CD19 CAR NK cells for use in methods of treating cancer

Abstract

The present disclosure provides, inter alia, methods and compositions for the treatment of cancer comprising administering CD19 CAR cord blood-derived natural killer (CB-NK) cells to a patient in need thereof.

Description

[Technical field]

[0001] Background technology Chimeric antigen receptor (CAR) T-cell therapy has been shown to be effective in treating certain cancers. Clinical trials have shown that 40-90% complete remission (CR) can be achieved in pediatric and adult patients treated with CD19-targeted CAR T cells. However, 30-60% of patients relapse after CD19 CAR T-cell therapy, and of those, 20-90% are CD19-negative relapses. Eligible treatment options for relapses after CAR are limited, making it even more difficult to achieve CR and improve survival. There is an unmet medical need for patients who relapse after previous CD19-targeted therapies. Summary of the Invention

[0002] The present application relates, at least in part, to the use of CD19-targeted genetically engineered NK cell immunotherapies as described herein (e.g., CD19 CAR+ NK cells, CD19 CAR+ viable NK cells and / or allogeneic cord blood-derived CD19 CAR NK cells). + This is based on the discovery that NK cells (NK cells) have efficacy in (i) patients with recurrent cancer that is CD19 positive, and (ii) patients with recurrent cancer that is CD19 negative and have a history of anti-CD19 therapy. Without wishing to be bound by theory, NK cells may be able to kill tumor cells upon downregulation of target antigens via innate immune receptor-mediated killing.

[0003] In one aspect, the invention provides a method of treating cancer in an individual comprising administering a therapeutically effective amount of live CD19-CAR+ cord blood natural killer (CB-NK) cells to the individual, wherein the individual has a history of anti-CD19 therapy.

[0004] In one aspect, the invention provides a method of treating cancer in an individual, comprising administering a therapeutically effective amount of live CD19-CAR+ cord blood natural killer (CB-NK) cells to the individual, where the individual has previously received anti-CD19 therapy.

[0005] In some embodiments, the individual has failed two or more prior systemic therapies prior to administration of the CD19-CAR+ viable CB-NK cells described herein.

[0006] In some embodiments, the therapeutically effective amount of immune cells will depend on the individual being treated, and the severity and type of cancer being treated.

[0007] In some embodiments, CD19-CAR+ viable CB-NK cells are 200×10 6 ~800×10 6 In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of between 200×10 6 , at least 300 x 10 6 , at least 400 x 10 6 , at least 500×10 6 , at least 600×10 6 , at least 700 x 10 6 , or at least 800×10 6 of CD19-CAR+viable CB-NK cells will be administered.

[0008] In some embodiments, CD19-CAR+ viable CB-NK cells are 800×10 6 In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of 700×10 6 In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of 600×10 6 In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of 500×10 6 In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of 400×10 6In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of 300×10 6 In some embodiments, the CD19-CAR+ viable CB-NK cells are administered at a dose of 200×10 6 of CD19-CAR+viable CB-NK cells will be administered.

[0009] In some embodiments, an anti-CD19 therapy (also referred to herein as a CD19-targeted therapy) is a therapy in which CD19-targeted chimeric antigen receptor (CAR) T cells, CD19-targeted antibody-drug conjugates, and / or CD19-targeted antibodies are administered to an individual. In one embodiment, the anti-CD19 therapy is a CD19-targeted chimeric antigen receptor (CAR) T therapy.

[0010] In some embodiments, the CD19-CAR+ viable CB-NK cells are derived from umbilical cord blood.

[0011] In some embodiments, the individual is administered lymphodepleting chemotherapy intravenously followed by administration of CD19-CAR+ viable CB-NK cells.

[0012] In some embodiments, the individual has undergone anti-CD19 CAR-T therapy 3 months or more prior to administration of the CD19-CAR+ viable CB-NK cells.

[0013] In some embodiments, the cancer is a solid tumor or is not a solid tumor.

[0014] In some embodiments, the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, cervix, or is a blood disease.

[0015] In some embodiments, the cancer is relapsed or refractory B-cell non-Hodgkin's lymphoma, including large B-cell lymphoma and low-grade non-Hodgkin's lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is acute lymphocytic leukemia (ALL). In some embodiments, the cancer is not limited to CD19 and CD20 double positive cancers.

[0016] In some embodiments, the individual is a human.

[0017] In some embodiments, the individual is administered one or more additional cancer therapies.

[0018] In some embodiments, the additional cancer therapy is surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

[0019] In some embodiments, the methods include diagnosing cancer in the individual.

[0020] In some embodiments, the methods include generating CD19-CAR+ viable CB-NK cells.

[0021] In some embodiments, the CD19-CAR+ viable CB-NK cells are autologous with respect to the individual.

[0022] In some embodiments, the CD19-CAR+ viable CB-NK cells are allogeneic with respect to the individual.

[0023] In some embodiments, the CD19-CAR+ viable CB-NK cells are administered in an individual intracranially, by injection, intravenously, intraarterially, intraperitoneally, intratumorally, intramuscularly, endoscopically, intralesional, intracranially, percutaneously, subcutaneously, topically, by perfusion, within the tumor microenvironment, or a combination thereof.

[0024] In some embodiments, the CD19-CAR+ surviving (NK) cells comprise one or more exogenously provided interleukins (ILs).

[0025] In some embodiments, the IL is selected from the group consisting of IL-12, IL-15, IL-21, IL-2, IL-18, IL-7, the p35 and p40 subunits of IL-12 artificially linked together with a linker, and combinations thereof.

[0026] In some embodiments, the IL is IL-15.

[0027] In some embodiments, the IL is secreted, tethered, or membrane bound within the cell.

[0028] In some embodiments, an exogenously provided IL is expressed from a vector intracellularly and / or the NK cells are cultured in the presence of one or more ILs.

[0029] In some embodiments, the NK cells comprise a suicide gene.

[0030] In some embodiments, the CD19-CAR+ surviving (NK) cells further comprise an iCaspase9 suicide gene.

[0031] In some embodiments, the NK cells are cord blood-derived or induced pluripotent stem cell (iPSC)-derived NK cells. In some embodiments, the NK cells are cord blood-derived NK cells. In some embodiments, the NK cells are iPSC-derived NK cells.

[0032] In some embodiments, the CD19-CAR+ viable CB-NK cells have been previously frozen and thawed.

[0033] In some embodiments, the NK cells are genetically engineered cord blood NK cells.

[0034] In some embodiments, the NK cells are engineered with a chimeric antigen receptor (CAR) that binds CD19. In some embodiments, the NK cells are not engineered to target tumor antigens other than CD19. In some embodiments, the CD19 CAR CB-NK cells described herein are engineered to target only CD19-expressing cells. In some embodiments, treatment with the CD19 CAR CB-NK described herein has reduced side effects on normal cells or cells expressing tumor antigens other than CD19. In some embodiments, the CD19 CAR CB-NK cells do not express the CD16 (hnCD16) Fc receptor.

[0035] In some embodiments, the genetically engineered cord blood NK cells comprise human cord blood-derived NK cells (CB-NK) transduced with a retroviral vector expressing iCaspase9, CD19-CAR, and IL-15. In some embodiments, the genetically engineered cord blood NK cells comprise human cord blood-derived NK cells (CB-NK) transduced with a non-viral vector expressing iCaspase9, CD19-CAR, and IL-15.

[0036] In some embodiments, the genetically engineered cord blood NK cells comprise a CD19-CAR that includes an anti-CD19 binding domain, e.g., the alpha, beta or zeta chain of the T cell receptor, a transmembrane domain such as 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, e.g., the intracellular signaling domains 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 a single chain antibody fragment, e.g., an scFv.

[0037] In some embodiments, 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, a CD-19 CAR can comprise an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is set forth in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is set forth in SEQ ID NO: 4), and can further comprise a suicide switch such as iCaspase 9 and / or IL-15.

[0038] In some embodiments, the genetically engineered cord blood NK cells comprise a nucleic acid molecule encoding a heavy chain variable region of an anti-CD19 binding domain and / or a nucleic acid molecule encoding a light chain variable region of an anti-CD19 binding domain.

