Dosing regimen for CD19 car NK cells in treating cancer

Abstract

The present disclosure provides, among other things, a method for treating cancer by administering two or more doses of cord blood derived natural killer cells expressing CD19 targeted chimeric antigen receptor to a subject in need thereof. The present disclosure provides a dosing regimen, for example, 800 million CD19 CAR NK cells administered in three doses for treating a cancer, for example, relapsed or refractory large B cell lymphoma.

Description

DOSING REGIMEN FOR CD19 CAR NK CELLS IN TREATING CANCERCROSS-REFERENCED APPLICATIONS[1] This application claims priority to, and the benefit of U.S. Provisional Application Serial No. 63 / 551,881 filed on February 9, 2024, the contents of which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING[2] The instant application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The sequence listing file entitled MIL-043WOl_SL.XML, was created on January 14, 2025, which is 16,384 bytes in size.BACKGROUND[3] Chimeric antigen receptor (CAR) T cell therapy has been shown to be effective in the treatment of certain cancers, especially B cell cancers, for example, large B cell lymphoma, and Indolent Non-Hodgkin’s lymphoma. However, CD19 CAR-T cells induce toxic effects and pose manufacturing challenges.[4] Further, although clinical trials have shown that 40-90% complete remission can be achieved in pediatric and adult subjects treated with CD19-directed CAR T-cells, 30- 60% of subjects relapse after CD 19 CAR T-cell treatment, and among those, 20-90% show CD 19-negative relapse. Eligible treatment options for post-CAR relapse are limited, making it more difficult to achieve complete remission and improve survival rate. There is an unmet clinical need for effective methods of treating subjects following relapse after exposure to at least one line of standard chemoimmunotherapy or targeted therapy, including CD19-targeted therapy.SUMMARY[5] The present disclosure provides, among other things, a method of treating cancer by administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to a subject in need thereof. The present disclosure provides, among other things, a dosing regimen for treating a cancer, for example, relapsed or refractory large B celllymphoma by administering to a subject in need thereof a therapeutically effective dose of about 800 million CD 19 CAR NK cells.[6] Without wishing to be bound by theory, CD 19 CAR NK cells kill tumor cells upon target antigen downregulation via innate immune receptor-mediated killing. The inventors of the present disclosure have discovered a highly efficacious, safe and tolerable treatment regimen for treating cancer by increasing efficacy, while minimizing toxicity.[7] Administration of large doses increases drug exposure, thereby increasing efficacy. As described in greater detail throughout the application, and in the examples below, the dosing regimen of the present disclosure, characterized by CD19 CAR NK administration intermittently on days 0, 7 and 14, wherein each dose is followed by days without drug treatment, may have a higher efficacy, increasing the therapeutic index, reducing toxicity and increasing tolerability and safety.[8] The present invention thus also provides a way to treat cancers that seem to respond well at first to other anti-cancer agents, but then the response stops and the tumors come back in a drug-resistant form, e.g. relapsed or refractory large B cell lymphoma.[9] In some aspects, provided herein is a method for treating cancer in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is administered in greater than one dose following a single lymphodepletion cycle.

[0010] In some embodiments, two doses are administered.

[0011] In some embodiments, three doses are administered.

[0012] In some embodiments, greater than three doses are administered.

[0013] In some embodiments, the subject has received one or more lymphodepletion cycles.

[0014] In some embodiments, the two or more doses are administered following the one or more lymphodepletion cycles.

[0015] In some embodiments, the three doses are administered on day 0, day 7 and day 14 following one or more lymphodepletion cycles.

[0016] In some embodiments, each dose is 300 million to 1.5 billion cells.

[0017] In some embodiments, each dose is 600 million cells.

[0018] In some embodiments, each dose is 800 million cells.

[0019] In some embodiments, each dose is 1 billion cells.

[0020] In some embodiments, each dose is 1.5 billion cells.

[0021] In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 2.

[0022] In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 2.

[0023] In some embodiments, the CD19-CAR comprises at least 95% identity to CDR sequences of CDR-L1 : RASQDISKYLN (SEQ ID NO: 8), CDR-L2: SRLHSGV (SEQ ID NO: 9), CDR-L3 : GNTLPYTFG (SEQ ID NO: 10), CDR-H1 : DYGVS (SEQ ID NO: 11), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) and CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0024] In some embodiments, the CD19-CAR comprises 100% identity to CDR sequences of CDR-L1 : RASQDISKYLN (SEQ ID NO: 8), CDR-L2: SRLHSGV (SEQ ID NO: 9), CDR-L3 : GNTLPYTFG (SEQ ID NO: 10), CDR-H1 : DYGVS (SEQ ID NO: 11), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) and CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0025] In some embodiments, the CD19-CAR comprises a CD28 domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 3.

[0026] In some embodiments, the CD19-CAR comprises a CD28 domain having 100% identity to the amino acid sequence of SEQ ID NO: 3.

[0027] In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

[0028] In some embodiments, the CD19-CAR comprises a CD3(^ domain having 100% identity to the amino acid sequence of SEQ ID NO: 4.

[0029] In some embodiments, the single lymphodepletion cycle comprises fludarabine and / or cyclophosphamide.

[0030] In some embodiments, the CD19-CAR CB-NK cells comprises one or more exogenously provided interleukin (IL).

[0031] In some embodiments, the one or more interleukin is selected from IL- 15, IL- 12, IL-21, IL-2, IL- 18, IL-7, and combinations thereof.

[0032] In some embodiments, the interleukin is IL-15.

[0033] In some embodiments, the IL-15 is secreted or membrane bound.

[0034] In some embodiments, the IL-15 is secreted from the cell.

[0035] In some embodiments, exogenously provided IL is expressed from a vector in the cells.

[0036] In some embodiments, the CD19-CAR CB-NK cell comprises a suicide gene.

[0037] In some embodiments, the suicide gene is an iCaspase9 suicide gene.

[0038] In some embodiments, the cancer is hematologic, lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, or cervix cancer.

[0039] In some embodiments, the cancer is a hematologic cancer.

[0040] In some embodiments, the hematologic cancer is a large B cell lymphoma(LBCL).

[0041] In some embodiments, the large B cell lymphoma is relapsed or refractory.

[0042] In some aspects, provided herein is a method for treating a relapsed or refractory large B cell lymphoma in a subject, the method comprising administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein each dose is 800 million cells.

[0043] In some embodiments, two doses are administered.

[0044] In some embodiments, three doses are administered.

[0045] In some embodiments, greater than three doses are administered.

[0046] In some embodiments, the subject has previously received one or more lymphodepletion cycles.

[0047] In some embodiments, the subject has previously received one lymphodepletion cycle.

[0048] In some embodiments, the two or more doses are administered following the one or more lymphodepletion cycles.

[0049] In some embodiments, the three doses are administered on day 0, day 7 and day 14 following the one or more lymphodepletion cycles.

[0050] In some embodiments, the subject has previously received surgery, radiation, chemotherapy, hormone therapy, immunotherapy, stem cell therapy, targeted therapy or a co bin a ti on th ereof .

[0051] In some embodiments, the subject has previously received at least one line of standard chemoimmunotherapy or targeted therapy.

[0052] In some embodiments, the targeted therapy comprises administration of one or more of CD19-targeted chimeric antigen receptor (CAR)-T cells, CD19-targeted antibodydrug conjugates and / or CD19-targeted antibodies.

[0053] In some embodiments, standard chemoimmunotherapy comprises administration of cyclophosphamide, doxorubicin, vincristine, and prednisone, and / or monoclonal antibody rituximab.

[0054] In some embodiments, the CD19-CAR CB-NK cells are autologous with respect to the subject.

[0055] In some embodiments, the CD19-CAR CB-NK cells are allogeneic with respect to the subject.

[0056] In some embodiments, the CD19-CAR CB-NK cells are administered intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion in a tumor microenvironment, or a combination thereof.

[0057] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and / or” unless stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.DEFINITIONS

[0058] Administering'. As used herein, the terms “administering,” or “introducing” are used interchangeably in the context of delivering a CD19-directed genetically modified NK cell immunotherapy described herein (e.g., CD 19 CAR viable NK cells, for example, autologous or allogenic cord blood derived CD 19 CAR NK cells) to a patient in need thereof. Various methods are known in the art for administering cells to patients, including forexample administering the cells to a patient in need thereof by intravenous or surgical methods.

