpoint protein can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control. In certain instances, the inhibitor reduces the activity of the checkpoint protein from an indirect or direct interaction. Non-limiting examples of checkpoint inhibitors include ipilimumab, pembrolizumab, nivolumab, talimogene laherparepvec, durvalumab, daclizumab, avelumab, and atezolizumab. [0060] Thus, a PD-1 inhibitor is a molecule that negatively affects (e.g., decreases) the activity or function of PD-1. In embodiments, the PD-1 inhibitor is pembrolizumab, nivolumab, or cemiplimab.

[0061] A PDL-1 inhibitor is a molecule that negatively affects (e.g., decreases) the activity or function of PDL-1. In embodiments, the PDL-1 inhibitor is atezolizumab, avelumab, or durvalumab.

[0062] A CTLA-4 inhibitor is a molecule that negatively affects (e.g., decreases) the activity or function of CTLA-4. In embodiments, the CTLA-4 inhibitor is ipilimumab or tremelimumab.

[0063] “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0064] According to the methods provided herein, the subject is administered an effective amount of one or more of the agents (e.g., a checkpoint inhibitor) provided herein. An “effective amount” is an amount sufficient for an active agent to accomplish a stated purpose relative to the absence of the active agent (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%,

15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. In embodiments, this increase or decrease for a given parameter may vary throughout the day (e.g. a peak percentage increase or decrease may differ from a percentage increase or decrease when therapeutic concentrations in circulating blood are at their peak or trough concentrations dependent on daily dosing patterns and individual pharmacokinetics). Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0065] For any compound described herein, the therapeutically effective amount can be initially determined from in vitro , e.g., cell culture, assays. Target concentrations will be those concentrations of active agent(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.

[0066] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0067] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5- fold, or more effect over a control.

[0068] Dosages may be varied depending upon the requirements of the patient and the active agent being employed. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered agent effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

[0069] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. In embodiments, the administering does not include administration of any active agent other than the recited active agent.

[0070] "Co-administer" it is meant that an active agent described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0071] As used herein, the term “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0072] As used herein, the term “allogeneic” refers to biological material from a subject that is genetically non-identical to biological material from another subject of the same species. For example, the term “allogeneic transplant” or “allogeneic transfusion” refers to the transfer of biological material to a recipient from a genetically non-identical donor of the same species. For example, a tissue or organ transplant may be an allogeneic transplant. An allogeneic transplant may include transfer of tissue, a group of cells or an organ to a recipient that is genetically non-identical to the donor. For example, the transplant may be a bone marrow transplant comprising stem cells from the donor, or islet cells from the donor.

[0073] A "cell" as used herein, refers to a cell carrying out metabolic or other functions sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect ( e.g ., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0074] As used herein, the terms “natural killer cells” and “NK cells” are used in accordance with their plain ordinary meaning and refer to a type of cytotoxic lymphocyte invol ved in the innate immune system. The role NK cells play is typically analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. Typically, immune cells detect major histocompatibility complex (MI 1C) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis. NK cells typically have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. [0075] As used herein, the terms ‘"activated NK-92 natural killer cells”, “aNK cells”, or “NK-92 cells” refers to cells of a cel! line having characteristics of natural killer cells and displaying cytotoxic properties to cancer cells. NK-92 cells are IL-2 dependent for optimal cytotoxic activity, and express activating receptors including NKp30, NKp46, 2B4, NKGD, and CD28 receptors. Further, NK-92 cells express molecules involved in the perforin- granzyme cytolytic pathway in addition to cytotoxic effector molecules including tumor necrosis factor (TNF)-superfamily members. Thus, aNK cells modulate the function of function of the immune system by secretion of cytokines, and are further are able to lyse target cells via secretion of enzymes (i.e. perforin and granzyme) and stimulation of apoptosis-initiation receptors. In contrast, NK-92 cells lack or express lower levels of inhibitory receptors expressed by normal NK cells. In embodiments, NK-92 cells are cells as described in U.S. Patent No. 7,618,817, which is incorporated herein by reference in its entirety.