[0039] In some embodiments, the genetically engineered cord blood NK cells are present in the pharmaceutical compositions and formulations at a concentration of 6M / mL to 120M / mL. In some embodiments, the genetically engineered cord blood NK cells are present at a concentration of 6M / mL to 200M / mL. In some embodiments, the genetically engineered cord blood NK cells are present at a concentration of 6M / mL to 25M / mL. In some embodiments, the genetically engineered cord blood NK cells are present at a concentration of 6M / mL to 120M / mL in a volume of medium ranging from 30 to 45mL. In some embodiments, the genetically engineered cord blood NK cells are present at a concentration of 6M / mL to 200M / mL in a volume of medium ranging from 30 to 45mL. In some embodiments, the genetically engineered cord blood NK cells are present at a concentration of 6M / mL to 25M / mL in a volume of medium ranging from 30 to 45mL.

[0040] In some embodiments, the CAR-NK cells are formulated at a concentration ranging from 100 million cells to 900 million cells and are present in a volume of 30-45 mL of medium. In some embodiments, the CAR-NK cells are present at a concentration of about 200 million cells in a volume of about 36 mL. In another embodiment, the CAR-NK cells are present at a concentration of about 800 million cells in a volume of about 36 mL.

[0041] In some embodiments, the NK cells are freshly isolated or from a cell line.

[0042] In some embodiments, the NK cells are derived from umbilical cord blood, peripheral blood, T cells, iPS cells. In some embodiments, the NK cells are derived from umbilical cord blood. In some embodiments, the NK cells are derived from peripheral blood. In some embodiments, the NK cells are derived from T cells. In some embodiments, the NK cells are derived from iPS cells.

[0043] In some embodiments, the NK cells are derived from umbilical cord blood.

[0044] In certain embodiments, the NK cells comprise human cord blood-derived NK cells (CB-NK) transduced with a retroviral vector expressing iCaspase9, CD19-CAR, and IL-15. In certain embodiments, the NK cells comprise human cord blood-derived NK cells (CB-NK) transduced with a non-retroviral vector expressing iCaspase9, CD19-CAR, and IL-15.

[0045] In some embodiments, engineered NK cell immunotherapy targeting CD19 (e.g., CD19 CAR+ viable NK cells and / or allogeneic cord blood derived CD19 CAR NK +The CD19 CAR (a CD19 CAR-derived NK cell) includes a cell that is an engineered cord blood NK cell comprising a CD19-CAR 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 sequence set forth in SEQ ID NO:2. In another embodiment, the CD19 CAR can comprise an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is set forth in SEQ ID NO:3), a CD3z signaling domain (an exemplary CD3z sequence is set forth in SEQ ID NO:4), and can further comprise a suicide switch, such as, for example, iCaspase9 and / or IL-15. In some embodiments, the engineered cord blood NK cell comprises a nucleic acid molecule encoding the heavy chain variable region of the anti-CD19 binding domain set forth in SEQ ID NO:2 and / or a nucleic acid molecule encoding the light chain variable region of the anti-CD19 binding domain set forth in SEQ ID NO:1.

[0046] In some embodiments, the genetically engineered cord blood NK cells comprise a nucleic acid encoding a heavy chain variable region of an anti-CD19 binding domain set forth in SEQ ID NO: 2 and / or a light chain variable region of an anti-CD19 binding domain set forth in SEQ ID NO: 1. In some embodiments, the genetically engineered cord blood NK cells comprise a nucleic acid encoding a heavy chain variable region of an anti-CD19 binding domain comprising SEQ ID NO: 2, a light chain variable region of an anti-CD19 binding domain comprising SEQ ID NO: 1, and a CD28 transmembrane domain comprising SEQ ID NO: 3, a CD3z signaling domain comprising SEQ ID NO: 4.

[0047] In some embodiments, the genetically engineered cord blood NK cells comprise nucleic acids encoding a heavy chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:2, a light chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:1, a transmembrane domain of CD28 comprising SEQ ID NO:3 or a functional fragment thereof, a CD3z signaling domain comprising SEQ ID NO:4 or a functional fragment thereof, and an IgG1 domain comprising SEQ ID NO:5 or a functional fragment thereof.

[0048] In some embodiments, the genetically engineered cord blood NK cells comprise a nucleic acid encoding a heavy chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:2, a light chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:1, a CD28 transmembrane domain comprising SEQ ID NO:3, a CD3z signaling domain comprising SEQ ID NO:4, and an IgG1 domain comprising SEQ ID NO:5.

[0049] In some embodiments, the genetically engineered cord blood NK cells comprise nucleic acids encoding a heavy chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:2, a light chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:1, a transmembrane domain of CD28 comprising SEQ ID NO:3 or a functional fragment thereof, a CD3z signaling domain comprising SEQ ID NO:4 or a functional fragment thereof, and an IgG1 domain comprising SEQ ID NO:5 or a functional fragment thereof.

[0050] In some embodiments, the genetically engineered cord blood NK cells comprise nucleic acids encoding a heavy chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:2, a light chain variable region of an anti-CD19 binding domain comprising SEQ ID NO:1, a CD28 transmembrane domain comprising SEQ ID NO:3, a CD3z signaling domain comprising SEQ ID NO:4, an IgG1 domain comprising SEQ ID NO:5, an IL15 comprising SEQ ID NO:6, and an iCaspase9 comprising SEQ ID NO:7.

[0051] In some aspects, a cell therapy formulation (e.g., a CAR-NK cell therapy formulation) comprising (a) a population of engineered NK cells comprising cord blood NK cells transduced with a retroviral vector expressing an anti-CD19 chimeric antigen receptor (CAR), IL-15, and iCaspase9, and (b) a pharma- ceutically acceptable carrier as described herein is provided for administration to a subject in need thereof.

[0052] In some embodiments, a cell therapy formulation suitable for administration to a subject in need of the cell therapy formulation comprises a population of CAR-NK cells expressing an anti-CD19 chimeric antigen receptor (CAR), IL-15, and iCaspase9 formulated in a medium comprising a cryoprotectant, a disaccharide, albumin, and a non-pyrogenic and isotonic crystalloid, wherein the population of cells comprises between 200 million and 800 million CAR-NK cells. In certain embodiments, a cell therapy formulation suitable for administration to a subject in need of the cell therapy formulation comprises between 200 million and 800 million CAR-NK cells expressing an anti-CD19 chimeric antigen receptor (CAR), IL-15, and iCaspase9 formulated in a medium for cryopreservation.

[0053] In some embodiments, the CD19-CAR NK cells comprise IL-15. In some embodiments, the IL-15 comprises SEQ ID NO:6 or a functional fragment thereof. In some embodiments, the IL-15 comprises SEQ ID NO:6.

[0054] In some embodiments, the CD19-CAR NK cells comprise iCaspase9. In some embodiments, the iCaspase9 comprises SEQ ID NO:7 or a functional fragment thereof. In some embodiments, the iCaspase9 comprises SEQ ID NO:7.

[0055] In some aspects, a cell therapy formulation is provided that comprises a population of CAR-NK cells comprising cord blood NK cells engineered to express CD-19 CAR, iCaspase9, and IL-15, formulated in a medium for cryopreservation.

[0056] In some embodiments, the cell concentration in the formulation is between about 6 million cells / mL and 200 million cells / mL. In some embodiments, the cell concentration in the formulation is between about 6 million cells / mL and 120 million cells / mL.

[0057] In some embodiments, the total live cells after thawing is about 200 million to about 800 million cells. In some embodiments, the total live cells after thawing is about 200 million cells. In some embodiments, the total live cells after thawing is about 300 million cells. In some embodiments, the total live cells after thawing is about 400 million cells. In some embodiments, the total live cells after thawing is about 500 million cells. In some embodiments, the total live cells after thawing is about 600 million cells. In some embodiments, the total live cells after thawing is about 700 million cells. In some embodiments, the total live cells after thawing is about 800 million cells.

[0058] In one aspect, the invention provides a method of treating cancer in an individual comprising administering a therapeutically effective amount of live CD19-CAR+ cord blood natural killer (CB-NK) cells to the individual, where the cancer is CD19 negative.

[0059] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is not a solid tumor.

[0060] In some embodiments, the cancer is a non-solid tumor.

[0061] In some embodiments, the cancer is large B-cell lymphoma.

[0062] In some embodiments, CD19-CAR+ viable CB-NK cells are at least 200×10 6 is administered at a dose of

[0063] Various aspects of the invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section may be applicable to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise indicated. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly indicates otherwise.