[0059] Adoptive Cell Therapy. As used herein interchangeably, the terms “adoptive cell therapy” or “adoptive cell transfer” or “cell therapy” or “ACT” refer to the transfer of cells, for example, a population of genetically modified cells (e.g. CD19 CAR NK cells), into a patient in need thereof. The cells can be derived and propagated from the patient in need thereof (i.e., autologous cells) or could have been obtained from a non-patient donor (i.e., allogeneic cells). In some embodiments, the cell is an immune cell, such as a lymphocyte. In some embodiments, the immune cell is a NK cell. Various cell types can be used for ACT including but not limited to, natural killer (NK) cells, T cells, CD8+ cells, CD4+ cells, deltagamma T-cells, regulatory T-cells, induced pluripotent stem cells (iPSCs), iPSC derived T cells, iPSC derived NK cells, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells.

[0060] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and / or a clone.

[0061] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a stated value of interest as well as value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0062] Allogeneic. As used herein, “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one ormore loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically

[0063] Autologous'. As used herein, the term “autologous” means from the same individual. For example, “autologous” in relation to donor and recipient means that the donor subject is the recipient subject.

[0064] Chimeric Antigen Receptor (CAR): As used herein, the term “chimeric antigen receptor” or “CAR” engineered receptors which can confer an antigen specificity onto cells (for example, immune cells such as NK cells including cord blood derived NK cells and iPSC derived NK cells (iNK cells)). CARs are also known as chimeric antigen receptors or chimeric immunoreceptors. For example, a CAR described herein may include one or more of an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain.

[0065] CD19-directed genetically modified NK cell immunotherapy. As used herein, the term “CD19-directed genetically modified NK cell immunotherapy” refers to compositions and formulations comprising CD 19 CAR+ NK cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises CD 19 CAR+viable NK cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises cord blood derived CD19 CAR NK+cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises allogenic cord blood derived CD19 CAR NK+cells.

[0066] Cell: As used herein, the term “cell” refers to any cell unless a specific type of cell is named. As non-limiting examples, a cell is a stem cell, progenitor cell, or somatic cell, e.g., adult stem cell, progenitor cell, or differentiated cell. In some embodiments, the cells are hematopoietic cell, e.g., a hematopoietic stem or progenitor cell. In some embodiments, the cells are immune cells and include B-cells, T cells, monocytes or progenitor cells. In some embodiments, the cells are NK cells, and in particular, CAR-NK cells.

[0067] Cryoprotectant'. As used herein, the term “cryoprotectant” means a substance used to protect biological tissue from freezing damage. Exemplary cryoprotectants include, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol and propanediol.

[0068] Engineered'. As used herein, the term “engineered” refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth.In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure.

[0069] Exogenous-. As used herein, the “exogenous” as used herein refers to a polynucleotide (such as one encoding a gene product or part of a gene product) that is not present endogenously in a mammalian cell, such as an immune cell, or is synthetically generated outside of a mammalian cell, such as by recombinant technology. In a specific case, a particular gene product may be provided to a cell exogenously, and the cell may or may not also express the corresponding endogenous gene product in the cell.

[0070] Ex vivo'. As used herein, the term “ex vivo" means a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube, in a culture bag, in a bioreactor).

[0071] Fresh cell or Rescued Fresh Cell: As used herein, the terms “fresh,” “fresh cell,” or “rescued fresh cell” refers to mammalian cells that have never been frozen and / or once frozen but subsequently restimulated, cultured in culture medium and then harvested as fresh cells.

[0072] Functional equivalent or derivative'. As used herein, the term “functional equivalent” or “functional derivative” denotes, in the context of a functional derivative of an amino acid sequence or any other molecule (e.g., a media formulation component) that retains an activity (either function or structural) that is substantially similar to that of the original molecule or sequence. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. Exemplary derivatives include those having chemico- physical properties which are similar to that of the original molecule or sequence. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.

[0073] Isotonic. As used herein, the term “isotonic” means having an osmotic pressure that is equal to or approximately the same as the osmotic pressure of a physiological fluid.

[0074] In vitro'. As used herein, the term “ / / / vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0075] In vivo: As used herein, the term “ / / / vivo" refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0076] Primary Cell'. The term, “primary cell,” refers to a cell that is directly isolated from a subject and subsequently propagated.

[0077] Polypeptide'. The term, “polypeptide,” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and / or modified.

[0078] Protein'. The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.

[0079] Refractory Cancer: “Refractory cancer” refers to cancer that did not respond to treatment. Complete remission (CR) is not reached because the chemotherapy drugs do not kill enough leukemia cells, neither is CRh or CRi reached at the response landmark, i.e., after 2 courses of intensive induction treatment or a defined landmark, e.g., 180 d after commencing less-intensive therapy.

[0080] Relapsed Cancer: “Relapsed cancer”, or recurrent cancer refers to cancer that has come back after treatment and remission. Relapsed lymphoma is associated with one or more symptoms and biomarkers such as bone marrow blasts of 5% or greater than 5%; or reappearance of blasts in the blood in at least two peripheral blood samples at least one week apart; or development of extramedullary disease.

[0081] Remission: As used herein, the term “remission” means that the signs and symptoms of cancer are reduced. Remission can be partial or complete. Measurable (or minimal) residual disease (MRD); cancer cells not destroyed by treatment) is a more sensitive measure of remission.

[0082] Complete Remission (CR): In a complete remission (CR), all signs and symptoms of hematologic cancer (e.g. LBCL or iNHL) have disappeared, for example, the bone marrow contains fewer than 5% blast cells and circulating blasts are absent. Extramedullary disease is absent. Blood cell counts return to within normal limits, for example, neutrophil count of 1.0 x 109 / L (1,000 / pL) or greater and platelet count of at least 100 x 109 / L [100,000 / pL]). If a person remains in complete remission for 5 years or more, the cancer is sometimes considered cured.

[0083] Partial Remission (PR): As used herein, in a partial remission (PR), all hematologic criteria of CR are met, but there is a decrease of bone marrow blast percentage to 5% to 25%, and decrease of pre-treatment bone marrow blast percentage by at least 50%.

[0084] Complete Remission with Partial Hematologic Recovery (CRh) : The term “CR with partial hematologic recovery (CRh)” has been introduced for patients with morphologic bone marrow blast clearance and partial recovery of both neutrophils (at least 0.5 x 109 / L [500 / pL]) and platelets (at least 50 x 109 / L [50,000 / pL]) because those represent clinical benefit to the patient; other CR criteria need to be met.

[0085] Complete Remission with Incomplete Hematologic Recovery (CRi)'. The term “CR with incomplete hematologic recovery (CRi)” has been introduced because CR criteria are met except for residual neutropenia of less than 1.0 x 109 / L (1,000 / pL) or thrombocytopenia of less than 100 x 109 / L (100,000 / pL).

[0086] Duration of Response: As used herein, “Duration of Response (DOR)” is defined as the time from the date of first documented CR, CRh, or CRi to the date of relapse or death.

[0087] Event-free Survival (EFS): As used herein, “Event-free Survival” is defined as the time from the date of first dose administration to the date of treatment failure, relapse or death, whichever comes first.

[0088] Overall Survival (OS): As used herein, “Overall Survival” is defined as the time from the date of the first dose administration to the date of death.

[0089] Subject. As used herein, the term “subject” refers to a human or any nonhuman animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeablywith “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0090] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0091] Suffering from'. An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0092] Sugar or Saccharide: The terms “sugar” and “ saccharide" herein have been used interchangeably, and generally refer to oligosaccharides such as monosaccharides, disaccharides, trisaccharides or polysaccharides, and the like. In some embodiments, the saccharide is one or more of glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, myoinositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltitol, methyl cellulose, carboxymethyl cellulose, dextran, glycogen, amylose, amylopectin, inulin, sodium alginate, ethyl cellulose, hydroxyethyl cellulose, raffinose, stachyose, xanthan gum, glucosamine, and galactosamine. In some embodiments, saccharide is a disaccharide. In some embodiments, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, or chitobiose. In some other embodiments, the disaccharide is trehalose. In some embodiments, one or more sugars includes trehalose, sucrose, mannitol, and / or dextran.