[0076] The term “N-803” as provided herein refers to an IL-15 superagonist mutant, complexed to a dimeric IL-15RaSushi-Fc fusion protein. Alternative names include ALT- 803, and IL-15N72D:IL-15RaSu/Fc fusion protein complex. N-803 has antibacterial, antiviral, and antineoplastic properties. In embodiments, N-803 is the complex as described in U.S. Patent Pub. No. 2017/0088597 or Knudson et ak, J Immunother Cancer , 2019 Mar 21;7(1):82, each of which is incorporated herein by reference in its entirety.

[0077] The term "CD8" as referred to herein is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein, see ENTREZ No. 925 and UNIPROT No. P01732, which are incorporated by reference herein.

[0078] A "PD-1 protein" or "PD-1" as referred to herein includes any of the recombinant or naturally-occurring forms of the Programmed cell death protein 1 (PD-1) also known as cluster of differentiation 279 (CD 279) or variants or homologs thereof that maintain PD-1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PD-1 protein). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-1 protein. In embodiments, the PD-1 protein is substantially identical to the protein identified by the UniProt reference number Q15116 or a variant or homolog having substantial identity thereto. In embodiments, the PD-1 protein is substantially identical to the protein identified by the UniProt reference number Q02242 or a variant or homolog having substantial identity thereto.

[0079] A "PD-L1 " or “PD-L1 protein” as referred to herein includes any of the recombinant or naturally-occurring forms of programmed death ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD 274) or variants or homologs thereof that maintain PD- L1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PD-L1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-L1 protein. In embodiments, the PD-L1 protein is substantially identical to the protein identified by the UniProt reference number Q9NZQ7 or a variant or homolog having substantial identity thereto.

[0080] The term "CTLA-4" or "CTLA-4 protein" as provided herein includes any of the recombinant or naturally-occurring forms of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or variants or homologs thereof that maintain CTLA-4 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CTLA- 4). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CTLA-4 polypeptide. In embodiments, CTLA-4 is the protein as identified by the NCBI sequence reference GI: 83700231, homolog or functional fragment thereof.

[0081] “Sensitize” as used herein refers to increasing responsiveness to a certain treatment or stimulus. For example, administration of aNK cells may sensitize a subject’s responsiveness to treatment with a checkpoint inhibitor.

[0082] “Refractory” as used herein refers to a disease that does not respond to treatment. For example, the disease may be resistant to treatment, or may develop resistance during treatment.

[0083] “Complete Response” or “CR” as used herein refers to a disappearance of all disease markers, for example target lesions, and/or normalization of a tumor marker level of target and/or non-target lesions in a patient. [0084] “Partial Response” or “PR” as used herein refers to at least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD.

[0085] “Progressive disease” or “PD” as used herein refers to at least a 30% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the onset of treatment or since the appearance of one or more new lesions. PD may also be used to refer to the appearance of one or more new non-target lesions and/or unequivocal progression of existing non-target lesions.

[0086] “Stable disease” or “SD” as used herein refers to neither sufficient target lesion shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since onset of treatment. SD may also be used to refer to the persistence of one or more non-target lesions and/or maintenance of tumor marker levels above a normal limit, such as that of a healthy subject.

Methods

[0087] In an aspect, a method of preparing a subject having cancer for treatment with a checkpoint inhibitor is provided. In an aspect, a method of treating cancer in a subject is provided. In an aspect, a method for sensitizing a cancer to a checkpoint inhibitor is provided. In an aspect, a method for increasing sensitivity of a cancer to a checkpoint inhibitor is provided. In an aspect, a method for treating a subject having a cancer that is refractory to a checkpoint inhibitor is provided.

[0088] In embodiments, the method may include administering to the subject an effective amount of activated natural killer (aNK) cells. In embodiments, the cancer is refractory to checkpoint inhibitor treatment prior to administration of the aNK cells. For example, a subject who was previously treated with a checkpoint inhibitor may be administered aNK cells when the checkpoint inhibitor treatment did not have an effect on the cancer, or had an effect that was lost during treatment.