[0064] definition Administration: As used herein, the terms "administering" or "introducing" refer to the administration of CD19-targeted engineered NK cell immunotherapy as described herein (e.g., CD19 CAR+ viable NK cells and / or allogeneic cord blood derived CD19 CAR NK cells). + The terms "cells" and "cell therapy" are used interchangeably in the context of delivering cells to a patient in need thereof. Various methods are known in the art for administering cells to a patient, including, for example, administering cells to a patient in need thereof by intravenous or surgical methods.

[0065] Adoptive Cell Therapy: As used interchangeably herein, the term "adoptive cell therapy" or "adoptive cell transfer" or "cell therapy" or "ACT" refers to the transfer of a population of cells, e.g., genetically engineered cells, to a patient in need thereof. The cells can be taken from the patient in need thereof and expanded (i.e., autologous cells) or obtained from a donor other than the patient (i.e., allogeneic cells). In some embodiments, the cells are immune cells, such as lymphocytes. In some embodiments, the immune cells are NK cells. A variety of cell types can be used for ACT, including, but not limited to, natural killer (NK) cells, T cells, CD8+ cells, CD4+ cells, γδ 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.

[0066] 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 animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0067] Approximately or about: As used herein, when applied to one or more subject values, the term "approximately" or "about" refers to a value similar to the subject's stated value as well as to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls 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 or smaller) of the stated reference value, unless otherwise specified or clear from the context (except when such number exceeds 100% of possible values).

[0068] Allogeneic: As used herein, "allogeneic" refers to any material derived from a different animal of the same species as the individual to which the material is introduced. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical. In some embodiments, allogeneic material derived from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0069] As used herein, the term "autologous" means derived from the same individual. For example, "autologous" in reference to a donor and a recipient means that the donor subject is the recipient subject.

[0070] Chimeric Antigen Receptor (CAR): As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that can confer antigen specificity to a cell (e.g., an immune cell such as an NK cell, including cord blood-derived NK cells and iPSC-derived NK cells (iNK cells)). CARs are also known as chimeric antigen receptors, or chimeric immune receptors. In various embodiments, the CARs described herein may include one or more of an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more costimulatory domains, and an intracellular signaling domain.

[0071] CD19-targeted engineered NK cell immunotherapy: As used herein, the term "CD19-targeted engineered NK cell immunotherapy" refers to compositions and formulations that include CD19 CAR+ NK cells. In some embodiments, the CD19-targeted engineered NK cell immunotherapy includes CD19 CAR+ viable NK cells. In some embodiments, the CD19-targeted engineered NK cell immunotherapy includes cord blood-derived CD19 CAR NK + In some embodiments, the CD19-targeted genetically engineered NK cell immunotherapy comprises allogeneic cord blood-derived CD19 CAR NK cells. + Contains cells.

[0072] Cell: As used herein, the term "cell" refers to any cell, unless a particular type of cell is specified. In some embodiments, the cell is a stem or progenitor cell. In certain embodiments, the cell is a somatic cell, e.g., an adult stem cell, progenitor cell, or differentiated cell. In some embodiments, the cell is a hematopoietic cell, e.g., a hematopoietic stem cell or hematopoietic progenitor cell. In some embodiments, the cell comprises a B cell, a T cell, a monocyte, or a progenitor cell. In some embodiments, the cell is a NK cell, particularly a CAR-NK cell.

[0073] Cryoprotectant: As used herein, the term "cryoprotectant" refers to a substance used to protect biological tissue from freezing damage. Exemplary cryoprotectants include, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propanediol.

[0074] Engineered: As used herein, the term "engineered" refers to entities produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, engineered entities are synthetic and contain elements that do not occur in nature or are configured in the manner utilized in this disclosure.

[0075] Exogenous: As used herein, "exogenous" refers to a polynucleotide (such as one that encodes a gene product or a portion of a gene product) that is not endogenously present in a mammalian cell, such as an immune cell, or that is synthetically produced outside the mammalian cell, such as by recombinant techniques. In certain cases, a particular gene product may be provided exogenously to a cell, and the cell may or may not also express the corresponding endogenous gene product within the cell.

[0076] In vitro: As used herein, the term "in vitro" refers to the process of removing cells from a living organism and growing them outside of the organism (e.g., in a test tube, in a culture bag, in a bioreactor).

[0077] Fresh cells or rescued fresh cells: As used herein, the terms "fresh," "fresh cells," or "rescued fresh cells" refer to mammalian cells that have not been frozen and / or mammalian cells that have been frozen but subsequently restimulated, cultured in medium, and then harvested as fresh cells.

[0078] Functional equivalent or derivative: As used herein, the term "functional equivalent" or "functional derivative" refers to a functional derivative of an amino acid sequence or other molecule (e.g., a media formulation component) that retains a substantially similar activity (either function or structure) as the original molecule or sequence. A functional derivative or equivalent may be a natural derivative or may be synthetically prepared. Exemplary derivatives include those that have chemical properties similar to those of the original molecule or sequence. Desirable similar chemical properties include similarity in charge, bulkiness, hydrophobicity, hydrophilicity, etc.

[0079] 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 physiological fluids.

[0080] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0081] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0082] Primary cells: The term "primary cells" refers to cells that are directly isolated from a subject and then expanded.

[0083] Polypeptide: The term "polypeptide" as used herein refers to a continuous chain of amino acids linked via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of skill in the art will understand that the term is not limited to long chains and can refer to a minimum chain comprising two amino acids linked together via a peptide bond. Polypeptides may be processed (processed) and / or modified, as known to those of skill in the art.

[0084] Protein: The term "protein" as used herein refers to one or more polypeptides that function as separate units. Where a single polypeptide is a separate functional unit and does not require permanent or temporary physical association with other polypeptides to form a separate functional unit, the terms "polypeptide" and "protein" may be used interchangeably. Where a separate functional unit is comprised of multiple polypeptides that are physically associated with each other, the term "protein" refers to multiple polypeptides that are physically associated and function together as a separate unit.

[0085] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human who visits a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0086] Substantially: As used herein, the term "substantially" refers to a qualitative state that exhibits all or nearly all extent or degree of a feature or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, reach perfection and / or progress to perfection or achieve or avoid certain results. The term "substantially" is therefore used to express the potential lack of perfection inherent in many biological and chemical phenomena.

[0087] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0088] Sugar or Saccharide: As used herein, the terms "sugar" and "saccharide" are used interchangeably and generally refer to an oligosaccharide, such as a monosaccharide, disaccharide, trisaccharide, or polysaccharide. In some embodiments, the saccharide is one or more of glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, myo-inositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltitol, methylcellulose, carboxymethylcellulose, dextran, glycogen, amylose, amylopectin, inulin, sodium alginate, ethylcellulose, hydroxyethylcellulose, raffinose, stachyose, xanthan gum, glucosamine, and galactosamine. In some embodiments, the saccharide is a disaccharide. In some embodiments, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, or chitobiose. In some embodiments, the disaccharide is trehalose. In some embodiments, the one or more sugars include trehalose, sucrose, mannitol, and / or dextran.

[0089] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent the onset of, and / or delay the onset of, a symptom(s) of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered by a dosing regimen that includes at least one unit dose. In some embodiments, as used herein, a therapeutically effective amount of adoptive cell therapy is a dosage of cells (e.g., a population of genetically engineered immune cells such as CAR-T or CAR-NK) in a particular formulation (e.g., a cryopreservation medium as described herein) administered to a subject in need thereof (e.g., a patient suffering from a B-cell malignancy). For example, in some embodiments, a therapeutically effective amount includes CAR-NK cells at a concentration of 6M / mL to 120M / mL in a volume of 10mL to 45mL. In some embodiments, a therapeutically effective amount comprises CAR-NK cells at a concentration of 5 M / mL to 25 M / mL in a volume of 10 mL to 45 mL. In some embodiments, a therapeutically effective amount comprises CAR-NK cells in an amount of about 200 million cells to about 800 million cells. In certain embodiments, the CAR-NK cells are engineered to express iCaspase9, IL-15, and the CD-19 chimeric antigen receptor.

[0090] In certain embodiments, the CAR-NK cells are engineered 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 with 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 with at least 95% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0091] Treating: As used herein, the terms "treat", "treatment" or "treating" refer to any method used to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease, with the intent of reducing the risk of developing pathology associated with the disease.

[0092] The recitation herein of numerical ranges by endpoints 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."