[0093] Therapeutically effective amount. As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen (e.g., comprising greater than one unit dose, for example, three doses). In some embodiments, a therapeutically effective amount of an adoptive cell therapy, as used herein, is a dosage ofcells (e.g., a population of genetically modified immune cells such as CAR-NK cells) in a certain formulation (e.g., a cry opreservation media described herein) administered to a subject in need thereof (e.g., a patient suffering from a B-cell malignancy, for example large B-cell lymphoma (LBCL)). For example, in some embodiments, a therapeutically effective dose comprises CAR-NK cells in an amount of about 300 million cells to about 1.5 billion cells, e.g. 800 million cells, administered in greater than one dose, for example, two doses or three doses or greater than three doses.

[0094] In some embodiments, the CAR-NK cells have been genetically modified to express a CD- 19 CAR comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2 or a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 2 and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or a sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0095] In some embodiments, the CAR-NK cells have been genetically modified to express a CD 19 CAR having at least 95% identity to CDR sequences of CDR-L1 : RASQDISKYLN (SEQ ID NO: 8), CDR-L2: SRLHSGV (SEQ ID NO: 9), CDR-L3: GNTLPYTFG (SEQ ID NO: 10), CDR-H1 : DYGVS (SEQ ID NO: 11), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) and CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0096] In some embodiments, the CAR-NK cells have been genetically modified to express a CD19 CAR having 100% identity to CDR sequences of CDR-L1 : RASQDISKYLN (SEQ ID NO: 8), CDR-L2: SRLHSGV (SEQ ID NO: 9), CDR-L3: GNTLPYTFG (SEQ ID NO: 10), CDR-H1 : DYGVS (SEQ ID NO: 11), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) and CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0097] Treating'. As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0098] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4 and 5).It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.”BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Drawings are for illustration purposes only; not for limitation.

[0100] FIG. 1A is a schematic of a dosing schedule indicating multiple doses of CD19 CAR NK cells at days 0, 7 and 14. Subjects are treated with fludarabine and cyclophosphamide (Flu / Cy) three to five days before dosing in a single cycle of lymphodepletion. Positron emission tomography / computed tomography (PET / CT) scans are carried out at day 28 as a predictor of progression-free survival (PFS) and overall survival (OS).

[0101] FIG. IB is a schematic of a dose escalation and expansion study from 200 million to 800 million CD 19 CAR NK cells in treating relapsed or refractory large B cell lymphoma (LBCL) or Indolent Non-Hodgkin’s lymphoma (iNHL). Cohorts were treated with either a single dose of 200 million CD 19 CAR NK cells, a single dose of 800 million CD 19 CAR NK cells or 3 doses each of 800 million CD19 CAR NK cells.DETAILED DESCRIPTION

[0102] CD 19 CAR+ NK cells are advantageous compared to currently available CD 19 CAR-T products and developing CD 19 CAR-T cells (e.g., iCAR-T), since NK cells have capability of innate immune receptor-mediated killing, are less toxic and have increased manufacturability. CD 19 CAR+ NK cells derived from cord blood cells do not require HLA selection See, e.g., Liu et al. Use of CAR-transduced Natural Killer Cells in CD19 Positive Lymphoid Tumors. NEJM (2020) 382-545). Therefore, CD 19 CAR+ CB-NK cells described herein, for example, in treating relapsed or refractory cancers are advantageous compared with allogenic CAR-T cells.

[0103] The present invention provides, among other things, a method for treating cancer in a subject, the method comprising a step of administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof. In some embodiments, three doses are administered. In some embodiments, the subject has received one or more lymphodepletion cycles. In some embodiments, three doses are administeredfollowing the one or more lymphodepletion cycles. In some embodiments, the three doses are administered on day 0, 7 and 14 following the one or more lymphodepletion cycles.

[0104] In some aspects, provided herein is a method for treating a relapsed or refractory large B cell lymphoma in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is 800 million cells.

[0105] In some embodiments, individuals with a history of prior treatment with standard chemotherapy or targeted therapy, e.g., anti-CD19 therapy (e.g., patients who have undergone CAR-T therapy or anti-CD19 monoclonal antibodies) are administered CARNK cells in the dosing regimen of the present invention.Dosing Regimen for CD19 CAR-NK Cell Compositions and Formulations

[0106] In some aspects, provided herein is a method for treating cancer in a subject, the method comprising a step of administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD19 targeted chimeric antigen receptor (CD19- CAR) (CD19-CAR CB-NK cells) to the subject in need thereof,.

[0107] In some embodiments, two doses are administered.

[0108] In some embodiments, three doses are administered.

[0109] In some embodiments, greater than three doses are administered.[HO] In some embodiments, the subject has received one or more lymphodepletion cycles.[Hl] In some embodiments, the two or more doses are administered following the one or more lymphodepletion cycles.

[0112] In some embodiments, the three doses are administered on day 0, day 7 and day 14 following the one or more lymphodepletion cycles.

[0113] In some embodiments, each dose is 300 million to 1.5 billion cells. In some embodiments, each dose is 300 million cells. In some embodiments, each dose is 400 million cells. In some embodiments, each dose is 500 million cells.

[0114] In some embodiments, each dose is 600 million cells. In some embodiments, each dose is 700 million cells. In some embodiments, each dose is 800 million cells. In some embodiments, each dose is 900 million cells. In some embodiments, each dose is 1 billion cells.

[0115] In some embodiments, each dose is 1.1 billion cells. In some embodiments, each dose is 1.2 billion cells. In some embodiments, each dose is 1.3 billion cells. In some embodiments, each dose is 1.4 billion cells. In some embodiments, each dose is 1.5 billion cells.

[0116] In some embodiments, the method comprises administering pharmaceutical compositions and formulations comprising CD19-directed genetically modified NK cells (e.g., CD19 CAR+ viable NK cells and / or “allogenic cord blood derived CD19 CAR NK+cells) in a dosing regimen of the present invention. CD 19 CAR+ NK cells described herein comprise CD 19 CAR and IL- 15, for example, secreted IL-15.

[0117] In some embodiments, CD 19 CAR+ NK cells are genetically engineered cord blood NK cells including a CD19-CAR comprising an anti-CD19 binding domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, CD19 CAR+ NK cells are genetically engineered cord blood NK cells including a CD19-CAR comprising an anti-CD19 binding domain comprising a light chain denoted by SEQ ID NO: 1 comprising CDR1, CDR2 and CDR3 and a heavy chain denoted by SEQ ID NO: 2 comprising CDR1, CDR2 and CDR3.

[0118] In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD-19 CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 98% identity to the amino acid sequence of SEQ ID NO: 2.

[0119] In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 2.

[0120] In some embodiments, the CAR-NK cells have been genetically modified to express a CD 19 CAR having at least 95% identity to CDR sequences of CDR-L1 : RASQDISKYLN (SEQ ID NO: 8), CDR-L2: SRLHSGV (SEQ ID NO: 9), CDR-L3: GNTLPYTFG (SEQ ID NO: 10), CDR-H1 : DYGVS (SEQ ID NO: 11), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) and CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0121] In some embodiments, the CAR-NK cells have been genetically modified to express a CD19 CAR having 100% identity to CDR sequences of CDR-L1 : RASQDISKYLN (SEQ ID NO: 8), CDR-L2: SRLHSGV (SEQ ID NO: 9), CDR-L3: GNTLPYTFG (SEQ ID NO: 10), CDR-H1 : DYGVS (SEQ ID NO: 11), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) and CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0122] In some embodiments, the genetically engineered cord blood NK cells include a nucleic acid molecule encoding the heavy chain variable region of an anti-CD19 binding domain and / or a nucleic acid molecule encoding the light chain variable region of an antiCD 19 binding domain.

[0123] In some embodiments, the genetically engineered cord blood NK cells include a CD19-CAR comprising an anti-CD19 binding domain, a transmembrane domain such as the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and an intracellular signaling domain such as an intracellular signaling domain FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3-zeta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. The CD- 19 binding domain can be a single chain antibody or single chain antibody fragment, such as an scFv. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the CD-19 CAR can include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 3), a CD3 zeta signaling domain (an exemplary CD3 zeta sequence is shown in SEQ ID NO: 4) and can further include IL-15 and / or a suicide switch, e.g., iCaspase9.

[0124] In some embodiments, the CD19-CAR comprises a CD28 domain. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 90% identity to theamino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 91% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 92% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 93% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 94% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD 19- CAR comprises a CD28 domain having at least 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 96% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 97% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 98% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 99% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD19-CAR comprises a CD28 domain having 100% identity to the amino acid sequence of SEQ ID NO: 3.

