Attorney Docket No. MIL-047WO1 CD19 TARGETING CAR NK CELLS IN TREATING SYSTEMIC LUPUS ERYTHEMATOSUS AND LUPUS NEPHRITIS CROSS-REFERENCE TO RELATED APPLICATIONS [1] This application claims priority to, and the benefit of U.S. Provisional Application Serial No.63/565,693 filed on March 15, 2024, the contents of which is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [2] The instant application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The sequence listing file entitled MIL-047WO1_SL.XML, was created on March 5, 2025, which is 16,384 bytes in size. BACKGROUND [3] Autoimmune diseases are diseases where cells, tissues and organs are attacked by cells or autoantibodies generated by the immune system. For example, in the autoimmune disease, systemic lupus erythematosus (SLE, or lupus), autoantibodies target joints, skin, brain, lungs, kidneys, and blood vessels, among other organs and tissues. [4] One particular manifestation of SLE is lupus nephritis (LN), which is an autoimmune disease in which the kidneys are affected by autoantibodies, leading to inflammation (swelling or scarring) of the kidney glomeruli (small blood vessels that filter waste), and could over time cause kidney failure, and can even be fatal. [5] Lupus is generally treated with immunosuppressants, e.g., corticosteroids, anti-malarial drugs or calcineurin inhibitors. In addition, monoclonal antibodies targeting B cells, e.g. rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes, or belimumab, which is directed against BAFF necessary for B-cell survival, or autologous stem cell transplants are therapeutic avenues. Symptoms are addressed using Angiotensin Converting Enzyme (ACE) inhibitors and angiotensin 2 receptor blockers (ARBs) to reduce proteinuria and relieve high blood pressure, diuretics and low salt diets to control edema and blood pressure. [6] Cyclophosphamide (CYP) and/or mycophenolate mofetil (MMF) are commonly used immunosuppressants for treatment, however, long term use of high dose
Attorney Docket No. MIL-047WO1 immunosuppressants is associated with toxicity (Yo et al., Open Access Rheumatol.2019; 11: 179–188.). There is also a problem of non-responsiveness and relapsing or refractory disease. SUMMARY [7] The present disclosure provides, among other things, a method of treating autoimmune diseases, for example, systemic lupus erythematosus (SLE), lupus nephritis, more specifically, relapsing or refractory SLE or relapsing or refractory lupus nephritis, by administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti-CD19- CAR CB-NK cells) to a subject in need thereof, wherein the therapeutically effective dose is administered in one or greater than one dose. [8] Prior to the present invention, autologous Chimeric Antigen Receptor (CAR) T cell therapy has also been shown to be effective in the treatment of certain patients with SLE and lupus nephritis in a small, open label study (Mackensen et al., Nat Med, 2022; 28(10), 2124-32). However, the clinical use of autologous CAR-T cells is accomplished with significant challenges, including logistics for personalized and timely cell product manufacturing and administration (Mackensen et al.2022). Allogeneic CAR-T cells with encouraging clinical results in early-stage development can help to improve the logistics challenges, but toxicity is still of concern (Locke et al., 2023; Blood, 142, 2095). Further, eligible treatment options for relapsing or refractory SLE and lupus nephritis were limited, making it more difficult to achieve durable remissions. There is an unmet clinical need for effective methods of treating subjects following relapse after exposure to at least one line of standard immunosuppressive therapy. [9] The present disclosure provides, among other things, a method of treating SLE or lupus nephritis, including relapsing or refractory SLE and relapsing or refractory lupus nephritis, by administering a therapeutically effective dose of natural killer cells (e.g., cord blood derived, peripheral blood derived or iPSC derived) expressing CD19 targeting chimeric antigen receptor to a subject in need thereof, wherein the therapeutically effective dose is administered in one or greater than one dose. [10] The present disclosure provides, among other things, a method of treating a SLE or lupus nephritis by administering to a subject in need thereof a therapeutically effective dose of about 800 million, anti-CD19 CAR-NK cells, administered in a single dose following a single lymphodepletion cycle. The present disclosure also provides, among other
Attorney Docket No. MIL-047WO1 things, a method of treating SLE or lupus nephritis by administering to a subject in need thereof a therapeutically effective dose of about 200 million anti-CD19 CAR-NK cells, administered in a single dose following a single lymphodepletion cycle. In some embodiments, more than one dose is administered (e.g., two, three or more doses). In some embodiments, the therapeutically effective dose is administered following a single lymphodepletion cycle. [11] The present invention thus also provides a way to treat SLE or lupus nephritis in subjects that seem to respond well at first to other immunosuppressive agents, but then the response stops and the disease comes back in a drug-resistant form, e.g., relapsing or refractory SLE or lupus nephritis. The inventors of the present disclosure provide an efficacious, safe and tolerable treatment for treating SLE or lupus nephritis, particularly relapsing or refractory SLE or relapsing or refractory lupus nephritis, by administering anti- CD19 CAR-NK cell therapy. [12] In some aspects, provided herein is a method for treating SLE or lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti-CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is administered following a single lymphodepletion cycle. [13] In some embodiments, the therapeutically effective dose is a single dose. [14] In some embodiments, the therapeutically effective dose is between 100 million to 2.4 billion cells. [15] In some embodiments, the therapeutically effective dose is between 100 million to 1 billion cells. [16] In some embodiments, the therapeutically effective dose is 1 billion cells. [17] In some embodiments, the therapeutically effective dose is 800 million cells. [18] In some embodiments, the therapeutically effective dose is 600 million cells. [19] In some embodiments, the therapeutically effective dose is 400 million cells. [20] In some embodiments, the therapeutically effective dose is 200 million cells. [21] In some embodiments, the therapeutically effective dose is 100 million cells. [22] In some embodiments, the therapeutically effective dose further comprises multiple therapeutically effective doses.
Attorney Docket No. MIL-047WO1 [23] In some embodiments, the method comprises administering two, three or more therapeutically effective doses. [24] In some embodiments, the therapeutically effective dose is administered following a single lymphodepletion cycle. [25] In some embodiments, the SLE or lupus nephritis is relapsing or refractory. [26] In some embodiments, the anti-CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. [27] In some embodiments, the anti-CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 2. [28] In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 9. [29] In some embodiments, the anti-CD19-CAR comprises a CD28 domain having 100% identity to the amino acid sequence of SEQ ID NO: 9. [30] In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 10. [31] In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having 100% identity to the amino acid sequence of SEQ ID NO: 10. [32] In some embodiments, the single lymphodepletion cycle comprises fludarabine and/or cyclophosphamide. [33] In some embodiments, the anti-CD19-CAR CB-NK cells are genetically modified to express one or more interleukin (IL). [34] In some embodiments, the one or more interleukin is selected from IL-15, IL- 12, IL-21, IL-2, IL-18, IL-7, and combinations thereof. [35] In some embodiments, the interleukin is IL-15. [36] In some embodiments, the IL-15 is secreted or membrane bound. [37] In some embodiments, the IL-15 is secreted from the cell. [38] In some embodiments, exogenously provided IL is expressed from a vector in the cells. [39] In some embodiments, the anti-CD19-CAR CB-NK cell comprises a suicide gene.
Attorney Docket No. MIL-047WO1 [40] In some embodiments, the suicide gene is an iCaspase9 suicide gene. [41] In some aspects, provided herein is a method for treating a relapsing or refractory lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti-CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is about 800 million administered in a single dose following a single lymphodepletion cycle. [42] In some embodiments, the subject has previously received at least one line of standard immunosuppressive therapy. [43] In some embodiments, the subject has previously received cyclophosphamide (CYP),mycophenolate mofetil (MMF), calcineurin inhibitors and/or elimumab. [44] In some embodiments, the subject has received corticosteroids in combination with cyclophosphamide, mycophenolate mofetil, calcineurin inhibitors and/or elimumab. [45] In some embodiments, the calcineurin inhibitors are sirolimus, tacrolimus, everolimus and/or voclosporin. [46] In some embodiments, the anti-CD19-CAR CB-NK cells are allogeneic with respect to the subject. [47] In some embodiments, the anti-CD19-CAR CB-NK cells are autologous with respect to the subject. [48] In some aspects, the anti-CD19-CAR CB-NK cells are administered intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion, or a combination thereof. [49] In some embodiments, the anti-CD19-CAR CB-NK cells are administered intravenously. [50] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
Attorney Docket No. MIL-047WO1 DEFINITIONS [51] Administering: As used herein, the terms “administering,” or “introducing” are used interchangeably in the context of delivering a CD19-directed genetically modified NK cell immunotherapy described herein (e.g., anti-CD19 CAR viable NK cells, for example, autologous or allogenic cord blood derived anti-CD19 CAR-NK cells) to a patient in need thereof. Various methods are known in the art for administering cells to patients, including for example administering the cells to a patient in need thereof by intravenous or surgical methods. [52] Adoptive Cell Therapy: As used herein interchangeably, the terms “adoptive cell therapy” or “adoptive cell transfer” or “cell therapy” or “ACT” refer to the transfer of cells, for example, a population of genetically modified cells (e.g. anti-CD19 CAR-NK cells), into a patient in need thereof. The cells can be derived and propagated from the patient in need thereof (i.e., autologous cells) or could have been obtained from a non-patient donor (i.e., allogeneic cells). In some embodiments, the cell is an immune cell, such as a lymphocyte. In some embodiments, the immune cell is a NK cell. Various cell types can be used for ACT including but not limited to, natural killer (NK) cells, T cells, CD8+ cells, CD4+ cells, delta-gamma T-cells, regulatory T-cells, induced pluripotent stem cells (iPSCs), iPSC derived T cells, iPSC derived NK cells, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells. [53] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone. [54] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a stated value of interest as well as value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either
Attorney Docket No. MIL-047WO1 direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [55] Allogeneic: As used herein, “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically. [56] Autologous: As used herein, the term “autologous” means from the same individual. For example, “autologous” in relation to donor and recipient means that the donor subject is the recipient subject. [57] Chimeric Antigen Receptor (CAR): As used herein, the term “chimeric antigen receptor” or “CAR” engineered receptors which can confer an antigen specificity onto cells (for example, immune cells such as NK cells including cord blood derived NK cells and iPSC derived NK cells (iNK cells)). CARs are also known as chimeric antigen receptors or chimeric immunoreceptors. For example, a CAR described herein may include one or more of an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain. [58] CD19-directed genetically modified NK cell immunotherapy: As used herein, the term “CD19-directed genetically modified NK cell immunotherapy” refers to compositions and formulations comprising anti-CD19 CAR+ NK cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises CD19 CAR+viable NK cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises cord blood derived anti-CD19 CAR-NK+ cells. In some embodiments, the CD19-directed genetically modified NK cell immunotherapy comprises allogenic cord blood derived anti-CD19 CAR-NK+ cells. [59] Cell: As used herein, the term “cell” refers to any cell unless a specific type of cell is named. As non-limiting examples, a cell is a stem cell, progenitor cell, or somatic cell, e.g., adult stem cell, progenitor cell, or differentiated cell. In some embodiments, the cells are hematopoietic cell, e.g., a hematopoietic stem or progenitor cell. In some embodiments, the cells are immune cells and include B-cells, T cells, monocytes or progenitor cells. In some embodiments, the cells are NK cells, and in particular, CAR-NK cells.
