CD19 car NK cells for use in methods of treating cancer

Abstract

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

Description

Background Art

[0001] 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-directed CAR T cells. However, 30-60% of patients relapse after CD19 CAR T cell therapy, of which 20-90% are CD19-negative relapses. Eligible treatment options for post-CAR relapse are limited, making it more difficult to achieve CR and improve survival. There is an unmet medical need for patients who relapse after exposure to prior CD19-targeted therapy. Summary of the invention

[0002] The present application is based at least in part on the following discovery: CD19-directed genetically modified NK cell immunotherapy described herein (e.g., CD19 CAR+ NK cells, CD19 CAR+ live NK cells and / or allogeneic umbilical cord blood-derived CD19 CARNK + Cells) are effective for (i) CD19-positive recurrent cancer and (ii) CD19-negative recurrent cancer patients with a history of anti-CD19 treatment. Without wishing to be bound by theory, NK cells can kill tumor cells via innate immune receptor-mediated killing after downregulation of target antigens.

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

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

[0005] In some embodiments, the individual has failed 2 or more prior lines of systemic therapy prior to administration of the CD19-CAR+ live CB-NK cells described herein.

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

[0007] In some embodiments, the CD19-CAR+ live CB-NK cells are expressed at 200×10 6 to 800×106 In some embodiments, the CD19-CAR+ live CB-NK cells are administered at a dose of at least 200×10 6 At least 300×10 6 At least 400×10 6 At least 500×10 6 At least 600×10 6 At least 700×10 6 or at least 800×10 6 The dose of CD19-CAR+ live CB-NK cells was administered.

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

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

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

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

[0012] In some embodiments, the individual has previously received anti-CD19 CAR-T therapy more than three months prior to administration of the CD19-CAR+ live 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 lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer, cervical cancer, or is a hematological cancer.

[0015] In some embodiments, the cancer is relapsed or refractory B cell non-Hodgkin's lymphoma (Non-Hodgkin Lymphoma), including large B cell lymphoma and indolent 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 not limited to CD19 and CD20 double positive cancer.

[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, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

[0019] In some embodiments, the method includes the step of diagnosing cancer in the individual.

[0020] In some embodiments, the method includes the step of generating CD19-CAR+ live CB-NK cells.

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

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

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

[0024] In some embodiments, the CD19-CAR+ live (NK) cells contain one or more exogenously provided interleukins (IL).

[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, 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 in the cell.

[0028] In some embodiments, the exogenously provided IL is expressed from a vector in the cell and / or wherein 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+ live (NK) cells further comprise an iCaspase9 suicide gene.

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

[0032] In some embodiments, the CD19-CAR+ live CB-NK cells are pre-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 genetically engineered with a chimeric antigen receptor (CAR) that binds CD19. In some embodiments, the NK cells are not genetically engineered to target tumor antigens other than CD19. In some embodiments, the CD19 CAR CB-NK cells described herein are genetically engineered to target only cells expressing CD19. 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 CD16 (hnCD16) Fc receptors.

[0035] In some embodiments, the genetically engineered cord blood NK cells include 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 include 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 include CD19-CAR, which includes an anti-CD19 binding domain; a transmembrane domain, such as the α, β or ζ chain of the T cell receptor, CD28, CD3 ε, 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 γ, FcR β, CD3 γ, CD3 δ, CD3-ζ, CD3 ε, 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 scFv.

[0037] In some embodiments, the anti-CD19 binding domain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1 and / or a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2. In another embodiment, the CD-19 CAR may include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is shown in SEQ ID NO: 4, and may further include a suicide switch such as iCaspase9 and / or IL-15.

[0038] In some embodiments, the genetically engineered umbilical cord blood NK cells include 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 a concentration of 6 M / mL to 120 M / mL in pharmaceutical compositions and formulations. In some embodiments, the genetically engineered cord blood NK cells are present in a concentration of 6 M / mL to 200 M / mL. In some embodiments, the genetically engineered cord blood NK cells are present in a concentration of 6 M / mL to 25 M / mL. In some embodiments, the genetically engineered cord blood NK cells are present in a concentration of 6 M / mL to 120 M / mL in a volume of medium in the range of 30-45 mL. In some embodiments, the genetically engineered cord blood NK cells are present in a concentration of 6 M / mL to 200 M / mL in a volume of medium in the range of 30-45 mL. In some embodiments, the genetically engineered cord blood NK cells are present in a concentration of 6 M / mL to 25 M / mL in a volume of medium in the range of 30-45 mL.

[0040] In some embodiments, the CAR-NK cells are formulated at a concentration ranging from 100×10 6 cells to 900×10 6 In some embodiments, the CAR-NK cells are present at about 200×10 6 In another embodiment, the CAR-NK cells are present at a concentration of about 800×10 cells in a volume of about 36 mL. 6 The concentration of cells exists.

[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 cord blood, peripheral blood, T cells, iPS cells. In some embodiments, the NK cells are derived from 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 include human umbilical cord blood-derived NK cells (CB-NK) transduced with a retroviral vector expressing iCaspase9, CD19-CAR and IL-15. In a specific embodiment, the NK cells include human umbilical cord blood-derived NK cells (CB-NK) transduced with a non-retroviral vector expressing iCaspase9, CD19-CAR and IL-15.

[0045] In some embodiments, the CD19-directed genetically modified NK cell immunotherapy (e.g., CD19CAR+ live NK cells and / or allogeneic cord blood-derived CD19 CAR NK + Cells) include the following cells, which are genetically engineered umbilical cord blood NK cells comprising CD19-CAR, the CD19-CAR including a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1 and / or a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2. In another embodiment, the CD-19 CAR may include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is shown in SEQ ID NO: 4 and may also include a suicide switch such as iCaspase9 and / or IL-15. In one embodiment, the genetically engineered umbilical cord blood NK cells include a nucleic acid molecule encoding a heavy chain variable region of an anti-CD19 binding domain as shown in SEQ ID NO: 2 and / or a nucleic acid molecule encoding a light chain variable region of an anti-CD19 binding domain as shown in SEQ ID NO: 1.

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

[0047] In some embodiments, the genetically engineered umbilical cord blood NK cells include nucleic acids encoding a heavy chain variable region comprising an anti-CD19 binding domain of SEQ ID NO: 2, a light chain variable region comprising an anti-CD19 binding domain of SEQ ID NO: 1, a CD28 transmembrane domain 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 umbilical cord blood NK cells include nucleic acid encoding a heavy chain variable region comprising an anti-CD19 binding domain of SEQ ID NO: 2, a light chain variable region comprising an anti-CD19 binding domain of 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 umbilical cord blood NK cells include nucleic acids encoding a heavy chain variable region comprising an anti-CD19 binding domain comprising SEQ ID NO: 2, a light chain variable region comprising an anti-CD19 binding domain comprising SEQ ID NO: 1, a CD28 transmembrane domain comprising SEQ ID NO: 3 or a functional fragment thereof, a CD3z signaling domain comprising SEQ ID NO: 4 or a functional fragment thereof, an IgG1 domain comprising SEQ ID NO: 5 or a functional fragment thereof, IL15 comprising SEQ ID NO: 6 or a functional fragment thereof, and iCaspase 9 comprising SEQ ID NO: 7 or a functional fragment thereof.

[0050] In some embodiments, the genetically engineered umbilical cord blood NK cells include nucleic acids encoding an anti-CD19 binding domain heavy chain variable region comprising SEQ ID NO: 2, an anti-CD19 binding domain light chain variable region 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, IL15 comprising SEQ ID NO: 6, and iCaspase 9 comprising SEQ ID NO: 7.