[0125] In some embodiments, the CD19-CAR comprises a CD3(^ domain. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 91% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 92% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 93% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 94% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 95% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 96% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 97% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 98% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having at least 99% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19-CAR comprises a CD3(^ domain having 100% identity to the amino acid sequence of SEQ ID NO: 4.

[0126] In some embodiments, CD 19 CAR+ NK cells described herein comprises an exogenous gene encoding a CD19 CAR (e.g., a polypeptide comprising SEQ ID Nos 1-5) and IL-15. In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO: 6. In some embodiments, the CD 19 CAR further comprises iCaspase9 comprising the amino acid sequence of SEQ ID NO: 7.Anti-CD19 Light chain variable fragment, VL:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKR (SEQ ID NO: 1)Anti-CD19 Heavy chain variable fragment, VH:EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTTVTVSSYVTVSSQDPA (SEQ ID NO: 2)CD28:FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRS (SEQ ID NO: 3)CD3RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PRGP (SEQ ID NO: 4)IgGl:EPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKKDPK (SEQ ID NO: 5)IL-15:MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 6) iCaspase9:MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESG GGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQAS HLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVAS TSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSG SWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS ASRA (SEQ ID NO: 7)Anti-CD19 CDR-L1: RASQDISKYLN (SEQ ID NO: 8)Anti-CD19 CDR-L2: SRLHSGV (SEQ ID NO: 9)Anti-CD19 CDR-L3: GNTLPYTFG (SEQ ID NO: 10)Anti-CD19 CDR-H1: DYGVS (SEQ ID NO: 11)Anti-CD19 CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 12) Anti-CD19 CDR-H3: YAMDYWG (SEQ ID NO: 13).

[0127] In some embodiments, subjects are administered CD 19 CARNK cells after a single lymphodepletion cycle.

[0128] In some embodiments, the single lymphodepletion cycle comprises fludarabine and / or cyclophosphamide. In some embodiments, the single lymphodepletion cycle comprises fludarabine. In some embodiments, the single lymphodepletion cycle comprises cyclophosphamide.

[0129] In some embodiments, the CD19-CAR CB-NK cells comprises one or more exogenously provided interleukin (IL).

[0130] In some embodiments, the one or more interleukin is selected from IL- 15, IL- 12, IL-21, IL-2, IL- 18, IL-7, and combinations thereof. In some embodiments, the CD 19- CAR CB-NK cells comprises IL-15. In some embodiments, the CD19-CAR CB-NK cells comprises IL-12. In some embodiments, the CD19-CAR CB-NK cells comprises IL-2. In some embodiments, the CD19-CAR CB-NK cells comprises IL-18. In some embodiments, the CD19-CAR CB-NK cells comprises IL-7. In some embodiments, the CD19-CAR CB-NK cells comprise a combination of one or more of IL-15, IL-12, IL-21, IL-2, IL-18 and IL-7. In some embodiments, the CD19-CAR CB-NK cells comprise two cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise three cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise four cytokines. In some embodiments, the CD 19- CAR CB-NK cells comprise five cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise six cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise greater than six cytokines.

[0131] In some embodiments, the interleukin is IL-15.

[0132] In some embodiments, the IL-15 is secreted or membrane bound. In some embodiments, the IL- 15 is secreted. In some embodiments, the IL- 15 is membrane bound.

[0133] In some embodiments, the IL-15 is secreted from the cell.

[0134] In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO: 6. In some embodiments, the IL- 15 has at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 91% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL- 15 has at least 92% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 93% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 94% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL- 15 has at least 96% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL- 15 has at least 97% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 98% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL- 15 has at least 99% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has 100% identity to the amino acid sequence of SEQ ID NO: 6.

[0135] In some embodiments, exogenously provided IL is expressed from a vector in the cells.

[0136] In some embodiments, the CD19-CAR CB-NK cell comprises a suicide gene.

[0137] In some embodiments, the NK cells are engineered to express one or more suicide genes. For example, in some examples the NK cells are engineered to express one or more of iCaspase9, non-secretable TNF alpha, herpes simplex virus thymidine kinase (HSV- TK), uracil phosphoribosyl transferase (UPRTase), or cytosine deaminase (CD).

[0138] Additional examples of suicide genes include engineered nonsecretable (including membrane bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see e.g., PCT / US2019 / 062009, which is incorporated by reference herein in its entirety), and they may be affected by delivery of an antibody that binds the TNF-alpha mutant. Non-limiting examples of suicide gene / prodrug combinations that may be used are herpes simplex virus- thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or 2’-deoxy-2’-fluoro-5-iodo-l-B-D- arabinofuranosyluracil (FIAU), oxidoreductase and cycloheximide, cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk-Tmk) and azidothymidine (AZT), and deoxycytidine kinase and cytosine arabinoside. The E. coll purine nucleosidephosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to toxic purine 6-methylpurine, may be utilized. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidases (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-a, g-lyase (MET), and thymidine phosphorylase (TP), among others.

[0139] In some embodiments, the NK cells are engineered to express one or more of iCaspase9. In some embodiments, the NK cells are engineered to express non-secretable TNF alpha. In some embodiments, the NK cells are engineered to express herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the NK cells are engineered to express uracil phosphoribosyl transferase (UPRTase). In some embodiments, the NK cells are engineered to express cytosine deaminase (CD). In some embodiments, the suicide gene is an iCaspase9 suicide gene. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 90% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 91% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 92% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 93% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 94% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 95% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 96% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 97% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 98% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 99% identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having 100% identity to an amino acid sequence of SEQ ID NO: 7.

[0140] In some embodiments, the CD19-CAR CB-NK cells are autologous with respect to the subject.

[0141] In some embodiments, the CD19-CAR CB-NK cells are allogeneic with respect to the subject.

[0142] In some embodiments, the CD19-CAR CB-NK cells are administered intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion in a tumor microenvironment, or a combination thereof. In some embodiments, the CD19-CAR CB-NK cells are administered intracranially. In some embodiments, the CD 19- CAR CB-NK cells are administered intravenously. In some embodiments, the CD19-CAR CB-NK cells are administered intraarterially. In some embodiments, the CD19-CAR CB-NK cells are administered intraperitoneally. In some embodiments, the CD19-CAR CB-NK cells are administered intratracheally. In some embodiments, the CD19-CAR CB-NK cells are administered intratumorally. In some embodiments, the CD19-CAR CB-NK cells are administered intramuscularly. In some embodiments, the CD19-CAR CB-NK cells are administered endoscopically. In some embodiments, the CD19-CAR CB-NK cells are administered intralesionally. In some embodiments, the CD19-CAR CB-NK cells are administered percutaneously. In some embodiments, the CD19-CAR CB-NK cells are administered subcutaneously. In some embodiments, the CD19-CAR CB-NK cells are administered by perfusion in a tumor microenvironment. In some embodiments, the CD 19- CAR CB-NK cells are administered by one or more of intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion in a tumor microenvironment, or a combination thereof, in any order and in any number of combinations.

[0143] In some embodiments, the NK cells are from primary cell isolates (e.g., NK cell derived from cord blood). In some embodiments, the NK cells are cord blood NK cells. In some embodiments, the NK cells are peripheral blood NK cells. In some embodiments, the NK cells are iPSC derived NK cells. In some embodiments, the NK cells are from a cell line. In some embodiments, the NK cells are fresh cells. In some embodiments, the NK cells were previously frozen and thawed.

[0144] In some embodiments the NK cell is gene edited to allow the cells to work more effectively in a tumor microenvironment. In some embodiments, the genes are one or more of TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, AD0RA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM 17, RPS6,4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7, or combinations thereof, in any order and in any number of combinations. In some embodiments, one or more of these genes are knocked out or knocked down in the cells.

[0145] In some embodiments, the NK cells are engineered to express CD19-CAR, IL- 15, and iCaspase9. An exemplary CAR-NK cell comprising CD19 IL-15, and iCaspase9 is described in Leukemia 32 (2018)520-531, incorporated herein by reference in its entirety.

[0146] In one aspect, the compositions described herein for use in treating LBCL or iNHL comprise CD19-directed genetically modified NK cell immunotherapy (e.g., CD 19 CAR+viable CB-NK cells) and a pharmaceutically acceptable carrier.

[0147] In some embodiments, compositions comprising CD19-directed genetically modified NK cell immunotherapy described herein comprises CAR-NK cells at a concentration of between 6 M / mL to 120 M / mL, 6 M / mL to 200 M / mL, 5 M / mL to 25 M / mL, 6 M / mL to 120 M / mL in a 36 mL volume or 5 M / mL to 25 M / mL in a 36 mL volume.