[0089] In embodiments, the method may include (a) providing or identifying a subject having a cancer that is refractory to checkpoint inhibitor treatment; and (b) administering to the subject an effective amount of activated natural killer (aNK) cells to potentiate the activity of the checkpoint inhibitor. For example, treatment with the aNK cells may result in increased sensitivity of the cancer to the checkpoint inhibitor. [0090] In embodiments, the effective amount of aNK cells may be between about lxlO⁷ cells per square meter of body surface area (cells/m²) and about 5xl0¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about 5xl0⁷ cells/m² and about 5xl0¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about lxlO⁸ cells/m² and about 5xl0¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about 5xl0⁸ cells/m² and about 5xl0¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about lxlO⁹ cells/m² and about 5xl0¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about 5xl0⁹ cells/m² and about 5xl0¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about lxlO⁷ cells/m² and about lxlO¹⁰ cells/m². In embodiments, the effective amount of aNK cells may be between about lxlO⁷ cells/m² and about 5xl0⁹ cells/m². In embodiments, the effective amount of aNK cells may be between about lxlO⁷ cells/m² and about lxlO⁹ cells/m². In embodiments, the effective amount of aNK cells may be between about lxlO⁷ cells/m² and about 5xl0⁸ cells/m². In embodiments, the effective amount of aNK cells may be about lxlO⁷ cells/m², about 5xl0⁷ cells/m², about lxlO⁸ cells/m², about 5xl0⁸ cells/m², about lxlO⁹ cells/m², about 5xl0⁹ cells/m², about lxlO¹⁰ cells/m², and about 5xl0¹⁰ cells/m². The amount may be any value or subrange within any range described herein, including endpoints.

[0091] In embodiments, the aNK cells may be administered between 1 and 7 days per week. In embodiments, the aNK cells may be administered one, two, three, four, five, six, or seven days per week. In embodiments, the aNK cells may be administered for a one-week, 2- week, 3-week, or 4-week treatment cycle. In embodiments, the aNK cells may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, 11 times, 12 times, 13 times, 14 times or more during the treatment cycle. In an embodiment, the aNK cells are administered on days 1 and 2 of a 2- week treatment cycle.

[0092] In embodiments, the method may include administering N-803 to the subject. In embodiments, the N-803 can be administered concurrently with the aNK cells. N-803 (ALT- 803) is an IL-15 superagonist mutant and dimeric IL-15RaSushi-Fc fusion protein complex. See, e.g. , U.S. Patent Pub. No. 2017/0088597; Knudson et ah, J Immunother Cancer , 2019 Mar 21;7(1):82; each of which is incorporated herein by reference for everything taught therein. [0093] In some instances, N-803 may be administered to a patient. The N-803 may be administered in conventional doses. For example, in some instances, N-803 is administered at a dose of about 1 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 2 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 3 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 4 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 5 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 6 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 7 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 8 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 9 pg/kg to about 10 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 9 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 8 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 7 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 6 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 5 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 4 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 3 pg/kg. In some instances, N-803 is administered at a dose of about 1 pg/kg to about 2 pg/kg.

[0094] In some instances, an immune checkpoint inhibitor may be administered to a patient. The immune checkpoint inhibitor may be administered in conventional doses. For example, in some instances, an immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 50 mg to about 500 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 150 mg to about 500 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 200 mg to about 500 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 240 mg to about 500 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 300 mg to about 500 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 50 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 100 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 150 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 200 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 240 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 250 mg.

In some instances, the immune checkpoint inhibitor is administered at a dose of about 300 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 350 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 400 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 450 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 480 mg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 500 mg.

[0095] In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 1.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 1.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 2.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 2.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 3.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 3.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 4.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 4.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 5.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 5.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 6.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 6.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 7.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 7.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 8.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 8.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 9.0 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 9.5 mg/kg to about 10 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 9 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 8 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 7 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 6 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 5 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 4 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 3 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 2 mg/kg. In some instances, the immune checkpoint inhibitor is administered at a dose of about 0.1 mg/kg to about 1 mg/kg.

[0096] In some instances, the immune checkpoint inhibitor is administered every 1 day to every 6 weeks. In some instances, the immune checkpoint inhibitor is administered every 1 week to every 6 weeks. In some instances, the immune checkpoint inhibitor is administered approximately every 1 week. In some instances, the immune checkpoint inhibitor is administered approximately every 2 weeks. In some instances, the immune checkpoint inhibitor is administered approximately every 3 weeks. In some instances, the immune checkpoint inhibitor is administered approximately every 4 weeks. In some instances, the immune checkpoint inhibitor is administered approximately every 5 weeks. In some instances, the immune checkpoint inhibitor is administered approximately every 6 weeks.