Attorney Docket No. MIL-047WO1 [60] Complete Renal Response: As used herein, “complete renal response (CRR)” is defined by meeting the following criteria: (a) a ratio of urinary protein to creatinine <0.5, an estimated glomerular filtration rate [eGFR] no worse than 10% below the preflare value [baseline] or ≥60 ml per minute per 1.73 m2, and no use of rescue therapy), (b) time to CRR, and (c) duration of CRR. [61] Cryoprotectant: As used herein, the term “cryoprotectant” means a substance used to protect biological tissue from freezing damage. Exemplary cryoprotectants include, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol and propanediol. [62] Engineered: As used herein, the term “engineered” refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. [63] Exogenous: As used herein, the “exogenous” as used herein refers to a polynucleotide (such as one encoding a gene product or part of a gene product) that is not present endogenously in a mammalian cell, such as an immune cell, or is synthetically generated outside of a mammalian cell, such as by recombinant technology. In a specific case, a particular gene product may be provided to a cell exogenously, and the cell may or may not also express the corresponding endogenous gene product in the cell. [64] Ex vivo: As used herein, the term “ex vivo” means a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube, in a culture bag, in a bioreactor). [65] Fresh cell or Rescued Fresh Cell: As used herein, the terms “fresh,” “fresh cell,” or “rescued fresh cell” refers to mammalian cells that have never been frozen and/or once frozen but subsequently restimulated, cultured in culture medium and then harvested as fresh cells. [66] Functional equivalent or derivative: As used herein, the term “functional equivalent” or “functional derivative” denotes, in the context of a functional derivative of an amino acid sequence or any other molecule (e.g., a media formulation component) that retains an activity (either function or structural) that is substantially similar to that of the original molecule or sequence. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. Exemplary derivatives include those having chemico- physical properties which are similar to that of the original molecule or sequence. Desirable
Attorney Docket No. MIL-047WO1 similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like. [67] Isotonic: As used herein, the term “isotonic” means having an osmotic pressure that is equal to or approximately the same as the osmotic pressure of a physiological fluid. [68] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. [69] In vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell- based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). [70] Primary Cell: The term, “primary cell,” refers to a cell that is directly isolated from a subject and subsequently propagated. [71] Polypeptide: The term, “polypeptide,” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified. [72] Protein: The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit. [73] Refractory disease: “Refractory disease” refers to disease that did not respond to treatment. E.g. Refractory SLE or lupus nephritis did not respond to standard immunosuppressive therapy.
Attorney Docket No. MIL-047WO1 [74] Relapsing disease: “Relapsing disease”, or recurrent disease refers to disease that has come back after treatment and remission. Relapsing SLE or lupus nephritis refers to SLE or lupus nephritis that has come back after treatment and remission. [75] Remission: As used herein, the term “remission” means that the signs and symptoms of SLE or lupus nephritis are reduced. Remission can be partial or complete. [76] Complete Remission (CR): In a complete remission (CR), all signs and symptoms of SLE or lupus nephritis have disappeared. [77] Subject: As used herein, the term “subject” refers to a human or any non- human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. [78] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. [79] Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition. [80] Sugar or Saccharide: The terms “sugar” and “saccharide” herein have been used interchangeably, and generally refer to oligosaccharides such as monosaccharides, disaccharides, trisaccharides or polysaccharides, and the like. In some embodiments, the saccharide is one or more of glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, myoinositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltitol, methyl cellulose, carboxymethyl cellulose, dextran, glycogen, amylose, amylopectin, inulin, sodium alginate, ethyl cellulose, hydroxyethyl cellulose, raffinose, stachyose, xanthan gum, glucosamine, and galactosamine. In some embodiments, saccharide
Attorney Docket No. MIL-047WO1 is a disaccharide. In some embodiments, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, or chitobiose. In some other embodiments, the disaccharide is trehalose. In some embodiments, one or more sugars includes trehalose, sucrose, mannitol, and/or dextran. [81] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen (e.g., comprising greater than one unit dose, for example, three doses). In some embodiments, a therapeutically effective amount of an adoptive cell therapy, as used herein, is a dosage of cells (e.g., a population of genetically modified immune cells such as CAR-NK cells) in a certain formulation (e.g., a cryopreservation media described herein) administered to a subject in need thereof (e.g., a patient suffering from a B-cell malignancy, for example large B-cell lymphoma (LBCL)). For example, in some embodiments, a therapeutically effective dose comprises CAR-NK cells in an amount of about 300 million cells to about 1.5 billion cells, e.g.800 million cells, administered in greater than one dose, for example, three doses. [82] In some embodiments, the CAR-NK cells have been genetically modified to express a CD19 CAR comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 or a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 2 and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or a sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 1. [83] In some embodiments, the CAR NK cells have been genetically modified to express a CD-19 CAR having at least 95% identity to CDR sequences of CDR-L1: RASQDISKYLN (SEQ ID NO: 3), CDR-L2: SRLHSGV (SEQ ID NO: 4), CDR-L3: GNTLPYTFG (SEQ ID NO: 5), CDR-H1: DYGVS (SEQ ID NO: 6), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 7) and CDR-H3: YAMDYWG (SEQ ID NO: 8). [84] In some embodiments, the CAR NK cells have been genetically modified to express a CD19 CAR having 100% identity to CDR sequences of CDR-L1: RASQDISKYLN (SEQ ID NO: 3), CDR-L2: SRLHSGV (SEQ ID NO: 4), CDR-L3: GNTLPYTFG (SEQ ID
Attorney Docket No. MIL-047WO1 NO: 5), CDR-H1: DYGVS (SEQ ID NO: 6), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 7) and CDR-H3: YAMDYWG (SEQ ID NO: 8). [85] Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. [86] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4 and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” BRIEF DESCRIPTION OF THE DRAWINGS [87] Drawings are for illustration purposes only; not for limitation. [88] FIG.1A-FIG.1F are bar graphs showing percent cytotoxicity of primary B cells by anti-CD19 CAR-NK cells from two exemplary cord blood donors (D004 and D005) at various effector: target ratio. Primary B cells from healthy donors (1, 218 and 366) are shown in FIG.1A-FIG.1C. Primary B cells from SLE donors (301, 001 and 602) are shown in FIG.2A-FIG.2C. [89] FIG.2A-FIG.2F showed , IFN-gamma cytokine was detected in supernatants from co-culturing anti-CD19 CAR-NK cells (from two independent donor cord blood units (D004 and D005)) with B cells from healthy and SLE donors. DETAILED DESCRIPTION [90] The present invention provides, among other things, a method for treating autoimmune disease, e.g., Systemic Lupus Erythematosus (SLE), or more specifically, lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of natural killer (NK) cells (e.g., cord blood derived, peripheral blood derived, or iPSC derived) expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti-CD19-CAR-NK cells) to the subject in need thereof, wherein the therapeutically
Attorney Docket No. MIL-047WO1 effective dose is administered (e.g., in a single dose) following lymphodepletion cycle (e.g., a single lymphodepletion cycle). [91] Anti-CD19 CAR+ NK cells derived from cord blood cells do not require HLA selection (See, e.g., Liu et al. Use of CAR-transduced Natural Killer Cells in CD19 Positive Lymphoid Tumors. NEJM (2020) 382-545), have innate immune receptor-mediated killing capability, are less toxic and have increased manufacturability. Therefore, anti-CD19 CAR+ CB-NK cells described herein, for example, in treating relapsing or refractory lupus nephritis are advantageous compared with currently available allogenic anti-CD19 CAR-T products and developing anti-CD19 CAR-T cells (e.g., iCAR-T). [92] In some aspects, provided herein is a method for treating SLE or lupus nephritis, including a relapsing or refractory SLE or lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD19 targeting chimeric antigen receptor (CD19- CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is about 800 million cells following a single lymphodepletion cycle. [93] In some embodiments, individuals that have relapsed or are refractory to standard immunosuppressive therapy are administered cord blood-derived anti-CD19 CAR- NK cells of the present invention. Autoimmune Diseases [94] In some embodiments, provided herein is a method of treating autoimmune diseases, the method comprising a step of administering a therapeutically effective dose of natural killer cells (e.g., cord blood derived, peripheral blood derived, or iPSC derived) expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti-CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is administered in a single dose following a single lymphodepletion cycle. [95] In some embodiments, provided herein a method of treating B-cell associated autoimmune diseases. Systemic Lupus Erythematosus (SLE) [96] In some embodiments, provided herein is a method of treating Systemic Lupus Erythematosus (SLE). SLE is a chronic illness that can affect many parts of the body, for example, joints, skin, kidneys, blood cells, brain, heart and lungs. Symptoms can vary from