[0148] In some embodiments, the total volume of a composition comprising CD 19- directed genetically modified NK cell immunotherapy in which the CAR-NK cells are suspended is between about 15 mL and 30 mL, about 30 mL and 45 mL, about 30 and 60 mL, or about 30 mL and 75 mL. In some embodiments, the total volume in which the NK cells are suspended is between about 15 mL and 30 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is between about 30 mL and 45 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is between about 30 mL and 60 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is between about 30 mL and 75 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL or 50 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 20 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 21 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 22 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 23 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 24 mL. In some embodiments, thetotal volume in which the CAR-NK cells are suspended is about 25 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 26 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 27 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 28 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 29 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 30 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 31 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 32 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 33 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 34 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 35 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 36 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 37 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 38 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 39 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 40 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 41 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 42 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 43 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 44 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 45 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 46 mL.

[0149] In some embodiments, a composition comprising CD19-directed genetically modified NK cell immunotherapy comprise CAR-NK cells at a concentration of about 300 million to 1.5 billion cells per 36 mL. In some embodiments, the composition comprises CAR-NK cells at a concentration of between about 200-800 million CAR-NK cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 100 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 200 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at aconcentration of about 300 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 400 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 500 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 600 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 700 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 800 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 900 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.1 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.2 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.3 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.4 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.5 billion cells per a 36 mL fill volume.

[0150] In some embodiments, the CAR-NK cell therapy product administered is an allogeneic cell therapy product comprised of human cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD- 19 CAR and IL-15.

[0151] In some embodiments, the CAR-NK cell therapy product comprises a population of between 300 million to 800 million cells formulated in a cryopreservation media described herein. In some embodiments, the CAR-NK cell therapy product comprises a population of 100 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 200 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 300 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 400 million cells. In some embodiments, the CAR- NK cell therapy product comprises a population of 500 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 600 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 700 million cells.In some embodiments, the CAR-NK cell therapy product comprises a population of 800 million cells.

[0152] In some embodiments, the CAR-NK cell therapy product comprises a population of between 900 million to 1.5 billion cells formulated in a cry opreservation media described herein. In some embodiments, the CAR-NK cell therapy product comprises a population of 900 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 1 billion cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 1.1 billion cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 1.2 billion cells. In some embodiments, the CAR- NK cell therapy product comprises a population of 1.3 billion cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 1.4 billion cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 1.5 billion cells.

[0153] In some embodiments, the CAR-NK cell therapy product is an allogeneic cell therapy product comprising 800 million human cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD-19 CAR and IL-15 and formulated in 36 mL of a medium for cryopreservation of Table 1 in a 50 mL AT vial. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 1 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 2 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 3 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 4 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 5 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 6 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 7 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 8 of Table 1.

[0154] In some embodiments, the CAR-NK cell therapy product is formulated in Cryopreservation Media 9 of Table 1.

[0155] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as the cells) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22ndedition, 2012), in the form of lyophilized formulations oraqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral- active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos.2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0156] In some embodiments, CD 19 CAR CB-NK cells are suspended in a medium for cry opreservation. In some embodiments, CD 19 CAR CB-NK cells are suspended in the cryopreservation media described in Table 1. To prepare the medium in Table 1, 25% w / v human serum albumin (HSA) and 400 mg / mL trehalose solution are used as components.Table 1: Cryopreservation MediaMethods of Treatment

[0157] The CAR-NK cell compositions described herein are suitable for adoptive cell therapy. Adoptive cell therapies can be used to treat various diseases, including, for example, cancer. In some embodiments, the CAR-NK cell compositions are useful for the treatment of a cancer or a tumor.

[0158] In some embodiments, the cancer is hematologic, lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, or cervix cancer.

[0159] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is blood cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is livercancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is head cancer. In some embodiments, the cancer is neck cancer. In some embodiments, the cancer is thoracic cancer. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is spleen cancer. In some embodiments, the cancer is gallbladder cancer. In some embodiments, the cancer is bone cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is cervical cancer.

[0160] In some embodiments, the cancer is not limited to CD 19 and CD20 double positive cancer.

[0161] In some embodiments, the cancer is a hematologic cancer.

[0162] In some embodiments, the hematologic (or blood) cancer is a B-cell malignancy (e.g., relapsed or refractory B-cell non-Hodgkin lymphoma (including large B- cell lymphoma and indolent non-Hodgkin lymphoma)). In some embodiments, the cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is acute lymphoblastic leukemia (ALL). In some embodiments, the cancer is a histologically proven B-cell NHL, including LBCL and iNHL (FL and MZL), including the types defined by the World Health Organization (WHO). In some embodiments, the hematologic cancer is a large B cell lymphoma (LBCL).

[0163] In some embodiments, the large B cell lymphoma is relapsed or refractory.

[0164] In some embodiments, the large B cell lymphoma is relapsed. In some embodiments, the large B cell lymphoma is refractory.

[0165] In some aspects, provided herein is a method for treating a relapsed or refractory large B cell lymphoma in a subject, the method comprising administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein each dose is 800 million cells.

[0166] In some embodiments, two doses are administered.

[0167] In some embodiments, three doses are administered.

[0168] In some embodiments, greater than three doses are administered.

[0169] In some embodiments, the subject has previously received one or more lymphodepletion cycles.

[0170] In some embodiments, the subject has previously received one lymphodepletion cycle.

[0171] In some embodiments, the three doses are administered following the one or more lymphodepletion cycles.

[0172] In some embodiments, the three doses are administered on day 0, 7 and 14 following the one or more lymphodepletion cycles.

[0173] In some embodiments, the CAR-NK cell compositions described herein are suitable for relapsed or refractory cancer in the individual, wherein the individual has previously received a CD 19 targeted therapy (including CD 19 targeted CAR T therapy and CD 19 targeted antibody therapy). In some embodiments, the relapsed or refractory cancer may be CD 19 positive or CD 19 negative.

[0174] In some embodiments, the cancer is a previously treated relapsed or refractory histologically proven Cluster of Differentiation (CD)19 expressing disease. In some embodiments, the cancer is LBCL, including the subtypes defined by the World Health Organization (WHO). In some embodiments, the cancer is Diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS). In some embodiments, the cancer is High-grade B- cell lymphoma (HGBL) with MYC and BCL2 and / or BCL6 rearrangement. In some embodiments, the cancer is HGBL NOS without translocations.

[0175] In some embodiments, the cancer is DLBCL arising from iNHL. In some embodiments, the cancer is follicular lymphoma (FL). In some embodiments, the cancer is marginal zone lymphoma (MZL). In some embodiments, the cancer is T-cell / histiocyte-rich LBCL. In some embodiments, the cancer is DLBCL associated with chronic inflammation. In some embodiments, the cancer is Epstein-Barr virus-positive DLBCL-NOS. In some embodiments, the cancer is Primary cutaneous DLBCL, leg type. In some embodiments, the cancer is Primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments, the cancer is FL Grade 3B.

[0176] In some embodiments, the cancer is iNHL. In some embodiments, the cancer is FL Grades 1, 2, 3 A. In some embodiments, the cancer is MZL (nodal, extranodal, and splenic). In some embodiments, the subject has previously received surgery, radiation, chemotherapy, hormone therapy, immunotherapy, stem cell therapy, targeted therapy or a combination thereof. In some embodiments, the subject has previously received surgery. In some embodiments, the subject has previously received radiation. In some embodiments, thesubject has previously received chemotherapy. In some embodiments, the subject has previously received hormone therapy. In some embodiments, the subject has previously received immunotherapy. In some embodiments, the subject has previously received stem cell therapy. In some embodiments, the subject has previously received targeted therapy. In some embodiments, the subject has previously received a combination of one or more of surgery, radiation, chemotherapy, hormone therapy, immunotherapy, stem cell therapy, targeted therapy, in any number and any combination.

[0177] In some embodiments, the subject has previously received at least one line of standard chemoimmunotherapy or targeted therapy. In some embodiments, the subject has previously received at least one line of standard chemoimmunotherapy. In some embodiments, the subject has previously received at least one line of targeted therapy.