[0093] The drawings are for illustrative purposes only and not for limiting purposes. [Brief description of the drawings]

[0094] [Figure 1] AB are exemplary graphs showing CAR-NK cytotoxicity (% killing) against Raji-CD19 KO cells (A) and Nalm6-CD19 KO cells (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] There are limited eligible treatment options for patients who relapse after anti-CD19 therapy, and there is a need for effective therapies for this relapsed population. There are two different types of patients who relapse, including CD19-positive relapse and CD19-negative relapse. In CD19-positive relapse patients, CD19 is still present on the surface of tumor cells, but the persistence of anti-CD19 therapy is limited and the efficacy of anti-CD19 T cell CAR therapy is low. For CD19-negative relapse, CD19 is not present on tumor cells, and despite the persistence of CAR T cells, the tumor escapes CAR-mediated recognition and clearance, resulting in ineffective CAR T cell therapy. CD19 CAR+NK cells are advantageous compared to currently available CD19 CAR-T formulations and CD19 CAR-T cells in development (e.g., iCAR-T) because NK cells have innate immune receptor-mediated killing capabilities. CD19 CAR+NK cells derived from umbilical 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, 382 (2020) 545-). Thus, the CD19 CAR+CB-NK cells described herein for patients with a history of anti-CD19 targeted therapy are advantageous compared to allogeneic CAR-T cells.

[0096] In some embodiments, individuals with a history of anti-CD19 therapy (e.g., patients who have received CAR-T therapy or anti-CD19 monoclonal antibody therapy) are allowed sufficient time to recover from the previous anti-CD19 therapy before administering the CAR NK cells of the present invention. In some embodiments, the previously received anti-CD19 targeted therapy was a CAR-T therapy. In some embodiments, the previously received anti-CD19 targeted therapy was a monoclonal antibody or antibody drug conjugate.

[0097] In some embodiments, the individual received an anti-CD19 targeted therapy within 5 years, within 4 years, within 3 years, within 2 years, within 1 year, within 11 months, within 10 months, within 9 months, within 8 months, within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 4 weeks, within 3 weeks, or within 2 weeks prior to administration of the CD19 CAR+CB-NK cells described herein.

[0098] In some embodiments, the individual received an anti-CD19 targeted therapy at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, at least 24 months, or at least 36 months prior to administration of the CD19 CAR+CB-NK cells described herein.

[0099] In some embodiments, the individual received anti-CD19 targeted therapy at least 3 months prior to administration of the CAR NK cells described herein. In some embodiments, the individual received anti-CD19 targeted therapy at least 6 months prior to administration of the CAR NK cells described herein.

[0100] In some embodiments, the individual received anti-CD19 targeted therapy at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, or at least 32 weeks prior to administration of the CAR NK cells described herein. In some embodiments, the individual received anti-CD19 targeted therapy at least 12 weeks prior to administration of the CAR NK cells described herein. In some embodiments, the individual received anti-CD19 targeted therapy at least 2 weeks prior to administration of the CAR CB-NK cells described herein.

[0101] In some embodiments, the individual has a minimum life expectancy of 12 weeks or greater.

[0102] In some embodiments, the individual has an Eastern Cooperative Oncology Group (ECOG) cancer status of 0 or 1.

[0103] In some embodiments, the individual has a cancer that expresses CD19 (e.g., CD19 positive (CD19+)). In some embodiments, the individual has a cancer that is CD19 negative (CD19-).

[0104] CAR-NK Cell Compositions and Formulations Provided herein are genetically engineered NK cell immunotherapies targeting CD19 (e.g., CD19 CAR+ viable NK cells and / or "allogeneic cord blood-derived CD19 CAR NK +In some embodiments, the CD19 CAR+ NK cells described herein comprise an exogenous gene encoding a CD19 CAR (e.g., a polypeptide comprising SEQ ID NOs: 1-5), IL-15, and iCaspase 9 (inducible caspase 9). In some embodiments, the amino acid sequence of IL-15 comprises SEQ ID NO:6. In some embodiments, the amino acid sequence of iCaspase 9 comprises SEQ ID NO:7.

[0105] In some embodiments, the CD19 CAR+ NK cells are engineered cord blood NK cells comprising a CD19-CAR that comprises 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, the CD19 CAR+ NK cells are engineered cord blood NK cells that comprise a CD19-CAR that comprises an anti-CD19 binding domain comprising the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1 and the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 2.

[0106] In some embodiments, the CD19 CAR+CB-NK cells comprise a CD28 domain. In some embodiments, the CD28 domain comprises SEQ ID NO: 3. In some embodiments, the CD19 CAR+CB-NK cells comprise a CD3 zeta domain. In some embodiments, the CD3 zeta domain comprises SEQ ID NO: 4. Anti-CD19 light chain variable fragment, VL: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKR (SEQ ID NO: 1) Anti-CD19 heavy chain variable fragment, VH: EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSQDPA (SEQ ID NO: 2) CD28: FWVLVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 3) CD3ζ: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGP (SEQ ID NO: 4) IgG1: EPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK (SEQ ID NO: 5) IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 6) iCasp9: MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASRA (SEQ ID NO: 7)

[0107] In some embodiments, the NK cells are derived from a primary cell isolate (e.g., NK cells derived from umbilical cord blood). In some embodiments, the NK cells are derived from a cell line. In some embodiments, the NK cells are fresh cells. In some embodiments, the NK cells have been previously frozen and thawed.

[0108] In some embodiments, the NK cells are engineered to express one or more cytokines. In some embodiments, the NK cells are engineered to express one or more of IL-15, a complex of IL-15 and IL-15Rα, IL-18, IL-12, IL-7, CCL19. Thus, in some embodiments, the NK cells are engineered to express IL-15. In some embodiments, the NK cells are engineered to express a complex of IL-15 and IL-15Rα. In some embodiments, the NK cells are engineered to express IL-18. In some embodiments, the NK cells are engineered to express IL-12. In some embodiments, the NK cells are engineered to express IL-7. In some embodiments, the NK cells are engineered to express CCL19.

[0109] In some embodiments, the NK cells are engineered to express one or more suicide genes, for example, in some instances, the NK cells are engineered to express one or more of iCaspase9, non-secreted TNFα, herpes simplex virus thymidine kinase (HSV-TK), uracil phosphoribosyltransferase (UPRTase), and cytosine deaminase (CD).

[0110] Further examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-alpha variant polypeptides (see, e.g., PCT / US2019 / 062009, incorporated herein by reference in its entirety), which may be affected by delivery of antibodies that bind to TNF-alpha variants. Examples of suicide gene / prodrug combinations that may be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Purine nucleoside phosphorylase of E. coli, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, a so-called suicide gene, may also be used. Other examples of suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-a,g-lyase (MET), and thymidine phosphorylase (TP).

[0111] In some embodiments, the NK cells are engineered to express one or more iCaspase9. In some embodiments, the NK cells are engineered to express non-secreted TNFalpha. 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 phosphoribosyltransferase (UPRTase). In some embodiments, the NK cells are engineered to express Cytosine Deaminase (CD).

[0112] In some embodiments, the NK cells are gene-edited to allow the cells to function more effectively in the tumor microenvironment. In some embodiments, the genes are one or more of TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7. In some embodiments, one or more of these genes are knocked out or knocked down in the cells.

[0113] In some embodiments, the NK cells are engineered to express CD19-CAR, IL-15, and iCaspase 9. Exemplary CAR-NK cells comprising CD19 IL-15, and iCaspase 9 are described in Leukemia, 32 (2018) 520-531, which is incorporated herein by reference in its entirety.

[0114] In some embodiments, the genetically engineered cord blood NK cells comprise a CD19-CAR comprising an anti-CD19 binding domain, e.g., 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, e.g., 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 a single chain antibody fragment, e.g., an scFv. In some embodiments, 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, a CD-19 CAR can comprise an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is set forth in SEQ ID NO:3), a CD3z signaling domain (an exemplary CD3z sequence is set forth in SEQ ID NO:4), and can further comprise a suicide switch such as iCaspase9 and / or IL-15.

[0115] In one embodiment, the genetically engineered cord blood NK cells comprise a nucleic acid molecule encoding a heavy chain variable region of an anti-CD19 binding domain and / or a nucleic acid molecule encoding a light chain variable region of an anti-CD19 binding domain.

[0116] In one aspect, the compositions described herein are directed to CD19-targeted genetically engineered NK cell immunotherapy (e.g., CD19 CAR+ viable CB-NK cells and / or "allogeneic cord blood-derived CD19 CAR NK + The present invention includes "a subject matter comprising a host cell" and a pharma- ceutically acceptable carrier.