Attorney Docket No. MIL-047WO1 mild to severe, come on quickly or slowly, and can get worse for a while before improving (flare). Symptoms can be concentrated on one area of the body or throughout the body. A characteristic sign of SLE is a rash over the cheeks and nose in the shape of a butterfly called “butterfly” rash or malar rash. [97] Symptoms of lupus include, but are not limited to arthritis, fever, fatigue, round scaly rashes, butterfly rash, sun sensitivity, hair loss, sores in the nose and mouth, change in color of fingers and toes to bluish-purple, white or red from cold or stress (Raynaud’s phenomenon), swollen glands, swelling in legs or around eyes, pain when breathing deeply or lying down, from inflammation of the lining around heart or lungs, headaches, dizziness, depression, confusion or seizures, or abdominal pain. [98] Lupus causes inflammation throughout the body, affecting one or more organs, for example, lupus nephritis affecting kidneys, seizures and memory problems due to changes in brain and central nervous system, heart problems, e.g., heart valve damage and scarring, pericarditis (inflammation of lining of heart), myocarditis (inflammation of heart muscle), vasculitis (inflammation of blood vessels). Other symptoms include blood clots due to high levels of autoantibodies called antiphospholipid antibodies, low blood cell counts, including red blood cells, white blood cells, and platelets, increased risk of cardiovascular disease (atherosclerosis, coronary artery disease), and pleurisy (inflammation of tissue surrounding the lungs, making it painful to breathe). [99] In some embodiments, provided herein is a method of treating one or more symptoms or indications of SLE (e.g., lupus nephritis). Lupus Nephritis [100] In some embodiments, provided herein is a method of treating lupus nephritis. Lupus nephritis is a type of kidney disease caused by SLE. Lupus nephritis is caused when kidneys are affected by autoantibodies, leading to inflammation (swelling or scarring) of the kidney glomeruli, small blood vessels that filter waste, and could over time cause kidney failure. Glomerular disease can cause glomeruli to leak blood into urine (hematuria). Some of the other symptoms of lupus nephritis include foamy urine due to excess protein (proteinuria), high blood pressure, edema (extra fluid causing swelling in legs, ankles, feet
Attorney Docket No. MIL-047WO1 and sometimes hands and face), and resulting weight gain, and high levels of creatinine waste product in blood. [101] Besides kidney failure, lupus nephritis can additionally cause hypertension, increased risk of heart and blood vessel problems, as well as increased risk of immune cancers, e.g., B cell lymphomas. Refractory lupus nephritis [102] In some embodiments, provided herein is a method of treating refractory SLE or lupus nephritis (LN). In some embodiments, refractory lupus nephritis is defined as adherent patients who fail to achieve adequate improvement within 12 weeks after treatment initiation. [103] Refractory lupus nephritis is a failure to attain clinical remission after appropriate immunosuppressive therapy and is associated with an increased risk of progression to end-stage kidney disease and mortality. Between 20% and 70% of patients with lupus nephritis are reported to be refractory to standard immunosuppressive therapy. [104] According to the European League Against Rheumatism (EULAR/ERA- EDTA), refractory SLE or lupus nephritis is defined as failure to achieve a partial response after 6–12 months. The guidelines from the American College of Rheumatology have defined refractory lupus nephritis (LN) as worsening nephritis by 3 months or, treatment failure as assessed by the treatment physician by 6 months. [105] Complete or partial renal response are measured based on clinical parameters including serum creatinine, proteinuria and estimated glomerular filtration rate (eGFR). The Kidney Disease and Improving Global Outcomes (KDIGO) guideline on glomerulonephritis defines complete remission as return of serum creatinine to previous baseline and a decline in the urine protein creatinine ratio (UPCR) to <500 mg/mmol. [106] Partial remission is defined by stabilization (±25%) or improvement of serum creatinine, but not to normal, and a >50% decrease in the UPCR. Partial remission is expected starting at 3–6 months, and clinical parameters are usually assessed every 4 weeks in the first 6 months. Renal histology is routinely not included as a component of assessment. Relapsing Lupus Nephritis [107] In some embodiments, provided herein is a method of treating relapsing SLE or lupus nephritis. Relapse is recurrence of disease following, for example, withdrawal of long-term immunosuppressive therapy. For example, cyclophosphamide withdrawal leads to
Attorney Docket No. MIL-047WO1 relapse in patients with lupus nephritis initially responsive to such a line of therapy. Approximately 37–45% of patients successfully treated at their initial episode have relapses of nephritis later as evidenced by recurrent renal flares, years after initial remission. Methods of Treatment [108] The anti-CD19 CAR-NK cell compositions (referred to interchangeably as CD19 CAR-NK cells or CD19 targeting CAR-NK cells) described herein are suitable for adoptive cell therapy. Adoptive cell therapies can be used to treat various diseases, including, for example, SLE or lupus nephritis. In some embodiments, the anti-CD19 CAR- NK cell compositions are useful for the treatment of SLE or lupus nephritis. In some embodiments, the anti-CD19 CAR-NK cell compositions are useful for the treatment of relapsing or refractory SLE or lupus nephritis. In some embodiments, the SLE or lupus nephritis is relapsing. In some embodiments, the SLE or lupus nephritis is refractory. [109] In some embodiments, provided herein is a method for treating SLE or lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of natural killer cells (e.g., cord blood derived, peripheral blood derived or iPSC derived) expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti- CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is administered following a single lymphodepletion cycle. [110] In some embodiments, the NK cells are cord blood derived. In some embodiments, the NK cells are peripheral blood derived. In some embodiments, the NK cells are iPSC derived. [111] In some embodiments, provided herein is a method for treating SLE or lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD19 targeting chimeric antigen receptor (anti-CD19-CAR) (anti-CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is administered following a single lymphodepletion cycle. In some embodiments, the therapeutically effective dose is a single dose. [112] In some embodiments, the single dose is 800 million cells. [113] In some embodiments, the single dose is 200 million cells. [114] In some embodiments, the therapeutically effective dose comprises two, three or more doses. In some embodiments, the therapeutically effective dose comprises two doses.
Attorney Docket No. MIL-047WO1 In some embodiments, the therapeutically effective dose comprises three doses. In some embodiments, the therapeutically effective dose comprises greater than three doses. [115] In some embodiments, the therapeutically effective dose is between 100 million to 1 billion cells. [116] In some embodiments, the therapeutically effective dose is 100 million cells. In some embodiments, the therapeutically effective dose is 200 million cells. In some embodiments, the therapeutically effective dose is 300 million cells. In some embodiments, the therapeutically effective dose is 400 million cells. In some embodiments, the therapeutically effective dose is 500 million cells. [117] In some embodiments, the therapeutically effective dose is 600 million cells. In some embodiments, the therapeutically effective dose is 700 million cells. In some embodiments, the therapeutically effective dose is 800 million cells. In some embodiments, the therapeutically effective dose is 900 million cells. In some embodiments, the therapeutically effective dose is 1 billion cells. In some embodiments, the therapeutically effective dose is 1.2 billion cells. In some embodiments, the therapeutically effective dose is 1.4 billion cells. In some embodiments, the therapeutically effective dose is 1.6 billion cells. In some embodiments, the therapeutically effective dose is 1.8 billion cells. In some embodiments, the therapeutically effective dose is 2 billion cells. In some embodiments, the therapeutically effective dose is 2.2 billion cells. In some embodiments, the therapeutically effective dose is 2.4 billion cells. [118] In some embodiments, the method comprises administering pharmaceutical compositions and formulations comprising anti-CD19-directed genetically modified NK cells (e.g., CD19 CAR+ viable NK cells and/or “allogenic cord blood derived anti-CD19 CAR - NK+ cells) in a dosing regimen of the present invention. Anti-CD19 CAR+ NK cells described herein comprise anti-CD19 CAR and IL-15, for example, secreted IL-15. [119] In some embodiments, the method comprises administering pharmaceutical compositions and formulations comprising CD19-directed genetically modified NK cells (e.g., anti-CD19 CAR+ viable NK cells and/or “allogenic cord blood derived anti-CD19
Attorney Docket No. MIL-047WO1 CAR-NK+ cells) in a dosing regimen of the present invention. Anti-CD19 CAR+ NK cells described herein comprise CD19 CAR and IL-15, for example, secreted IL-15. Anti-CD19 CAR-NK Cell Compositions and Formulations [120] In some embodiments, CD19 CAR+ NK cells are genetically engineered cord blood NK cells including a CD19-CAR comprising an anti-CD19 binding domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, CD19 CAR+ NK cells are genetically engineered cord blood NK cells including a CD19-CAR comprising an anti-CD19 binding domain comprising a light chain denoted by SEQ ID NO: 1 comprising CDR-L1 set forth in SEQ ID NO: 3, CDR- L2 set forth in SEQ ID NO: 4 and CDR-L3 set forth in SEQ ID NO: 5, and a heavy chain denoted by SEQ ID NO: 2 comprising CDR-H1 set forth in SEQ ID NO: 6, CDR-H2 set forth in SEQ ID NO: 7 and CDR-H3 set forth in SEQ ID NO: 8. [121] In some embodiments, the CAR-NK cells have been genetically modified to express a CD19 CAR having at least 95% identity to CDR sequences of CDR-L1: RASQDISKYLN (SEQ ID NO: 3), CDR-L2: SRLHSGV (SEQ ID NO: 4), CDR-L3: GNTLPYTFG (SEQ ID NO: 5), CDR-H1: DYGVS (SEQ ID NO: 6), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 7) and CDR-H3: YAMDYWG (SEQ ID NO: 8). [122] In some embodiments, the CAR-NK cells have been genetically modified to express a CD19 CAR having 100% identity to CDR sequences of CDR-L1: RASQDISKYLN (SEQ ID NO: 3), CDR-L2: SRLHSGV (SEQ ID NO: 4), CDR-L3: GNTLPYTFG (SEQ ID NO: 5), CDR-H1: DYGVS (SEQ ID NO: 6), CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 7) and CDR-H3: YAMDYWG (SEQ ID NO: 8). [123] In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD19 CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 95% identity to the amino acid
Attorney Docket No. MIL-047WO1 sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 98% identity to the amino acid sequence of SEQ ID NO: 2. [124] In some embodiments, the CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 2. [125] In some embodiments, the genetically engineered cord blood NK cells include a nucleic acid molecule encoding the heavy chain variable region of an anti-CD19 binding domain and/or a nucleic acid molecule encoding the light chain variable region of an anti- CD19 binding domain. [126] In some embodiments, the genetically engineered cord blood NK cells include a CD19-CAR comprising an anti-CD19 binding domain, a transmembrane domain such as the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and an intracellular signaling domain such as an intracellular signaling domain FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3-zeta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. The CD19 binding domain can be a single chain antibody or single chain antibody fragment, such as an scFv. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and/or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the CD19 CAR can include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 9), a CD3 zeta signaling domain (an exemplary CD3 zeta sequence is shown in SEQ ID NO: 10) and can further include IL-15 and/or a suicide switch, e.g., iCaspase9. [127] In some embodiments, the CD19-CAR comprises a CD28 domain. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 91% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 92% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 93% identity to the amino acid sequence of SEQ