[0178] In some embodiments, the targeted therapy comprises administration of one or more of CD19-targeted chimeric antigen receptor (CAR)-T cells, CD19-targeted antibodydrug conjugates and / or CD19-targeted antibodies. In some embodiments, the targeted therapy comprises administration of CD19-targeted chimeric antigen receptor (CAR)-T cells, CD 19- targeted antibody-drug conjugates and CD19-targeted antibodies. In some embodiments, the targeted therapy comprises administration of CD19-targeted chimeric antigen receptor (CAR)-T cells and CD19-targeted antibody-drug conjugates. In some embodiments, the targeted therapy comprises administration of CD19-targeted chimeric antigen receptor (CAR)-T cells and CD19-targeted antibodies. In some embodiments, the targeted therapy comprises administration of CD19-targeted antibody-drug conjugates and CD19-targeted antibodies.

[0179] In some embodiments, standard chemoimmunotherapy comprises administration of cyclophosphamide, doxorubicin, vincristine, and prednisone, and / or monoclonal antibody rituximab. In some embodiments, standard chemoimmunotherapy comprises administration of cyclophosphamide. In some embodiments, standard chemoimmunotherapy comprises administration of doxorubicin. In some embodiments, standard chemoimmunotherapy comprises administration of vincristine. In some embodiments, standard chemoimmunotherapy comprises administration of prednisone. In some embodiments, standard chemoimmunotherapy comprises administration of monoclonal antibody rituximab.

[0180] In some embodiments, the patient has measurable disease, defined as at least one lesion per the Lugano classification. In some embodiments, the lesions situated in apreviously irradiated area are considered measurable if radiographic progression has been documented in such lesions following completion of radiation therapy. In some embodiments, the LBCL have positron emission tomography (PET) positive disease per the Lugano classification.

[0181] In some embodiments, the patient has a disease that is relapsed or refractory (r / r) after at least two prior lines of systemic therapy. In some embodiments, the patient with r / r LBCL has received an anti-CD20 monoclonal antibody (mAb) and an anthracycline containing chemotherapy regimen and failed or is ineligible for high-dose chemotherapy and autologous stem cell transplantation (ASCT).

[0182] In some embodiments, the patient with iNHL has received an anti-CD20 mAb and an alkylating agent (e.g., bendamustine or cyclophosphamide). In some embodiments, preinduction salvage chemotherapy and ASCT is considered one line of therapy. In some embodiments, the patient consolidation / maintenance therapy after a chemotherapy regimen (without intervening relapse) is considered one line of therapy. In some embodiments, maintenance antibody therapy is not considered a line of therapy. In some embodiments, the single-agent anti-CD20 mAb therapy is not considered a line of therapy.

[0183] In some embodiments, the patient has adequate bone marrow function. In some embodiments, adequate bone marrow function is defined as absolute neutrophil count >500 / pL and / or platelet count of >50,000 / pL at screening. In some embodiments, patients with transfusion-dependent thrombocytopenia are excluded.

[0184] In some embodiments, the patient has adequate renal, hepatic, cardiac, and pulmonary function. In some embodiments, adequate renal, hepatic, cardiac, and pulmonary function comprises one or more of: a) Estimated glomerular filtration rate (GFR; Modification of Diet in Renal Disease equation [MDRD]) >30 mL / min; b) Serum alanine aminotransferase / aspartate aminotransferase <5 times the upper limit of normal range (ULN), as long as participant is asymptomatic; c) Total bilirubin <2 mg / dL. Participants with Gilbert’s syndrome may have a bilirubin level >2 x ULN, per discussion between the investigator and the medical monitor;d) Left ventricular ejection fraction (LVEF) >40% as determined by an echocardiogram (ECHO) or multigated acquisition (MUGA) scan performed within 1 month of determination of eligibility; e) No evidence of clinically relevant pericardial effusion, and no acute clinically significant electrocardiogram (ECG) findings; f) Absence of Grade >2 pleural effusion. Grade 1 stable pleural effusions are allowed; and / or g) Baseline oxygen saturation >92% on room air.

[0185] In some embodiments, the previously received CD 19 targeted therapy is a CAR T therapy selected from the group consisting of axicabtagene ciloleucel, tisagenlecleucel, brexucabtagene autoleucel and or lisocabtagene maraleucel. In some embodiments, the previously administered CD 19 targeted therapy is a CD 19 targeted antibody therapy selected from the group consisting of tafasitamab and blinatumomab. In some embodiments, the previously administered CD19 targeted therapy is a CD19-targeted antibody-drug-conjugate. In some embodiments, the CD19-targeted antibody-drug-conjugate is loncastuximab tesirine.

[0186] In some embodiments, the CAR-NK cells are frozen prior to administration. In some embodiments, the CAR-NK cells are not washed prior to administering to a subject in need thereof. In some embodiments, the CAR-NK cells are washed prior to administering to a subject in need thereof. In some embodiments, the frozen cells are thawed and administered into a patient in need thereof within about 30 minutes and 2 hours from thawing the cells. In some embodiments, the rate of intravenous infusion into a subject is between about 2-3 minutes. In some embodiments, administration is at a patient’s bedside. In some embodiments, the thawed cells are administered directly without further dilution.Viability Assessment

[0187] Viability of CAR-NK cells can be assessed in vitro using various methods known in the art. In some embodiments, the in vitro cell viability test includes the Trypan Blue exclusion assay. In some embodiments, other analytical methods can be used to assess the cell viability of cells, for example, flow cytometry-based viability markers and the like. A person of ordinary skill in the art can opt for any analytical method to assess the viability of CAR NK cells that can be applied to assess the cell viability cells described herein.

[0188] Phenotype and function of CAR-NK cells can be assessed in vitro using various methods known in the art. In some embodiments, the in vitro cell phenotyping tests includes flow cytometry assays. In some embodiments, the in vitro cell function test includes cytokine production, cytotoxicity, proliferation and other analytical methods.

[0189] Efficacy of CAR-NK cells in vivo can be assessed using animal studies known in the art. In some embodiments, the in vivo cell phenotyping tests immunodeficiency mice- based tumor models.

[0190] The CAR-NK cells described herein retain high viability (e.g., greater than 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) and retain physiological characteristics of their native state, which allows the cells to be used for a variety of applications, such as for genetic manipulation of the cells, and for cell therapy purposes such as, for example, in adoptive cell therapy applications.EXAMPLES

[0191] Other features, objects, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples.Example 1: Multi-dose Dosing Regimen for Administration of CD19 CAR NK cells in Treating Cancer

[0192] This example describes an exemplary dosing regimen for administration of CAR NK cell to a subject suffering from cancer, for example, a subject who has Relapsed or Refractory (r / r) Large B-cell Lymphoma (LBCL) or Indolent Non-Hodgkin’s Lymphoma (iNHL).

[0193] Exemplary CAR-NK cells used in this example comprised CD 19 CAR NK cells. Exemplary CD 19 CAR-NK cells used in these examples were genetically engineered cord blood derived NK cells including a CD19-CAR comprising an anti-CD19 binding domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or a heavy chain variable region comprising the amino acid sequenceset forth in SEQ ID NO: 2. CAR-NK cells used herein were formulated in suitable media for cryopreservation (e.g., a cryopreservation media described in Table 1).

[0194] Subjects with relap sed / refractory B-cell lymphoma who were previously treated with CD19-targeted therapy, receiving the chimeric antigen receptor (CAR)-NK cell therapy targeting CD 19 were evaluated for efficacy of the CAR-NK cells (cord blood derived NK cells comprising exogenously transduced anti-CD19 CAR comprising sequences represented by SEQ ID Nos: 1-5, IL-15 represented by SEQ ID No: 6). For the frozen formulation, each exemplary cell sample was formulated in 40% PLASMA-LYTE A, 50% CS10, 10% HSA and 30 mM trehalose (e.g., 37.7 % v / v PLASMA-LYTE A + 50% v / v CS10 +9.4% v / v HSA + 2.8% v / v trehalose).

[0195] Subjects include those previously administered anti-CD19 therapy (e.g., CD19-targeted chimeric antigen receptor (CAR) T cells or monoclonal antibodies) at least 3 months prior to administration of CAR NK cells described herein.

[0196] Subjects include those with histologically proven B-cell NHL, including LBCL and iNHL, including the types defied by WHO, who have failed 1 or more prior lines of systemic therapy (i.e., 2L or 3L+).