[0117] In some embodiments, compositions comprising CD19-targeted genetically engineered NK cell immunotherapy described herein include CAR-NK cells at a concentration of 6M / mL to 120M / mL in a volume of 36mL, 6M / mL to 200M / mL, 5M / mL to 25M / mL, 6M / mL to 120M / mL, or 5M / mL to 25M / mL in a volume of 36mL.

[0118] In some embodiments, the total volume of the composition comprising the CD19-targeted engineered NK cell immunotherapy in which the CAR-NK cells are suspended is between about 15 mL and 30 mL, between about 30 mL and 45 mL, between about 30 mL and 60 mL, or between 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 of suspending the CAR-NK cells 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 of suspending the CAR-NK cells is about 20 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 21 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 22 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 23 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 24 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 25 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 26 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 27 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 28 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 29 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 30 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 31 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 32 mL.In some embodiments, the total volume of suspending the CAR-NK cells is about 33 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 34 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 35 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 36 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 37 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 38 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 39 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 40 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 41 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 42 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 43 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 44 mL. In some embodiments, the total volume of suspending the CAR-NK cells is about 45 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 46 mL.

[0119] In some embodiments, the composition comprising a CD19-targeted genetically engineered NK cell immunotherapy comprises CAR-NK cells at a concentration of about 200-800 million cells per 36 mL. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 100-1 billion CAR-NK cells per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 200-800 million cells per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 100 million per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 200 million per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 300 million per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 400 million per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 500 million per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 600 million per 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 700 million per 36 mL of fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 800 million per 36 mL of fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1 billion per 36 mL of fill volume.

[0120] In some embodiments, the CAR-NK cell therapy formulation is an allogeneic cell therapy formulation composed of human umbilical cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD-19 CAR, and IL-15.

[0121] In some embodiments, the CAR-NK cell therapy formulation comprises 1×10 NK cells formulated in the cryopreservation medium described herein. 6 ~5×10 9 In some embodiments, the CAR-NK cell therapy formulation comprises a population of cells of 2×10 6 ~8×10 6 The cell population comprises:

[0122] In some embodiments, the CAR-NK cell therapy formulation comprises 200×10 CAR-NK cells transduced with retroviral vectors expressing iCaspase9, CD-19CAR, and IL-15 and formulated in 36 mL of cryopreservation medium of Table 1 in a 50 mL AT vial. 6 ~800×10 6 In some embodiments, the CAR-NK cell therapy formulation is formulated in cryopreservation medium 1 of Table 1. In some embodiments, the CAR-NK cell therapy formulation is formulated in cryopreservation medium 2 of Table 1. In some embodiments, the CAR-NK cell therapy formulation is formulated in cryopreservation medium 3 of Table 1. In some embodiments, the CAR-NK cell therapy formulation is formulated in cryopreservation medium 4 of Table 1. In some embodiments, the CAR-NK cell therapy formulation is formulated in cryopreservation medium 5 of Table 1.

[0123] In some embodiments, the CAR-NK cell therapy formulation is formulated in cryopreservation medium 9 of Table 1.

[0124] In some embodiments, the CAR-NK cell therapy formulation comprises 200×10 NK cells transduced with retroviral vectors expressing iCaspase9, CD-19 CAR, and IL-15 and formulated in 36 mL of cryopreservation medium in a 50 mL AT vial. 6 ~800×10 6 It is an allogeneic cell therapy product composed of live NK cells derived from human umbilical cord blood.

[0125] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredient (e.g., cells) having the desired purity, in the form of a lyophilized formulation or an aqueous solution, with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersion agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0126] In some embodiments, the CD19 CAR CB-NK cells are suspended in a medium for cryopreservation. In some embodiments, the CD19 CAR CB-NK cells are suspended in a cryopreservation medium as described in Table 1. To prepare the medium in Table 1, 25% w / v HSA (human serum albumin) and 400 mg / mL trehalose solution are used as components.

[0127] [Table 1]

[0128] Treatment method The CAR-NK cell compositions described herein are suitable for adoptive cell therapy. Adoptive cell therapy can be used to treat a variety of diseases, including, for example, cancer. In certain embodiments, the CAR-NK cell compositions are useful for treating cancer or tumors. In certain embodiments, the cancer includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and liver. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is not limited to CD19 and CD20 double positive cancers.

[0129] In some embodiments, the hematological cancer is a B-cell malignancy (e.g., relapsed or refractory B-cell non-Hodgkin's lymphoma (including large B-cell lymphoma and low-grade non-Hodgkin's 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 histologically proven B-cell NHL, including LBCL and iNHL (FL and MZL), including types defined by the World Health Organization (WHO). In some embodiments, the CAR-NK cell compositions described herein are suitable for relapsed or refractory cancer in an individual, where the individual has previously received a CD19-targeted therapy (including CD19-targeted CAR T therapy and CD19-targeted antibody therapy). In some embodiments, the relapsed or refractory cancer may be CD19-positive or CD19-negative.

[0130] In some embodiments, the cancer is a previously treated r / r histologically proven cluster of differentiation (CD) 19 expressing disease. In some embodiments, the cancer is LBCL, including subtypes defined by the World Health Organization (WHO). In some embodiments, the cancer is diffuse large B-cell lymphoma of unspecified (NOS) (DLBCL). In some embodiments, the cancer is high-grade B-cell lymphoma (HGBL) with MYC and BCL2 and / or BCL6 rearrangements. In some embodiments, the cancer is HGBL NOS without translocations.

[0131] 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 NHL is primary mediastinal large B cell lymphoma (PMBCL). In some embodiments, the cancer is FL grade 3B.

[0132] In some embodiments, the cancer is iNHL. In some embodiments, the cancer is FL grade 1, 2, 3A. In some embodiments, the cancer is MZL (nodal, extranodal, and splenic).

[0133] In some embodiments, the patient has measurable disease, defined as at least one lesion according to the Lugano classification. In some embodiments, lesions located within previously irradiated fields are considered measurable if such lesions have radiographically documented progression after completion of radiation therapy. In some embodiments, the LBCL has positron emission tomography (PET) positive disease according to the Lugano classification.

[0134] In some embodiments, patients have disease that is r / r after at least two prior systemic therapies. In some embodiments, patients with r / r LBCL have received a chemotherapy regimen containing an anti-CD20 monoclonal antibody (mAb) and an anthracycline, and have failed or are ineligible for high-dose chemotherapy and autologous stem cell transplantation (ASCT).

[0135] In some embodiments, the iNHL patient is receiving an anti-CD20 mAb and an alkylating agent (e.g., bendamustine or cyclophosphamide). In some embodiments, pre-induction salvage chemotherapy and ASCT are considered one line of therapy. In some embodiments, consolidation / maintenance therapy of a patient following a chemotherapy regimen (without intervening relapse) is considered one line of therapy with the preceding combination therapy. In some embodiments, maintenance antibody therapy is not considered a line of therapy. In some embodiments, a patient's single agent anti-CD20 mAb therapy is not considered a line of therapy.

[0136] In some embodiments, patients have adequate bone marrow function. In some embodiments, adequate bone marrow function is defined as an absolute neutrophil count >500 / μL and / or a platelet count >50,000 / μL at screening. In some embodiments, patients with transfusion-dependent thrombocytopenia are excluded.

[0137] In some embodiments, the patient has adequate renal, hepatic, cardiac, and pulmonary function. In some embodiments, adequate renal, hepatic, cardiac, and pulmonary function is 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 (ULN) (as long as the participant is asymptomatic); c) total bilirubin ≤ 2 mg / dL. Participants with Gilbert syndrome may have bilirubin levels > 2 × ULN, per discussion between the investigator and medical monitor; d) Left ventricular ejection fraction (LVEF) ≥ 40% as determined by echocardiogram (ECHO) or multigated cardiac scan (MUGA) performed within 1 month of eligibility determination; e) no evidence of clinically relevant pericardial effusion and no acute clinically significant electrocardiogram (ECG) findings; f) No grade ≥2 pleural effusion. Grade 1 stable pleural effusion is permitted; and / or g) Baseline oxygen saturation >92% on room air.

[0138] In some embodiments, the previously administered CD19-targeted therapy is a CAR T therapy selected from the group consisting of axicabtagene siloreucel, tisagenlecleucel, brexcabtagene autorueucel, and / or lysocabtagene malareucel. In some embodiments, the previously administered CD19-targeted therapy is a CD19-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 tesirin.