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

[0052] In some embodiments, a cell therapy product suitable for administration to a subject in need thereof comprises a CAR-NK cell population formulated in a medium comprising a cryoprotectant, a disaccharide, an albumin, and a pyrogen-free and isotonic crystalloid solution, wherein the CAR-NK cell population expresses an anti-CD19 chimeric antigen receptor (CAR), IL-15, and iCaspase9, wherein the cell population comprises 200×10 6 CAR-NK cells up to 800×10 6 In a specific embodiment, a cell therapy product suitable for administration to a subject in need thereof comprises 200-800×10 6 The invention relates to a CAR-NK cell expressing anti-CD19 chimeric antigen receptor (CAR), IL-15 and iCaspase9.

[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 iCaspase 9. In some embodiments, the iCaspase 9 comprises SEQ ID NO: 7 or a functional fragment thereof. In some embodiments, the iCaspase 9 comprises SEQ ID NO: 7.

[0055] In some aspects, a cell therapy product is provided, comprising a CAR-NK cell population formulated in a cryopreservation medium, wherein the CAR-NK cell population comprises umbilical cord blood NK cells genetically modified to express CD-19 CAR, iCaspase, and IL-15.

[0056] In some embodiments, the concentration of cells in the product is between about 6×10 6 cells / mL and 200×10 6 In some embodiments, the concentration of cells in the product is between about 6×106 cells / mL and 120×10 6 cells / mL.

[0057] In some embodiments, the total viable cells after thawing are about 200×10 6 About 800×10 6 In some embodiments, the total viable cells after thawing are about 200×10 6 In some embodiments, the total viable cells after thawing are about 300×10 6 In some embodiments, the total viable cells after thawing are about 400×10 6 In some embodiments, the total viable cells after thawing are about 500×10 6 In some embodiments, the total viable cells after thawing are about 600×10 6 In some embodiments, the total viable cells after thawing are about 700×10 6 In some embodiments, the total viable cells after thawing are about 800×10 6 cells.

[0058] In one aspect, the present invention provides a method of treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of CD19-CAR+ live cord blood natural killer (CB-NK) cells, wherein 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, the CD19-CAR+ live CB-NK cells are at least 200×10 6 dosage administration.

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

[0064] definition

[0065] Administration: As used herein, the term "administering" or "introducing" refers to delivering the CD19-directed genetically modified NK cell immunotherapy described herein (e.g., CD19 CAR+ live NK cells and / or allogeneic umbilical cord blood-derived CD19 CAR NK cells) to a patient in need thereof. + Various methods for administering cells to a patient are known in the art, including, for example, administering cells to a patient in need thereof by intravenous or surgical methods.

[0066] Adoptive cell therapy: As used interchangeably herein, the terms "adoptive cell therapy" or "adoptive cell transfer" or "cell therapy" or "ACT" refer to the transfer of cells, e.g., a population of genetically modified cells, into a patient in need thereof. The cells may be derived from and proliferated in a patient in need thereof (i.e., autologous cells), or may be obtained from a non-patient donor (i.e., allogeneic cells). In some embodiments, the cells are immune cells, such as lymphocytes. In some embodiments, the immune cells are NK cells. Various cell types can be used for ACT, including but not limited to natural killer (NK) cells, T cells, CD8+ cells, CD4+ cells, δ-γ T cells, regulatory T cells, induced pluripotent stem cells (iPSC), iPSC-derived T cells, iPSC-derived NK cells, hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), and peripheral blood mononuclear cells.

[0067] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0068] About or approximately: As used herein, the term "about" or "approximately" refers to the stated value of interest and a value similar to the stated reference value when applied to one or more values ​​of interest. In certain embodiments, unless otherwise specified or otherwise apparent from the context (except that such number exceeds 100% of the possible value), the term "about" or "approximately" refers to a range of values ​​in either direction (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value.

[0069] Allogeneic: As used herein, allogeneic refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0070] Autologous: As used herein, the term "autologous" means from the same individual. For example, "autologous" with respect to a donor and a recipient means that the donor subject is the recipient subject.

[0071] Chimeric antigen receptor (CAR): As described herein, the term "chimeric antigen receptor" or "CAR" engineered receptor can confer antigen specificity to cells (e.g., immune cells, such as NK cells, including NK cells derived from cord blood and NK cells derived from iPSC (iNK cells)). CAR is also referred to as a chimeric antigen receptor or chimeric immune receptor. In various embodiments, the CAR described herein may include one or more of the following: an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain.

[0072] CD19-directed genetically modified NK cell immunotherapy: As used herein, the term "CD19-directed genetically modified NK cell immunotherapy" refers to compositions and preparations comprising CD19 CAR+ NK cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises CD19 CAR+ live NK cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises umbilical cord blood-derived CD19 CAR NK + In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises allogeneic umbilical cord blood-derived CD19 CAR NK + cell.

[0073] Cell: As described herein, the term "cell" refers to any cell unless a specific type of cell is specified. In some embodiments, the cell is a stem cell or a progenitor cell. In certain embodiments, the cell is a somatic cell, such as an adult stem cell, a progenitor cell, or a differentiated cell. In some embodiments, the cell is a hematopoietic cell, such as a hematopoietic stem cell or a progenitor cell. In some embodiments, the cell includes a B cell, a T cell, a monocyte, or a progenitor cell. In some embodiments, the cell is a NK cell and in particular a CAR-NK cell.

[0074] 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 propylene glycol.

[0075] Engineered: As used herein, the term "engineered" refers to an artificially produced entity, 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 used in the present disclosure.

[0076] Exogenous: As used herein, "exogenous" as used herein refers to a polynucleotide (e.g., a polynucleotide encoding 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 of a mammalian cell, such as by recombinant technology. In certain cases, a particular gene product may be provided exogenously to a cell, and the cell may or may not express the corresponding endogenous gene product in the cell.

[0077] Ex vivo: As used herein, the term "ex vivo" means a process in which cells are removed from a living organism and propagated outside the organism (eg, in a test tube, in a culture bag, in a bioreactor).

[0078] Fresh cells or rescued fresh cells: As used herein, the terms "fresh," "fresh cells," or "rescued fresh cells" refer to mammalian cells that have never been frozen and / or have been frozen but then restimulated in culture, cultured, and then harvested as fresh cells.

[0079] Functional equivalents or derivatives: As used herein, the term "functional equivalents" or "functional derivatives" in the context of functional derivatives of an amino acid sequence or any other molecule (e.g., a medium formulation component) means retaining substantially similar activity (function or structure) as the original molecule or sequence. Functional derivatives or equivalents may be natural derivatives or prepared synthetically. Exemplary derivatives include those having chemical and physical properties similar to the original molecule or sequence. Desirable similar chemical and physical properties include charge similarity, bulkiness, hydrophobicity, hydrophilicity, etc.

[0080] Isotonic: As used herein, the term "isotonic" refers to an osmotic pressure that is equal to or approximately equal to the osmotic pressure of physiological body fluids.

[0081] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0082] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0083] Primary cells: The term "primary cells" refers to cells that are isolated directly from a subject and subsequently proliferated.

[0084] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and may refer to a minimal chain comprising two amino acids linked together via a peptide bond. As known to those skilled in the art, polypeptides may be processed and / or modified.

[0085] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. If a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form a discrete functional unit, the terms "polypeptide" and "protein" are used interchangeably. If the discrete functional unit is composed of more than one polypeptide physically associated with each other, the term "protein" refers to multiple polypeptides that are physically coupled and function together as a discrete unit.