[0197] Patients meeting any of the following inclusion criteria are enrolled in the study:1. Patients aged >18 years or meeting country definition of adult, whichever is older, at the time of signing the informed consent.2. Patients who have a life expectancy >12 weeks.3. Patients who have an Eastern Cooperative Oncology Group performance status of 0 or 1.4. Patients with a diagnosis of previously treated r / r histologically proven CD19-expressing disease of the following types: a. LBCL, including the following subtypes defined by the WHO: i. Diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS). ii. High-grade B-cell lymphoma (HGBL) with MYC and BCL2 and / or BCL6 rearrangement. iii. HGBL NOS without translocations. iv. DLBCL arising from iNHL including FL or MZL.v. T-cell / histiocyte-rich large B-cell lymphoma. vi. DLBCL associated with chronic inflammation. vii. Epstein-Barr virus-positive DLBCL-NOS. viii. Primary cutaneous DLBCL, leg type. ix. Primary mediastinal large B-cell lymphoma. x. FL Grade 3B. b. iNHL, including the following subtypes defined by the WHO: i. FL Grade 1, 2, 3A. ii. MZL (nodal, extranodal, and splenic).5. Patients who have measurable disease, defined as at least 1 lesion per the Lugano classification. Lesions situated in a previously irradiated area are considered measurable if radiographic progression has been documented in such lesions following completion of radiation therapy. LBCL should have a positronemission tomography-positive disease per the Lugano classification.6. Patients who have r / r LBCL or iNHL after >2 prior lines of systemic therapy, or r / r LBCL after 1 prior line of systemic therapy (Part 1 Cohort 1C only): a. Patients with r / r LBCL must have received an anti-CD20 mAb and an anthracycline-containing chemotherapy regimen and failed or be ineligible for high-dose chemotherapy and autologous stem cell transplantation (ASCT). b. Patients with iNHL must have received an anti-CD20 mAb and an alkylating agent (eg, bendamustine or cyclophosphamide). c. Preinduction salvage chemotherapy and ASCT should be considered 1 therapy. d. Any consolidation / maintenance therapy after a chemotherapy regimen (without intervening relapse) should be considered 1 line of therapy with the preceding combination therapy. Maintenance antibody therapy should not be considered a line of therapy. e. Single-agent anti-CD20 mAb therapy should not be considered a line of therapy. f. Bridging chemotherapy given just prior to CAR cell therapy treatment should be considered one line of therapy with cell therapy.g. Patients who have received prior CD19-targeting CAR-T cell therapy must have achieved at least a partial response to the most recent CD19-targeting CAR-T cell therapy.7. Patients who have adequate bone marrow function defined as follows: a. Absolute neutrophil count >500 / pL. b. Platelet count of >50,000 / pL at screening. Patients with transfusion-dependent thrombocytopenia are excluded.8. Patients who have adequate renal, hepatic, cardiac, and pulmonary function as defined in the study protocol.

[0198] Patients meeting any of the following exclusion criteria are not enrolled in the study:1. Patients with total body weight of <40 kg.2. Patients with primary or secondary central nervous system (CNS) involvement by lymphoma. Patients with a history of secondary CNS involvement by lymphoma without evidence of CNS involvement at screening may be included.3. Patients with Burkitt lymphoma, mantle cell lymphoma, lymphoplasmocytic lymphoma, or transformation from CLL / small lymphocytic lymphoma (Richter transformation).4. Patients with a history of malignancy other than nonmelanoma skin cancer, carcinoma in situ (e.g., cervix, bladder, breast), low-grade tumors deemed to be cured and not treated with systemic therapy (e.g., by gastro-endoscopy curatively removed gastric cancer) or unless disease free for >3 years at screening.5. Patients who have undergone autologous or allogeneic transplant or CAR-T or CAR-NK cell therapy within 3 months of planned enrollment. Patients after allogeneic transplant have to be off systemic immunosuppressive therapy and without the evidence of clinically relevant acute or chronic GvHD at the time of enrollment.6. Treatment with any investigational products or any systemic anticancer treatment within 14 days or 2 half-lives of the treatment (whichever is longer) before conditioning therapy. For rituximab, a half-life of 22 days should be considered.7. Patients with clinical evidence of active infection, including fungal, bacterial, viral, or other infection that is uncontrolled or requires IV antimicrobials for management within 3 days before enrollment.8. Patients with active HIV, HBV, or HCV, infection at screening (positive DNA / RNA test).9. Patients with a history or presence of active or clinically relevant CNS disorder, such as seizure, encephalopathy, cerebrovascular ischemia / hemorrhage, severe dementia, cerebellar disease, or any autoimmune disease with CNS involvement. For CNS disorders that recover or are in remission, patients without recurrence within 2 years of planned study enrollment may be included.10. Patients with any of the following within 6 months of enrollment: myocardial infarction, cardiac angioplasty or stenting, unstable angina, symptomatic congestive heart failure (i.e., New York Heart Association Class II or greater), clinically significant arrythmia (including uncontrolled atrial fibrillation), or any other clinically significant cardiac disease.11. Patients who have received a live vaccine <6 weeks before the start of the conditioning regimen

[0199] Subjects receive a single cycle of lymphodepletion therapy (e.g., fludarabine and cyclophosphamide as per standard of care) at least 3-5 days prior to administration of CD 19 CAR NK cells.

[0200] CD19 CAR NK cells are administered in three doses on days 0, 7 and 14 after the single cycle of lymphodepletion therapy. FIG. 1A is a schematic of a dosing schedule indicating multiple doses of CD19 CARNK cells at days 0, 7 and 14. Subjects are treated with fludarabine and cyclophosphamide (Flu / Cy) three to five days before dosing in a single cycle of lymphodepletion. Positron emission tomography / computed tomography (PET / CT) scans are carried out at day 28 as a predictor of progression-free survival (PFS) and overall survival (OS).

[0201] FIG. IB is a schematic of a dose escalation and expansion study from 200 million to 800 million CD 19 CAR NK cells in treating relapsed or refractory large B cell lymphoma (LBCL) or Indolent Non-Hodgkin’s lymphoma (iNHL). Cohorts are treated with either a single dose of 200 million CD 19 CAR NK cells, a single dose of 800 million CD 19 CAR NK cells or 3 doses each of 800 million CD 19 CAR NK cells.

[0202] Dose Escalation: Patients will receive lymphodepleting chemotherapy consisting of IV fludarabine (30 mg / m2 body surface area [BSA] per day) and cyclophosphamide (300 mg / m2 BSA per day) on Days -5, -4, and -3 before a single IV administration of CD19 CAR NK cells at a dose of 200 million (±30%) or 800 million (± 25%) CAR+ NK cells on Day 0 of the study. Dose Expansion Cohort 1 A (LBCL 3L+) and Cohort 2A (iNHL 3L+): Patients will receive the lymphodepleting chemotherapy on Days -5, -4, and -3 before the single IV administration of CD 19 CAR NK cells at a dose of 200 million (±30%) or 800 million (±25%) CAR+ viable NK cells on Day 0 of the study.

[0203] Dose Expansion Cohort IB (LBCL 3L+), Cohort 1C (LBCL 2L), and Cohort 2B (iNHL 3L+): Patients will receive the lymphodepleting chemotherapy on Days -5, -4, and -3 before the 3 IV administrations of CD 19 CAR NK cells, each at a dose of 800 million (± 25%) CAR+ NK cells, on Days 0, 7, and 14 of the study.

[0204] Outcomes are assessed up to 3 months after administration.

[0205] Primary Outcome Measures: The primary outcome measures for evaluating the effect of CD 19 CAR-NK cell administration include monitoring for Adverse Events (AEs), clinically significant changes in Laboratory Parameters (e.g., include hematology, clinical chemistry, serum immunoglobulin and urinalysis tests), clinically significant changes in vital signs (e.g., body temperature (oral or tympanic measurement), sitting blood pressure (after the participant has rested for at least 5 minutes), and pulse rate (bpm)) and / or Overall Response Rate (ORR) per Independent Review Committee (IRC). ORR is defined as the percentage of participants with complete response (CR) or partial response (PR) as best response to treatment, determined by the IRC per the Lugano 2014 criteria after CD 19 CARNK cells administration.

[0206] Secondary Outcome Measures include:

[0207] ORR per the Lugano 2014 criteria after CD19 CAR-NK cells administration

[0208] Complete Response (CR) per Investigator (CR is determined per Lugano 2014 criteria as percentage of participants with target nodes / nodal masses must regress to <1.5 cm in the longest transverse diameter of all lesions and no extralymphatic sites of disease)

[0209] Complete Response (CR) Per IRC (per Lugano criteria as percentage of participants with target nodes / nodal masses must regress to <1.5 cm in the longest transverse diameter of all lesions and no extralymphatic sites of disease).