[0097] In embodiments, the checkpoint inhibitor may be administered after administration of the aNK cells. In embodiments, the checkpoint inhibitor may not be administered concurrently with the aNK cells. For example, the checkpoint inhibitor may be administered some time after the aNK treatment (e.g., aNK treatment cycle) ends. In embodiments, the checkpoint inhibitor may be administered between 1 week and 5 years after the aNK treatment (e.g., aNK treatment cycle) ends. In embodiments, the checkpoint inhibitor may be administered between 1 month and 5 years after the aNK treatment (e.g., aNK treatment cycle) ends. In embodiments, the checkpoint inhibitor may be administered between 6 months and 5 years after the aNK treatment (e.g., aNK treatment cycle) ends. In embodiments, the checkpoint inhibitor may be administered between 1 month and 12 months after the aNK treatment (e.g., aNK treatment cycle) ends, or between 1 month and 10 months, or between 1 month and 8 months, or between 1 month and 6 months, or between 1 month and 4 months, or between 1 month and 2 months. In embodiments, the checkpoint inhibitor may be administered between 2 months and 12 months after the aNK treatment (e.g., aNK treatment cycle) ends, or between 4 months and 12 months, or between 6 months and 12 months, or between 8 months and 12 months, or between 10 months and 12 months. In embodiments, the checkpoint inhibitor may be administered between 1 year and 5 years after the aNK treatment (e.g., aNK treatment cycle) ends. For example, the checkpoint inhibitor may be administered not less than 1 week, not less than 2 weeks, not less than 3 weeks, not less than 4 weeks, not less than 5 weeks, not less than 6 weeks, not less than 7 weeks, not less than 8 weeks, not less than 9 weeks, not less than 10 weeks, not less than 11 weeks, or not less than 12 weeks after administration of the aNK cells. In another example, the checkpoint inhibitor may be administered not less than 1 month, not less than 2 months, not less than 3 months, not less than 4 months, not less than 5 months, not less than 6 months, not less than 7 months, not less than 8 months, not less than 9 months, not less than 10 months, not less than 11 months, or not less than 12 months after administration of the aNK cells. In another example, the checkpoint inhibitor may be administered not less than 1 year, not less than 2 years, not less than 3 years, not less than 4 years, not less than 5 years, or even more than 5 years after administration of the aNK cells. The amount of time may be any value or subrange within any range described herein, including endpoints.

[0098] In embodiments, the cancer may be a skin cancer. In embodiments, the skin cancer may be Merkel cell carcinoma.

[0099] In embodiments, the checkpoint inhibitor may be a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In embodiments, the checkpoint inhibitor is a PD-L1 inhibitor. In embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In embodiments, the checkpoint inhibitor may be ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, camrelizumab toripalimab, sintilimab, or spartalizumab. In embodiments, the checkpoint inhibitor is pembrolizumab. In embodiments, the checkpoint inhibitor is avelumab.

[0100] In embodiments, the checkpoint inhibitor may be a therapeutic antibody. [0101] In embodiments, the aNK cells can be allogeneic to the subject. In embodiments, the aNK cells can be autologous to the subject.

[0102] In embodiments, the subject may have been treated with a checkpoint inhibitor before administration of the aNK cells. The checkpoint inhibitor can be the same checkpoint inhibitor that is administered to (or intended to be administered to) the subject after administration of the aNK cell. Alternatively, the checkpoint inhibitor can be a different checkpoint inhibitor than is administered to (or intended to be administered to) the subject after administration of the aNK cell.

[0103] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EMBODIMENTS

[0104] Embodiment 1. A method of preparing a subject having cancer for treatment with a checkpoint inhibitor, the method comprising administering to the subject an effective amount of activated natural killer (aNK) cells, wherein the cancer is refractory to the checkpoint inhibitor treatment prior to administration of the aNK cells.

[0105] Embodiment 2. A method of treating cancer in a subject, comprising administering to the subject an effective amount of activated natural killer (aNK) cells, wherein the cancer is refractory to checkpoint inhibitor treatment prior to administration of the aNK cells.