[0086] 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). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a human who visits a medical provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may have or be susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0087] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a full or near full degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, are completed and / or performed to perfection or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to record the potential lack of completeness inherent in many biological and chemical phenomena.

[0088] Suffering from: An individual who is "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.

[0089] Sugar or saccharide: The terms "sugar" and "saccharide" are used interchangeably herein and generally refer to oligosaccharides, such as monosaccharides, disaccharides, trisaccharides or polysaccharides, etc. In some embodiments, the saccharide is one or more of the following: glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, inositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltitol, methylcellulose, carboxymethylcellulose, polydextrose, glycogen, straight-chain starch, 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 other embodiments, the disaccharide is trehalose. In some embodiments, the one or more sugars include trehalose, sucrose, mannitol, and / or polydextrose.

[0090] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent and / or delay the onset of symptoms of a disease, disorder and / or illness when administered to a subject suffering from or susceptible to a disease, disorder and / or illness. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. In some embodiments, a therapeutically effective amount of adoptive cell therapy as used herein is a cell (e.g., a genetically modified immune cell population, such as CAR-T or CAR-NK) in a certain preparation (e.g., a cryopreservation medium as described herein) administered to a subject in need (e.g., a patient with a B-cell malignancy). For example, in some embodiments, the therapeutically effective amount comprises CAR-NK cells at a concentration of 6 M / mL to 120 M / mL in a volume between 10 mL and 45 mL. In some embodiments, the therapeutically effective amount comprises CAR-NK cells at a concentration of 5 M / mL to 25 M / mL in a volume between 10 mL and 45 mL. In some embodiments, the therapeutically effective amount comprises approximately 200×10 6 cells to about 800×10 6 In a specific embodiment, the CAR-NK cells are genetically modified to express iCaspase, IL-15 and CD-19 chimeric antigen receptors.

[0091] In a specific embodiment, the CAR-NK cell is genetically modified to express a CD-19 CAR comprising a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2 or a sequence having at least 95% identity with the sequence as shown in SEQ ID NO: 2 and / or a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1 or a sequence having at least 95% identity with the amino acid sequence as shown in SEQ ID NO: 1.

[0092] Treatment: 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 can be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease to reduce the risk of developing lesions associated with the disease.

[0093] The recitation of numerical ranges by endpoints herein includes all numbers and fractions within that range (eg, 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 assumed to be modified by the term "about." BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The drawings are for illustration purposes only; they are not limiting.

[0095] Figure 1A-1B It is to show the effect of CAR-NK on Raji-CD19 KO cells ( Figure 1A ) and Nalm6-CD19 KO cells ( Figure 1B ) is an exemplary graph of cytotoxicity (% killing) of . DETAILED DESCRIPTION

[0096] Patients who relapse after anti-CD19 treatment have limited eligible treatment options, and effective therapies for this relapse population are needed. There are two different types of relapse patients, 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 anti-CD19 T cell CAR therapy is low in efficacy. For CD19-negative relapse, CD19 is not present on tumor cells, resulting in the ineffectiveness of CAR T cell therapy because tumors escape CAR-mediated recognition and clearance despite the persistence of CAR T cells. Compared with currently available CD19 CAR-T products and CD19 CAR-T cells in development (such as iCAR-T), CD19 CAR+ NK cells have advantages 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 CD19Positive Lymphoid Tumors. NEJM 382(2020)545-). Therefore, compared with allogeneic CAR-T cells, the CD19 CAR+ CB-NK cells described herein have advantages for patients with a history of anti-CD19 targeted therapy.

[0097] In some embodiments, individuals with a history of anti-CD19 treatment (e.g., patients who have undergone CAR-T therapy or anti-CD19 monoclonal antibodies) are allowed enough time to recover from previous anti-CD19 treatment before administering the CAR NK cells of the present invention. In some embodiments, the previously received anti-CD19 targeted therapy is CAR-T therapy. In some embodiments, the previously received anti-CD19 targeted therapy is a monoclonal antibody or an antibody drug conjugate.

[0098] In some embodiments, the individual received 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.

[0099] In some embodiments, the individual received 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.

[0100] In some embodiments, the individual receives anti-CD19 targeted therapy at least 3 months before administering the CAR NK cells described herein. In some embodiments, the individual receives anti-CD19 targeted therapy at least 6 months before administering the CAR NK cells described herein.

[0101] In some embodiments, the individual receives 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 before administering the CAR NK cells described herein. In some embodiments, the individual receives anti-CD19 targeted therapy at least 12 weeks before administering the CAR NK cells described herein. In some embodiments, the individual receives anti-CD19 targeted therapy at least 2 weeks before administering the CAR CB-NK cells described herein.

[0102] In some embodiments, the individual has a minimum life expectancy of ≥ 12 weeks.

[0103] In some embodiments, the individual has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0104] In some embodiments, the individual has a cancer that expresses CD19 (e.g., CD19 positive (CD19+)). In some embodiments, the individual has a CD19 negative (CD19-) cancer.

[0105] CAR-NK cell compositions and preparations

[0106] Provided herein are methods for immunotherapy comprising CD19-directed genetically modified NK cells (e.g., CD19 CAR+ live NK cells and / or "allogeneic cord blood-derived CD19 CAR NK +Cells”). The CD19 CAR+ NK cells described herein comprise CD19 CAR, IL-15, and iCaspase9 (inducible caspase 9). In some embodiments, the CD19 CAR+ NK cells described herein comprise exogenous genes encoding CD19 CAR (e.g., a polypeptide comprising SEQ ID No 1-5), IL-15, and iCaspase9 (inducible caspase 9). In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO: 6. In some embodiments, the iCaspace9 amino acid sequence comprises SEQ ID NO: 7.

[0107] In some embodiments, the CD19 CAR+ NK cells are genetically 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 shown in SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the CD19 CAR+ NK cells are genetically engineered cord blood NK cells comprising a CD19-CAR comprising 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.

[0108] 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 CD3ζ domain. In some embodiments, the CD3ζ domain comprises SEQ ID NO: 4.

[0109] Anti-CD19 light chain variable fragment, VL:

[0110] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKR (SEQ ID NO: 1)

[0111] Anti-CD19 heavy chain variable fragment, VH:

[0112] EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSQDPA (SEQ ID NO: 2)

[0113] CD28:

[0114] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 3)

[0115] CD3ζ:

[0116] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGP (SEQ ID NO: 4)

[0117] IgG1:

[0118] EPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK (SEQ ID NO: 5)

[0119] IL-15:

[0120] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 6)

[0121] iCasp9:

[0122] MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLE LAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASRA (SEQ ID NO: 7)

[0123] In some embodiments, the NK cells are from primary cell isolates (e.g., NK cells derived from umbilical cord blood). In some embodiments, the NK cells are from cell lines. In some embodiments, the NK cells are fresh cells. In some embodiments, the NK cells are pre-frozen and thawed.

[0124] 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. Therefore, 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.

[0125] 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-secretory TNFα, herpes simplex virus thymidine kinase (HSV-TK), uracil phosphoribosyltransferase (UPRTase), and cytosine deaminase (CD).

[0126] Additional examples of suicide genes include engineered non-secretable (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see, e.g., PCT / US2019 / 062009, which is incorporated herein by reference in its entirety), and they may be affected by the delivery of antibodies that bind to TNF-α mutants. Examples of suicide gene / prodrug combinations that can 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 cytarabine. Escherichia coli (E. coli) purine nucleoside phosphorylase, a so-called suicide gene, can be used to convert prodrug 6-methyl purine deoxynucleosides into toxic purine 6-methyl purines. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine-α,g-lyase (MET), and thymidine phosphorylase (TP) as examples.