Attorney Docket No. MIL-047WO1 ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 94% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19- CAR comprises a CD28 domain having at least 95% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 96% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 97% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 98% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having at least 99% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD19-CAR comprises a CD28 domain having 100% identity to the amino acid sequence of SEQ ID NO: 9. [128] In some embodiments, the CD19-CAR comprises a CD3ζ domain. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 91% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 92% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 93% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 94% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 95% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 96% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 97% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 98% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having at least 99% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD19-CAR comprises a CD3ζ domain having 100% identity to the amino acid sequence of SEQ ID NO: 10. [129] In some embodiments, anti-CD19 CAR+ NK cells described herein comprises an exogenous gene encoding a CD19 CAR (e.g., a polypeptide comprising SEQ ID Nos 1 and 2) and IL-15. In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO:
Attorney Docket No. MIL-047WO1 12. In some embodiments, the anti-CD19 CAR further comprises iCaspase9 comprising the amino acid sequence of SEQ ID NO: 13. Anti-CD19 Light chain variable fragment, VL: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKR (SEQ ID NO: 1) Anti-CD19 Heavy chain variable fragment, VH: EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTTVTVSSYVTVSSQDPA (SEQ ID NO: 2) Anti-CD19 binding domain CDR Sequences : CDR-L1: RASQDISKYLN (SEQ ID NO: 3) CDR-L2: SRLHSGV (SEQ ID NO: 4) CDR-L3: GNTLPYTFG (SEQ ID NO: 5) CDR-H1: DYGVS (SEQ ID NO: 6) CDR-H2: VIWGSETTYYNSALKS (SEQ ID NO: 7) CDR-H3: YAMDYWG (SEQ ID NO: 8) CD28: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRS (SEQ ID NO: 9) CD3ζ: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PRGP (SEQ ID NO: 10) IgG1: EPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKKDPK (SEQ ID NO: 11) IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 12)
Attorney Docket No. MIL-047WO1 iCaspase9: MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESG GGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNID CEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQAS HLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVAS TSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSG SWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS ASRA (SEQ ID NO: 13) [130] In some embodiments, subjects are administered anti-CD19 CAR-NK cells after a single lymphodepletion cycle. [131] In some embodiments, the single lymphodepletion cycle comprises fludarabine and/or cyclophosphamide. In some embodiments, the single lymphodepletion cycle comprises fludarabine. In some embodiments, the single lymphodepletion cycle comprises cyclophosphamide. [132] In some embodiments, the anti-CD19-CAR CB-NK cells comprises one or more exogenously provided interleukin (IL). [133] In some embodiments, the one or more interleukin is selected from IL-15, IL- 12, IL-21, IL-2, IL-18, IL-7, and combinations thereof. In some embodiments, the CD19- CAR CB-NK cells comprises IL-15. In some embodiments, the CD19-CAR CB-NK cells comprises IL-12. In some embodiments, the CD19-CAR CB-NK cells comprises IL-2. In some embodiments, the CD19-CAR CB-NK cells comprises IL-18. In some embodiments, the CD19-CAR CB-NK cells comprises IL-7. In some embodiments, the CD19-CAR CB-NK cells comprise a combination of one or more of IL-15, IL-12, IL-21, IL-2, IL-18 and IL-7. In some embodiments, the CD19-CAR CB-NK cells comprise two cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise three cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise four cytokines. In some embodiments, the CD19- CAR CB-NK cells comprise five cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise six cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise greater than six cytokines. [134] In some embodiments, the interleukin is IL-15. [135] In some embodiments, the IL-15 is secreted or membrane bound. In some embodiments, the IL-15 is secreted. In some embodiments, the IL-15 is membrane bound. [136] In some embodiments, the IL-15 is secreted from the cell.
Attorney Docket No. MIL-047WO1 [137] In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO: 6. In some embodiments, the IL-15 has at least 90% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 91% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 92% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 93% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 94% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 96% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 97% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 98% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has at least 99% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-15 has 100% identity to the amino acid sequence of SEQ ID NO: 6. [138] In some embodiments, exogenously provided IL is expressed from a vector in the cells. [139] In some embodiments, the CD19-CAR CB-NK cell comprises a suicide gene. [140] In some embodiments, the NK cells are engineered to express one or more suicide genes. For example, in some examples the NK cells are engineered to express one or more of iCaspase9, non-secretable TNFalpha, herpes simplex virus thymidine kinase (HSV- TK), uracil phosphoribosyl transferase (UPRTase), or cytosine deaminase (CD). [141] Additional examples of suicide genes include engineered nonsecretable (including membrane bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see e.g., PCT/US2019/062009, which is incorporated by reference herein in its entirety), and they may be affected by delivery of an antibody that binds the TNF-alpha mutant. Non-limiting examples of suicide gene/prodrug combinations that may be used are herpes simplex virus- thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or 2’-deoxy-2’-fluoro-5-iodo-1-B-D- arabinofuranosyluracil (FIAU), oxidoreductase and cycloheximide, cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk-Tmk) and azidothymidine (AZT), and deoxycytidine kinase and cytosine arabinoside. The E. coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to toxic purine 6-methylpurine, may be utilized. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450
Attorney Docket No. MIL-047WO1 enzymes (CYP), carboxypeptidases (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-a, g-lyase (MET), and thymidine phosphorylase (TP), among others. [142] In some embodiments, the NK cells are engineered to express one or more of iCaspase9. In some embodiments, the NK cells are engineered to express non-secretable TNF alpha. In some embodiments, the NK cells are engineered to express herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the NK cells are engineered to express uracil phosphoribosyl transferase (UPRTase). In some embodiments, the NK cells are engineered to express cytosine deaminase (CD).In some embodiments, the suicide gene is an iCaspase9 suicide gene. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 90% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 91% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 92% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 93% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 94% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 95% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 96% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 97% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 98% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 99% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having 100% identity to an amino acid sequence of SEQ ID NO: 13. [143] In some embodiments, the anti-CD19-CAR CB-NK cells are autologous with respect to the subject. [144] In some embodiments, the anti-CD19-CAR CB-NK cells are allogeneic with respect to the subject. [145] In some embodiments, the anti-CD19-CAR CB-NK cells are administered intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally,
Attorney Docket No. MIL-047WO1 intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion in a tumor microenvironment, or a combination thereof. In some embodiments, the CD19-CAR CB-NK cells are administered intracranially. In some embodiments, the CD19- CAR CB-NK cells are administered intravenously. In some embodiments, the CD19-CAR CB-NK cells are administered intraarterially. In some embodiments, the CD19-CAR CB-NK cells are administered intraperitoneally. In some embodiments, the CD19-CAR CB-NK cells are administered intratracheally. In some embodiments, the CD19-CAR CB-NK cells are administered intratumorally. In some embodiments, the CD19-CAR CB-NK cells are administered intramuscularly. In some embodiments, the CD19-CAR CB-NK cells are administered endoscopically. In some embodiments, the CD19-CAR CB-NK cells are administered intralesionally. In some embodiments, the CD19-CAR CB-NK cells are administered percutaneously. In some embodiments, the CD19-CAR CB-NK cells are administered subcutaneously. In some embodiments, the CD19-CAR CB-NK cells are administered by perfusion (e.g., in a tumor microenvironment). In some embodiments, the CD19-CAR CB-NK cells are administered by one or more of intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion, or a combination thereof, in any order and in any number of combinations. [146] In some embodiments, the NK cells are from primary cell isolates (e.g., NK cell derived from cord blood). In some embodiments, the NK cells are cord blood derived NK cells. In some embodiments, the NK cells are peripheral blood NK cells. In some embodiments, the NK cells are iPSC derived NK cells. In some embodiments, the NK cells are from a cell line. In some embodiments, the NK cells are fresh cells. In some embodiments, the NK cells were previously frozen and thawed. [147] In some embodiments the NK cell is gene edited to allow the cells to work more effectively. In some embodiments, the genes are one or more of TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM 17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7, or combinations thereof, in any order and in any number of combinations. In some embodiments, one or more of these genes are knocked out or knocked down in the cells.