[0139] In some embodiments, the subject is administered a composition comprising a CAR-NK cell. In some embodiments, the CAR-NK cell comprises an anti-CD19 CAR gene and an IL-15 gene. In some embodiments, the CAR-NK cell comprises an anti-CD19 CAR gene, an IL-15 gene, and iCaspase9.

[0140] In some embodiments, the CAR-NK cells are frozen prior to administration. In some embodiments, the CAR-NK cells are not washed prior to administration to a subject in need thereof. In some embodiments, the CAR-NK cells are washed prior to administration to a subject in need thereof. In some embodiments, the frozen cells are thawed and administered to a patient in need thereof within about 30 minutes to 2 hours after thawing the cells. In some embodiments, the rate of intravenous infusion into a subject is between about 2 to 3 minutes.

[0141] In some embodiments, adoptive cell therapy is used in combination with one or more additional cancer treatments, such as, for example, lymphodepleting chemotherapy. Thus, in some embodiments, a subject with cancer undergoes lymphodepleting chemotherapy prior to administration of a CAR-NK cell therapy formulation formulated in a cryopreservation medium described herein.

[0142] In some embodiments, CAR-NK cell therapy is administered to a patient for the treatment of a B-cell malignancy. In some embodiments, the CAR-NK cell therapy formulation comprises a CD19-CAR that includes an anti-CD19 binding domain and 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 the 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 a single chain antibody fragment, such as an scFv.

[0143] In some embodiments, 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, a CD-19 CAR can comprise an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is set forth in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is set forth in SEQ ID NO: 4), and can further comprise a suicide switch such as iCaspase 9 and / or IL-15.

[0144] In one embodiment, the CAR-NK cell therapy formulation comprises a nucleic acid molecule encoding the heavy chain variable region of the anti-CD19 binding domain and / or a nucleic acid molecule encoding the light chain variable region of the anti-CD19 binding domain.

[0145] CD19-CAR NK cell concentration and volume In some embodiments, the cell therapy formulation is a population of CD19-CAR NK cells further comprising IL-15 and iCaspase9. In some embodiments, the cell therapy formulation comprises CD19-CAR NK cells at a concentration of about 6-120 million cells per milliliter. In some embodiments, the cell therapy formulation comprises CD19-CAR NK cells in a 50 mL container at a concentration of about 6-120 million cells per milliliter. In some embodiments, the cell therapy formulation comprises CD19-CAR NK cells in a 50 mL container at a concentration of about 3-150 million cells per milliliter. In some embodiments, the cell therapy formulation comprises CD19-CAR NK cells in a 50 mL container at a concentration of about 1-250 million cells per milliliter. In some embodiments, the cell therapy formulation comprises CD19-CAR NK cells in a 50 mL container at a concentration of about 1 million to 350 million cells per milliliter. In some embodiments, the cell therapy formulation comprises CD19-CAR NK cells in a 50 mL container at a concentration of about 1 million to 500 million cells per milliliter.

[0146] In some embodiments, the cell therapy formulation is about 20×10 in a 50 mL container. 6 ~100×10 7 In some embodiments, the cell therapy formulation comprises about 100×10 cells in a 50 mL container. 6 ~900×10 6 In some embodiments, the cell therapy formulation comprises about 50×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 100×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 200×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 200×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 300×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 400×10 cells in a 50 mL container.6 In some embodiments, the cell therapy formulation comprises about 500×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 600×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 700×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 800×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 900×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 1000×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 1500×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 2000×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 2500×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 3000×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 3500×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 4000×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 4500×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy formulation comprises about 5000×10 cells in a 50 mL container. 6 Contains cells of.

[0147] In some embodiments, the cell therapy formulation is contained in a 50 mL container with a fill volume of about 20-45 mL. In some embodiments, the cell therapy formulation is contained in a 50 mL container with a fill volume of about 36 mL. In some embodiments, the NK cells are engineered to include one or more transgenes, e.g., a chimeric antigen receptor (CAR). In some embodiments, the cells are CAR-NK+ cells. In some embodiments, the cell therapy formulation includes a CD19-CAR, an IL-15 transgene, and iCaspase9. In some embodiments, the cell therapy formulation is contained in a 50 mL container with a fill volume of about 100×10 6 ~900×10 6 In some embodiments, the current cell therapy formulation comprises about 200×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is approximately 300×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is approximately 400×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is about 500×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is approximately 600×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is approximately 700×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is approximately 800×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the current cell therapy formulation is approximately 900×10 CAR-NK+ cells in a 50 mL container. 6 These are CAR-NK+ cells.

[0148] Survival assessment The viability of CAR-NK cells can be assessed in vitro using various methods known in the art. In some embodiments, in vitro cell viability tests include trypan blue exclusion assays. In some embodiments, other analytical methods can be used to assess the cell viability of the cells, such as flow cytometry-based viability markers. The skilled artisan can select any analytical method for assessing the viability of CAR NK cells that can be applied to assess the cell viability of the cells described herein.

[0149] The phenotype and function of CAR-NK cells can be assessed in vitro using a variety of methods known in the art. In some embodiments, in vitro cell phenotyping tests include flow cytometry assays. In some embodiments, in vitro cell function tests include cytokine production, cytotoxicity, proliferation, and other analytical methods.

[0150] The efficacy of CAR-NK cells in vivo can be assessed using animal studies known in the art, hi some embodiments, in vivo cell phenotyping examines tumor models based on immunodeficient mice.

[0151] The CAR-NK cells described herein retain high viability (e.g., 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) and retain the physiological properties of their native state, thereby allowing the cells to be used for a variety of applications, e.g., for genetic engineering of cells and for cell therapy purposes, such as, for example, adoptive cell therapy applications.

[0152] Combination therapy In certain embodiments, the compositions and methods of the present embodiments include an immune cell population (e.g., a CAR NK cell population) in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic virus, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0153] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic drug. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of a treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy may be one or more of chemotherapeutic agents known in the art.

[0154] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, the individual has been provided, can be provided, and / or is provided with certain additional therapies for cancer, including one or more of surgery, radiation, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like.

[0155] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy. Administration may occur at intervals ranging from simultaneous to minutes, days, or weeks. In embodiments in which immune cell therapy is provided to a patient separately from the additional therapeutic agent, it is generally ensured that no significant period of time passes between the respective deliveries so that the two compounds can still beneficially exert their combined effect on the patient. In such instances, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 hours or 72 hours of each other, and more specifically within about 6-12 hours of each other. In some circumstances, it may be desirable to extend the period of time for treatment sufficiently such that several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between each administration. EXAMPLES

[0156] Other features, objects, and advantages of the present invention will become apparent in the following examples. However, it should be understood that the examples, while illustrating embodiments of the present invention, are given by way of illustration only, not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0157] The CAR-NK cells used in these examples included a CD19 CAR, IL-15, and iCaspase 9. The exemplary CAR-NK cells used in these examples were engineered cord blood NK cells comprising 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 sequence set forth in SEQ ID NO: 2. The CAR-NK cells used herein are formulated in a medium for cryopreservation (e.g., a cryopreservation medium described in Table 1).

[0158] Example 1: Administration of CAR NK Cells to an Individual This example describes an exemplary patient population and administration of CAR NK cells to cancer patients, such as patients with relapsed or refractory (r / r) B-cell non-Hodgkin's lymphoma (NHL). Included are patients with a history of anti-CD19 therapy (e.g., CD19-targeted chimeric antigen receptor (CAR) T cells or monoclonal antibodies). Patients with a history of anti-CD19 therapy (e.g., patients who have undergone CAR-T therapy) are allowed sufficient time (e.g., at least 3 months) to recover from the previous anti-CD19 therapy before administering the CAR NK cells described herein.