[0127] In some embodiments, the NK cells are engineered to express one or more iCaspase 9. In some embodiments, the NK cells are engineered to express non-secretory TNFα. 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).

[0128] In some embodiments, the NK cells are edited by genes to allow cells to work more effectively in a tumor microenvironment. In some embodiments, the gene is TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM 17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5 and CD7. One or more of these genes are knocked out or knocked down in cells.

[0129] 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.

[0130] In some embodiments, the genetically engineered cord blood NK cells include CD19-CAR, which includes an anti-CD19 binding domain; a transmembrane domain, such as the α, β or ζ chain of the T cell receptor, CD28, CD3 ε, 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 γ, FcR β, CD3 γ, CD3 δ, CD3-ζ, CD3 ε, 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 scFv. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1 and / or a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2. In another embodiment, the CD-19 CAR may include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is shown in SEQ ID NO: 4) and may further include a suicide switch such as iCaspase9 and / or IL-15.

[0131] In one embodiment, the genetically engineered umbilical cord blood NK cells include 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.

[0132] In one aspect, the compositions described herein comprise CD19-directed genetically modified NK cell immunotherapy (e.g., CD19 CAR+ live CB-NK cells and / or "allogeneic cord blood-derived CD19 CAR NK + cells) and a pharmaceutically acceptable carrier.

[0133] In some embodiments, a composition comprising a CD19-directed genetically modified NK cell immunotherapy described herein comprises CAR-NK cells at a concentration of 6 M / mL to 120 M / mL, 6 M / mL to 200 M / mL, 5 M / mL to 25 M / mL, 6 M / mL to 120 M / mL, or 5 M / mL to 25 M / mL in a volume of 36 mL.

[0134] In some embodiments, the total volume of the composition of the CD19-directed genetically modified NK cell immunotherapy containing the suspended CAR-NK cells is between about 15 mL and 30 mL, about 30 mL and 45 mL, about 30 and 60 mL, or about 30 mL and 75 mL. In some embodiments, the total volume of the suspended NK cells is between about 15 mL and 30 mL. In some embodiments, the total volume of the suspended CAR-NK cells is between about 30 mL and 45 mL. In some embodiments, the total volume of the suspended CAR-NK cells is between about 30 mL and 60 mL. In some embodiments, the total volume of the suspended CAR-NK cells is between about 30 mL and 75 mL. In some embodiments, the total volume of the suspended 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 the suspended CAR-NK cells is about 20 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 21 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 22 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 23 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 24 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 25 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 26 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 27 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 28 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 29 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 30 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 31 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 32 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 33 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 34 mL.In some embodiments, the total volume of the suspended CAR-NK cells is about 35 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 36 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 37 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 38 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 39 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 40 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 41 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 42 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 43 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 44 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 45 mL. In some embodiments, the total volume of the suspended CAR-NK cells is about 46 mL.

[0135] In some embodiments, the composition comprising a CD19-directed genetically modified NK cell immunotherapy comprises a concentration of about 200×10 6 Up to 800×10 6 cells / 36 mL of CAR-NK cells. In some embodiments, the composition comprises a concentration of about 100-1000×10 6 CAR-NK cells / 36 mL of filling volume of CAR-NK cells. In some embodiments, the composition comprises a concentration of about 200-800×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 100×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 200×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 300×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 400×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 500×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 600×106 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 700×10 6 cells / 36 mL of filling volume. In some embodiments, the composition comprises a concentration of about 800×10 6 cells / 36 mL of fill volume. In some embodiments, the composition comprises a concentration of about 1000×10 6 cells / 36 mL filling volume of CAR-NK cells.

[0136] In some embodiments, the CAR-NK cell therapy product is an allogeneic cell therapy product composed of human umbilical cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD-19 CAR and IL-15.

[0137] In some embodiments, the CAR-NK cell therapy product comprises 1×10 6 Up to 5X10 9 In some embodiments, the CAR-NK cell therapy product comprises 2×10 6 Up to 800X10 6 of cell populations.

[0138] In some embodiments, the CAR-NK cell therapy product comprises 200×10 6 Up to 800X10 6 Allogeneic cell therapy products of human umbilical cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD-19 CAR, and IL-15 and formulated in 36 mL of the cryopreservation medium of Table 1 in a 50 mL AT vial. In some embodiments, the CAR-NK cell therapy product is formulated in cryopreservation medium 1 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in cryopreservation medium 2 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in cryopreservation medium 3 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in cryopreservation medium 4 of Table 1. In some embodiments, the CAR-NK cell therapy product is formulated in cryopreservation medium 5 of Table 1.

[0139] In some embodiments, the CAR-NK cell therapy product is formulated in the cryopreservation medium 9 of Table 1.

[0140] In some embodiments, the CAR-NK cell therapy product is composed of 200×10 6 Up to 800X10 6 An allogeneic cell therapy product composed of live human umbilical cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD-19 CAR, and IL-15 and formulated in 36 mL cryopreservation medium in a 50 mL AT vial.

[0141] Pharmaceutical compositions and formulations as described herein can be prepared by mixing an active ingredient (e.g., cells) having a desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012) in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; chloride); phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX ® , Baxter International Company). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0142] In some embodiments, the CD19 CAR CB-NK cells are suspended in a cryopreservation medium. In some embodiments, the CD19 CAR CB-NK cells are suspended in a cryopreservation medium 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.

[0143] Table 1: Cryopreservation media

[0144]

[0145] Treatment

[0146] The CAR-NK cell compositions described herein are suitable for adoptive cell therapy. Adoptive cell therapy can be used to treat various diseases, including, for example, cancer. In certain embodiments, the CAR-NK cell compositions can be used to treat cancer or tumors. In certain embodiments, the cancers include 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 tumors. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is not limited to CD19 and CD20 double positive cancers.

[0147] In some embodiments, the blood cancer is a B-cell malignancy (e.g., relapsed or refractory B-cell non-Hodgkin's lymphoma (including large B-cell lymphoma and indolent 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 confirmed 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 composition described herein is suitable for an individual's relapsed or refractory cancer, wherein the individual has previously received therapy targeting CD19 (including CAR T therapy targeting CD19 and antibody therapy targeting CD19). In some embodiments, the relapsed or refractory cancer may be CD19 positive or CD19 negative.

[0148] In some embodiments, the cancer is a previously treated r / r histologically confirmed disease expressing cluster of differentiation (CD) 19. 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 (DLBCL) of non-specific type (NOS). 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 translocation.

[0149] In some embodiments, the cancer is DLBCL caused by 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 LBCL rich in T cells / tissue cells. In some embodiments, the cancer is DLBCL associated with chronic inflammation. In some embodiments, the cancer is Epstein-Barr virus (Epstein-Barrvirus) positive DLBCL-NOS. In some embodiments, the cancer is primary skin DLBCL, leg type. In some embodiments, the cancer is primary mediastinal large B cell lymphoma (PMBCL). In some embodiments, the cancer is FL grade 3B.

[0150] 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 (lymph node, extra-lymph node, and spleen).

[0151] In some embodiments, the patient has measurable disease, defined as at least 1 lesion according to the Lugano classification. In some embodiments, a lesion located in a previously irradiated area is considered measurable if radiographic progression has been documented in such lesion after completion of radiation therapy. In some embodiments, the LBCL has positron emission tomography (PET) positive disease according to the Lugano classification.