[0210] Duration of Response (DOR) per Investigator (DOR is defined as the time from the date of first documented objective response to the date of first documented disease progression, determined by investigator per Lugano 2014 criteria classification or death, whichever comes first, for participants who experience an objective response).

[0211] Duration of Response (DOR) per IRC (DOR is defined as the time from the date of first documented objective response to the date of first documented disease progression, determined by the IRC Lugano 2014 criteria classification or death, whichever comes first, for participants who experience an objective response).

[0212] Progression-free Survival (PFS) per Investigator (PFS is defined as time from enrollment date to the date of disease progression, determined by the investigator per Lugano 2014 criteria classification or death from any cause, whichever comes first).

[0213] Progression-free Survival (PFS) per IRC (PFS is defined as time from enrollment date to the date of disease progression, determined by the IRC per Lugano 2014 criteria classification or death from any cause, whichever comes first).

[0214] Overall Survival (OS) (OS is defined as time from enrollment to the date of death from any cause).

[0215] Cmax - Maximum Observed Blood Concentration of CD19 CARNK cells

[0216] Tmax - Time of First Occurrence of Cmax of CD 19 CAR NK cells

[0217] Tlast - Time of Last Measurable Concentration Above the Lower Limit of Quantitation of CD 19 CAR NK cells

[0218] AUC last - Area Under the Concentration-time Curve From Time 0 to Time of the Last Quantifiable Concentration of CD 19 CAR-NK cells

[0219] Concentration of Interleukin (IL)- 15 and Other Soluble Immune Factors in Plasma Over Time

[0220] Concentration of IL-15 and soluble immune factors (e.g., Interferon (IFN)- gamma (y), IL-1 beta (P), IL- 2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, Tumor necrosis factor (TNF) alpha (a), Granulocyte-macrophage colony-stimulating factor (GM-CSF)) in plasma over time will be reported.

[0221] Percentage of Participants with B-cell Aplasia Before and After CD 19 CARNK cells Administration

[0222] Percentage of Participants with Detectable Anti-human Leukocyte Antigen (HLA) and Anti-chimeric Antigen Receptor (CAR) Antibodies Before (Prevalence) and After (Incidence) CD19 CAR NK cells Administration Over Time

[0223] Percentage of Participants with Positive Replication Competent Retrovirus (RCR) Test Results Before (Prevalence) and After (Incidence) CD19 CAR NK cells Administration Over Time

[0224] Example 2: Exposure-Response Modeling & Quantitative Systems Pharmacology Modeling (using fresh product)

[0225] The exposure-response analysis using the fresh formulation indicated that the probability of response (measured as CR and ORR) increases with higher exposures of CD 19 CAR+NK cells. Similarly, the trial simulation results using the mechanistic quantitative system pharmacology (QSP) model also indicated that increased in response rate at higher exposures achieved using multiple doses (2 doses or 3 doses of 800 * 106CD 19 CAR CBNK cells following 1 cycle of lymphodepletion) compared to a single dose of 800 x io6CD19 CAR CBNK cells. The QSP model exploring the effects of CD 19 CAR-NK treatment response in CD 19-positive lymphoid tumors was developed using the preclinical and clinical data of CD 19 CARCB-NK cells for fresh and cryopreserved formulations. The developed fit- for-purpose model adequately describes the effects of CAR-NK treatment response in CD 19- positive lymphoid tumors. In summary, the increase in exposures of cryopreserved CD 19 CAR+ NK cells with multiple doses (e.g., 2 doses delivered on days 0, 3, or 0,7 and 3 doses delivered on days 0, 7, 14) is expected to result in improved efficacy. The proposed multiple dosing frequency is also optimized to deliver the last dose of CAR+ NK cells prior to functional immune reconstitution following the lymphodepletion cycle.EQUIVALENTS AND SCOPE

[0226] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWe claim:

1. A method for treating cancer in a subject comprising a step of administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof.

2. The method of claim 1, wherein two doses are administered.

3. The method of claim 1, wherein three doses are administered.

4. The method of claim 1, wherein greater than three doses are administered.

5. The method of claim 1, wherein the subject has received one or more lymphodepletion cycles.

6. The method of claim 5, wherein the two or more doses are administered following the one or more lymphodepletion cycles.

7. The method of any one of the preceding claims, wherein each dose is 300 million to 1.5 billion cells.

8. The method of any one of the preceding claims, wherein each dose is 600 million cells.

9. The method of any one of the preceding claims, wherein each dose is 800 million cells.

10. The method of any one of the preceding claims, wherein each dose is 1 billion cells.

11. The method of any one of the preceding claims, wherein each dose is 1.5 billion cells.

12. The method of any one of the preceding claims, wherein the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 2.

13. The method of claim 12, wherein the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 2.

14. The method of any one of the preceding claims, wherein the CD19-CAR comprises a CD28 domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 3.

15. The method of claim 14, wherein the CD19-CAR comprises a CD28 domain having 100% identity to the amino acid sequence of SEQ ID NO: 3.

16. The method of any one of the preceding claims, wherein the CD19-CAR comprises a CD3^ domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

17. The method of claim 16, wherein the CD19-CAR comprises a CD3(^ domain having 100% identity to the amino acid sequence of SEQ ID NO: 4.

18. The method of any one of the preceding claims, wherein the single lymphodepletion cycle comprises fludarabine and / or cyclophosphamide.

19. The method of any one of the preceding claims, wherein the CD19-CAR CB-NK cells comprises one or more exogenously provided interleukin (IL).

20. The method of claim 19, wherein the one or more interleukin is selected from IL- 15, IL- 12, IL-21, IL-2, IL- 18, IL-7, and combinations thereof.

21. The method of claim 20, wherein the interleukin is IL-15.

22. The method of claim 21, wherein the IL- 15 is secreted or membrane bound.

23. The method of claim 21, wherein the IL-15 is secreted from the cell.

24. The method of any one of claims 19-23, wherein exogenously provided IL is expressed from a vector in the cells.

25. The method of any one of the preceding claims, wherein the CD19-CAR CB-NK cell comprises a suicide gene.

26. The method of claim 25, wherein the suicide gene is an iCaspase9 suicide gene.

27. The method of any one of the preceding claims, wherein the cancer is hematologic, lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, or cervix cancer.

28. The method of claim 27, wherein the cancer is a hematologic cancer.

29. The method of claim 28, wherein the hematologic cancer is a large B cell lymphoma (LBCL).

30. The method of claim 29, wherein the large B cell lymphoma is relapsed or refractory.

31. A method for treating a relapsed or refractory large B cell lymphoma in a subject, the method comprising administering two or more doses of cord blood derived natural killer (CB-NK) cells expressing CD 19 targeted chimeric antigen receptor (CD 19- CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein each dose is 800 millioncells.

32. The method of claim 31, wherein two doses are administered.

33. The method of claim 31, wherein three doses are administered.

34. The method of claim 31, wherein greater than three doses are administered.

35. The method of any one of claims 31-34, wherein the subject has previously received one or more lymphodepletion cycles.

36. The method of claim 35, wherein the subject has previously received one lymphodepletion cycle.

37. The method of any one of the preceding claims, wherein the subject has previously received surgery, radiation, chemotherapy, hormone therapy, immunotherapy, stem cell therapy, targeted therapy or a combination thereof.

38. The method of any one of the preceding claims, wherein the subject has previously received at least one line of standard chemoimmunotherapy or targeted therapy.

39. The method of claim 38, wherein the targeted therapy comprises administration of one or more of CD19-targeted chimeric antigen receptor (CAR)-T cells, CD 19- targeted antibody-drug conjugates and / or CD19-targeted antibodies.

40. The method of claim 38, wherein standard chemoimmunotherapy comprises administration of cyclophosphamide, doxorubicin, vincristine, and prednisone, and / or monoclonal antibody rituximab.

41. The method of any one of the preceding claims, wherein the CD19-CAR CB-NK cells are autologous with respect to the subject.

42. The method of any one of claims 1-40, wherein the CD19-CAR CB-NK cells are allogeneic with respect to the subject.

43. The method of any one of the preceding claims, wherein the CD19-CAR CB-NK cells are administered intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion in a tumor microenvironment, or a combination thereof.

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