[0106] Embodiment 3. A method of treating cancer in a subject, comprising (a) providing a subject having a cancer that is refractory to checkpoint inhibitor treatment; and (b) administering to the subject an effective amount of activated natural killer (aNK) cells to potentiate the activity of the checkpoint inhibitor.

[0107] Embodiment 4. A method of treating cancer in a subject, comprising (a) identifying a subject having a cancer that is refractory to checkpoint inhibitor treatment; and (b) administering to the subject an effective amount of activated natural killer (aNK) cells to potentiate the activity of a checkpoint inhibitor.

[0108] Embodiment 5. The method of any one of Embodiments 1 to 4, further comprising administering N-803 to the subject.

[0109] Embodiment 6. The method of Embodiment 5, wherein the N-803 is administered concurrently with the aNK cells.

[0110] Embodiment 7. The method of any one of Embodiments 1 to 6, further comprising administering a checkpoint inhibitor to the subject.

[0111] Embodiment 8. The method of Embodiment 7, wherein the checkpoint inhibitor is administered after administration of the aNK cells.

[0112] Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the cancer is a skin cancer.

[0113] Embodiment 10. The method of Embodiment 9, wherein the skin cancer is Merkel cell carcinoma.

[0114] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0115] Embodiment 12. The method of Embodiment 11, wherein the checkpoint inhibitor is ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, spartalizumab.

[0116] Embodiment 13. The method of Embodiment 12, wherein the checkpoint inhibitor is pembrolizumab.

[0117] Embodiment 14. The method of any one of the above Embodiments, wherein the aNK cells are allogenic to the subject.

[0118] Embodiment 15. The method of any one of the above Embodiments, wherein the subject was treated with the checkpoint inhibitor before administration of the aNK cells. [0119] Embodiment 16. The method of any one of the above Embodiments, wherein the checkpoint inhibitor is not administered concurrently with the aNK cells.

EXAMPLES

[0120] One skilled in the art would understand that descriptions of making and using the particles described herein is for the sole purpose of illustration, and that the present disclosure is not limited by this illustration.

Example 1. Phase 2 Study Using Off-the-Shelf Activated Natural Killer (aNK) Cells in Combination with N-803, an IL-15 Superagonist, in Patients with Metastatic Merkel Cell Carcinoma (MCC)

[0121] Approximately 2500 cases of Merkel Cell Carcinoma (MCC) occur annually in the United States. MCC presents as cutaneous malignancy with neuroendocrine features. Presentation is often “unimpressive,” but the course of the disease is usually aggressive. The current mortality rate for MCC is about 45%, and 5-year overall survival rate for Stage IV MCC is less than 20% (Lemos 2010 J Am Derm).

[0122] MCC can be caused by MCC polyoma virus (MCPyV) or UV-induced damage, and can be categorized as Virus-Positive MCC and UV-Induced MCC. MCC is associated with UV-induced high mutational load (neoantigens), and immune exhaustion of tumor- infiltrating leukocytes (TILs) which may be reversible with immune checkpoint inhibitors (ICIs).

[0123] Immune checkpoint inhibitors (ICIs) induce high response rates in MCC. For example, it has been reported that avelumab produced an objective response rate (ORR) of 32% (N=88) (Kaufman et al 2016 Lancet Oncol), pembrolizumab produced an ORR of 56% (N=50) (Nghiem P, Bhatia S et al 2019 JCO), and nivolumab produced an ORR of 64% (N=22) (Topalian S, Bhatia S et al 2017 AACR). ICI responses are impressively durable, with many patients experiencing complete or partial remission, as reported by Nghiem et al and Kaufman. However, many patients do not respond to ICI, and relapse is common in patients who do respond. Anecdotal reports of progression in ICI responders are emerging.

[0124] MHC-I downregulation is a prevalent mechanism for immune evasion. However, allogeneic aNK cells have been shown to kill a broad range of tumor cells with deficient MHC-I, and N-803 increases the number and cytotoxicity of NK and memory CD8+ T cells. [0125] Natural killer cells play a central role in immune surveillance. For example, NK cells mediate early defenses against tumor cells through direct cytolytic activity, antibody- dependent cellular cytotoxicity (ADCC), and cytokine release. NK cell activity is determined by the balance of activating and inhibitory NK cell receptors. Although NK cells are potent cytotoxic effector cells for cancer therapy, there are challenges to obtaining functionally active NK cells from a patient’s blood including low numbers of NK cells within lymphocytes that are often dysfunctional.