[0152] In some embodiments, the patient has disease that is r / r after at least 2 prior lines of systemic therapy.In some embodiments, patients with r / r LBCL have received anti-CD20 monoclonal antibodies (mAbs) and anthracycline-containing chemotherapy regimens, and have failed or are not eligible for high-dose chemotherapy and autologous stem cell transplantation (ASCT).

[0153] In some embodiments, patients with iNHL have received anti-CD20 mAb and alkylating agents (e.g., bendamustine or cyclophosphamide). In some embodiments, pre-induction salvage chemotherapy and ASCT are considered as 1 therapy. In some embodiments, patient consolidation / maintenance therapy after chemotherapy regimen (without intervention relapse) is considered as first-line therapy with previous combination therapy. In some embodiments, maintenance antibody therapy is not considered a treatment line. In some embodiments, a single-agent anti-CD20 mAb therapy for a patient is not considered a treatment line.

[0154] In some embodiments, the patient has 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.

[0155] In some embodiments, the patient has adequate renal, hepatic, cardiac, and pulmonary function. In some embodiments, adequate renal, hepatic, cardiac, and pulmonary function comprises one or more of the following:

[0156] a) Estimated glomerular filtration rate (GFR; Modification of Diet in Renal Disease [MDRD]) ≥ 30 mL / min;

[0157] b) serum alanine aminotransferase / aspartate aminotransferase ≤ 5 times the upper limit of normal range (ULN), as long as the participant is asymptomatic;

[0158] c) Total bilirubin ≤2 mg / dL. Based on discussion between the investigator and the medical monitor, participants with Gilbert's syndrome may have bilirubin levels > 2 × ULN;

[0159] d) left ventricular ejection fraction (LVEF) ≥ 40%, determined by echocardiogram (ECHO) or multi-gated acquisition (MUGA) scan performed within 1 month of determining eligibility;

[0160] e) no evidence of clinically relevant pericardial effusion and no acute clinically significant electrocardiographic (ECG) findings;

[0161] f) No Grade ≥ 2 pleural effusion is present. Grade 1 stable pleural effusion is permitted; and / or

[0162] g) Baseline oxygen saturation >92% on room air.

[0163] In some embodiments, the previously accepted CD19 targeted therapy is a CAR T therapy selected from the group consisting of axicabtageneciloleucel, tisagenlecleucel, brexucabtagene autoleucel and or lisocabtagene maraleucel. In some embodiments, the previously administered CD19 targeted therapy is a CD19 targeted antibody therapy selected from tafasitamab and blinatumomab. In some embodiments, the previously administered CD19 targeted therapy is an antibody-drug conjugate targeting CD19. In some embodiments, the antibody-drug conjugate targeting CD19 is loncastuximab tesirine.

[0164] In some embodiments, a composition comprising CAR-NK cells is administered to an individual. In some embodiments, the CAR-NK cells comprise anti-CD19 CAR genes and IL-15 genes. In some embodiments, the CAR-NK cells comprise anti-CD19 CAR genes, IL-15 genes, and iCaspase9.

[0165] In some embodiments, the CAR-NK cells are frozen before administration. In some embodiments, the CAR-NK cells are not washed before being administered to a subject in need. In some embodiments, the CAR-NK cells are washed before being administered to a subject in need. In some embodiments, the frozen cells are thawed and administered to a patient in need within about 30 minutes and 2 hours after thawing the cells. In some embodiments, the rate of intravenous infusion into the subject is about 2-3 minutes.

[0166] In some embodiments, adoptive cell therapy is used in combination with one or more additional cancer treatments, such as lymphocyte-depleting chemotherapy. Thus, in some embodiments, a subject with cancer receives lymphocyte-depleting chemotherapy prior to administration of a CAR-NK cell therapy product formulated in a cryopreservation medium as described herein.

[0167] In some embodiments, the CAR-NK cell therapy is administered to a patient to treat B cell malignancies. In some embodiments, the CAR-NK cell therapy product comprises a CD19-CAR comprising an anti-CD19 binding domain; a transmembrane domain such as an α, β or ζ chain of a T cell receptor, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; and an intracellular signaling domain such as an intracellular signaling domain FcR γ, FcR β, CD3 γ, CD3 δ, CD3-ζ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d. The CD-19 binding domain may be a single-chain antibody or a single-chain antibody fragment, such as scFv.

[0168] In one embodiment, the anti-CD19 binding domain comprises a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1 and / or a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2. In another embodiment, the CD-19 CAR may include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is shown in SEQ ID NO: 4, and may further include a suicide switch such as iCaspase9 and / or IL-15.

[0169] In one embodiment, the CAR-NK cell therapy product comprises 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.

[0170] Concentration and volume of CD19-CAR NK cells

[0171] In some embodiments, the cell therapy product is a CD19-CARNK cell population that further comprises IL-15 and iCaspase 9. In some embodiments, the cell therapy product comprises a concentration between about 6 and 120×10 6 In some embodiments, the cell therapy product comprises a concentration of between about 6 and 120×10 6 In some embodiments, the cell therapy product comprises a concentration of between about 3 and 150×10 6In some embodiments, the cell therapy product comprises a concentration of between about 1 and 250×10 6 In some embodiments, the cell therapy product comprises a concentration of between about 1 and 350×10 6 In some embodiments, the cell therapy product comprises a concentration of between about 1 and 500×10 6 CD19-CAR NK cells between 10 cells / mL.

[0172] In some embodiments, the cell therapy product contains about 20×10 6 With 100X10 7 In some embodiments, the cell therapy product contains about 100×10 6 With 900×10 6 In some embodiments, the cell therapy product contains about 50×10 6 In some embodiments, the cell therapy product contains about 100×10 6 In some embodiments, the cell therapy product contains about 200×10 6 In some embodiments, the cell therapy product contains about 200×10 6 In some embodiments, the cell therapy product contains about 300×10 6 In some embodiments, the cell therapy product contains about 400×10 6 In some embodiments, the cell therapy product contains about 500×10 6 In some embodiments, the cell therapy product contains about 600×10 6 In some embodiments, the cell therapy product contains about 700×10 6 In some embodiments, the cell therapy product contains about 800×10 6 In some embodiments, the cell therapy product contains about 900×10 6In some embodiments, the cell therapy product contains about 1000×10 6 In some embodiments, the cell therapy product contains about 1500×10 6 In some embodiments, the cell therapy product contains about 2000×10 6 In some embodiments, the cell therapy product contains about 2500×10 6 In some embodiments, the cell therapy product contains about 3000×10 6 In some embodiments, the cell therapy product contains about 3500×10 6 In some embodiments, the cell therapy product contains about 4000×10 6 In some embodiments, the cell therapy product contains about 4500×10 6 In some embodiments, the cell therapy product contains about 5000×10 6 cells.

[0173] In some embodiments, the cell therapy product is contained in a 50 mL container with a fill volume between about 20-45 mL. In some embodiments, the cell therapy product is contained in a 50 mL container with a fill volume of about 36 mL. In some embodiments, the NK cells are engineered to contain one or more transgenes, such as a chimeric antigen receptor (CAR). In some embodiments, the cells are CAR-NK+ cells. In some embodiments, the cell therapy product comprises CD19-CAR, IL-15 transgene, and iCaspase9. In some embodiments, the cell therapy product comprises about 100×10 6 With 900×10 6 In some embodiments, the cell therapy product is present as 200×10 6 In some embodiments, the cell therapy product is present as 300×10 CAR-NK+ cells in a 50 mL container. 6 In some embodiments, the cell therapy product is present as 400×10 6 In some embodiments, the cell therapy product is present as 500×10 CAR-NK+ cells in a 50 mL container.6 In some embodiments, the cell therapy product is present as 600×10 6 In some embodiments, the cell therapy product is present as 700×10 6 In some embodiments, the cell therapy product is present as 800×10 6 In some embodiments, the cell therapy product is present as 900×10 6 CAR-NK+ cells.