[0159] Patients who meet any of the following exclusion criteria will not be enrolled in the study. 1. Patients with a total body weight of less than 40 kg. 2. Patients with primary or secondary central nervous system (CNS) involvement from lymphoma. Patients with a history of secondary CNS involvement from lymphoma who do not have evidence of CNS involvement at screening may be included. 3. Patients with Burkitt lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, or transformation from CLL / small lymphocytic lymphoma (Richter transformation). 4. Patients with a history of malignancy other than non-melanoma skin cancer, carcinoma in situ (e.g., cervix, bladder, breast), low-grade tumors considered curable and not treated with systemic therapy (e.g., gastric cancer curatively resected by gastroscopy), or who have not been in remission for ≥3 years at the time of screening. 5. Patients who have undergone autologous or allogeneic transplantation or CAR-T therapy within 3 months of planned enrollment. Patients after allogeneic transplantation must have discontinued systemic immunosuppressive therapy at the time of enrollment and must not have clinically relevant acute or chronic GvHD. 6. Treatment with any investigational or systemic anticancer agent within 14 days or two drug half-lives (whichever is longer) prior to conditioning therapy. 7. Patients with clinical evidence of active infection, including fungal, bacterial, viral, or other infections, that are uncontrolled or require IV antibiotics for management within 3 days prior to enrollment. 8. Patients with active HIV, HBV, or HCV infection at screening (positive DNA / RNA test). 9. Patients with a history of or presence of active or clinically relevant CNS disorders such as seizures, encephalopathy, cerebrovascular ischemia / hemorrhage, severe dementia, cerebellar disease, or any autoimmune disease involving the CNS. For CNS disorders that are in remission or remission, patients without relapse 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 stent placement, unstable angina, symptomatic congestive heart failure (i.e., New York Heart Association class II or greater), clinically significant arrhythmias (including uncontrolled atrial fibrillation), or any other clinically significant cardiac disease. 11. Patients have received a live vaccine within 6 weeks prior to starting the conditioning regimen.

[0160] The CD19 CAR-NK cells described herein will be investigated to evaluate safety and tolerability in adult participants with r / r B-cell NHL. The study has two parts: Part 1 (dose escalation and dose expansion) and Part 2, and will enroll approximately 242 patients.

[0161] In Part 1, dose escalation and dose expansion cohort participants will receive CD19 CAR-NK cells as follows:

[0162] Part 1: Dose escalation: CD19 CAR-NK cells: 200 × 10 6 of CD19-CAR+ viable CB-NK cells (±30%)

[0163] Part 1: Dose escalation: CD19 CAR-NK cells: 800 × 10 6 of CD19-CAR+ viable CB-NK cells (±25%)

[0164] Part 1: Dose expansion: r / rLBCL: CD19 CAR-NK cells: 200 × 10 6 / 800×10 6 viable NK cells

[0165] Part 1: Dose expansion: r / riNHL: CD19 CAR-NK cells: 200 × 10 6 / 800×10 6 viable NK cells

[0166] Based on the Part 1 data, the sponsor and investigators will select a dose level of the single CD19 CAR-NK cells as the recommended phase 2 dose (RP2D). Once the RP2D is determined, participants will be enrolled into Part 2 of the study in the following cohorts:

[0167] Cohort 1: CD19 CAR-NK cells (LBCL)

[0168] Cohort 2: CD19 CAR-NK cells (iNHL)

[0169] Overall enrollment in the study will be for five years. Participants will have multiple clinic visits and will be enrolled in a separate long-term follow-up study for continued safety evaluation for up to 15 years after CD19 CAR-NK cell administration.

[0170] Efficacy endpoints for evaluating the efficacy of CD19 CAR-NK cell administration include monitoring of adverse events (AEs), clinically significant changes in laboratory parameters (including, e.g., hematology, clinical chemistry, serum immunoglobulins, and urinalysis), clinically significant changes in vital signs (e.g., temperature (oral or tympanic measurements), sitting blood pressure (after the participant rests for at least 5 minutes), and pulse rate (bpm)), and / or overall response rate (ORR) as determined by an Independent Review Committee (IRC). ORR is defined as the proportion of participants after CD19 CAR-NK cell administration who experience a complete response (CR) or partial response (PR) as their best response to treatment as determined by the IRC according to the Lugano 2014 criteria.

[0171] Secondary efficacy measures included: ORR according to Lugano 2014 criteria after administration of CD19 CAR-NK cells Complete response (CR) as determined by the investigator (CR is determined as the proportion of participants with regression of target lymph nodes / nodal masses to ≤1.5 cm in the longest transverse diameter of all lesions and no extranodal sites of disease according to the Lugano 2014 criteria) Complete response (CR) by IRC (proportion of participants with regression of target lymph nodes / nodal masses to ≤1.5 cm in the longest transverse diameter of all lesions and no extranodal sites of disease by Lugano criteria). Investigator-determined duration of response (DOR) (DOR is defined as the time from the date of first documented objective response to the date of first documented disease progression as determined by the investigator according to the Lugano 2014 criteria classification, or death, whichever occurs first, for participants who achieved an objective response). Duration of response (DOR) by IRC (DOR is defined as the time from the date of first documented objective response to the date of first documented disease progression as determined by the IRC Lugano 2014 criteria classification, or death, whichever occurs first, for participants who experienced an objective response). Investigator-determined progression-free survival (PFS) (PFS is defined as the time from enrollment to the date of disease progression as determined by the investigator according to the Lugano 2014 criteria or death from any cause, whichever occurs first). Progression-free survival (PFS) by IRC (PFS is defined as the time from enrollment to the date of disease progression as determined by IRC according to the Lugano 2014 criteria or death from any cause, whichever occurs first). Overall survival (OS) (OS is defined as the time from enrollment to death from any cause). Cmax-Maximum observed blood concentration of CD19 CAR-NK cells ·Tmax-Time to first occurrence of Cmax of CD19 CAR-NK cells Time to last measurable concentration above the lower limit of quantification of Tlast-CD19 CAR-NK cells AUClast - Area under the concentration-time curve from time 0 to the time of the last quantifiable concentration of CD19 CAR-NK cells - Plasma interleukin (IL)-15 and other soluble immune factors concentrations over time Concentrations of IL-15 and soluble immune factors (e.g., interferon (IFN)-gamma (γ), IL-1 beta (β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor (TNF) alpha (α), granulocyte-macrophage colony-stimulating factor (GM-CSF)) over time in plasma will be reported. Proportion of participants with B-cell dysplasia before and after CD19 CAR-NK cell administration Proportion of participants with detectable anti-human leukocyte antigen (HLA) and anti-chimeric antigen receptor (CAR) antibodies before (prevalence) and after (incidence) administration of CD19 CAR-NK cells over time Proportion of participants with reactive retroviral (RCR) test results before (prevalence) and after (incidence) administration of CD19 CAR-NK cells over time

[0172] Patients with relapsed / refractory B-cell lymphoma previously treated with CD19-targeted therapy and receiving CD19-targeted chimeric antigen receptor (CAR)-NK cell therapy were evaluated as described above in a single-arm, open-label, multicenter, international, Phase 2 clinical trial to evaluate the efficacy of CAR-NK cells (cord blood-derived NK cells exogenously transduced with an anti-CD19 CAR comprising sequences represented by SEQ ID NOs: 1-5, IL-15 represented by SEQ ID NO: 6, and iCasp9 represented by SEQ ID NO: 7). For frozen preparations, each cell sample was formulated in 40% Plasmalyte A, 50% CS10, 10% HSA, 30 mM trehalose (e.g., 37.7% v / v Plasmalyte A + 50% v / v CS10 + 9.4% v / v HSA + 2.8% v / v trehalose).

[0173] An exemplary responding patient is 800×10 6The patient showed a partial response within one month after treatment with CD19 CAR+ viable NK cells. The patient had iNHL and had previously received eight prior therapies, including two CD19-targeted CAR-T cells (axicabtagenecilloreucel and tisagenlecleucel). The partial response converted to a complete response after receiving CAR-NK cell infusion (genetically engineered cord blood-derived natural killer cells (NK cells) transduced with CAR19-CD28-zeta-2A-IL15 and inducible caspase-9) for three months.

[0174] The efficacy of CAR-NK cells in patients with negative CD19 expression was analyzed by immunohistochemistry (IHC). Screening biopsies of exemplary responding patients showed CD19-negative LBCL by immunohistochemistry. Patients received 800×10 6 The patient received CD19 CAR+live NK cells and achieved a partial response within one month of treatment.

[0175] Example 2. Evaluation of in vitro cytotoxicity against CD19 KO cancer cells This example shows an in vitro cytotoxicity analysis against CD19 KO cancer cells to evaluate the CD19-independent activity of CAR-NK cells, which are cord blood-derived NK cells exogenously transduced with anti-CD19 CARs comprising sequences represented by SEQ ID NOs: 1-5, IL-15 represented by SEQ ID NO: 6, and iCasp9 represented by SEQ ID NO: 7. The in vitro cytotoxicity of CAR-NK cells was evaluated against two CD19-negative (CD19-, Raji CD19 knockout [KO], NALM6 CD19 KO) target cell lines. The target cancer cell lines used in these studies expressed the NKG2D stress ligand. The two cell types were evaluated using a flow cytometry-based dead cell assessment method (CAR-NK cell lots examined included Donor A, which had a CAR% of 75.28%, and Donor B, which had a CAR% of 80.08%).