[0126] Thus, the cytotoxic natural killer cell line NK-92 was derived from a patient with a rare NK-cell lymphoma. NK-92 is an IL-2 dependent NK cell line, lacks expression of most killer cell inhibitor receptors (KIRs), and has a broad cytotoxic range. NK-92 cells further express Fc receptors, which may bind to antibodies with high affinity. NK-92 cells are irradiated before administration to patients.

[0127] Interleukin- 15 (IL-15) regulates NK and CD8+ T cell differentiation, proliferation, homeostatis and cytotoxicity, and thus has potential antiviral and anticancer functionalities. Via transpresentation, a cell surface IL-15 bound to a heterodimeric IL-15 receptor complex is delivered to an opposing lymphocyte through a cell-cell interaction. N- 803, an IL-15 superagonist including an IL-15/IL-15Ra/IgGl Fc fusion protein complex, has been found to modulate the immune response for treatment of various cancers. N-803 includes a N72D mutation in its IL-15 domain, which enhances binding to the receptor IL- 2RP, driving proliferation and activation of NK and T cells without expansion of Tregs. The IL-15Ra domain allows transpresentation selectively to only IL-2RPy chain, and the IgGl Fc increases half-life and lymphoid recycling and distribution. N-803 promotes NK and CD8+ T cell expansion and activation without expanding immunosuppressive regulatory T cells. N- 803 further enhances NK cell-mediated antibody-dependent cellular cytotoxicity, and has 30- fold greater activity and 10-fold longer half-life than IL-15.

[0128] QUILT-3.009 is a Phase 2 study including treatment of activated NK-92 natural killer cells (aNK) infusions in combination with ALT-803 (IL-15 superagonist) in patients with Stage III (MB) or Stage IV MCC. Seven patients were enrolled, three of which received aNK monotherapy and four of which received aNK and N-803. Two-week treatment cycles were performed (see FIG. 1), with aNK (2 x 10⁹ cells) administered intravenously on days 1 and 2 of each 2 week cycle, and N-803 administered on day 1 at a dose of 10 pg/kg subcutaneously. [0129] Key eligibility criteria included unresectable stage III or IV MCC, and < 2 prior systemic therapies. Endpoints included objective response rate (ORR) by RECIST 1.1, progression-free survival, safety, and immunologic changes in the tumor microenvironment.

[0130] Table 1 below gives the demographics and cancer history, and Table 2 gives the investigational product exposure, of the QUILT-3.009 study. Age and previous therapies show median (minimum, maximum).

[0131] Table 1

[0132] Table 2

[0133] Results: Seven subjects were enrolled; 3 received aNK monotherapy, and 4 received aNK plus N-803. Two of 7 subjects had objective responses (ORR=29%; one partial response [PR] and one radiologic complete response [CR]), and one had stable disease lasting 5.5 months. Treatment was safe, without any immune-related adverse events (AEs), treatment-related AEs of grade > 3, or serious AEs. Intriguing changes were noted clinically in superficial tumors in several patients within hours of aNK infusion.

[0134] One heavily pretreated subject, Patient 02-02, was refractory to pembrolizumab despite high PD-L1 expression (FIGs. 2-4), experienced an impressive clinical and radiologic CR at ~4 months with aNK monotherapy (FIG. 5) before being found to have low-volume progressive disease (PD) at 6 months, resulting in treatment discontinuation (FIG. 6, left photo). Subsequently, rechallenging with pembrolizumab resulted in a durable CR ongoing at over 30 months (FIG. 6, right photo). Serial tumor biopsies after aNK treatment revealed increased CD8+ and CD4+ T-cell infiltration and upregulation (FIG. 7), and increased expression of markers for immune cells, antigen presentation genes, and IFN-gamma genes. Another subject treated with combination therapy had a PR followed by apparent PD and treatment discontinuation. However, continued tumor regression, in the absence of additional treatment, indicated pseudoprogression. This patient subsequently experienced a near CR (ongoing for over 26 months).