[0174] Vitality Assessment

[0175] 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 (Trypan Blue) exclusion assays. In some embodiments, other analytical methods can be used to assess the cell viability of cells, such as flow cytometry-based viability markers, etc. Those of ordinary skill in the art can select any analytical method that can be used to assess the cell viability of cells described herein to assess the viability of CAR-NK cells.

[0176] Various methods known in the art can be used to assess the phenotype and function of CAR-NK cells in vitro. In some embodiments, in vitro cell phenotype testing includes flow cytometry. In some embodiments, in vitro cell function testing includes cytokine production, cytotoxicity, proliferation and other analytical methods.

[0177] Animal studies known in the art can be used to evaluate the efficacy of CAR-NK cells in vivo. In some embodiments, in vivo cell phenotype testing is based on a tumor model in immunodeficient mice.

[0178] The CAR-NK cells described herein maintain high viability (e.g., greater than 70%, 75%, 80%, 85%, 90%, 95% or more) and retain the physiological characteristics of their native state, which allows the cells to be used in various applications, such as genetic manipulation of cells, and for achieving cell therapy purposes, such as in adoptive cell therapy applications.

[0179] Combination therapy

[0180] In certain embodiments, the compositions and methods of the embodiments of the present invention relate to a combination of an immune cell colony (e.g., a CARNK cell colony) and at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic virus, or aforesaid combination. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0181] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is the administration of a side effect limiter (e.g., an agent intended to reduce the occurrence and / or severity of treatment side effects, such as an anti-nausea agent, etc.). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy 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 chemotherapeutic agents known in the art.

[0182] In certain embodiments, in addition to the inventive cell therapy disclosed herein, an individual may have been, may be, or will be provided with certain additional therapies for cancer, including one or more of surgery, radiation, immunotherapy (in addition to the cell therapy disclosed herein), hormone therapy, gene therapy, chemotherapy, and the like.

[0183] Immune cell therapy can be administered in various combinations before, during, after or relative to additional cancer therapy. The time interval for administration can vary from simultaneously to several minutes to several days to several weeks. In the embodiment where immune cell therapy and additional therapeutic agents are provided separately to the patient, it is generally ensured that there is no long period of time between each delivery time, so that the two compounds can still play a combined effect to the patient. In such cases, it is expected that antibody therapy and anticancer therapy can be provided to the patient within about 12 hours to 24 hours or 72 hours, and more specifically, antibody therapy and anticancer therapy are provided to the patient within about 6-12 hours. In some cases, it may be necessary to significantly extend the treatment time period, wherein a few days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) are spaced between individual administrations.

[0184] Example

[0185] Other features, objects and advantages of the present invention are apparent in the following examples. However, it should be understood that the examples, although indicating embodiments of the present invention, are given by way of illustration only and not limitation. Based on the examples, various changes and modifications within the scope of the present invention will become apparent to those skilled in the art.

[0186] The CAR-NK cells used in these embodiments include CD19 CAR, IL-15 and iCaspase 9. The exemplary CAR-NK cells used in these embodiments are genetically engineered cord blood NK cells including CD19-CAR, wherein the CD19-CAR includes an anti-CD19 binding domain, wherein the anti-CD19 binding domain includes a light chain variable region including an amino acid sequence shown in SEQ ID NO: 1 and / or a heavy chain variable region including an amino acid sequence shown in SEQ ID NO: 2. The CAR-NK cells used herein are formulated in a cryopreservation medium (e.g., a cryopreservation medium described in Table 1).

[0187] Example 1: Administration of CAR NK cells to an individual

[0188] This embodiment describes an exemplary patient population and CAR NK cells administered to cancer patients, such as patients with relapsed or refractory (r / r) B-cell non-Hodgkin lymphoma (NHL). Patients with a history of anti-CD19 treatment (e.g., chimeric antigen receptor (CAR) T cells or monoclonal antibodies targeting CD19) are included. Patients with a history of anti-CD19 treatment (e.g., patients who have received CAR-T therapy) are allowed enough time to recover from previous anti-CD19 treatment (e.g., at least 3 months) before administering the CAR NK cells described herein.

[0189] Patients who met any of the following exclusion criteria were not included in the study:

[0190] 1. Patients with a total body weight < 40 kg.

[0191] 2. Patients with primary or secondary central nervous system (CNS) involvement from lymphoma. Patients with a history of secondary CNS involvement from lymphoma but without evidence of CNS involvement at screening may be included.

[0192] 3. Patients with Burkitt lymphoma, mantle cell lymphoma, lymphoplasmacytic lymphoma, or CLL / small lymphocytic lymphoma transformation (Richter transformation).

[0193] 4. Patients with a history of non-melanoma skin cancer, carcinoma in situ (e.g., cervical cancer, bladder cancer, breast cancer), malignancy other than low-grade tumors that were considered cured and who have not received systemic treatment (e.g., gastric cancer that was curatively resected by gastric endoscopy) or who have been disease-free for ≥3 years at the time of screening.

[0194] 5. Patients who have received autologous or allogeneic transplantation or CAR-T therapy within 3 months of enrollment are planned to be enrolled. Patients after allogeneic transplantation must stop systemic immunosuppression and have no clinically relevant evidence of acute or chronic GvHD at the time of enrollment.

[0195] 6. Treatment with any investigational product or any systemic anticancer therapy within 14 days prior to conditioning therapy or 2 half-lives of treatment, whichever is longer.

[0196] 7. Patients with clinical evidence of active infection within 3 days prior to enrollment, including fungal, bacterial, viral or other infections that cannot be controlled or require IV antimicrobial drugs for management.

[0197] 8. Patients with active HIV, HBV, or HCV, infection at screening (positive DNA / RNA test).

[0198] 9. Patients with a history of or presence of active or clinically relevant CNS disease, such as seizures, encephalopathy, cerebrovascular ischemia / hemorrhage, severe dementia, cerebellar disease, or any autoimmune disease involving the CNS. For CNS disorders that are in recovery or remission, patients who have not relapsed within 2 years after planned study enrollment may be included.

[0199] 10. Patients with any of the following within 6 months of enrollment: myocardial infarction, cardiac angioplasty or stenting, unstable angina, symptomatic congestive heart failure (i.e., New York Heart Association class II or higher), clinically significant arrhythmia (including uncontrolled atrial fibrillation), or any other clinically significant heart disease.

[0200] 11. Patients who have received live vaccines ≤ 6 weeks before the start of the conditioning regimen

[0201] The CD19 CAR-NK cells described herein are being tested to evaluate the safety and tolerability in adult participants with r / r B-cell NHL. The study will consist of 2 parts: Part 1 (dose escalation and dose expansion) and Part 2, with approximately 242 patients.