[0176] Cytolytic activity of CAR-NK cell lines generated from two independent donor cord blood units (donor A and donor B) was assessed with Raji CD19 KO (Figure 1A) and NALM6 CD19 KO (Figure 1B) tumor cells after 20 hours of co-culture. Raji CD19 KO and NALM6 CD19 KO cell lines were freshly thawed, resuspended, labeled with CellTrace Violet, and plated at 30,000 cells / 100 μL / well. Frozen CAR-NK cells from donor A and frozen CAR-NK cells from donor B (for frozen preparations, each cell sample was formulated in a formulation consisting of 40% Plasmalyte A, 50% CS10, 10% HSA, 30 mM trehalose) were thawed, resuspended in growth medium, and viable cells were counted. CAR-NK cells were co-cultured with the respective target cells at seven different effector (e.g., CAR-NK cells) to target cell ratios (10:1, 5:1, 2.5:1, 1.25:1, 0.63:1, 0.3:1, 0.16:1). Target cells were pre-labeled with a fluorescent dye (CellTrace Violet) to allow differentiation from effector cells. After a 20-h incubation period at 37° C., cells were washed with staining buffer, resuspended in phosphate-buffered saline (PBS) premixed with a fixable viability dye (eFluor 780), and incubated for 30 min at 40° C. in the dark. The cells were then washed twice before measuring the killing activity of the different effector cell treatments by flow cytometry. Target cells alone were used as a baseline of viable cells without killing. Data represent the average of duplicate wells. Target cell killing or specific lysis was calculated as follows: Target cell killing or specific lysis (%)=100-([number of CellTrace Violet positive target cells in wells co-cultured with effector cells / mean number of CellTrace Violet positive cells in target cell alone controls]*100).

[0177] CAR-NK cells from two donors demonstrated killing of both cell types in an E:T cell ratio-dependent manner. CAR-NK cells demonstrated killing of both CD19-negative (Raji-CD19KO and NALM6-CD19KO) cancer cells examined, consistent with natural NK receptor binding of stress ligands expressed on cancer cells.

[0178] Example 3: Combination Therapy of CAR NK Cells to an Individual This example exemplarily describes the administration of chemotherapy agents and intravenous administration of CD19 CAR NK cells, where NK cells are derived from core blood, to cancer patients, such as relapsed or refractory (r / r) B-cell non-Hodgkin's lymphoma (NHL) patients. Patients with a history of anti-CD19 therapy (e.g., CD19-targeted chimeric antigen receptor (CAR) T cells or monoclonal antibodies) are included. Patients are administered CD19 CAR-NK cells in combination with chemotherapy agents (e.g., fludarabine and cyclophosphamide according to standard of care).

[0179] Experiment: Part 1: Dose Escalation: CD19 CAR-NK cells - 200x10^6 CD19-CAR+ viable CB-NK cells. Participants will receive lymphodepleting chemotherapy intravenously once daily, followed by a single dose of 200x10^6 anti-CD19 chimeric antigen receptor (CD19-CAR+) viable natural killer (NK) cells intravenously once on day 0.

[0180] Experiment: Part 1: Dose escalation: CD19 CAR-NK cells - 800x10^6 CD19-CAR+ viable CB-NK cells. Participants will receive lymphodepleting chemotherapy intravenously once daily followed by a single dose of CD19 CAR-NK cells - 800x10^6 CD19-CAR+ viable CB-NK cells intravenously once on day 0.

[0181] Experiment: Part 1: Dose expansion: CD19 CAR-NK cells - 200x10^6 / 800x10^6 CD19-CAR+viable CB-NK cells. Participants with r / r large B-cell lymphoma (LBCL) receive lymphodepleting chemotherapy intravenously once daily followed by a single dose of CD19 CAR-NK cells (200x10^6 / 800x10^6 CD19-CAR+viable CB-NK cells) intravenously once on day 0 to determine RP2D.

[0182] Experiment: Part 1: Dose expansion: iNHL: CD19 CAR-NK cells - 200x10^6 / 800x10^6 CD19-CAR+viable CB-NK cells. Participants with r / r indolent non-Hodgkin lymphoma (iNHL) receive lymphodepleting chemotherapy intravenously once daily followed by a single dose of CD19 CAR-NK cells (200x10^6 / 800x10^6 CD19-CAR+viable CB-NK cells) intravenously once on day 0 to determine RP2D.

[0183] Experimental: Part 2: Cohort 1 - LBCL Participants with LBCL will be enrolled in this cohort to receive lymphodepleting chemotherapy administered intravenously once daily followed by CD19 CAR-NK cells administered intravenously once on day 0 at RP2D.

[0184] Experiment: Part 2: Cohort 2-iNHL

[0185] Participants with iNHL will be enrolled in this cohort to receive lymphodepleting chemotherapy administered intravenously once daily, followed by CD19 CAR-NK cells administered intravenously once on day 0 at RP2D.

[0186] The results are evaluated as described in Example 1.

[0187] Equivalents and Scope 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 invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

1. A pharmaceutical composition for treating cancer in a subject, comprising CD19-CAR + umbilical cord blood natural killer (CB-NK) live cells, wherein the subject has previously received CD19-targeted CAR T therapy.

2. The pharmaceutical composition according to claim 1, wherein the subject has previously received at least two types of standard chemoimmunotherapy or targeted therapy.

3. The pharmaceutical composition according to claim 1 or 2, wherein the subject is administered lymphocyte depletion chemotherapy intravenously, and subsequently CD19-CAR+ viable CB-NK cells are administered.

4. The pharmaceutical composition according to claim 1, wherein the subject has already received anti-CD19 CAR-T therapy at least three months prior to the administration of the CD19-CAR+ viable CB-NK cells.

5. The pharmaceutical composition according to claim 1, wherein the cancer is a solid tumor.

6. The pharmaceutical composition according to claim 1, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, or cervix, or is a blood disorder.

7. The pharmaceutical composition according to claim 1, wherein the cancer is recurrent or refractory B-cell non-Hodgkin lymphoma.

8. A pharmaceutical composition for treating cancer in a subject, comprising CD19-CAR + umbilical cord blood natural killer (CB-NK) living cells, wherein the cancer is CD19-negative.

9. The pharmaceutical composition according to claim 8, wherein the cancer is a solid tumor.

10. The pharmaceutical composition according to claim 8, wherein the cancer is large cell B-cell lymphoma.

11. CD19-CAR+ surviving CB-NK cells number at least 200 × 10⁶ ６ The pharmaceutical composition according to claim 1 or 8, administered in the dose of [amount].

12. The pharmaceutical composition according to claim 1 or 8, wherein the subject is a human.

13. The pharmaceutical composition according to claim 1 or 8, wherein the subject is administered one or more additional cancer therapies.

14. The pharmaceutical composition according to claim 13, wherein the additional cancer therapy is surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

15. The pharmaceutical composition according to claim 1 or 8, wherein the cells are autologous with respect to the subject.

16. The pharmaceutical composition according to claim 1 or 8, wherein the cells are allogeneic with respect to the subject.

17. The pharmaceutical composition according to claim 1 or 8, wherein the population of cells is administered to the subject intracranially, by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, subcutaneously, locally, by perfusion, into the tumor microenvironment, or in combination thereof.

18. The pharmaceutical composition according to claim 1 or 8, comprising one or more interleukins (ILs) that provide CD19-CAR+ live (NK) cells to exogenously.

19. The pharmaceutical composition according to claim 18, wherein the IL is selected from the group consisting of IL-12, IL-15, IL-21, IL-2, IL-18, IL-7, IL-12 containing p35 and p40 subunits artificially linked together with a linker, and combinations thereof.

20. The pharmaceutical composition according to claim 18, wherein the IL is IL-15.

21. The pharmaceutical composition according to claim 18, wherein the IL is secreted, anchored, or membrane-bound within the cell.

22. The pharmaceutical composition according to claim 18, wherein exogenously supplied IL is expressed from the intracellular vector, and / or the NK cells are cultured in the presence of one or more IL.

23. The pharmaceutical composition according to claim 1 or 8, wherein the NK cells contain a suicide gene.

24. The pharmaceutical composition according to claim 23, wherein the suicide gene is the iCaspace9 suicide gene.

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