Attorney Docket No. MIL-047WO1 [148] In some embodiments, the NK cells are engineered to express CD19-CAR, IL- 15, and iCaspase9. An exemplary CAR-NK cell comprising CD19 IL-15, and iCaspase9 is described in Leukemia 32 (2018)520-531, incorporated herein by reference in its entirety. [149] In one aspect, the compositions described herein for use in treating lupus nephritis, including relapsing or refractory lupus nephritis, comprise anti-CD19-directed genetically modified NK cell immunotherapy (e.g., anti-CD19 CAR+viable CB-NK cells) and a pharmaceutically acceptable carrier. [150] In some embodiments, compositions comprising anti-CD19-directed genetically modified NK cell immunotherapy described herein comprises CAR-NK cells at a concentration of between 6 M/mL to 25 M/mL, 5 M/mL to 23 M/mL, 5 M/mL to 24 M/mL in a 36 mL volume or 5 M/mL to 25 M/mL in a 36 mL volume. [151] In some embodiments, the total volume of a composition comprising anti- CD19-directed genetically modified NK cell immunotherapy in which the CAR-NK cells are suspended is between about 15 mL and 30 mL, about 30 mL and 45 mL, about 30 and 60 mL, or about 30 mL and 75 mL. In some embodiments, the total volume in which the NK cells are suspended is between about 15 mL and 30 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is between about 30 mL and 45 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is between about 30 mL and 60 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is between about 30 mL and 75 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL or 50 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 20 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 21 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 22 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 23 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 24 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 25 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 26 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 27 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is
Attorney Docket No. MIL-047WO1 about 28 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 29 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 30 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 31 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 32 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 33 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 34 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 35 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 36 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 37 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 38 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 39 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 40 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 41 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 42 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 43 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 44 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 45 mL. In some embodiments, the total volume in which the CAR-NK cells are suspended is about 46 mL. [152] In some embodiments, a composition comprising CD19-directed genetically modified NK cell immunotherapy comprise CAR-NK cells at a concentration of about 300 million to 1.5 billion cells per 36 mL. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 200 or 800 million CAR-NK cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 100 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 200 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 300 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 400 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 500 million cells per a 36 mL fill volume. In some embodiments, the composition
Attorney Docket No. MIL-047WO1 comprises CAR-NK cells at a concentration of about 600 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 700 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 800 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 900 million cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.1 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.2 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.3 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.4 billion cells per a 36 mL fill volume. In some embodiments, the composition comprises CAR-NK cells at a concentration of about 1.5 billion cells per a 36 mL fill volume. [153] In some embodiments, the CAR-NK cell therapy product administered is an allogeneic cell therapy product comprised of human cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, anti-CD19 CAR and IL-15. [154] In some embodiments, the CAR-NK cell therapy product comprises a population of between 300 million to 800 million cells formulated in a cryopreservation media described herein. In some embodiments, the CAR-NK cell therapy product comprises a population of 100 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 200 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 300 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 400 million cells. In some embodiments, the CAR- NK cell therapy product comprises a population of 500 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 600 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 700 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 800 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 900 million cells. In some embodiments, the CAR-NK cell therapy product comprises a population of 1 billion cells.
Attorney Docket No. MIL-047WO1 [155] In some embodiments, the CAR-NK cell therapy product comprises a population of between 100 million to 1 billion cells formulated in a cryopreservation media described herein. In some embodiments, the CAR-NK cell therapy product comprises a population of 800 million cells. [156] In some embodiments, anti-CD19 CAR+ NK cells are genetically engineered cord blood NK cells including an anti-CD19-CAR comprising an anti-CD19 binding domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, anti-CD19 CAR+ NK cells are genetically engineered cord blood NK cells including an anti-CD19-CAR comprising an anti-CD19 binding domain comprising a light chain denoted by SEQ ID NO: 1 comprising CDR1, CDR2 and CDR3 and a heavy chain denoted by SEQ ID NO: 2 comprising CDR1, CDR2 and CDR3. [157] In some embodiments, the anti-CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD19 CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti- CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having at least 98% identity to the amino acid sequence of SEQ ID NO: 2. [158] In some embodiments, the anti-CD19-CAR comprises an anti-CD19 binding domain comprising a light chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having 100% identity to the amino acid sequence of SEQ ID NO: 2. [159] In some embodiments, the genetically engineered cord blood NK cells include a nucleic acid molecule encoding the heavy chain variable region of an anti-CD19 binding
Attorney Docket No. MIL-047WO1 domain and/or a nucleic acid molecule encoding the light chain variable region of an anti- CD19 binding domain. [160] In some embodiments, the genetically engineered cord blood NK cells include an anti-CD19-CAR comprising an anti-CD19 binding domain, a transmembrane domain such as the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and an intracellular signaling domain such as an intracellular signaling domain FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3-zeta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. The anti-CD19 binding domain can be a single chain antibody or single chain antibody fragment, such as an scFv. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and/or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In another embodiment, the anti-CD19 CAR can include an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO: 9), a CD3 zeta signaling domain (an exemplary CD3 zeta sequence is shown in SEQ ID NO: 10) and can further include IL-15 and/or a suicide switch, e.g., iCaspase9. [161] In some embodiments, the anti-CD19-CAR comprises a CD28 domain. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19- CAR comprises a CD28 domain having at least 91% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 92% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 93% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 94% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 95% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 96% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 97% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 98% identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having at least 99% identity to the amino acid sequence of SEQ
Attorney Docket No. MIL-047WO1 ID NO: 9. In some embodiments, the anti-CD19-CAR comprises a CD28 domain having 100% identity to the amino acid sequence of SEQ ID NO: 9. [162] In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti- CD19-CAR comprises a CD3ζ domain having at least 91% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 92% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 93% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 94% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 95% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 96% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 97% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 98% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having at least 99% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD19-CAR comprises a CD3ζ domain having 100% identity to the amino acid sequence of SEQ ID NO: 10. [163] In some embodiments, anti-CD19 CAR+ NK cells described herein comprises an exogenous gene encoding an anti-CD19 CAR (e.g., a polypeptide comprising SEQ ID Nos 1 and 2) and IL-15. In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO: 12. In some embodiments, the anti-CD19 CAR further comprises iCaspase9 comprising the amino acid sequence of SEQ ID NO:13. [164] In some embodiments, the CD19-CAR CB-NK cells comprises one or more exogenously provided interleukin (IL). [165] In some embodiments, the one or more interleukin is selected from IL-15, IL- 12, IL-21, IL-2, IL-18, IL-7, and combinations thereof. In some embodiments, the CD19- CAR CB-NK cells comprises IL-15. In some embodiments, the CD19-CAR CB-NK cells comprises IL-12. In some embodiments, the CD19-CAR CB-NK cells comprises IL-2. In some embodiments, the CD19-CAR CB-NK cells comprises IL-18. In some embodiments, the CD19-CAR CB-NK cells comprises IL-7. In some embodiments, the CD19-CAR CB-NK
Attorney Docket No. MIL-047WO1 cells comprise a combination of one or more of IL-15, IL-12, IL-21, IL-2, IL-18 and IL-7. In some embodiments, the CD19-CAR CB-NK cells comprise two cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise three cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise four cytokines. In some embodiments, the CD19- CAR CB-NK cells comprise five cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise six cytokines. In some embodiments, the CD19-CAR CB-NK cells comprise greater than six cytokines. [166] In some embodiments, the interleukin is IL-15. [167] In some embodiments, the IL-15 is secreted or membrane bound. In some embodiments, the IL-15 is secreted. In some embodiments, the IL-15 is membrane bound. [168] In some embodiments, the IL-15 is secreted from the cell. [169] In some embodiments, the IL-15 amino acid sequence comprises SEQ ID NO: 12. In some embodiments, the IL-15 has at least 90% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 91% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 92% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 93% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 94% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 95% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 96% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 97% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 98% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has at least 99% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-15 has 100% identity to the amino acid sequence of SEQ ID NO: 12. [170] In some embodiments, exogenously provided IL is expressed from a vector in the cells. [171] In some embodiments, the CD19-CAR CB-NK cell comprises a suicide gene. [172] In some embodiments, the NK cells are engineered to express one or more suicide genes. For example, in some examples the NK cells are engineered to express one or more of iCaspase9, non-secretable TNFalpha, herpes simplex virus thymidine kinase (HSV- TK), uracil phosphoribosyl transferase (UPRTase), or cytosine deaminase (CD).