[0135] Conclusions: aNK (with/without N-803) therapy was well tolerated, led to clinical benefit in several patients, and reversed refractoriness to PD- 1 -blockade in one patient. This trial has provided proof-of-concept for aNK-based therapy in MCC and has facilitated an upcoming registrational trial (NCT03853317) using cryopreserved NK cells (not requiring on-site expansion) plus N-803 plus avelumab in patients with advanced MCC refractory to treatment with checkpoint inhibitors.

Example 2. A Phase 2 Study of Combination Therapy with N-803, Off-the-Shelf CD16- Targeted Natural Killer Cells (haNK), and Avelumab without Cytotoxic Chemotherapy in Subjects with MCC That Has Progressed on or after Treatment with a Checkpoint Inhibitor

[0136] QUILT-3.063 is a Phase 2 study of combination therapy with N-803, haNK cells, and avelumab without cytotoxic chemotherapy, in subjects with MCC that has progressed on or after treatment with a checkpoint inhibitor. haNK cells have been engineered to express a high-affinity variant of the CD 16 receptor. This binds with greater affinity to IgG antibodies, increasing ADCC. Like aNK cells they are also lacking most inhibitory receptors.

[0137] The study is designed as a Phase 2, Single-Arm Combination Therapy of investigational products N-803 and haNK, and FDA-approved product avelumab (BAVENCIO®). Subjects must have progressed on or within six months of checkpoint inhibitor therapy with a single-event avelumab or pembrolizumab.

[0138] QUILT-3.063 aims to evaluate the efficacy of combination therapy of N-803, haNK and avelumab for patients with histologically-confirmed metastatic MCC, who have progressed on, or within 6 months of completing treatment with either pembrolizumab or avelumab. The patients must also have at least 1 measurable lesion of size greater than or equal to 1 cm.

REFERENCES

[0139] Lemos & al. (2010) 7 Am. Derm. 63(5):751-61.

[0140] Kaufman & al. (2016) Lancet Oncol. 17(10): 1374—85.

[0141] Nghiem & al. (2019) J. Clin. Oncol. 37(9):693-702.

[0142] Topalian & al. (2017) Proceedings AACR 201777(13S):CT074.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a subject having cancer for treatment with a checkpoint inhibitor, the method comprising administering to the subject an effective amount of activated natural killer (aNK) cells, wherein the cancer is refractory to the checkpoint inhibitor treatment prior to administration of the aNK cells.

2. A method of treating cancer in a subject, comprising administering to the subject an effective amount of activated natural killer (aNK) cells, wherein the cancer is refractory to checkpoint inhibitor treatment prior to administration of the aNK cells.

3. A method of treating cancer in a subject, comprising (a) providing a subject having a cancer that is refractory to checkpoint inhibitor treatment; and (b) administering to the subject an effective amount of activated natural killer (aNK) cells to potentiate the activity of the checkpoint inhibitor.

4. A method of treating cancer in a subject, comprising (a) identifying a subject having a cancer that is refractory to checkpoint inhibitor treatment; and (b) administering to the subject an effective amount of activated natural killer (aNK) cells to potentiate the activity of a checkpoint inhibitor.

5. The method of claim 1, further comprising administering N-803 to the subject.

6. The method of claim 5, wherein the N-803 is administered concurrently with the aNK cells.

7. The method of claim 1, further comprising administering a checkpoint inhibitor to the subject.

8. The method of claim 7, wherein the checkpoint inhibitor is administered after administration of the aNK cells.

9. The method of claim 1, wherein the cancer is a skin cancer.

10. The method of claim 9, wherein the skin cancer is Merkel cell carcinoma.

11. The method of claim 1, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

12. The method of claim 11, wherein the checkpoint inhibitor is ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, spartalizumab.

13. The method of claim 12, wherein the checkpoint inhibitor is pembrolizumab.

14. The method of claim 1, wherein the aNK cells are allogenic to the subject.

15. The method of claim 1, wherein the subject was treated with the checkpoint inhibitor before administration of the aNK cells.

16. The method of claim 3, wherein the checkpoint inhibitor is not administered concurrently with the aNK cells.