[0202] In Part 1, participants in the dose-escalation and dose-expansion cohorts received CD19 CAR-NK cells as follows:

[0203] Part 1: Dose escalation: CD19 CAR-NK cells - 200 × 10 6 CD19-CAR+ live CB-NK cells (±30%)

[0204] Part 1: Dose escalation: CD19 CAR-NK cells - 800 × 10 6 CD19-CAR+ live CB-NK cells (±25%)

[0205] Part 1: Dose expansion: r / r LBCL: CD19 CAR-NK cells - 200×10 6 / 800×10 6 Live NK cells

[0206] Part 1: Dose expansion: r / r iNHL: CD19 CAR-NK cells - 200×10 6 / 800×10 6 Live NK cells

[0207] Based on the data in Part 1, the sponsor and investigators selected a single CD19 CAR-NK cell dose level as the recommended Phase 2 dose (RP2D). Once the RP2D is determined, participants will participate in Part 2 of the study in the following cohorts:

[0208] Cohort 1: CD19 CAR-NK cells (LBCL)

[0209] Cohort 2: CD19 CAR-NK cells (iNHL)

[0210] The total duration of participation in this study is 5 years. Participants will have multiple clinic visits and will participate in a separate long-term follow-up study for ongoing safety assessments up to 15 years after CD19 CAR-NK cell administration.

[0211] Outcome measures to evaluate the effect of CD19 CAR-NK cell administration include monitoring adverse events (AEs), clinically significant changes in laboratory parameters (e.g., including hematology, clinical chemistry, serum immunoglobulins, and urinalysis tests), clinically significant changes in vital signs (e.g., temperature (oral or tympanic membrane measurement), sitting blood pressure (after the participant has rested for at least 5 minutes), and pulse rate (bpm)), and / or overall response rate (ORR) according to the Independent Review Committee (IRC). ORR is defined as the percentage of participants with complete response (CR) or partial response (PR) as the best response to treatment, as determined by the IRC according to the Lugano 2014 criteria after CD19 CAR-NK cell administration.

[0212] Secondary outcome measures included:

[0213] ● ORR after CD19 CAR-NK cell administration according to Lugano 2014 criteria

[0214] ● Complete response (CR) according to the investigator (CR is the percentage of participants with target lymph nodes / nodal masses that must regress to ≤1.5 cm in the longest transverse diameter of all lesions and no extralymphatic disease sites as determined by the Lugano 2014 criteria)

[0215] ● Complete response (CR) by IRC (percentage of participants with target lymph nodes / nodal masses that must regress to ≤1.5 cm in the longest transverse diameter of all lesions and no extralymphatic disease sites according to the Lugano criteria)

[0216] ● Duration of response (DOR) according to the investigator (For participants who experienced an objective response, DOR was defined as the time from the date of the first documented objective response to the date of the first documented disease progression as determined by the investigator classification according to the Lugano 2014 criteria or death, whichever came first)

[0217] ● Duration of response (DOR) according to IRC (For participants who experienced an objective response, DOR was defined as the time from the date of the first documented objective response to the date of the first documented disease progression, as classified by the IRC Lugano 2014 criteria or death, whichever occurred first)

[0218] ● Progression-free survival (PFS) according to the investigator (PFS is defined as the time from the date of enrollment to the date of disease progression as determined by the investigator according to the Lugano 2014 criteria or death from any cause, whichever comes first)

[0219] ● Progression-free survival (PFS) according to IRC (PFS is defined as the time from the date of enrollment to the date of disease progression as determined by the IRC classification according to the Lugano 2014 criteria or death from any cause, whichever comes first)

[0220] ● Overall survival (OS) (OS is defined as the time from enrollment to the date of death from any cause.)

[0221] ● Cmax - Maximum observed blood concentration of CD19 CAR-NK cells

[0222] ● Tmax - the time when CD19 CAR-NK cells first reach Cmax

[0223] ● Tlast - the time when the last measurable concentration is above the lower limit of quantification of CD19 CAR-NK cells

[0224] ● AUCLast - the area under the concentration-time curve from time 0 to the last quantifiable concentration time of CD19 CAR-NK cells

[0225] ● Plasma concentrations of interleukin (IL)-15 and other soluble immune factors over time

[0226] ● Concentrations of IL-15 and soluble immune factors (e.g., interferon (IFN)-γ (γ), IL-1β (β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor (TNF) α (α), granulocyte-macrophage colony-stimulating factor (GM-CSF)) in plasma will be reported over time.

[0227] ● Percentage of participants with B-cell aplasia before and after CD19 CAR-NK cell administration

[0228] ● Percentage 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

[0229] ● Percentage of participants with positive replication-competent retroviral (RCR) test results before (prevalence) and after (incidence) CD19 CAR-NK cell administration over time

[0230] Patients with relapsed / refractory B-cell lymphoma who had previously received CD19-targeted therapy received chimeric antigen receptor (CAR)-NK cell therapy targeting CD19, as described above, in a single-arm, open-label, multicenter, global Phase 2 clinical trial evaluating the efficacy of CAR-NK cells (NK cells derived from umbilical cord blood exogenously transduced with an anti-CD19 CAR comprising a sequence 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% PLASMA-LYTE A, 50% CS10, 10% HSA, and 30 mM trehalose (e.g., 37.7% v / v PLASMA-LYTE A + 50% v / v CS10 + 9.4% v / v HSA + 2.8% v / v trehalose).

[0231] In use 800 × 10 6 Within one month after CD19 CAR+ live NK cell treatment, an exemplary response patient showed a partial response. The patient suffered from iNHL and had previously received 8 previous treatment lines, including two CAR-T cells targeting CD19 (Akiren and Tesalen). After receiving CAR-NK cell infusion (natural killer cells (NK cells) derived from umbilical cord blood transduced with CAR19-CD28-ζ-2A-IL15 and inducible caspase-9), the partial response was converted to a complete response 3 months later.

[0232] The efficacy of CAR-NK cells in patients with negative CD19 expression was analyzed by immunohistochemistry (IHC). Screening biopsy of an exemplary responding patient showed CD19-negative LBCL by immunohistochemistry. The patient received 800 × 10 6 CD19 CAR+ live NK cells were detected in the patients and a partial response was achieved within one month after treatment.

[0233] Example 2. In vitro cytotoxicity assessment of CD19 KO cancer cells

[0234] This embodiment shows in vitro cytotoxicity analysis to evaluate the non-CD19 dependent activity of CAR-NK cells on CD19 KO cancer cells, wherein the CAR-NK cells are NK cells derived from umbilical cord blood transduced by anti-CD19 CAR exogenously, and the anti-CD19CAR comprises a sequence represented by SEQ ID No: 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 for two CD19 negative (CD19-; Raji CD19 knockout [KO], NALM6 CD19 KO) target cell lines. The target cancer cell lines used in these studies express NKG2D stress ligands. Two cell types were evaluated using a dead cell evaluation method based on flow cytometry (many CAR-NK cells tested included: donor A, whose CAR% was 75.28%; and donor B, whose CAR% was 80.08%).