Attorney Docket No. MIL-047WO1 [173] Additional examples of suicide genes include engineered nonsecretable (including membrane bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see e.g., PCT/US2019/062009, which is incorporated by reference herein in its entirety), and they may be affected by delivery of an antibody that binds the TNF-alpha mutant. Non-limiting examples of suicide gene/prodrug combinations that may be used are herpes simplex virus- thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or 2’-deoxy-2’-fluoro-5-iodo-1-B-D- arabinofuranosyluracil (FIAU), oxidoreductase and cycloheximide, cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk-Tmk) and azidothymidine (AZT), and deoxycytidine kinase and cytosine arabinoside. The E. coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to toxic purine 6-methylpurine, may be utilized. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidases (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-a, g-lyase (MET), and thymidine phosphorylase (TP), among others. [174] In some embodiments, the NK cells are engineered to express one or more of iCaspase9. In some embodiments, the NK cells are engineered to express non-secretable TNF alpha. In some embodiments, the NK cells are engineered to express herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the NK cells are engineered to express uracil phosphoribosyl transferase (UPRTase). In some embodiments, the NK cells are engineered to express cytosine deaminase (CD). In some embodiments, the suicide gene is an iCaspase9 suicide gene. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 90% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 91% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 92% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 93% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 94% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 95% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 96% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 97%
Attorney Docket No. MIL-047WO1 identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 98% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having at least 99% identity to an amino acid sequence of SEQ ID NO: 13. In some embodiments, the iCaspase9 gene encodes an iCaspase9 having 100% identity to an amino acid sequence of SEQ ID NO: 13. [175] Also provided by the present disclosure are anti-CD19 CAR described in WO2012/079000A1 and WO2014/031687A1, each of which is incorporated herein by reference in entirety. [176] In some aspects, provided herein is a method for treating a relapsing or refractory SLE or lupus nephritis in a subject, the method comprising a step of administering a therapeutically effective dose of cord blood derived natural killer (CB-NK) cells expressing CD19 targeting chimeric antigen receptor (CD19-CAR) (CD19-CAR CB-NK cells) to the subject in need thereof, wherein the therapeutically effective dose is about 800 million following a single lymphodepletion cycle. [177] In some embodiments, the subject has previously received at least one line of standard immunosuppressive therapy. In some embodiments, the subject has previously received cyclophosphamide (CYP) and/or mycophenolate mofetil (MMF). In some embodiments, the subject has previously received cyclophosphamide (CYP). In some embodiments, the subject has previously received mycophenolate mofetil (MMF). In some embodiments, the subject has previously received cyclophosphamide (CYP) and mycophenolate mofetil (MMF). [178] In some embodiments, the anti-CD19-CAR CB-NK cells are allogeneic with respect to the subject. [179] In some embodiments, the anti-CD19-CAR CB-NK cells are autologous with respect to the subject. [180] In some embodiments, the anti-CD19-CAR CB-NK cells are administered intracranially, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, by perfusion t, or a combination thereof. [181] In some embodiments, the anti-CD19-CAR CB-NK cells are administered intravenously.
Attorney Docket No. MIL-047WO1 [182] In some embodiments, the patient has a disease that is relapsing or refractory (r/r) after at least two prior lines of therapy. [183] In some embodiments, the anti-CD19 CAR-NK cells are frozen prior to administration. In some embodiments, the CAR-NK cell therapy product is an allogeneic cell therapy product comprising between 100 million to about 1 billion human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 1 billion human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 900 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 800 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 700 million human cord blood- derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 600 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 500 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 400 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 300 million human cord blood- derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product comprising about 200 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. In some embodiments, the CAR-NK cell therapy product is an allogenic cell therapy product
Attorney Docket No. MIL-047WO1 comprising about 100 million human cord blood-derived NK cells transduced with a vector (e.g., a retroviral vector) expressing anti-CD19 CAR and IL-15. [184] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as the cells) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral- active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases. [185] In some embodiments, anti-CD19 CAR CB-NK cells are suspended in a medium for cryopreservation. In some embodiments, compositions comprising anti-CD19 CAR CB-NK cells as described herein are formulated in a solution comprising a cryopreservation medium. For example, cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw. For example, compositions comprising immune effector cells as described herein may be cryopreserved in a medium discussed in the PCT application publication WO2022/173866A1 (the contents of which are
Attorney Docket No. MIL-047WO1 incorporated herein by reference in their entirety). In some embodiments, the cryopreservation medium comprises a non-pyrogenic and isotonic crystalloid solution, a disaccharide, a cryoprotectant and an albumin. In some embodiments, the non-pyrogenic and isotonic crystalloid solution is present at a concentration of 25% v/v to 50% v/v. In some embodiments, the non-pyrogenic and isotonic crystalloid solution is present at a concentration of about 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the non- pyrogenic and isotonic crystalloid solution is present at a concentration of 30% v/v to 40% v/v. [186] In some embodiments, the non-pyrogenic and isotonic crystalloid solution is present at a concentration of 37.1% v/v, 37.3% v/v, 37.5% v/v, 37.7% v/v, or 37.9% v/v. [187] In some embodiments, the non-pyrogenic and isotonic crystalloid solution is present at a concentration of 37.7% v/v. [188] In some embodiments, the non-pyrogenic and isotonic crystalloid solution is present at a concentration of 38.0% v/v, 38.2% v/v, 38.4% v/v, 38.6% v/v, 38.8% v/v, or 39.0% v/v. [189] In some embodiments, the non-pyrogenic and isotonic crystalloid solution is present at a concentration of 38.6% v/v. [190] In some embodiments, the disaccharide is selected from the group consisting of sucrose, lactose, maltose, trehalose, cellobiose, and chitobiose. Accordingly, in some embodiments, the disaccharide is sucrose. In some embodiments, the disaccharide is lactose. In some embodiments, the disaccharide is maltose. In some embodiments, the disaccharide is trehalose. In some embodiments, the disaccharide is cellobiose. In some embodiments, the disaccharide is chitobiose. [191] In some embodiments, a cryopreservation medium provided herein comprises one or more of sodium chloride, potassium chloride, magnesium chloride hexahydrate, sodium acetate trihydrate, sodium gluconate, adenosine, dextran-40, lactobionic acid, HEPES, sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate; potassium phosphate, dextrose, sucrose, mannitol, calcium chloride dihydrate, magnesium chloride, sodium hydroxide, potassium hydroxide, DMSO, human serum albumin and trehalose. [192] In some embodiments, a cryopreservation medium provided herein comprises about 2.35% w/v human serum albumin (HSA). In some embodiments, the cryopreservation
Attorney Docket No. MIL-047WO1 medium comprises between about 2.0% w/v and 5.0% w/v human serum albumin (HSA). In some embodiments, the cryopreservation medium comprises between about 2.0% w/v and 3.0% w/v human serum albumin (HSA). In some embodiments, a cryopreservation medium provided herein comprises about 1% v/v to 10% v/v human serum albumin (HSA). In some embodiments, a cryopreservation medium provided herein comprises about 5% v/v to 10% v/v human serum albumin (HSA). In some embodiments, the cryopreservation medium comprises 9.4% v/v human serum albumin. [193] In some embodiments, the cryopreservation medium comprises between about 10 mM – 100 mM trehalose. In some embodiments, the cryopreservation medium comprises between about 10 mM – 50 mM trehalose. In some embodiments, the cryopreservation medium comprises between about 20 mM – 40 mM trehalose. In some embodiments, the cryopreservation medium comprises about 30 mM trehalose. [194] In some embodiments, a cryopreservation medium is provided comprising: human serum albumin (HSA), sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, magnesium chloride, dimethyl sulfoxide (DMSO), and a trehalose. [195] In some embodiments, a cryopreservation medium is provided, the medium comprising: a non-pyrogenic and isotonic crystalloid solution, a cryoprotectant, an albumin, and a disaccharide. In some embodiments, a cryopreservation medium is provided, the medium comprising: PLASMA-LYTE A, a cryoprotectant, human serum albumin (HSA), and trehalose. In some embodiments, the cryoprotectant is DMSO. In some embodiments, a cryopreservation medium is provided, the medium comprising 37.7% PLASMA-LYTE A, 50% DMSO, 2.35% w/v HSA and 30 mM trehalose. [196] In some embodiments, anti-CD19 CAR CB-NK cells are suspended in the cryopreservation media described in Table 1. To prepare the medium in Table 1, 25% w/v human serum albumin (HSA) and 400 mg/mL trehalose solution are used as components. Table 1: Cryopreservation Media Cryopreservation Media Composition 1 50% v/v PLASMA-LYTE-HEPES + 35% v/v Dextran/Dextrose + 10% v/v HSA + 5% v/v DMSO 2 40% v/v PLASMA-LYTE A+ 50% v/v CS10 +10% v/v HSA
Attorney Docket No. MIL-047WO1 3 50% v/v MEM-HEPES + 35% v/v Dextran/Dextrose + 10% v/v HSA + 5% v/v DMSO 4 40% v/v MEM + 50% v/v CS10 +10% v/v HSA 5 38.6% v/v PLASMA-LYTE A + 50% v/v CS10 +10% v/v HSA + 0.8% v/v AA+ Vitamin (Baxter; 0.2% vial 1 and 0.4% vial 2) + 30 mM trehalose 6 40% v/v MEM + 50% v/v CS10 +10% v/v HSA + 1.5 mM Glutathione 7 40% v/v PLASMA-LYTE A+ 50% v/v CS10 +10% v/v HSA + 1.5 mM Glutathione 8 38.6% v/v PLASMA-LYTE A+ 50% v/v CS10 +10% v/v HSA + 0.8% v/v Amino Acids + 0.6% v/v Vitamins +1.5 mM Glutathione 9 40% PLASMA-LYTE A + 50% CS10 +10% HSA + 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) [197] PLASMA-LYTE A (Baxter) is a sterile, nonpyrogenic isotonic solution containing 526 mg of Sodium Chloride; 502 mg of Sodium Gluconate; 368 mg of Sodium Acetate Trihydrate; 37 mg of Potassium Chloride; and 30 mg of Magnesium Chloride per 100 ml. at pH 7.4 (6.5 to 8.0). One liter of PLASMA-LYTE A has an ionic concentration of 140 mEq sodium, 5 mEq potassium, 3 mEq magnesium, 98 mEq chloride, 27 mEq acetate, and 23 mEq gluconate. The osmolarity is 294 mOsmol/L (calc). (280 to 310 mOsmol/L). [198] In some embodiments, the anti-CD19 CAR-NK cells are directly administered to a subject in need thereof. In some embodiments, the anti-CD19 CAR-NK cells are not washed prior to administering to a subject in need thereof. In some embodiments, the anti- CD19 CAR-NK cells are washed prior to administering to a subject in need thereof.
Attorney Docket No. MIL-047WO1 [199] In some embodiments, the frozen cells are thawed and administered into a patient in need thereof within about 30 minutes and 2 hours from thawing the cells. In some embodiments, the rate of intravenous infusion into a subject is between about 2-3 minutes. In some embodiments, administration is at a patient’s bedside. In some embodiments, the thawed cells are administered directly without further dilution. Viability Assessment [200] Viability of anti-CD19 CAR-NK cells can be assessed in vitro using various methods known in the art. In some embodiments, the in vitro cell viability test includes the Trypan Blue exclusion assay. In some embodiments, other analytical methods can be used to assess the cell viability of cells, for example, flow cytometry-based viability markers and the like. A person of ordinary skill in the art can opt for any analytical method to assess the viability of anti-CD19 CAR-NK cells that can be applied to assess the cell viability cells described herein. [201] Phenotype and function of anti-CD19 CAR-NK cells can be assessed in vitro using various methods known in the art. In some embodiments, the in vitro cell phenotyping tests includes flow cytometry assays. In some embodiments, the in vitro cell function test includes cytokine production (e.g., interferon gamma), cytotoxicity, proliferation and other analytical methods. [202] Efficacy of anti-CD19 CAR-NK cells in vivo can be assessed using animal studies known in the art. [203] The anti-CD19 CAR-NK cells described herein retain high viability (e.g., greater than 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) and retain physiological characteristics of their native state, which allows the cells to be used for a variety of applications, such as for genetic manipulation of the cells, and for cell therapy purposes such as, for example, in adoptive cell therapy applications. EXAMPLES [204] Other features, objects, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the present invention, are given by way of illustration only, not
Attorney Docket No. MIL-047WO1 limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples. Example 1: Administration of cord-blood derived anti-CD19 CAR-NK cells in Treating Refractory Lupus Nephritis [205] This example describes a Phase 1b, multicenter, open-label study to evaluate the safety, tolerability, cellular kinetics, pharmacodynamics, and efficacy of cord blood- derived CD19 targeting CAR-NK cells in adult subjects with refractory lupus nephritis (LN). [206] About 20 subjects will be enrolled in a multi-site study. Subjects will be enrolled having a diagnosis of Systemic Lupus Erythematosus (SLE) and refractory Lupus Nephritis with an inadequate response, defined as failure to improve within 12 weeks, based on investigator discretion, to at least two standard-of-care treatments for SLE (including glucocorticoids and immunosuppressive agents) OR at least one biologic treatment for SLE. Enrolled subjects must have met the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 SLE classification criteria per Inclusion Criterion. [207] Enrollment Criteria: Subjects are required to have met the below criteria for enrollment: [208] Positive for antinuclear antibody (ANA) (by immunofluorescent assay [IFA] with a titer ≥1:80) and at least one of anti–double-stranded DNA (anti-dsDNA) or anti-Smith (anti-Sm) autoantibodies at screening or by documented medical history. [209] Active disease as defined by a Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) total score ≥6. [210] Histology-proven glomerulonephritis (proliferative LN class III or IV, with or without the presence of class V, according to 2018 International Society of Nephrology/Renal Pathology Society [ISN/RPS] criteria). [211] Inadequate response, defined as failure to improve within 12 weeks, based on investigator discretion, to at least two standard-of-care treatments for systemic lupus erythematosus (SLE) (including glucocorticoids and immunosuppressive agents), or at least one biologic treatment for SLE. [212] A single dose of 800 million allogeneic cord-blood derived anti-CD19 CAR- NK cells will be administered intravenously on day 1 following a single cycle of
Attorney Docket No. MIL-047WO1 lymphodepleting chemotherapy. The proposed dose demonstrated a favorable safety profile in clinical studies with lymphoma patients (NCT05020015). [213] A lower dose of 200 million cord-blood derived anti-CD19 CAR-NK cells administered on day 1 following a single cycle of lymphodepleting chemotherapy will also be evaluated in a dose de-escalation study, as also staggered dosing, safety monitoring, necessary stopping for risk mitigation and management. [214] 800 million cells is a starting dose as well as maximum dose/exposure for the study, with 200 million cells being the minimum dose. Subjects will be followed for a total of 24 months (including primary and secondary follow-up). [215] Cord blood-derived CAR-NK cell administration will follow lymphodepleting chemotherapy (fludarabine 30 mg/m2 body surface area [BSA] per day intravenously on Days -5, -4, and -3 and cyclophosphamide 300 mg/m2 BSA per day intravenously on Days -5, -4, and -3). At least two days must elapse between the end of lymphodepleting chemotherapy and administration of cord blood-derived CD19 targeting CAR-NK cell therapy. [216] The primary analyses of the primary, secondary, and selected exploratory endpoints will be performed at a time when all subjects have had the opportunity to be evaluated for at least 6 months after administration of cord blood-derived CD19 targeting CAR-NK cell therapy. Analyses of the primary, secondary, and exploratory endpoints will also be assessed in the final analysis, if applicable. [217] Primary endpoints of the study include (a) Incidence of treatment-emergent adverse events (TEAEs) and (b) Incidence of dose-limited toxicity (DLTs). [218] Secondary objectives of the study include the following: [219] To characterize the cellular kinetics of cord blood-derived CD19 targeting CAR-NK cells in adult subjects with refractory Lupus Nephritis. [220] To assess pharmacodynamic biomarkers such as B cell counts and cytokines in adult subjects with refractory Lupus Nephritis. [221] To determine preliminary efficacy of cord blood derived CD19 targeting CAR -NK cells in adult subjects with refractory Lupus Nephritis. [222] To evaluate the immunogenicity of cord blood derived CD19 targeting CAR - NK cells in adult subjects with refractory Lupus Nephritis. [223] Secondary Endpoints include the following:
Attorney Docket No. MIL-047WO1 [224] Cellular kinetics of cord-blood derived CAR-NK cells assessed by maximum observed concentration (Cmax), time of first occurrence of Cmax (Tmax), persistence (time of last measurable concentration above the lower limit of quantitation [Tlast]), area under the concentration-time curve from time 0 to time of the last quantifiable concentration [AUClast]), and by other parameters as appropriate. [225] Change in CD19+ B cell counts and duration of B cell depletion from baseline. [226] Change from baseline in cytokine levels (e.g., interleukin-15). [227] Change from baseline in physician’s global assessment scores. [228] Proportion of subjects achieving a reduction in Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) relative to baseline. [229] Proportion of subjects achieving complete renal response (CRR) (defined by the following [all criteria must be met]: (a) a ratio of urinary protein to creatinine <0.5, an estimated glomerular filtration rate [eGFR] no worse than 10% below the preflare value [baseline] or ≥60 ml per minute per 1.73 m2, and no use of rescue therapy), (b) time to CRR, and (c) duration of CRR. [230] Proportion of subjects achieving Lupus Low Disease Activity State (LLDAS), time to LLDAS, and duration of LLDAS. [231] Proportion of subjects meeting the definition of remission in SLE (DORIS) criteria, time to DORIS, and duration of DORIS remission. [232] Change from baseline levels of clinical biomarkers such as anti-dsDNA, ANA, proteinuria, creatinine, eGFR, and complement (C3, C4). [233] Development of anti-drug antibodies (ie, anti-HLA, anti-CAR). [234] Prevalence and incidence of replication-competent retrovirus positive test results. [235] A separate, long-term, follow-up study for continued safety assessments for up to 15 years will subsequently be carried out per the 2020 FDA Guidance on Long-Term Follow-Up After Administration of Human Gene Therapy Products. [236] Inclusion Criteria for subjects: To be eligible to participate in the study, a subject must meet all the following criteria at screening: [237] Age and Reproductive Status: 1. The subject must be aged 18 to 75 years (inclusive) at the time of consent.
Attorney Docket No. MIL-047WO1 2. Subjects with female reproductive anatomy who: a. Are postmenopausal for at least 1 year before the screening visit, or b. Are surgically sterile for at least 6 weeks, or c. Are of childbearing potential: i. Agree to practice one highly effective method of contraception and one additional effective (barrier) method at the same time, from the time of signing the informed consent through at least 24 months following cord-blood derived anti-CD19 CAR-NK administration, or ii. Agree to practice true abstinence, when this is in line with the preferred and usual lifestyle of the subject (periodic abstinence [e.g., calendar, ovulation, symptothermal, postovulation methods], withdrawal, spermicides only, and lactational amenorrhea are not acceptable methods of contraception). Female and male condoms should not be used together, and iii. Agree not to donate an egg or eggs (ova) or breastfeed a baby during the study and until at least 24 months following cord blood-derived anti-CD19 CAR-NK cell administration. 3. Subjects with male reproductive anatomy, even if surgically sterilized (i.e, status post vasectomy), who: a. Agree to practice effective barrier contraception from the time of signing the informed consent through at least 6 months following cord blood-derived CAR-NK cell administration, or b. Agree to practice true abstinence, when this is in line with the preferred and usual lifestyle of the subject (periodic abstinence [e.g., calendar, ovulation, symptothermal, postovulation methods], withdrawal, spermicides only, and lactational amenorrhea are not acceptable methods of contraception), and c. Agree not to donate sperm during the study and at least 6 months following cord- blood derived anti-CD19 CAR-NK cell administration. [238] Organ function: [239] 1. The subject must have adequate bone marrow function defined as follows: (a) Absolute neutrophil count ≥1000/µl, (b) Platelet count ≥75,000/µl.