[0235] After 20 hours of co-culture, in Raji CD19 KO ( Figure 1A ) and NALM6 CD19 KO ( Figure 1B ) tumor cells were evaluated by two independent donor umbilical cord blood units (donor A and donor B) produced CAR-NK cell lytic activity. Raji CD19 KO and NALM6 CD19 KO cell lines were freshly thawed, resuspended, labeled with CellTrace purple and spread with 30,000 cells / 100 μL / well. Frozen CAR-NK cells derived from donor A and frozen CAR-NK cells derived from donor B (for frozen preparations, each cell sample was prepared in a preparation consisting of 40% PLASMA-LYTE A, 50% CS10, 10% HSA and 30 mM trehalose) were thawed, resuspended in growth medium, and viable cells were counted. CAR-NK cells were co-incubated with individual target cells at seven different ratios of effector cells (e.g., CAR-NK cells) to target cells (10: 1, 5: 1, 2.5: 1, 1.25: 1, 0.63: 1, 0.3: 1, and 0.16: 1). The target cells were pre-labeled with a fluorescent dye (CellTraceViolet) to distinguish them from effector cells. After incubation at 37 ° C for 20 hours, the cells were washed with staining buffer, resuspended in phosphate-buffered saline (PBS) premixed with a fixable active dye (eFluor 780), and incubated at 40 ° C in the dark for 30 minutes. The cells were then washed twice, and the killing activity of different effector cell treatments was determined by flow cytometry. Target cells alone were used as a baseline for live cells without killing. The data represent the average of duplicate wells. The killing or specific dissolution of target cells is calculated as follows:

[0236] Killing or specific lysis of target cells (%) = 100 - ([number of CellTrace Purple-positive target cells in wells co-cultured with effector cells / average number of CellTrace Purple-positive cells in target cell alone controls] × 100).

[0237] 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 CD19-negative (Raji-CD19 KO and NALM6-CD19 KO) cancer cells tested, consistent with innate NK receptor engagement of stress ligands expressed on cancer cells.

[0238] Example 3: Combination therapy of CAR NK cells for individuals

[0239] This embodiment describes the exemplary administration of chemotherapeutic agents and intravenous CD19 CAR NK cells to cancer patients, such as patients with relapsed or refractory (r / r) B cell non-Hodgkin lymphoma (NHL), wherein NK cells are derived from cord blood. Patients with anti-CD19 treatment history (e.g., chimeric antigen receptor (CAR) T cells or monoclonal antibodies targeting CD19) are included. A combination of CD19 CAR-NK cells and chemotherapeutic agents (e.g., according to standard of care, fludarabine and cyclophosphamide) is administered to the patient.

[0240] Experimental: Part 1: Dose escalation: CD19 CAR-NK cells - 200×10^6 CD19-CAR+ live CB-NK cells. Participants received lymphodepleting chemotherapy intravenously daily, followed by 200×10^6 anti-CD19 chimeric antigen receptor (CD19-CAR+) live natural killer (NK) cells, a single dose, intravenously, once on day 0.

[0241] Experimental: Part 1: Dose escalation: CD19 CAR-NK cells - 800 × 10^6 CD19-CAR + live CB-NK cells. Participants received lymphodepleting chemotherapy intravenously daily, followed by CD19 CAR-NK cells - 800 × 10^6 CD19-CAR + live CB-NK cells, a single dose, intravenously, once on day 0.

[0242] Experimental: Part 1: Dose expansion: LBCL: CD19 CAR-NK cells - 200×10^6 / 800×10^6 CD19-CAR + live CB-NK cells. Participants with r / r large B-cell lymphoma (LBCL) received lymphodepleting chemotherapy intravenously daily, followed by CD19 CAR-NK cells - 200×10^6 / 800×10^6 CD19-CAR + live CB-NK cells, single dose, intravenously, once on day 0 to determine the RP2D.

[0243] Experimental: Part 1: Dose expansion: iNHL: CD19 CAR-NK cells - 200×10^6 / 800×10^6 CD19-CAR + live CB-NK cells. Participants with r / r indolent non-Hodgkin lymphoma (iNHL) received lymphocyte-depleting chemotherapy intravenously daily, followed by CD19 CAR-NK cells - 200×10^6 / 800×10^6 CD19-CAR + live CB-NK cells, single dose, intravenously, once on day 0 to determine the RP2D.

[0244] Experimental: Part 2: Cohort 1 - LBCL. Participants with LBCL were enrolled in this cohort to receive lymphodepleting chemotherapy intravenously daily, followed by CD19 CAR-NK cells, RP2D, intravenously, once on day 0.

[0245] Experiment: Part 2: Cohort 2-iNHL

[0246] Participants with iNHL enrolled in this cohort received lymphodepleting chemotherapy intravenously daily, followed by CD19 CAR-NK cells, RP2D, intravenously, once on day 0.

[0247] The results were evaluated as described in Example 1.

[0248] Equivalents and scope

[0249] 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 set forth in the following claims.

Claims

1. A method for treating cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of CD19-CAR+ live cord blood natural killer (CB-NK) cells, wherein the individual has previously received CAR T therapy targeting CD19.

2. The method of claim 1, wherein the patient has previously received at least 2 lines of standard chemoimmunotherapy or targeted therapy.

3. The method of claim 1 or 2, wherein lymphocyte-depleting chemotherapy is administered intravenously to the individual, followed by administration of CD19-CAR+ live CB-NK cells.

4. The method of any of the preceding claims, wherein the individual has previously received anti-CD19 CAR-T therapy more than three months prior to administration of the CD19-CAR+ live CB-NK cells.

5. The method of any one of the preceding claims, wherein the cancer is a solid tumor or is not a solid tumor.

6. The method of any of the preceding claims, wherein the cancer is lung cancer, brain cancer, breast cancer, blood cancer, skin cancer, pancreatic cancer, liver cancer, colon cancer, head and neck cancer, kidney cancer, thyroid cancer, stomach cancer, spleen cancer, gallbladder cancer, bone cancer, ovarian cancer, testicular cancer, endometrial cancer, prostate cancer, rectal cancer, anal cancer, cervical cancer, or is a hematological cancer.

7. The method of any one of the preceding claims, wherein the cancer is relapsed or refractory B-cell non-Hodgkin's lymphoma.

8. A method for treating cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of CD19-CAR+ live umbilical cord blood natural killer (CB-NK) cells, wherein the cancer is CD19 negative.

9. The method of claim 8, wherein the cancer is a solid tumor or is not a solid tumor.

10. The method of claim 8, wherein the cancer is large B-cell lymphoma.

11. The method of any one of the preceding claims, wherein the number of CD19-CAR+ live CB-NK cells is at least 200×10 6 dosage administration.

12. The method of any one of the preceding claims, wherein the individual is a human.

13. The method of any of the preceding claims, wherein the individual is administered one or more additional cancer therapies.

14. The method of claim 13, wherein the additional cancer therapy is surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

15. The method of any one of the preceding claims, further comprising the step of diagnosing cancer in the individual.

16. The method according to any one of the preceding claims, further comprising the step of generating the CD19-CAR+ live CB-NK cells.

17. The method of any one of the preceding claims, wherein the cells are autologous to the individual.

18. The method of any one of the preceding claims, wherein the cells are allogeneic to the individual.

19. The method of any of the preceding claims, wherein the cell population is administered to the individual intracranially, by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, transdermally, subcutaneously, topically, by perfusion, in a tumor microenvironment, or a combination thereof.

20. The method of any of the preceding claims, wherein the CD19-CAR+ live (NK) cells comprise one or more exogenously provided interleukins (IL).

21. The method of claim 20, wherein the IL is selected from the group consisting of IL-12, IL-15, IL-21, IL-2, IL-18, IL-7, p35 and p40 subunits of IL-12 artificially linked together with a linker, and combinations thereof.

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

23. The method of any one of claims 20-22, wherein the IL is secreted, tethered or membrane-bound in the cell.

24. The method of any one of claims 20-23, wherein the exogenously provided IL is expressed from a vector in the cells and / or wherein the NK cells are cultured in the presence of one or more ILs.

25. The method of any of the preceding claims, wherein the NK cells comprise a suicide gene. The method according to claim 25 , wherein the suicide gene is iCaspase9 suicide gene.

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