Attorney Docket No. MIL-047WO1 [240] 2. The subject must have adequate renal, hepatic, cardiac, and pulmonary function defined as follows: [241] eGFR ≥30 mL/min, as calculated using the chronic kidney disease epidemiology collaboration equation. [242] Serum alanine aminotransferase/aspartate aminotransferase ≤5 times the upper limit of normal range (ULN), and subject must be asymptomatic. [243] Total bilirubin ≤2 mg/dL. Subjects with Gilbert’s syndrome may have a bilirubin level >2 × ULN, per discussion between the investigator and the medical monitor. [244] Left ventricular ejection fraction ≥40% as determined by an echocardiogram or multigated acquisition scan performed within 1 month of enrollment. [245] No evidence of clinically relevant pericardial effusion. [246] Absence of Grade ≥2 pleural effusion. Grade 1 stable pleural effusions are allowed. [247] Baseline oxygen saturation >92% on room air. [248] Exclusion criteria: [249] SLE-Related Exclusions: [250] 1. The subject has a history of drug-induced SLE. [251] 2. The subject has a current diagnosis of active or unstable neuropsychiatric lupus (eg, cerebritis, cerebrovascular accident [CVA], and seizures). However, subjects with mononeuritis multiplex or polyneuropathy can be included in the study. [252] 3. The subject has a history or current diagnosis of other autoimmune diseases or current inflammatory joint or skin disease other than SLE that, in the opinion of the investigator and per sponsor assessment, could interfere with the inflammatory arthritis or skin assessments and confound the disease activity assessments (e.g., rheumatoid arthritis, multiple sclerosis, scleroderma, uncontrolled type 1 diabetes, uncontrolled type 2 diabetes, psoriasis, inflammatory bowel disease, and psoriatic arthritis). [253] 4. The subject has a history of catastrophic antiphospholipid syndrome or saddle embolism (antiphospholipid syndrome adequately controlled by anticoagulant therapy for at least 3 months is acceptable). [254] Noninfectious Disorders Exclusions: [255] 1. The subject has any clinically significant medical condition, evidence of an
Attorney Docket No. MIL-047WO1 unstable clinical condition (e.g., cardiovascular, renal, hepatic, hematologic, gastrointestinal, endocrine, pulmonary, or immunologic), or vital signs/physical/laboratory/electrocardiogram abnormality that would, in the opinion of the investigator and per sponsor assessment, put the subject at undue risk or interfere with interpretation of study results. These include but are not limited to: [256] (a) The subject has a history of a known or suspected condition/illness with compromised immunity, including but not limited to congenital or acquired immunodeficiency and/or splenectomy. [257] (b) Subjects with a history or presence of active or clinically relevant central nervous system (CNS) disorder, such as seizure, encephalopathy, cerebrovascular ischemia/hemorrhage, severe dementia, cerebellar disease, or any autoimmune disease with CNS involvement. For CNS disorders that recover or are in remission, subjects without recurrence within 24 months of planned study enrollment may be included. [258] (c) Subjects with a history of solid organ transplantation. [259] (d) The subject has unstable or uncontrolled cardiovascular disease, a clinical cardiovascular event (including but not limited to acute coronary syndrome, myocardial infarction, stroke, rapid atrial fibrillation, or other dysrhythmia), symptomatic congestive heart failure (i.e., New York Heart Association Class II or greater), poorly-controlled or severe hypertension, or a cardiac hospitalization (e.g., revascularization procedure, pacemaker implantation) within 3 months before screening. [260] (e) The subject has a history of cancer or lymphoproliferative disease within 5 years before planned enrollment. Note: Subjects with successfully treated nonmetastatic cutaneous squamous cell or basal cell carcinoma and/or localized carcinoma in situ of the cervix are not to be excluded. [261] (f) The subject has a non-SLE concomitant illness, as determined by medical judgment, that is likely to require additional systemic corticosteroid therapy during the study (e.g., uncontrolled asthma). [262] (g) The subject has a history of any significant/uncontrolled psychiatric illness (including but not limited to active suicidal ideation at screening or at time of planned enrollment) for which participation in the trial would, in the opinion of the investigator, put the subject at undue risk or would interfere with interpretation of study results. [263] (i) Per medical judgment, the subject has a known history of clinically
Attorney Docket No. MIL-047WO1 significant drug or alcohol abuse, excluding marijuana/tetrahydrocannabinol/cannabidiol use, within 12 months before screening. [264] (j) The subject has had any major surgery within the last 30 days before planned enrollment or has any surgery planned during the course of the study. [265] Overall, the results from this study will demonstrate the safety, tolerability and efficacy of anti-CD19 CB CAR-NK therapy in treating refractory lupus nephritis. [266] Example 2. In vitro cytotoxicity assessment on primary B cells from both healthy donors and SLE patient donors [267] This example demonstrates in vitro cytotoxicity of exemplary anti-CD19 CAR -NK cells on primary B cells from healthy donors and SLE patient donors. [268] The anti-CD19 CAR-NK cells used in this example are cord blood derived NK cells exogenously transduced with anti-CD19 CAR having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; comprising light chain CDR sequences represented by SEQ ID NO: 3, 4 and 5; and heavy chain CDR sequences represented by SEQ ID NO: 6, 7 and 8. The anti-CD19 CAR additionally included a CD28 transmembrane domain (SEQ ID NO: 9), a CD3 zeta signaling domain (SEQ ID NO: 10), an IgG1 Fc spacer (SEQ ID NO: 11) and IL-15 (SEQ ID NO: 12) and iCaspase9 (SEQ ID NO: 13). [269] The anti-CD19 CAR-NK cells were generated from two independent donor cord blood units (D004 and D005) and cytotoxicity was evaluated against primary B cells from three independent healthy donors (healthy donor 1, 219 and 366) and three independent SLE donors (301, 001 and 602), respectively after 20 hours of co-culture, using a flow cytometry-based cell viability assay (FIG.1A-FIG.1F). [270] Briefly, primary B cells were isolated from frozen PBMC samples by negative selection (Miltenyi Biotec, Inc. [Waltham, MA, USA]), and labeled with CellTrace Violet (CTV) (Thermo Fisher Scientific, Inc.) for traceability. Labeled primary B cells were plated at 20,000 cells/100 μL/well. Frozen anti-CD19 CAR-NK cells derived from donor 004 and frozen anti-CD19 CAR-NK cells derived from donor 005 (formulated in 40% PLASMA- LYTE A, 50% CS 10, 10% HSA and 30 mM trehalose) were thawed, resuspended in growth medium and viable cells counted. [271] The anti-CD19 CAR-NK cells (effector cells) were co-incubated with respective primary B cells (target cells) at seven different effector to target cell ratios (10: 1,
Attorney Docket No. MIL-047WO1 5: 1, 2.5: 1, 1.25: 1, 0.63: 1, 0.3: 1, and 0.16: 1), respectively. After a 20-hour incubation period at 37°C, 100 µL cell supernatant was collected and saved at -80 °C for IFN-γ detection.100 µL of diluted propidium iodide (PI) was then added to each well to achieve a final concentration of 10 µg/mL. [272] Cells were resuspended and used for flow cytometry analysis with fixed volume (100 μL) stopping gate using an Attune NxT acoustic focusing cytometer (Thermo Fisher Scientific, Inc.). The number of primary B cells (target) alone was used as the baseline of viable cells with no killing. Data represent mean of duplicate wells. Killing or specific lysis of target cells was calculated as follows: % ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ = 100 100
[273] The results from this study show cytoxicity of anti-CD19 CAR-NK cells from two exemplary cord blood donors. FIG.1A-FIG.1C are bar graphs depicting primary B cell killing from three exemplary healthy donors at various Effector : Target ratios. FIG.1D-FIG. 1F are bar graphs depicting primary B cell killing from three exemplary SLE donors at various Effector : Target ratios. [274] The results showed that anti-CD19 CAR-NK cells from the two cord blood donors demonstrated killing of primary B cells from both healthy donors and SLE patient donors. Example 3. Detection of IFN-gamma cytokine secretion level in co-culture supernatant [275] This example demonstrates in vitro production of cytokines (e.g., IFN-gamma) by exemplary anti-CD19 CAR-NK cells co-cultured with primary B cells from healthy donors and SLE patient donors. [276] In this study, MSD V-PLEX Human IFN-gamma (IFN-g) Kit was used for cytokine analysis. Supernatant from each co-culture group in Example 2 with three exemplary Effector : Target ratios (2.5:, 1.25:1 and 0.625:1) was transferred to a dilution plate and diluted 10-fold with assay diluent. After pre-coated MSD plates were washed three times with wash buffer, 50 µL of standards, controls or diluted samples were transferred to the MSD plates for 2 hours incubation at room temperature with shaking at 700 RPM. After 2 hours of sample incubation, the MSD plates were washed three times and 25 µL of detection antibody solution were added to each well. MSD plates were incubated for another 2 hours at room temperature in dark with shaking at 700 RPM. After 2 hours of detection antibody
Attorney Docket No. MIL-047WO1 incubation, MSD plates were washed three times, followed by addition of 150 µL of 2x read buffer T and analysis on an MSD instrument. [277] The results are shown in FIG.2A-FIG.2F. As shown in FIG.2A-FIG.2C, IFN-gamma cytokine was detected in supernatants from co-culturing anti-CD19 CAR-NK cells (from two independent donor cord blood units (D004 and D005)) with B cells from healthy donors. As shown in FIG.2D-FIG.2F, IFN-gamma cytokine was detected in supernatants from co-culturing anti-CD19 CAR-NK cells (from two independent donor cord blood units (D004 and D005)) with B cells from SLE donors. [278] The results from this study showed that anti-CD19 CAR-NK cells from the two cord blood donors demonstrated IFNg secretion when co-cultured with primary B cells from both healthy donors and SLE patient donors. EQUIVALENTS AND SCOPE Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: