WO2025122947A1 - Methods and compositions for enhancing adoptive cell therapy - Google Patents

Abstract

Aspects of the disclosure include methods of monitoring and/or prognosing an adoptive cell therapy in a subject, comprising determining a level of endogenous SCF in a biological sample from the subject, in which the detection of a presence, absence, and/or amount of endogenous SCF is informative of the in vivo activation and/or expansion of the adoptive immune cells, the anti-tumor effect of the adoptive cell therapy, and/or the need for a treatment modification and/or extension. The disclosure also provides a method of treating a disease by administering at least one c-kit agonist (e.g., SCF) simultaneously or sequentially with an adoptive cell therapy.

Description

METHODS AND COMPOSITIONS FOR ENHANCING ADOPTIVE CELL THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/607,808, filed December 8, 2023. The entire contents of the above-identified application are hereby fully incorporated herein by reference.

FIELD OF DISCLOSURE

[0002] The present disclosure relates generally to methods for monitoring and/or prognosing adoptive cell therapies, and methods for treating diseases with such therapies using one or more c-kit agonists (e.g., stem cell factor) as an adjunctive therapy.

BACKGROUND OF THE DISCLOSURE

[0003] Adoptive cell therapies (ACTs) have remarkable clinical potential in the treatment of cancers, autoimmune diseases, and other diseases. From the initial infusion of autologous lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes (TILS) to the subsequent engineered T cell receptor (TCR)-T and chimeric antigen receptor (CAR)-T cell therapies, many novel strategies for cancer treatment have been developed. Engineered immune cell therapies such as CAR-T cells have revolutionized the field of ACTs, particularly for hematologic malignancies, and researchers have more recently focused on the application of CAR engineering technology to other types of immune cells. Consequently, several new cell therapies based on CAR technology have been developed, including CAR-NK, CAR-macrophage, CAR- 76T, and CAR-NKT. Unfortunately, however, the therapeutic efficacy of these new approaches still faces challenges such as insufficient responses in certain patient populations and/or variations in response among different patients. Therefore, there is a continuing need for new and better approaches of monitoring and/or prognosing adoptive cell therapies to optimize and adjust ongoing treatment plans, and to provide adjunctive therapy to enhance patients’ response to such therapies.

SUMMARY OF DISCLOSURE

[0004] The present invention addresses this unmet need in the art with an informative marker for evaluating a patient’s response to an adoptive cell therapy, including an engineered immune cell therapy, and determining whether to adjust, extend and/or add to the patient’s therapy. As demonstrated herein for the first time, determining the level of endogenous stem cell factor (SCF) in a patient receiving an adoptive cell therapy can inform the level of in vivo activation and/or expansion of the adoptive immune cells, the anti-tumor effect of the adoptive cell therapy, and/or the need for a treatment modification and/or extension. Moreover, adjunctive therapies are also contemplated comprising administering at least one c-kit agonist to a patient undergoing an adoptive cell therapy to enhance the in vivo activation and/or expansion of the adoptive immune cells and/or improve the efficacy of the adoptive cell therapy.

[0005] As detailed herein, the present invention is generally applicable to any adoptive cell therapy, including both engineered and non-engineered immune cells. Tn embodiments, the adoptive cell therapy comprises non-engineered immune cells such as, e.g., LAKs, TILs, virusspecific T cells (VSTs), and the like. In embodiments, the adoptive cell therapy comprises engineered immune cells such as, e.g., TCR-T cells, CAR-T cells, CAR-NK cells, CAR-M cells, CAR-y8T cells, CAR-NKT cells, and the like.

[0006] In one aspect, the present disclosure provides a method for monitoring and/or prognosing an adoptive cell therapy in a subject, comprising determining a level of endogenous stem cell factor (SCF) in a biological sample from the subject, wherein the detection of a presence, absence, and/or amount of endogenous SCF is informative of the in vivo activation and/or expansion of the adoptive immune cells, the anti-tumor effect of the adoptive cell therapy, and/or the need for a treatment modification and/or extension.

[0007] In embodiments, the biological sample is obtained from the subject after administering a lymphodepletion (LD) regimen to the subject and prior to administering a first dose of the adoptive cell therapy to the subject; preferably at least 24, 48, 72, 96, or 120 hours after administration of the LD regimen. In embodiments, biological sample is obtained from the subject pre-infusion on the same day as the first dose of the adoptive cell therapy.

[0008] In embodiments, the treatment modification comprises adjusting a dosage level (e.g., increasing or decreasing the dosage of the adoptive cell therapy), adjusting a previously planned dosing schedule, and/or administering one or more adjunctive or alternative therapies. In embodiments, the one or more alternative therapies comprises administering a different adoptive cell therapy. In embodiments, the one or more adjunctive therapies comprises administering at least one c-kit agonist to the subject in conjunction with administering the adoptive cell therapy. In embodiments, the c-kit agonist comprises recombinant SCF, a nucleic acid encoding SCF, or an immune cell engineered to stably express SCF, wherein the immune cell engineered to stably express SCF may be the same or different than the adoptive cell therapy.

[0009] In embodiments, the method further comprises determining a level of endogenous SCF in at least one additional biological sample obtained from the subject after administration of the adoptive cell therapy, and determining a dose, or a continuing dose, of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in the at least one additional sample. In embodiments, detecting an amount of endogenous SCF greater than 500 pg/ml in a serum sample from the subject is supportive of a positive prognosis.

[0010] In embodiments, the treatment extension comprises administering a second dose of the adoptive cell therapy to the subject at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, or at least one month after the first dose, with or without administering an additional LD regimen.

[0011] In embodiments, the adoptive cell therapy comprises T cells, NK cells, and/or macrophages that are engineered to stably express one or more antigen recognition moieties; preferably wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen.

[0012] In another aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising a) administering to the subject a lymphodepletion (LD) regimen; b) administering to the subject a first dose of an adoptive cell therapy at least 5 days after administering the LD regimen; and c) simultaneously or sequentially administering a therapeutically effective amount of at least one c-kit agonist to the subject to enhance in vivo expansion of the engineered immune cells. In embodiments, the adoptive cell therapy is an engineered immune cell therapy comprising T cells, NK cells and/or macrophages that are engineered to stably express one or more antigen recognition moieties; preferably wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen.

[0013] In embodiments, the at least one c-kit agonist is administered after the administration of the adoptive cell therapy. [0014] In embodiments, the method of treating a disease further comprises determining a level of endogenous SCF in a biological sample obtained from the subject after administration of the LD regimen and before administration of the adoptive cell therapy, and determining a dose of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in the sample.

[0015] In embodiments, the biological sample is obtained from the subject at least 24, 48, 72, 96, or 120 hours after administration of the LD regimen; preferably wherein the biological sample is obtained from the subject prior to infusion on the same day as the first dose of the adoptive cell therapy.

[0016] In embodiments, the method of treating a disease further comprises determining a level of endogenous SCF in at least one biological sample obtained from the subject after administration of the adoptive cell therapy, and determining a dose of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in the sample.

[0017] In embodiments, the method of treating a disease further comprises determining a level of endogenous SCF in at least one additional biological sample obtained from the subject after administration of the adoptive cell therapy, and determining a dose, or a continuing dose, of the c- kit agonist based on the presence, absence and/or amount of endogenous SCF in the at least one additional sample.

[0018] In embodiments, the method of treating a disease further comprises administering a second dose of the adoptive cell therapy to the subject at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, or at least one month after the first dose, with or without administering an additional LD regimen.

[0019] In embodiments, the LD regimen comprises administration of fludarabine at about 30 mg/m2/day plus cyclophosphamide at about 500 mg/m2/day for three days. In embodiments, the LD regimen comprises administration of fludarabine at about 30 mg/m2/day for four days, plus cyclophosphamide at about 1000 mg/m2/day for three days.

[0020] In embodiments, the c-kit agonist comprises recombinant SCF, a nucleic acid encoding SCF, or an immune cell engineered to stably express SCF, wherein the immune cell engineered to stably express SCF may be the same or different than the adoptive cell therapy. [0021] In another aspect, the present disclosure provides a composition comprising T cells, NK cells and/or macrophages that are engineered to stably express one or more antigen recognition moieties and at least one c-kit agonist; preferably wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen, and the c-kit agonist is hSCF.

[0022] In embodiments, the T cells, NK cells and/or macrophages are engineered to express two or more antigen recognition moieties, preferably wherein the two or more antigen recognition moieties are different, and wherein each different antigen recognition moiety is engineered to recognize different epitopes of the same antigen or to recognize different epitopes of different antigens.

[0023] In embodiments, the T cells, NK cells, and/or macrophages are further engineered to stably express at least one c-kit agonist; preferably wherein the at least one c-kit agonist comprises human SCF (hSCF).

[0024] In another aspect, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding at least one c-kit agonist. In embodiments, the c-kit agonist is hSCF.

[0025] In embodiments, the CAR comprises an affinity binding entity comprising an antigen binding domain that specifically binds to (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).

[0026] In embodiments, the antigen recognition moiety is selected from the group consisting of a TCR, <x(3 TCR, y8 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigenbinding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide- MHC complex).

[0027] In embodiments, the CAR further comprises a hinge domain; optionally wherein the hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or receptor-derived hinge. In embodiments, the receptor-derived hinge is a CD8 alpha hinge domain.

[0028] In embodiments, the CAR further comprises a transmembrane (TM) domain; optionally wherein the TM domain comprises a TM region of 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, or CD 154, CD 100 (SEMA4D), CD 103, CD 160 (BY 5), CD 18, CD 19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD11c, CD1 Id, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM- 1, Ig alpha (CD79a), IL-2Rbeta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 ; CDl la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB- A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. In embodiments, the TM domain comprises a TM domain of CD8, preferably wherein the CD8 TM domain is a TM domain of CD8 alpha.

[0029] In embodiments, the CAR further comprises at least one costimulatory domain; optionally wherein the costimulatory domain comprises a costimulatory domain of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8a, CD8[3, CDl la, CDl lb, CDl lc, CDl ld, IL2RP, IL2y, IL7Ra, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (0X40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD 11 a/CD 18), FcR, FcyRI, FcyRII, FcyRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, LylO8), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG/Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, or TRANCE/RANKL, or a portion thereof, or combinations thereof. In embodiments, the costimulatory domain is a 4-1BB costimulatory domain.

[0030] In embodiments, the CAR and/or the c-kit agonist further comprises a signal peptide. In embodiments, the c-kit agonist is operably linked to a nucleic acid sequence encoding a signal peptide.

[0031] In embodiments, the isolated polynucleotide further comprises a nucleic acid sequence encoding at least one multi ci stronic linker region; optionally wherein the multi ci stronic region encodes a cleavage sequence and/or an internal ribosomal entry site (IRES). In embodiments, the cleavage sequence is selected from T2A, F2A, P2A, E2A, furin, and furin-P2A (FP2A).

[0032] In another aspect, the present disclosure provides an expression vector comprising the isolated polynucleotide described herein, operably linked to a cis-acting regulatory element.

[0033] In another aspect, the present disclosure provides a cell comprising the isolated polynucleotide, and/or the expression vector described herein. In another aspect, the present disclosure provides a modified immune cell, comprising the isolated polynucleotide, and/or the expression vector described herein. In embodiments, the modified immune cell is a y5 T cell, a y5 NKT cell, an up T cell, aNK cell, aNKT cell, or a macrophage. In embodiments, the modified immune cell is a y5 T cell; optionally wherein the y5 T cell is a 51, a 82, a 53, or a 64 y5 T cell, preferably a 52‘ y5 T cell, more preferably a 51 y5 T cell. INCORPORATION BY REFERENCE

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows a summary of the study in Example 1. AEs= Adverse events; Cy= Cyclophosphamide; DLBCL=Diffuse large B-cell lymphoma; DL= Dose level; DLT= Dose limiting toxicity; D0R= Duration of response; ECOG= Eastern Cooperative Oncology Group; Flu= Fludarabine; GLEAN= Gamma Delta adoptive therapy for Nhl-1; OS= Overall survival; PFS= Progression-free survival; R/R= Relapsed or refractory; TTP= Time to progression.

[0036] FIGS. 2A-2E show cellular kinetics of ADI-001. ADI-001 was measured by (FIG. 2A) droplet digital PCR (ddPCR) with (FIG. 2B) cellular kinetic parameters summarized for DL3 and DL4 showing mean Cmax, Area under the curve (AUCO-28), Tmax and Day 28 persistence. Mean DL3 and DL4 Cmax by ddPCR aligned with what was previously reported by internal flow cytometry analysis. AUCO-28 was calculated using a model-based cellular kinetics analysis for CAR T cells for the first 28 days after infusion (units of days*CAR copies/ug DNA). CAR cells/pL were derived from ddPCR and considered product VCN. LLOQ (64.5 CAR copies/ pg) and LOD (49.5 CAR copies/pg) are shown (y-axis, dashed lines). N= 20 patients were assessed by ddPCR; N = 16 patients were assessed for AUCO-28. (FIG. 2C) Quantitative SNP profiling of cell product (AlloCell) was assessed in 24 patients and plotted as Mean ±SEM. (FIG. 2D) Whole blood (WB) from 24 patients across multiple timepoints was evaluated for the presence of CAR+ V81+ T cells by flow cytometry and expressed as absolute cell counts (cells/pL blood). The lower limit of quantification (LLOQ) for this assay is 3.2 cells/pL blood with a lower limit of detection (LOD) of 0.18 cells/pL blood. CAR+ V61+ T cells detected below LLOQ were graphed at ’A the LOD of 0.18 cells/pL blood; internal paired flow analysis subject to verification. (FIG. 2E) The gross dynamic range of average AD 1-001 genomes (measured by AlloCell and expressed as % AD 1-001 cells per total nucleated cells) across all dose levels is shown versus the average recovery of patient CD8+ T cells by flow cytometry (N = 24). For all measures, detection of ADI-001 returns to <LOD by the month 3 assessment. [0037] FIGS. 3A-3D show the relationship of ADI-001 cellular kinetics with dose and clinical response. Relationship of (FIG. 3 A) Cmax and (FIG. 3B) AUCO-28 with dose level. (FIG. 3C and FIG. 3D) Relationship of Cmax and AUCO-28 with best overall response (BOR) in patients. Those subjects whose BOR was complete response (CR) or partial response (PR) appear to associate with higher (FIG. 3C) Cmax (Wilcoxon Rank Sum test, two-sided P-value, N=20, P=0.0757) and (FIG. 3D) AUCO-28 (Mann- Whitney test, N = 16, P=0.1804) than those who had a stable disease (SD) or progressed disease (PD) as BOR. DL1 was not assessed by ddPCR due to insufficient or unavailable sample material; N = 20 patients total were assessed by ddPCR. AUCO- 28 was calculated using a model -based cellular kinetics analysis for CAR T cells for the first 28 days after infusion (units of days*CAR copies/ug DNA).

[0038] FIGS. 4A-4B show the association of serum SCF and IL-15 cytokine levels in the 24 DLT (dose limiting toxicity) evaluable patients. (FIG. 4A) Subjects whose BOR was CR/PR showed a trend of higher stem cell factor (SCF) production on Day 1 pre-infusion than subjects who had a NR/PD as BOR Mann-Whitney, p=0.0565. (FIG. 4B) Interleukin- 15 (IL-15) serum levels (y-axis left side, pg/mL) were examined across multiple timepoints, and immune cell reconstitution was assessed using flow cytometry (y-axis right side, cells/pL). Each timepoint represents the Mean ± SEM. Following lymphodepletion, there was a transient increase in the homeostatic cytokine IL-15, coinciding with ADI-001 expansion and host-mediated immune cell recovery, which dropped close to baseline levels between D14 and D28 in all patients and for all LD regimens evaluated.

DETAILED DESCRIPTION

Definitions

[0039] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. [0040] About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0041] As used herein, “w/v” refers to the weight of the component in a given volume of solution.

[0042] “Ranges”: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0043] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal amenable to the methods described herein. In certain nonlimiting embodiments, the patient, subject or individual is a human.

[0044] As used herein, the term “agent” refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell e.g., cell engineered to express a chimeric antigen receptor). Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional agent induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject suffering from cancer, or other disease/condition.

[0045] The term “diagnosis”, or “diagnosing” as used herein refers to the process of identifying a disease, such as cancer, by its signs, symptoms, and/or results of various tests. A conclusion reached through such a process is a diagnosis. Forms of testing commonly performed include blood tests, medical imaging, urinalysis, biopsy, and the like. [0046] The term “prognosis” refers to a forecast as to the probable outcome of a disease, the prospect as to recovery from a disease, or the potential recurrence of a disease as indicated by the nature and symptoms of the case.

[0047] The term “positive prognosis” refers to a situation in which the predicted outcome for a particular patient is improved in comparison to an average patient with the same disease.

Typical examples of a positive prognosis include a better than average cure rate, a lower propensity for metastasis, a longer than expected life expectancy, etc. For example, if a prognosis is that a patient has a 50% probability of being cured of a particular cancer after treatment, while the average patient with the same cancer has only a 25% probability of being cured, then that patient exhibits a positive prognosis.

[0048] The term "therapeutically effective amount", or simply “effective amount” refers to the amount of an agent or composition (e.g., composition comprising an agent) that will elicit a biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of an agent, or a composition comprising an agent, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease (e.g., hematological or solid tumor) being treated. The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0049] To "treat" a disease as the term is used herein, means to decrease or reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or condition by decreasing one or more signs or symptoms associated with the disease or condition, for example as compared to the response in the absence of the therapy. For example, administration of a therapeutic agent may provide an anti-tumor effect that decreases one or more signs or symptoms associated with cancer.

[0050] As used herein, the term “administration” means to provide or give a subject one or more agents, such as an agent that treats one or more signs or symptoms associated with a condition/disorder or disease including but not limited to cancer (e.g., lymphoma), viral infection, bacterial infection, etc., by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Administration “in combination with ” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0051] The term "pharmaceutically acceptable", as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulatory agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate. For example, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an, e.g., immune cell such as a y8 T cell, preferably a immune cell engineered to express a CAR directed to CD20, as described herein.

[0052] As used herein, the term “pharmacodynamic (PD) biomarker” and/or “pharmacokinetic (PK) biomarker” refers to one or more measurable indicators associated with administration of a therapeutic agent to a subject. Broadly speaking, a PK marker relates to how the body affects a therapeutic agent, whereas a PD marker relates to how the therapeutic agent affects a subject. [0053] The term “cancer” as used herein refers to a physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Neoplasia, malignancy, cancer, and tumor may be used interchangeably and refer to abnormal growth of a tissue or cells that results from excessive cell division.

[0054] Cancers include hematologic cancers and solid tumors. Hematologic cancers include cancers originating in the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0055] In embodiments, a cancer is relapsed/refractory B cell malignancy. As used herein, the term “relapsed/refractory B cell malignancy” encompasses any B cell lymphoma that is ultimately non-responsive to treatment including but not limited to Non-Hodgkin lymphoma (NHL); chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), acute lymphocytic leukemia (ALL), and acute myeloid leukemia (AML). Accordingly in embodiments the B cell malignancy may be selected from the group comprising or consisting of NHL, CLL, ALL and/or AML. In embodiments the B cell malignancy may be a form of NHL selected from the group comprising or consisting of diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), transformed follicular lymphoma (tFL), primary mediastinal (thymic) large B cell lymphoma (PMBCL), high-grade B cell lymphomas, Burkitt lymphoma, follicular lymphoma (FL), and marginal zone lymphoma (MZL).

[0056] Solid tumors include tumors that comprise a tumor mass of at least about 10 or at least about 100 tumor cells. The solid tumor can be a soft tissue tumor, a primary solid tumor, or a metastatic lesion. Examples of solid tumors include, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), pancreas, prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0057] The term “autoimmune disease” as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriately excessive response to an antigen associated with an autoimmune disease (e.g., a self-antigen). Examples of autoimmune diseases include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis/scleroderma, polymyositis/dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCTD), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson’s, Alzheimer’s, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott- Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto’s thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenia purpura, dermatomyositis, Sydenham’ a chorea, myasthenia gravis, polyglandular syndromes, bullous pemphigoid, Henoch-Schonlein purpura, poststreptococcalnephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu’s arteritis, Addison’s disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture’s syndrome, thromboangitisubiterans, Sjogren’s syndrome, primary biliary cirrhosis, Hashimoto’s thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pamphigus vulgaris, Wegener’s granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, polymyalgia, peraiciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosing alveolitis.

[0058] ‘Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. [0059] The term “adoptive cell therapy” generally refers to the transfer of cells into a patient. The cells may have originated from the patient or from another individual. The cells are most commonly derived from the immune system with the goal of improving immune functionality and characteristics. See, e.g., Rohaan MW, Wilgenhof S, Haanen JBAG. Adoptive cellular therapies: the current landscape. Virchows Arch. 2019 Apr;474(4):449-461. doi: 10.1007/s00428-018-2484- 0. Zhang, P., Zhang, G. & Wan, X. Challenges and new technologies in adoptive cell therapy. J Hematol Oncol 16, 97 (2023). https://doi.org/10.1186/sl3045-023-01492-8

[0060] The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including proteins or peptides, can serve as an antigen. An antigen may be, for example, a peptide, a protein, a hapten, a lipid, a carbohydrate, bacteria, a pathogen, or a virus. In embodiments, an antigen may be a disease associated antigen, which comprises an epitope that may be presented by the MHC I or MHC II complexes on the surface of a cell (e g., a tumor cell). An epitope can be the portion of the antigen that is expressed on the cell surface and recognized by an antigen recognition moiety described herein.

[0061] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic specificity, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, diabodies, single domain antibodies (sdAbs), as long as they exhibit the desired biological or immunological activity, Fv, Fab and F(ab), as well as single chain antibodies and humanized antibodies (Harlow et ah, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY: Harlow et ah, 1989, In; Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et ah, 1988, Proc. Nat Acad. Sci. USA 85:5879-5883: Bird et ah, 1988, Science 242:423- 426). [0062] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0063] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.

[0064] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, K and light chains refer to the two major antibody light chain isotypes.

[0065] By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0066] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant capable of specific binding to an immunoglobulin or receptor, for example a T-cell receptor. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0067] The term "specifically binds”, as used herein refers to a receptor (which can include but is not limited to an antibody or antibody fragment) which recognizes a specific molecule/ligand, but does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a molecule from one species may also bind to that molecule from one or more other species. But, such cross-species reactivity does not itself alter the classification as specific. In another example, a receptor that specifically binds to a molecule may also bind to different allelic forms of the molecule. However, such cross reactivity does not itself alter the classification as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of a protein (or a peptide) with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, a receptor recognizes and binds to a specific a structure rather than to proteins generally. If receptor is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the receptor, will reduce the amount of labeled A bound to the receptor.

[0068] In embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about IxlO'⁸ M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0069] The term "anti-tumor effect" as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in metabolic activity of the tumor cells (e.g., PET signal), a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.

[0070] As used herein, the term "autologous" is meant to refer to any material derived from an individual which is later to be re-introduced into the same individual.

[0071] As used herein, the term "allogeneic" refers to material derived from an animal which is later introduced into a different animal of the same species.

[0072] The term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells may be of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. The term “engineered immune cell” refers to an immune cell that is genetically modified. In embodiments, an immune cell is an aP T cell. In embodiments, an immune cell is a y5 T cell. In embodiments, an immune cell is a natural killer (NK) cell. In embodiments, an immune cell is a y5 NKT cell. In embodiments, an immune cell is a NKT cell. In embodiments, an immune cell is a macrophage. [0073] The term “immune cell therapy” refers to an immune cell population and/or a cell line, e.g., as described herein, expanded and/or manufactured outside of a subject. The cells may be autologous or allogeneic, and may comprise immune effector cells. The cell population and/or cell line may comprise NK cells. The cell population and/or cell line may comprise T cells (e.g., aP T cells or y5 T cells). The cell population and/or cell line may comprise macrophages. In embodiments, the immune cell therapy is an engineered immune cell therapy comprising immune cells having one or more genetic modifications as disclosed herein. In embodiments, the engineered immune cell therapy comprises immune cells (e.g., T cells (such as aP T cells or yb T cells) or NK cells) engineered to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR) comprising a binding domain that specifically binds to an antigen on a host (e.g., tumor) cell.

[0074] The term "chimeric antigen receptors (CARs)" refers to artificial T-cell receptors, T- bodies, single-chain immunoreceptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell or NK cell, thereby allowing a large number of specific T or NK cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a disease associated antigen, (e.g., a tumor associated antigen, an autoimmune associated antigen, or a pathogenic antigen). In some embodiments, CARs comprise an intracellular activation domain (allowing the T or NK cell to activate upon engagement of targeting moiety with target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain that may vary in length and comprises a disease- or disorder-associated, e.g., a tumor-antigen binding region. In embodiments, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co- stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP 10/12, and/or 0X40, ICOS, TLRs, etc. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T or NK cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0075] The term “Natural killer (NK) cell” refers to CD56 CD3 granular lymphocytes that play important roles in immunity against viruses and in the immune surveillance of tumors, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53 : 1666-1676). NK cells express a remarkably diverse repertoire of inhibitory and activating receptors on their cell surface, which regulates their immune responses. NK cells can kill transformed or infected cells by the release of perforin and granzymes or by using effector molecules of the tumor necrosis factor (TNF) family, such as TNF, TNF-related apoptosis inducing ligand (TRAIL), and Fas ligand, which induce apoptosis in the target cells. Additionally, upon activation NK cells rapidly produce chemokines and cytokines, including interferon (IFN)-y, GM- CSF, and IL-10, that recruit and affect the function of hematopoietic and nonhematopoietic cells in the host. Unlike cytotoxic CD8⁺ T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can also eradicate MHC-I-negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23 :427-431). NK cells are considered fairly safe effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan R A, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter D L, et al. N Engl J Med 2011 365:725-733), and on-target, off-tumor effects. NK cells can be obtained from an allogeneic or an autologous donor. The NK cells can be partially or entirely purified, or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in Becker et al., (2016) Cancer Immunol. Immunother. 65(4): 477-84). The expansion may be performed before or after, or before and after, a CAR is introduced into the NK cell(s). Briefly, and without limitation, expansion of NK cells can include the use of engineered feeder cells, cytokine cocktails (e.g., IL-2, IL-15), and/or aAPCs (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0076] The term “aP T cells” or “alpha beta T cells” refers to T cells expressing a and chains of the TCR as part of a complex with CD3 chain molecules. Each a and P chain contains one variable and one constant domain. aP T cells primarily recognize peptide antigens presented by major histocompatibility complex (MHC) class I and class II molecules, where most of the receptor diversity is contained within the third complementarity determining region (CDR3) of the TCR a and P chains. [0077] The term " y8 T cells” or “gamma delta T cells" as used herein refers to a subset of T cells that express a distinct T-cell receptor (TCR), namely y8 TCR, on their surface, composed of one y-chain and one 8-chain. The term “y8 T cells” specifically includes all subsets of y8 T cells, including, without limitation, V61, V62, and V63 y6 T cells, as well as naive, effector memory, central memory, and terminally differentiated y8 T cells. As a further example, the term “y8 T cells” includes V84, V65, V67, and V68 y6 T cells, as well as Vy2, Vy3, Vy5, Vy8, Vy9, VylO, and Vyl 1 y8 T cells.

[0078] The term “Natural killer T (NKT) cells” are T lineage cells that share morphological and functional characteristics with both T cells and NK cells. NKT cells are rapid responders of the innate immune system and mediate potent immunoregulatory and effector functions in a variety of disease settings. Ligand recognition in NKT cells leads to rapid secretion of proinflammatory cytokines (such as IFN-y and TNF-a) and anti-inflammatory cytokines (such as IL-4, IL-10, and IL-13) that enhance the immune response to e.g., cancer by directly targeting tumor cells and by indirectly modulating the antitumor response through the release of diverse cytokines or by altering the TME. Following activation, NKT cells can immediately commence cytokine secretion without first having to differentiate into effector cells. The rapidity of their response makes NKT cells important players in the very first lines of innate defense against some types of bacterial and viral infections. In addition, many of the cytokines secreted by NKT cells have powerful effects on a|3 T cell differentiation and function, linking NKT cells to adaptive defense. NKT cells bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id. NKT cells can be obtained from an allogeneic or an autologous donor. The NKT cells can be partially or entirely purified, or not purified, and expanded ex vivo. Briefly and without limitation, NKT cells can be expanded via the use of ex vivo IL-2, and/or monoclonal antibodies specific for the TCR a-chain CDR3 loop (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0079] The term “y8 natural killer T cells” or “y8 NKT cells” refers to iPSC-derived cells that express y8 TCRs and NK receptors, but lack the expression of hallmark y8 T cell markers (Cortes- Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59). These cells have been shown to have anti-tumor activity against a broad number of cancer cell lines, but not against normal cells, and showed more potent killing than donor-derived y5 T cells or donor-derived NK cells (Zeng J et al., (2019) PLoS ONE 14(5): e0216815). CARs can be expressed in yo NKT cells, in embodiments herein, for use in accordance with the methods disclosed herein.

[0080] The term “myeloid cells” refers to a subgroup of leukocytes represented by granulocytes, monocytes, macrophages, and dendritic cells (DCs). They circulate through the blood and lymphatic system and are rapidly recruited to sites of tissue damage and infection via various chemokine receptors. Within the tissues they are activated for phagocytosis as well as secretion of inflammatory cytokines, thereby playing major roles in protective immunity. Myeloid cells can also be found in tissues under steady-state condition, where they control development, homeostasis, and tissue repair.

[0081] The term “macrophages” refers to highly plastic innate cells with functional and phenotypic signatures that can be shaped in response to various stimuli. Macrophage polarization is broadly simplified into two different states, either a Ml phenotype (classically activated) in response to factors such as lipopolysaccharide (LPS) or IFN-y, or a M2 phenotype in response to cytokines such as IL-4, IL-5, and IL-13. An example of Ml -like macrophages express iNOS and proinflammatory cytokines such as TNF-a, 1L1-P, IL-6, IL-12, and IL-23. An example of M2 macrophages exhibit increased expression of CD209, CD200R, CDla, and CDlb in humans, and have been implicated in wound healing and antitumor responses. The ability of macrophages to infiltrate solid tumors and be reprogrammed, as well as the antitumor effects associated with a switch to the Ml phenotype, render macrophages relevant to the present disclosure in terms of engineered macrophages that express a CAR described herein. For example, it has been shown that macrophages can be reprogrammed towards antitumor Ml phenotype cells that are capable of producing nitric oxide and inducing IL-12-dependentNK-mediated antitumor effects by inhibiting NK-KB signaling in a murine model of ovarian cancer (Zhang F et al., (2019) Nat Commun 10: 3974).

[0082] Macrophages can be obtained/derived from an allogeneic or an autologous donor. The macrophages can be partially or entirely purified, or not purified, and cultured ex vivo (see, e.g., Davies JQ and Gordon A (2005) Methods Mol Biol 290: 105016). In embodiments, the present disclosure encompasses macrophages derived from hESCs (Karlsson, KR et al., (2008) Exp Hematol 36: 1 167-1 175), or iPSC-derived macrophages (Takata K. et al., (2017) Immunity 47: 183-198).

[0083] The term “TCR” or “T cell receptor” refers to a dimeric heterologous cell surface signaling protein forming an alpha-beta or gamma-delta receptor or combinations thereof. aPTCRs recognize an antigen presented by an MHC molecule, whereas ybTCR can recognize an antigen independently of MHC presentation.

[0084] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encodes cell-surface antigen-presenting proteins. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. Herein, the abbreviations MHC or HLA are used interchangeably.

[0085] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0086] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0087] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. [0088] “Expression cassette” refers to a nucleic acid comprising expression control sequences operatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression cassettes can be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be in a host cell, such as an immune cell e.g., an aP T cell, a 76 T cell, or a NK cell.

[0089] The term “stem cell factor (SCF)” refers to a cytokine that that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Endogenous SCF is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. It is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a role in mast cell development, gametogenesis, and melanogenesis. SCF through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions (see e.g., Mazzoldi, E.L., et al. A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer. Cell Death Dis 10, 412 (2019)). In embodiments, SCF includes a soluble form SCF (e.g., comprising or consisting of

EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, (human SCF based on NP_000890, first 165 aa of the mature peptide without signal peptide, SEQ ID NO: 1)). In embodiments, SCF includes a transmembrane form SCF (e.g., comprising or consisting of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRNDSSSSNRKAKN PPGD S SLHWAAM ALP AL F SLIIGF AFGAL YWKKRQP SLTRAVENIQINEEDNEISMLQEK EREFQEV (human SCF based on NP 000890, mature peptide without signal peptide, SEQ ID NO: 2), see also Aderson DM et al. Cell Growth Differ. 1991 Aug;2(8):373-8)), or comprising or consists of

EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SID AFKDF VVASETSDC VVS STL SPEKGK AKNPPGD S SLHWAAMALP ALF SLIIGF AFGA LYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (human SCF based on NP NP 003985, mature peptide without signal peptide), SEQ ID NO: 3, see also Johan Lennartsson et al. Physiol Rev. 2012 Oct;92(4): 1619-49). Examples of soluble and transmembrane forms of SCF also include those described in J G Flanagan et al., Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sid mutant Cell. 1991 Mar 8;64(5): 1025-35; DM Anderson et al., Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24, Cell Growth Differ. 1991 Aug;2(8):373-8; and Johan Lennartsson et al., Stem cell factor receptor/c-Kit: from basic science to clinical implications, Physiol Rev. 2012 Oct; 92(4): 1619-49, each of which is incorporated by reference in its entirety.

[0090] The term “c-kit agonist” refers to a molecule that binds and activates the receptor tyrosine kinase protein c-kit (also known as tyrosine-protein kinase KIT, CD117 (cluster of differentiation 117) or mast/stem cell growth factor receptor (SCFR)). Examples of c-kit agonists include small molecule compounds, antibodies or fragments thereof, polypeptides, and nucleic acids, and other types of molecules that bind and activate c-kit.

[0091] In embodiments, a c-kit agonist is a cell engineered to stably express SCF. The cell may be an immune cell. In some examples, the SCF-expressing immune cell is the same as the cell in the immune cell therapy administered to a subject. In some examples, the SCF-expressing immune cell is different from the cell in the immune cell therapy administered to a subject.

[0092] In embodiments, a c-kit agonist is stem cell factor, e.g., a natural form, or a recombinant or synthetic stem cell factor, such as ancestim, a 166-amino-acid protein produced by E. coli bacteria into which a gene has been inserted for soluble human stem cell factor (MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSL TDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIF NRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, SEQ ID NO: 4) as described in McNiece IK and Briddel RA, The Cytokine Handbook (Fourth Edition), 2003); and MG da Silva et al. Ancestim (recombinant human stem cell factor, SCF) in association with filgrastim does not enhance chemotherapy and/or growth factor-induced peripheral blood progenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection, Bone Marrow Transplant. 2004 Oct;34(8):683-91, each of which is incorporated by reference herein in its entirety.

[0093] Additional examples of recombinant and synthetic stem cell factor molecules include those described in WO1991005795 (titled “Stem cell factor”); US6020469 (titled “Stem cell factor formulations and methods”); US6759215 (titled “Method of preparing human stem cell factor polypeptide”); and US6218148 (titled “DNS encoding stem cell factor”), each of which is incorporated by reference herein in its entirety.

[0094] Additional examples of c-kit agonists also include stem cell factor analogs, e.g., those described in US5885962 (titled “Stem cell factor analog compositions and method”); and Tilayov T. et al. Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies, Molecules. 2020 Oct 21;25(20):4850, each of which incorporated herein in its entirety.

[0095] In embodiments, a c-kit agonist is SCF-encoding or SCF-analog-encoding nucleic acid (e.g., DNA or mRNA), including those described in the references incorporated herein.

[0096] Additional examples of c-kit agonists include those described in Sara Abdalrazzaq M Noraldeen et al., Involving sternness factors to improve CAR T-cell-based cancer immunotherapy, Med Oncol. 2023 Oct 1 ;40(l 1):313; Shoichi Iriguchi et al., A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy, Nat Commun. 2021 Jan 18; 12(l):430; Clinical trial NCT01016795 (Stem Cell Factor (SCF) Priming of Haematopoietic Stem Cell Grafts in Malignant Lymphoma (SCF980266)”, Drug: r-metHuSCF and Filgrastim); Clinical Trial NCT00001398 (Stem Cell Factor Medication for Aplastic Anemia); Clinical Trial NCT00005783 (A Phase Eli Trial of Recombinant-Methionyl Human Stem Cell Factor (SCF) in Adult Patients With Sickling Disorders); Clinical Trials NCT00001398 (Stem Cell Factor Medication for Aplastic Anemia); Clinical Trial NCT02501811 (Combination of Mesenchymal and C-kit+ Cardiac Stem Cells as Regenerative Therapy for Heart Failure (CONCERT -HF)); Tricot G et al., Superior mobilization of peripheral blood progenitor cells (PBPC) with r-metHuSCF (SCF) and r-metHuG-CSF (fdgrastim) in heavily pretreated multiple myeloma (MM) patients, Blood, 88 (1996), p. 388a; E J Shpall et al., A randomized phase 3 study of peripheral blood progenitor cell mobilization with stem cell factor and filgrastim in high-risk breast cancer patients, Blood. 1999 Apr 15;93(8):2491-501; Kurzrock R et al., Trilineage responses seen with stem cell factor (STEMGEN, SCF) and filgrastim (G-CSF) treatment in aplastic anemia (AA) patients (Pts) Br J Haematol. 1998; Laura Breda et al., In vivo hematopoietic stem cell modification by mRNA delivery, Science. 2023 Jul 28;381(6656):436-443; Dorota Wilamowska-Kokoszko et al., Assessment of stem cell factor expression and its c-KIT receptor in patients with vitiligo, Postepy Dermatol Alergol. 2022 Aug;39(4):762-767; Yuquan Xiong et al., c-Kit signaling potentiates CAR T cell efficacy in solid tumors by CD28- and IL-2-independent co-stimulation; Nat Cancer. 2023 Jul;4(7):1001-1015; each of which is incorporated by reference in its entirety.

[0097] The term “lymphodepletion (LD)” or “lymphodepletion regimen” refers to the administration to a subject of one or more agents (e.g., lymphodepletion agents) capable of reducing endogenous lymphocytes in the subject for immunotherapy; e.g., a reduction of one or more lymphocytes (e.g., B cells, T cells, and/or NK cells) by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% relative to a control (e.g., relative to a starting amount in the subject undergoing treatment, relative to a pre-determined threshold, or relative to an untreated subject).

[0098] The LD may be performed by administering a biological LD agent, a chemotherapeutic LD agent, or a combination thereof. The term “biological LD agent” refers to a biological material, such an antibody, antibody fragment, antibody conjugate, or the like, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy. In some cases, such biological lymphodepletion agents can have specificity for antigens present on lymphocytes; e.g., CD52 or CD 19. As used herein, the term “chemotherapeutic LD agents” refers to non-biological materials, such as small molecules, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy. In some examples, the chemotherapeutic lymphodepl eting agent can be lymphodepleting but non-myeloablative. In some examples, the chemotherapeutic lymphodepletion agents may be cyclophosphamide, fludarabine, or a combination thereof.

Antigen recognition moieties

[0099] The immune cells herein may comprise (e.g., stably express) one or more antigen recognition moieties, which recognizes a disease associated antigen. The antigen recognition moiety may be any moiety that recognizes an antigen. Examples of antigen recognition moieties include a TCR, u.p TCR, yo TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide- MHC complex).

[00100] An engineered immune cell may express one, or multiple antigen recognition moieties. In embodiments, an engineered immune cell expresses two or more antigen recognition moieties. In some examples, each antigen recognition moiety recognizes a different epitope of the same antigen. In some examples, each antigen recognition moiety recognizes different epitopes of different antigens.

[00101] Two or more antigen recognition moieties may be expressed in the immune cell from genetically different, substantially different, or substantially identical, TCR polynucleotides stably expressed from the engineered immune cell or from genetically distinct TCR polynucleotides stably incorporated in the engineered immune cell. In the case of genetically distinct TCR(s), TCR(s) recognizing different antigens associated with the same condition may be utilized. In one preferred embodiment, an immune cell is engineered to express different TCRs, from human or mouse origin, from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In another preferred embodiment, an immune cell is engineered to express one TCR and two or more antibodies directed to the same or different peptides from a given antigen complexed with different MHC haplotypes. In some cases, expression of a single TCR by an engineered immune cell facilitates proper TCR pairing. An engineered immune cell that expresses different TCRs can provide a universal allogeneic engineered immune cell. In a second preferred embodiment, an immune cell is engineered to express one or more different antibodies directed to peptide-MHC complexes, each directed to the same or different peptide complexed with the same or different MHC haplotypes. In some cases, an antigen recognition moiety can be an antibody that binds to peptide-MHC complexes. [00102] An immune cell can be engineered to express TCRs from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In some cases, an engineered immune cell is designed to express a single TCR, or a TCR in combination with a CAR to minimize the likelihood of TCR mispairing within the engineered cell. The anitgen recognition moieties expressed from two or more expression cassettes preferably have different polynucleotide sequences, and encode antigen recognition moieties that recognize different epitopes of the same target, e.g., in the context of different HLA haplotypes. An engineered immune cell that expresses such different TCRs or CARs can provide a universal allogeneic engineered immune cell.

[00103] In some cases, an immune cell is engineered to express two or more antigen recognition moieties. The two or more antigen recognition moieties may be expressed from genetically identical, or substantially identical, antigen-specific chimeric (CAR) polynucleotides engineered in the immune cell. Two or more antigen recognition moieties may be expressed from genetically distinct CAR polynucleotides engineered in the immune cell. The genetically distinct CAR(s) may be designed to recognize different antigens associated with the same condition.

[00104] An antigen recognition moiety may be engineered to recognize an antigen with certain avidity. For instance, an antigen recognition moiety encoded by a TCR or CAR construct may recognize an antigen with a dissociation constant of at least at least 10 fM, at least 100 fM, at least 1 picomolar (pM), at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 7 pM, at least 80 pM, at least 90 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nanomolar (nM), at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nm, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1 mM, at least 2 mM, at least 3 pM, at least 4 pM, at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 70 pM, at least 80 pM, at least 90 pM, or at least 100 pM. [00105] In some instances, an antigen recognition moiety may be engineered to recognize an antigen with a dissociation constant of at most 10 fM, at most 100 fM, at most 1 picomolar (pM), at most 10 pM, at most 20 pM, at most 30 pM, at most 40 pM, at most 50 pM, at most 60 pM, at most 7 pM, at most 80 pM, at most 90 pM, at most 100 pM, at most 200 pM, at most 300 pM, at most 400 pM, at most 500 pM, at most 600 pM, at most 700 pM, at most 800 pM, at most 900 pM, at most 1 nanomolar (nM), at most 2 nM, at most 3 nM, at most 4 nM, at most 5 nM, at most 6 nM, at most 7 nM, at most 8 nM, at most 9 nM, at most 10 nM, at most 20 nM, at most 30 nM, at most 40 nM, at most 50 nm, at most 60 nM, at most 70 nM, at most 80 nM, at most 90 nM, at most 100 nM, at most 200 nM, at most 300 nM, at most 400 nM, at most 500 nM, at most 600 nM, at most 700 nM, at most 800 nM, at most 900 nM, at most 1 mM, at most 2 mM, at most 3 pM, at most 4 pM, at most 5 pM, at most 6 pM, at most 7 pM, at most 8 pM, at most 9 pM, at most 10 pM, at most 20 pM, at most 30 pM, at most 40 pM, at most 50 pM, at most 60 pM, at most 70 pM, at most 80 pM, at most 90 pM, or at most 100 pM.

[00106] Examples of suitable tumor antigens include, but are not limited to, CD19, CD20, CD30, CD22, CD37, CD38, CD56, CD33, CD 138, CD123, CD79b, CD70, CD75, CA6, GD2, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2/Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c-Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, LI CAM, GPC2, GPC3, folate binding protein/receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3Ra2, SLTRK6, gplOO, MARTI, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3. MAGEA4), KKLC1, mutated ras, VRaf, p53, MHC class I chain- related molecule A (MICA), or MHC class I chain-related molecule B (MICB), or one or more antigens ofHPV, CMV, or EBV, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[00107] An antigen recognition moiety may recognize an antigen associated with an autoimmune disease. Such antigens include endogenous antigens that stimulate the production of an autoimmune response, such as production of autoantibodies. Antigens associated with autoimmune diseases also include antigens from a normal tissue that is the target of a cell mediated or an antibody-mediated immune response that may result in the development of an autoimmune disease. Examples of antigens associated with autoimmune diseases include aggrecan, alanyl- tRNA syntetase (PL-12), alpha beta crystallin, alpha fodrin (Sptan 1), alpha-actinin, al antichymotrypsin, al antitripsin, al microglobulin, aldolase, aminoacyl-tRNA synthetase, an amyloid, an annexin, an apolipoprotein, aquaporin, bactericidal/permeability-increasing protein (BPI), P-globin precursor BP 1, P-actin, P-lactoglobulin A, P-2-gly coprotein I, p2-microglobulin, a blood group antigen, C reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalase, cathepsin B, a centromere protein, chondroitin sulfate, chromatin, collagen, a complement component, cytochrome C, cytochrome P450 2D6, cytokeratins, decorin, dermatan sulfate, DNA topoisomerase I, elastin, Epstein-Barr nuclear antigen 1 (EBNA1), elastin, entaktin, an extractable nuclear antigen, Factor I, Factor P, Factor B, Factor D, Factor H, Factor X, fibrinogen, fibronectin, formiminotransferase cyclodeaminase (LC-1), gp210 nuclear envelope protein, GP2 (major zymogen granule membrane glycoprotein), a glutenin, glycoprotein gpIIb/IIIa, glial fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), haptoglobin A2, heat shock proteins, hemocyanin, heparin, a histone, histidyl-tRNA synthetase (Jo-1), a hordein, hyaluronidase, immunoglobulins, an integrin, interstitial retinol -binding protein 3, intrinsic factor, Ku (p70/p80), lactate dehydrogenase, laminin, liver cytosol antigen type 1 (LC1), liver/kidney microsomal antigen 1 (LKM1), lysozyme, melanoma differentiation- associated protein 5 (MDAS), Mi-2 (chromodomain helicase DNA binding protein 4), a mitochondrial protein, muscarinic receptors, myelin-associated glycoprotein, myosin, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein, myeloperoxidase (MPO), rheumatoid factor (IgM anti-IgG), neuron-specific enolase, nicotinic acetylcholine receptor A chain, nucleolin, a nucleoporin, nucleosome antigen, PM/Scl lOO, PM/Scl 75, pancreatic 3-cell antigen, pepsinogen, peroxiredoxin 1, phosphoglucose isomerase, phospholipids, phosphatidyl inositol, platelet derived growth factors, polymerase beta (POLB), potassium channel KIR4.1, proliferating cell nuclear antigen (PCNA), proteinase-3, proteolipid protein, proteoglycan, prothrombin, recoverin, rhodopsin, ribonuclease, a ribonucleoprotein, ribosomes, a ribosomal phosphoprotein, RNA, an Sm protein, SplOO nuclear protein, SRP54 (signal recognition particle 54 kDa), a selectin, smooth muscle proteins, sphingomyelin, streptococcal antigens, superoxide dismutase, synovial joint proteins, T1F1 gamma collagen, threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid stimulating hormone receptor, transferrin, triosephosphate isomerase, tubulin, tumor necrosis factor-alpha, topoisomerase, Ul- dnRNP 68/70 kDa, Ul-snRNP A, Ul-snRNP C, U-snRNP B/B', ubiquitin, vascular endothelial growth factor, vimentin, and vitronectin. [00108] In embodiments, an antigen recognition moiety recognizes a disease associated antigen expressed on an immune cell. In some examples, such immune cell is a B cell, including at all phases of development including terminal differentiation into plasma cells such as, e.g. pro B cells, pre B cells, transitional/immature B cells, naive B cells, GC B cells, memory B cells, plasmablasts, and plasma cell. In some examples, such immune cell is a T cell such as a Thl/Thl7 T cell, CD4+ T cell, keratinocyte, and the cells described in Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat Rev Drug Discov. 2021 Mar;20(3): 179-199, which is incorporated by reference in its entirety.

[00109] In embodiments, the antigen recognition moiety binds to one or more of CD 19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In embodiments, the antigen recognition moiety binds to a B cell antigen selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In embodiments, the antigen recognition moiety binds to a plasma cell antigen selected from the group comprising or consisting of CD38, CD 138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB. In embodiments, the antigen recognition moiety binds to a T cell antigen selected from the group comprising or consisting of CD52, CD40L, alpha4 integrin, CXCR6, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L.

[00110] An antigen recognition moiety may recognize a pathogenic antigen. A pathogenic antigen may be a bacterial, viral, or fungal molecule, such as a bacterial, viral, or fungal protein. In embodiments, an antigen presenting cell may internalize pathogenic molecules (e.g., pathogenic proteins, nucleic acids, lipids, or fragments produced by a pathogenic organism such as a bacterium or a virus), for instance with phagocytosis or by receptor-mediated endocytosis, and display a fragment of the antigen bound to an appropriate MHC molecule. For instance, various 9 mer fragments of a pathogenic protein may be displayed by an APC. Engineered, enriched immune cell populations of the disclosure may be designed to recognize various antigens and antigen fragments of a pathogenic bacterium or a virus. [00111] Examples of pathogenic bacteria can be found in the: a) Bordetella genus, such as Bordetella pertussis species; b) Borrelia genus, such Borrelia burgdorferi species; c) Brucelia genus, such as Brucella abortus, Brucella canis, Brucela meliterisis, and/or Brucella suis species; d) Campylobacter genus, such as Campylobacter jejuni species; e) Chlamydia and Chlamydophila genuses, such as Chlamydia pneumonia, Chlamydia trachomatis, and/or Chlamydophila psittaci species; f) Clostridium genus, such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani species; g) Corynebacterium genus, such as Corynebacterium diphtheria species; h) Enterococcus genus, such as Enterococcus faecalis, and/or Enterococcus faecium species; i) Escherichia genus, such as Escherichia coli species; j) Francisella genus, such as Francisella tularensis species; k) Haemophilus genus, such as Haemophilus influenza species; 1) Helicobacter genus, such as Helicobacter pylori species; m) Legionella genus, such as Legionella pneumophila species; n) Leptospira genus, such as Leptospira interrogans species; o) Listeria genus, such as Listeria monocytogenes species; p) Mycobacterium genus, such as Mycobacterium leprae, mycobacterium tuberculosis, and/or mycobacterium ulcerans species; q) Mycoplasma genus, such as Mycoplasma pneumonia species; r) Neisseria genus, such as Neisseria gonorrhoeae and/or Neisseria meningitidia species; s) Pseudomonas genus, such as Pseudomonas aeruginosa species; t) Rickettsia genus, such as Rickettsia rickettsii species; u) Salmonella genus, such as Salmonella typhi and/or Salmonella typhimurium species; v) Shigella genus, such as Shigella sonnei species; w) Staphylococcus genus, such as Staphylococcus aureus, Staphylococcus epidermidis, and/or Staphylococcus saprophyticus spedes; x) Streptpcoccus genus, such as Streptococcus agalactiae, Streptococcus pneumonia, and/or Streptococcus pyogenes species; y) Treponema genus, such as Treponema pallidum species; z) Vibrio genus, such as Vibrio cholera; and/or aa) Yersinia genus, such as Yersinia pestis spedes.

[00112] Examples of pathogenic viruses can be found in the following families of viruses and are illustrated with exemplary spedes: a) Adenoviridae family, such as Adenovirus spedes; b) Herpesviridae family, such as Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus type 8 species; c) Papillomaviridae family, such as Human papillomavirus species; d) Polyomaviridae family, such as BK vims, JC vims species; e) Poxviridae family, such as Smallpox species; f) Hepadnaviridae family, such as Hepatitis B vims species; g) Parvoviridae family, such as Human bocavims, Parvovirus B19 species; h) Astroviridae family, such as Human astrovims spedes; i) Caliciviridae family, such as Norwalk virus species; j) Flaviviridae family, such as Hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae family, such as Rubella virus species; 1) Hepeviridae family, such as Hepatitis E virus species; m) Retroviiidae family, such as Human immunodeficiency virus (HIV) species; n) Orthomyxoviridaw family, such as Influenza virus species; o) Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, and/or Sabia virus species; p) Bunyaviridae family, such as Crimean-Congo hemorrhagic fever virus species; q) Filoviridae family, such as Ebola virus and/or Marburg virus spedes; Paramyxoviridae family, such as Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Hendra virus and/or Nipah virus spedes; r) Rhabdoviridae genus, such as Rabies virus species; s) Reoviridae family, such as Rotavirus, Orbivirus, Coltivirus and/or Banna virus species. In some examples, a virus is unassigned to a viral family, such as Hepatitis D.

Chimeric Antigen Receptor Constructs

[00113] Aspects of the invention include CARs, nucleic acids encoding CARs, and constructs and vectors containing such nucleic acids. The present disclosure further provides an isolated polynucleotide (used exchangeable with “nuclei acid” herein) comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding at least one c- kit agonist. In some cases, the nucleic acid is a, e.g., heterologous, component of an expression cassette. In embodiments, the nucleic acid is a, e.g., heterologous, component of a retroviral vector. In embodiments, the nucleic acid is a, e.g., heterologous, component of an immune cell such as an aP T cell, a d T cell, an NK cell, or a macrophage. In embodiments, the nucleic acid is a, e.g., heterologous, component of an aP T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of a NK cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of a macrophage. In embodiments, the nucleic acid is a, e.g., heterologous, component of a yd T cell. In some embodiments, the nucleic acid is a, e.g., heterologous, component of a y⁺ T cell and/or a 5⁺ T cell.

[00114] A CAR may comprise an antigen binding domain operably linked to another domain of the CAR, for example a transmembrane domain, a costimulatory domain and/or an intracellular signaling domain, as described herein. The antigen binding domains described herein can be combined with any of the transmembrane, costimulatory, and/or intracellular signaling domain(s) described herein, and/or any of the other domains described herein that may be included in a CAR herein. A CAR may also include a hinge domain as described herein. A CAR may also include at least one spacer domain as described herein.

Antigen Binding Domain

[00115] The antigen binding domain can include any domain that binds to a disease associated antigen (e g., the antigens associated with tumors and autoimmune diseases and pathogenic antigens described hereinabove). In embodiments, the antigen binding domain binds to a tumor antigen. For example, the antigen binding domain may bind to CD20, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[00116] The antigen binding domain may comprise a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a bi specific antibody, and any fragment thereof. In embodiments, the antigen binding domain may be an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a F(ab)2, or any combination thereof. In embodiments, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen binding domain may depend upon the type and number of antigens that are present on the surface of a target cell.

CD 20 binding domains

[00117] In embodiments, the binding domain is a CD20 binding domain, such as a CD20 binding domain described in U.S. Patent Appl. No. 2009/0035322 and WO 2020/072536A9, and WO 2023/102264, the contents of each of which are incorporated by reference in the entirety and for all purposes and in particular for the binding domains, antibodies, antibody fragments, complementarity determining regions, polypeptides containing said complementarity determining regions, nucleic acids encoding for said complementarity determining regions, and epitope specificities and assays for determining epitope specificity described therein. Typically, the region encoding the binding domain is 5’ of a linker region (e.g., a region encoding a CD8a hinge domain). In embodiments, the immune cell herein may be a yd T cell expressing anti-CD20 CAR.

[00118] Exemplary CD20 binding domains include but are not limited to binding domains that selectively bind to an epitope within CD20 bound by, or that competes for binding with, 3B9, 3H7, 2B7, 9C11, or 10F2; or 3B9, 3H7, 2B7, or 9C 11; or 3H7, as described in WO 2023/102264. Additionally or alternatively, the CD20 binding domain can comprise the complementary determining regions of an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, 9C11, and 10F2; selected from the group consisting of 3B9, 3H7, 2B7, and 9C11; or comprise the complementary determining regions of an anti-CD20 antibody selected from the group consisting of 3H7. The present disclosure also contemplates CD20 binding domains that compete for binding with a sequence provided herein.

[00119] One can determine whether a CD20 binding domain binds to the same epitope as, or competes for binding with, a reference antibody or binding domain by using known methods. For example, to determine if a test antibody binds to the same epitope as a reference binding domain, the reference binding domain can be allowed to bind to CD20 under saturating conditions. Next, the ability of a test binding domain to bind to CD20 molecule can be assessed. If the test binding domain is able to bind to CD20 following saturation binding with the reference binding domain, it can be concluded that the test binding domain binds to a different epitope than the reference binding domain. On the other hand, if the test binding domain is not able to bind to CD20 following saturation binding with the reference binding domain, then the test binding domain may bind to the same epitope as the epitope bound by the reference binding domain.

[00120J To determine if a binding domain competes for binding with a reference binding domain, the above-described binding methodology is performed in two orientations: In a first orientation, the reference binding domain is allowed to bind to CD20 under saturating conditions followed by assessment of binding of the test binding domain to the CD20 molecule. In a second orientation, the test binding domain is allowed to bind to a CD20 molecule under saturating conditions followed by assessment of binding of the reference binding domain to the CD20 molecule. If, in both orientations, only the first (saturating) binding domain is capable of binding to the CD20 molecule, then it is concluded that the test binding domain and the reference binding domain compete for binding to CD20. As will be appreciated by a person of ordinary skill in the art, a binding domain that competes for binding with a reference binding domain may not necessarily bind to the identical epitope as the reference binding domain, but may sterically block binding of the reference binding domain by binding an overlapping or adjacent epitope.

[00121] Two binding domains bind to the same or an overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one binding domain inhibits binding of the other by at least 50%, for example, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50: 1495-1502). Alternatively, two binding domains have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one binding domain also reduce or eliminate binding of the other. Two binding domains have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one binding domain also reduce or eliminate binding of the other.

[00122J Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test binding domain is in fact due to binding to the same epitope as the reference binding domain or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative binding assay available in the art.

[00123] In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from a CD20 epitope bound by rituximab. In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from a CD20 epitope bound by ocrelizumab. In some embodiments, a CD20 binding domain as herein disclosed binds a same or overlapping CD20 epitope bound by ocrelizumab. In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from a CD20 epitope bound by ofatumumab, or obinutuzumab, or veltuzumab. In embodiments, a CD20 binding domain as herein disclosed bind a same or overlapping CD20 epitope bound by ofatumumab, or obinutuzumab, or veltuzumab. In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from one or more anti-CD20 mAbs described by Luo et al. (2021) Scientific Reports, 11(3255). In embodiments, a CD20 binding domain as herein disclosed binds a same or overlapping CD20 epitope bound by one or more anti-CD20 mAbs described by Luo et al. (2021) Scientific Reports, 11(3255). In embodiments, a CD20 binding domain as herein disclosed binds a CD20 epitope that is different from one or more anti-CD20 mAbs described by Casan et al. (2018) Hum Vaccin Immunother, 14(12): 2820-2841. In embodiments, a CD20 binding domain as herein disclosed binds a same or overlapping CD20 epitope bound by one or more anti-CD20 mAbs described by Casan et al. (2018) Hum Vaccin Immunother,, 14(12): 2820-2841. [00124] The present disclosure provides antibodies and CARs with “substantial identity” or “substantial similarity” to the sequences provided herein in the CDR or framework regions. The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), there is nucleotide sequence identity in %, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[00125] As applied to polypeptides, the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity. In some aspects, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet el al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[00126] Sequence identity and/or similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Sequences also can be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.

[00127] Provided herein are anti-CD20 CARs comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more substitutions (e.g., conservative substitutions). For example, the present disclosure includes anti-CD20 CARs having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR (e.g., HCDR1 , HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, an anti-CD20 CAR can comprise 20, 19, 18, 17, 16, 15, 14 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (e.g., conservative amino acid substitutions) relative to any of the HCVR, LCVR, and/or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, orLCDR3) amino acid sequences disclosed herein.

[00128J In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain complementary determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 and LCDR3 are selected from the group consisting of SEQ ID NO: 5 (AKDPSYGSGSYHSYYGMDV) and 6 (QQRFNWPLT); 7 (VKDFHYGSGSNYGMDV) and 8 (QQSNDWPLT); and 9 (TKDGSYGHFYSGLDV) and 10 (QQRYYWPLT).

[00129] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are selected from the group consisting of SEQ ID NO: 11 (EEQLVESGGDLVQPGRSLRLSCAASGFTFHDYTMH

WVRQAPGKGLEWVSGISWNSGSLGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAL YYCAKDPSYGSGSYHSYYGMDVWGQGTTVTVSS) and 12 (EIVLTQSPATLSLSPGE RATLSCWASQSISRYLVWYQQKCGQAPRLLIYEASKRATGIPVRFSGSGSGTDFTLTISSL ESEDFAVYYCQQRFNWPLTFGGGTKVEIK); 13 (EVQLAESGGDLVQSGRSLRLSCAAS GITFHDYAMHWVRQPPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKKSLYLQM NSLRPDDTALYYCVKDFHYGSGSNYGMDVWGQGTTVTVSP) and 14 (EIVMTQSPATL SMSPGERATLSCRASQSVSRNLAWYQQKVGQAPRLLISGASTRATGIPARFSGSGSGTEF TLTINSLQSEDFAVYYCQQSNDWPLTFGQGTRLEIK); and 15 (EVQLVESGGGLVQPGR SLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSDTIGYADSVKGRFTISRDN AKNSLYLQMNSLRAEDTALYYCTKDGSYGHFYSGLDVWGQGTTVTVSS) and 16 (EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYVASNRATGIPA RFSGSGSGTDFTLTISSLEPDDFAVYYCQQRYYWPLTFGGGTKVEIK).

[00130] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain complementary determining region 3 (HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain comprises an amino acid sequence of the formula XI— X2— X3— X4— X5— X6— X7— X8— X9— X10— XI 1—X12—X13— X14— X15— X16— X17— X18— X19, wherein X1=A, V or T; X2=K; X3=D; X4=P, F or G; X5=S or H; X6=Y; X7=G; X8=S or H; X9=G or F; X10=S or Y; XI 1=Y, N or S; X12=Y, G or H; X13=G, L or S; X14=Y, M or D; X15=Y, D or V; X16 =G, V or absent; X17=M or absent; X18=D or absent; X I 9= V or absent; and the LCDR3 domain comprises an amino acid sequence of the formula XI— X2— X3— X4— X5— X6— X7— X8— X9 , wherein X1=Q; X2=Q; X3=R or S; X4=N, Y or F; X5=N, D, or Y; X6=W; X7=P; X8=L; X9=T.

[00131] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are SEQ ID NO: 17 (EVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGY IGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQ GTTVTVSS) and 18 (EIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPR LLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEI).

[00132] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds the same epitope as, competes with, or is an anti-CD20 binding domain having heavy chain complementarity determining regions (HCDR) and a light chain complementarity determining regions (LCDR), wherein the HCDR and LCDR sequences are the HCVR sequences of SEQ ID NO: 17 and the LCVR sequences of SEQ ID NO: 18 respectively.

[00133] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds the same epitope as, competes with, or is an anti-CD20 binding domain having an HCDR1 that is or comprises SEQ ID NO: 19 (GFTFYDYA), an HCDR2 that is or comprises SEQ ID NO: 20 (ISWNSGYI), and/or an HCDR3 that is or comprises SEQ ID NO: 21 (AKDNSYGKFYYGLDV). In some embodiments, the isolated nucleic acid encodes an anti- CD20 binding domain that binds the same epitope as, competes with, or is an anti-CD20 binding domain having an LCDR1 that is or comprises SEQ ID NO: 22 (QSVSSN), an LCDR2 that is or comprises SEQ ID NO: 23 (GAS), and/or an LCDR3 that is or comprises SEQ ID NO: 24 (QQYNNWPIT). In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds the same epitope as, competes with, or is an anti-CD20 binding domain having an HCDR1 that is or comprises SEQ ID NO: 19, an HCDR2 that is or comprises SEQ ID NO:20, an HCDR3 that is or comprises SEQ ID NO: 21, an LCDR1 that is or comprises SEQ ID NO: 22, an LCDR2 that is or comprises SEQ ID NO: 23, and an LCDR3 that is or comprises SEQ ID NO: 24. In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having an HCDR1 comprising SEQ ID NO: 19, an HCDR2 comprising SEQ ID NO:20, an HCDR3 comprising SEQ ID NO: 21, an LCDR1 comprising SEQ ID NO: 22, an LCDR2 comprising SEQ ID NO: 23, and an LCDR3 comprising SEQ ID NO: 24.

[00134J Exemplary binding domains described herein typically comprise, in order from the amino to carboxy terminus, a heavy chain region followed by a light chain region (VH-VL). Where a certain order of of VH and VL region in the binding domain is explicitly or implicitly described, the present disclosure is also understand to describe the alternate embodiment in which the order of VH and VL regions are reversed, e.g., in an scFV or a CAR comprising an scFv binding domain. Thus, description of a VH-VL order also describes the alternate VL-VH order, e.g., in an scFV or a CAR comprising an scFv binding domain. Moreover, description of a VL- VH order also describes the alternate VH-VL order, e.g., in an scFV or a CAR comprising an scFv binding domain.

[00135J Additional examples of antigen binding domains included those described in PCT/US2019/054132, PCT/US2019/054144, PCT/US2023/034227, PCT/US2023/029047, PCT/US2023/030115, each of which is incorporated by reference in its entirety.

[00136]

Transmembrane domains

[00137] In embodiments, the CAR encoding nucleic acids described herein include an extracellular linker portion that encodes a peptide linker that links the binding domain to a transmembrane domain. Exemplary linker portions include, without limitation, a linker portion that encodes the CD8a hinge domain, e.g., SEQ ID NO: 25 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 26 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY), or

IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 27). Typically, the region encoding the peptide linker (e.g., CD8a hinge domain) is 3’ of the region encoding the binding domain and 5’ of a region encoding a transmembrane domain. [00138] In embodiments, the CAR comprises a transmembrane domain. The transmembrane domain can link an extracellular antigen binding domain, e.g., and hinge, to one or more intracellular signaling components. For example, the transmembrane domain can link an antigen binding domain, e.g., and hinge, to a CD3(^ signaling domain and optionally with one or two costimulation endodomains. Exemplary transmembrane domains include without limitation a CD8a transmembrane domain, e.g., SEQ ID NO: 27 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain (e.g., CD8a transmembrane domain) is 3’ of the region encoding the peptide linker (e.g., CD8a hinge domain) and 5’ of a region encoding one or more cytoplasmic domains.

[00139] For example, a transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, or CD 154, CD 100 (SEMA4D), CD 103, CD 160 (BY55), CD 18, CD 19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[00140] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in a CAR.

Hinge region

[00141] In embodiments, the transmembrane domain further comprises a hinge region. The hinge region of the CAR may be a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, CD8[3 hinge, CD28 hinge, 4-1BB hinge, CD7 hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4). Naturally- occurring hinge domains may be used as wild-type hinge regions or the molecules may be altered.

[00142] In embodiments, a CAR of the present disclosure includes a hinge region that couples the antigen binding domain with the transmembrane domain, which, in turn, couples to one or more intracellular domain(s). The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations. In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[00143] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

[00144] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[00145] For example, hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 28) and (GGGS)n (SEQ ID NO: 29), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 30), GGSGG (SEQ ID NO: 31), GSGSG (SEQ ID NO: 32), GSGGG (SEQ ID NO: 33), GGGSG (SEQ ID NO: 34), GSSSG (SEQ ID NO: 35), and the like.

[00146] In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et ah, Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et ah, Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 36); CPPC (SEQ ID NO: 37); CPEPKSCDTPPPCPR (SEQ ID NO: 38) (see, e g., Glaser et al., J. Biol. Chem. (2005) 280:41494- 41503); ELKTPLGDTTHT (SEQ ID NO: 39); KSCDKTHTCP (SEQ ID NO: 40); KCCVDCP (SEQ ID NO: 41); KYGPPCP (SEQ ID NO: 42); EPKSCDKTHTCPPCP (SEQ ID NO: 43) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 44) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 45) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 46) (human IgG4 hinge); and the like.

[00147] The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 47); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897.

[00148] In certain embodiments, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof. In certain embodiments, the CAR comprises a CD8 alpha hinge sequence comprising the amino acid sequence set forth in SEQ ID NO: 48 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). In certain embodiments, the CAR comprises a hinge and transmembrane domain sequence comprising the amino acid sequence set forth in SEQ ID NO: 49

(TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLV1TLYC).

Cytoplasmic region

[00149] In embodiments, the CAR comprises a cytoplasmic region containing one or more cytoplasmic domains. A nucleic acid sequence region encoding the cytoplasmic region is typically 3 ’ of the region encoding the transmembrane domain in the nucleic acid molecule. The cytoplasmic domains are typically signaling domains that provide an activating signal for immune cell proliferation, cytotoxic activity, and/or pro-inflammatory cytokine expression (e.g., TNF-a or IFNy). An exemplary cytoplasmic domain is a CD3^ signaling domain. In some embodiments, the CD3(^ signaling domain is or comprises SEQ ID NO: 50 (RVKF SRS AD AP A YQQGQNQL YNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the CD3^ signaling domain is or comprises SEQ ID NO: 51

(RVKFSRSADAP A YQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the CD3^ signaling domain is or comprises RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 52). In some embodiments, the CD3(^ signaling domain is or comprises RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE GLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 53).

[00150] In embodiments, the cytoplasmic region contains multiple (e.g., 2, 3, 4, 5, or 6) signaling domains, such as multiple (e.g., 2, 3, 4, 5, or 6) CD3(^ signaling domains, e.g., each independently selected from SEQ ID NO: 24 and 25. In some embodiments, the cytoplasmic region contains multiple (e.g., 2, 3, 4, 5, or 6) non- CD3(^ signaling domains and a CD3(^ signaling domain. In some embodiments, the cytoplasmic region contains a non- CD3^ signaling domain and multiple (e.g., 2, 3, 4, 5, or 6) CD3(^ signaling domains.

[00151] Additional examples of the signaling domains include repeat (e.g., 2-5) DAP 10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRy, FcR0, CD3y, CD38, CD3s, CD79a, CD79b, CD5, CD22, FcsRI, CD66d, and the like. It is within the scope of this disclosure that the endodomain of a disclosed CAR can include a plurality (e.g., 2, 3, 4, or more) of intracellular signaling domains. In a case where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same, or they may be different.

[00152] The cytoplasmic region can contain one or more costimulatory domains. A nucleic acid region encoding one or more costimulatory domains can be 5’ or 3’ of a region encoding a signaling domain. In embodiments, the region encoding one or more costimulatory endodomains is 5’ of the region encoding a signaling domain. In embodiments, a region encoding one or more costimulatory endodomains is 5’ of a signaling domain and an additional region encoding one or more costimulatory endodomains is 3’ of the signaling domain. Exemplary costimulation endodomains include, without limitation, CD28; CD137 (4-1BB); CD278 (ICOS); CD27; CD134 (0X40); Dap 10; Dap 12; DNAm-1; 2B4; a SLAM domain; and TLR2 costimulation endodomains, , as well as costimulatory domains derived from one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8a, CD80, CD1 la, CD1 lb, CD11c, CD1 Id, IL2RP, TL2y, IL7Ra, IL4R, IL7R, IL15R, IL21R, CD 18, CD 19, CD 19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (0X40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CDl la/CD18), FcR, FcyRI, FcyRII, FcyRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, LylO8), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG/Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, IAML, CD150, PSGL1, TSLP, TNFR2, and TRANCE/RANKL, or a portion/combination thereof, and combinations thereof.

[00153] In embodiments, the construct encodes at least one 4-1BB costimulation endodomain, and optionally a second costimulation endodomain selected from a 4- IBB, 2B4, ICOS, CD28, and CD27 costimulation endodomain. In some embodiments, the construct encodes at least two 4- 1BB costimulation endodomains, or two 4-1BB costimulation endodomains in combination with one, two, three, or four, or more, costimulation endodomains selected from a 4- IBB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB costimulation endodomain comprises SEQ ID NO: 26 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

[00154] In some embodiments, the construct encodes one CD27 costimulation endodomain, and optionally a second costimulation endodomain selected from a 4-1BB, ICOS, CD28, and CD27 costimulation endodomain. In some embodiments, the construct encodes a CD27 costimulation endodomain, and a 4-1BB costimulation endodomain. In some embodiments, the construct encodes two CD27 costimulation endodomains. In some embodiments, the CD27 costimulation endodomain comprises SEQ ID NO: 54

(QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[00155] In embodiments, an isolated nucleic acid encodes a signal peptide (also referred to as secretion signal) operably linked to facilitate directing of the one or more additional polypeptides to the secretory pathway. Such one or more additional polypeptides can be those that reside inside certain organelles, are secreted from the host cell, or are inserted into cellular membranes. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 55 (MSVPTQVLGLLLLWLTDARC) In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 56 (MALPVTALLLPLALLLHAARP). In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 57 (MLLLVTSLLLCELPHPAFLLIP). In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 58 (MGRGLLRGLWPLHIVLWTRIAS). In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 59 (MLLPWATSAPGLAWGPLVLGLFGLLAASQP). In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 60 (MGAGATGRAMDGPRLLLLLLLGVSLGGA). In embodiments, the signal peptide comprises or consists of the amino acid sequence MKKTQTWTLTCIYLQLLLFNPLVKT (SEQ ID NO: 61). In embodiments, the construct encodes a signal peptide, e.g., SEQ ID NO: 62 (MALPVTALLLPLALLLHAARP) operably linked to facilitate secretion of a C-terminal polypeptide, such as a cytokine that supports the activation, cytotoxicity, and/or persistence of a T cell (e.g., CAR-T cell). In embodiments, the construct encodes a secretion signal, e.g., SEQ ID NO: 28 operably linked to facilitate secretion of a common gamma chain cytokine such as IL- 15 or an active fragment thereof, e.g., SEQ ID NO: 63 (NWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSS NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Exemplary common gamma chain cytokines include IL-2 and IL-15. In some embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In some embodiments, the common gamma chain cytokine is IL-15. IL-15 sequences, including codon optimized nucleic acid sequences encoding sIL15, are disclosed herein and in WO 2007/037780.

[00156] In embodiments, the construct encodes one or more multi -ci stronic linker regions, e.g., between a signaling domain and/or costimulation endodomain and a secretion signal operably linked to facilitate secretion of a cytokine. In embodiments, the multi-ci stronic region encodes a cleavage sequence and/or an internal ribosomal entry site (IRES). A multi -ci stronic linker region is a region of polypeptide sequence or RNA sequence that facilitates the production of multiple discrete polypeptides from a single transcription product. In embodiments, the multi -ci stronic linker region encodes a cleavage sequence. Suitable cleavage sequences include self-cleavage sequences such as a P2A, F2A, E2A, or T2A cleavage sequence and/or sequences that are cleaved by an endogenous protease, such as furin.

[00157] In embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a furin cleavage sequence. In embodiments, the cleavage sequences are a P2A and a furin cleavage sequence. In embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 63 (SGSGATNFSLLKQAGDVEENPGP). In embodiments, the cleavage sequence is a furin cleavage sequence of SEQ ID NO: 64 (RAK.R). In embodiments, the cleavage sequence is a P2A+furin cleavage sequence of SEQ ID NO: 65 (RAKRSGSGATNFSLLKQAG DVEENPGP).

[00158] In embodiments, the cleavage sequence is or comprises a P2A cleavage sequence of SEQ ID NO: 66 (ATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is or comprises an F2A cleavage sequence of SEQ ID NO: 67 (VKQTLNNFDLLKLAGDVESNPGP). In embodiments, the cleavage sequence is or comprises an E2A cleavage sequence of SEQ ID NO: 68 (QCTNYALLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises an T2A cleavage sequence of SEQ ID NO: 69 (EGRSLLTCGDVEENPGP). In certain aspects, multiple self-cleavage sequences can be encoded carboxy terminal to a signaling and/or costimulatory domain and amino-terminal to an encoded secreted cytokine (e.g., common gamma chain cytokine such as IL-15), preferably wherein the multiple self cleavage sequences are independently selected from the group consisting of a P2A cleavage sequence, a T2A cleavage sequence, an E2A cleavage sequence, and an F2A cleavage sequence. In certain aspects, one or more self-cleavage sequences and one or more sequences cleaved by an endogenous protease are encoded in a construct described herein. In certain embodiments, an endogenous protease recognition site is encoded amino terminal to a self cleavage sequence.

[00159] In embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by SEQ ID NO: 70 (CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGT TATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGT CTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCT GTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTC TGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCG GCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCAC GTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAAC AAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCC TCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCC GAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATA).

[00160] Another exemplary internal ribosome entry site is encoded by SEQ ID NO: 71 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTC CAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGC GAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCC TCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATG TGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAA CTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAA

CACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTT TAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATT ACTGACCAC).

[00161] Further suitable internal ribosome entry sites include, but are not limited to, those described in Nucleic Acids Res. 2010 Jan;38(Database issue):D131-6. doi: 10.1093/nar/gkp981. Epub 2009 Nov 16, those described at iresite.org, those described in WO 2018/215787, the sequence described in GenBank accession No. KP019382.1, and the IRES element disclosed in GenBank accession No. LT727339.1.

[00162] Additional multi-cistronic linker regions, including cleavage, self-cleavage, and IRES elements, are disclosed in US 2018/0360992 and U.S. 8,865,467.

[00163] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 72 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQ KPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFG QGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMH WVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR), a 3H7-CD8-BBz polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, and a CD3(^ signaling domain.

[00164] In embodiments, the isolated nucleic acid encoding a 3H7-CD8-BBz polypeptide comprises the sequence of SEQ ID NO: 73

(ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAG AACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCT

TCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCAC TGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTCACCAT CAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTG GCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAGGTGGATCTG GAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACTGGTGGAGTCTGGGGGAGG CTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT TATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGT CTCAGGTATTAGTTGGAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGGCCG ATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCT GAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAACCA CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGG GCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT CCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA

GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA).

[00165] In embodiments, the isolated nucleic acid comprises a codon optimized sequence encoding a CD8a hinge region. Exemplary codon optimized CD8a hinge region nucleic acid sequences include, without limitation, SEQ ID NO: 74

(ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCA CAGCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCA TACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8a hinge region is encoded by the following sequence SEQ ID NO: 75

(ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGA GGGGGCTGGACTTCGCCTGTGAT).

[00166] In embodiments, the isolated nucleic acid encodes a 3B9 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 74. In some embodiments, the isolated nucleic acid encodes a 2B7 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 74. In some embodiments, the isolated nucleic acid encodes a 9C11 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 74. In some embodiments, the isolated nucleic acid encodes a 3H7 binding domain and comprises the following sequence encoding a CD8a hinge domain SEQ ID NO: 75.

[00167] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 76 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQ KPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFG QGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMH WVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLL HAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), an anti-CD20-CAR polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, a CD3^ signaling domain, a P2A cleavage domain (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 77), a secretion signal, and a sIL15 domain.

[00168] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 78 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQ KPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFG QGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMH WVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMRISKPHLRSISIQCYL CLLLNSHFLTE AGIHVFILGCF S AGLPKTE ANWVNVISDLKKIEDLIQ SMHID ATLYTESD VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEEL EEKNIKEFLQSFVHIVQMFINTS*), an anti-CD20-CAR polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, a CD3^ signaling domain, a P2A cleavage domain of SEQ ID NO: 77, a secretion signal of SEQ ID NO: 79 (MRISKPHLRSISIQCYLCLLLNSHFLTEAG IHVFILGCFSAGLPKTEA), and a sIL15 domain.

[00169] In embodiments, the isolated nucleic acid encoding an anti-CD20 CAR + sIL15 polypeptide comprises the sequence of SEQ ID NO: 80

(ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAG AACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCT TCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCAC TGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTCACCAT CAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTG GCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAGGTGGATCTG GAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACTGGTGGAGTCTGGGGGAGG CTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT TATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGT

CTCAGGTATTAGTTGGAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGGCCG

ATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCT GAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAACCA

CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGG GCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT

CCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAG

TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT

GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGTAGC

GGGGCTACGAACTTCTCCCTTCTTAAACAAGCGGGAGACGTGGAAGAAAATCCCGG

ACCTATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC GCCAGGCCGAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTAT TCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGC

AAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCC

GGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAG TTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGG AGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCA

TCAACACTTCTTGA).

[00170] In embodiments, the isolated nucleic acid encoding an anti-CD20 CAR + sIL15 polypeptide comprises the sequence of SEQ ID NO: 81 (ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAG

AACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCT

TCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCAC

TGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTCACCAT

CAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTG

GCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAGGTGGATCTG

GAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACTGGTGGAGTCTGGGGGAGG

CTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT

TATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGT

CTCAGGTATTAGTTGGAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGGCCG

ATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCT

GAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT

TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAACCA

CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG

TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGG

GCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT

CCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCT

GTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG

GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAG

TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA

CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC

GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT

GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA

AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA

GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGTAGC

GGGGCTACGAACTTCTCCCTTCTTAAACAAGCGGGAGACGTGGAAGAAAATCCCGG

ACCTATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTG

TTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGC

TGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGAT

TTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACG

GAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAG TTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAAT CTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGA TGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTT TGTACATATTGTCCAAATGTTCATCAACACTTCTTGA).

[00171] In embodiments, the isolated nucleic acid encodes SEQ ID NO: 82 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQ KPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFG QGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMH WVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAL YYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR*), an anti-CD20-CAR polypeptide comprising the following domains in order: a 3H7 binding domain, a CD8a hinge and transmembrane domain, a 4-1BB costimulation endodomain, and a CD3(^ signaling domain; and, via an internal ribosome entry site (e.g., encoded by SEQ ID NO: 37) 3’of the region encoding SEQ ID NO: 58, the isolated nucleic acid further encodes SEQ ID NO: 83

(MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS*), a secretion signal of SEQ ID NO: 62 and a sIL15 domain.

[00172] In embodiments, the isolated nucleic acid encoding an anti-CD20 CAR + sIL15 polypeptide comprises the sequence of SEQ ID NO: 84

(ATGTCCGTGCCTACCCAGGTGCTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAG ATGCGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAG AACCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCT TCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCAC TGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCATTCTCACCAT CAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTG GCCGATCACCTTCGGCCAAGGGACACGGCTGGAGATTAAAGGTGGAGGTGGATCTG GAGGAGGAGGATCCGGTGGAGGAGGTGAAGTGCAACTGGTGGAGTCTGGGGGAGG CTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT

TATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGT

CTCAGGTATTAGTTGGAATAGTGGTTACATAGGCTATGCGGACTCTGTGAAGGGCCG

ATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCT

GAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAACAGCTATGGAAAGT

TCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAACCA

CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG

TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGG

GCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT

CCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCT

GTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG

GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAG

TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA

CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC

GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCT

GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA

AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA

GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAGAGT

ACTGCGGCCGCTACGTAAATTCCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGC

CGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATAT

TGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCA

TTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGA

AGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTT

GCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGT

GTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGAT

AGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGG

ATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCT

TTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGAC

GTGGTTTTCCTTTGAAAAACACGATGATATTAATTAAGCCACCGCCATGGCCTTACC

AGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGAACTG

GGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATAT TGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAAT GAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTAT TCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGG GAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTA AAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA).

[00173] In embodiments, the isolated nucleic acid is a linear nucleic acid. In embodiments, the isolated nucleic acid is a circular nucleic acid. In embodiments, the isolated nucleic acid is a vector, such as a plasmid vector, an adenoviral vector, an adeno-associated viral vector, a viral vector, a retroviral vector, or a lentiviral vector. In embodiments, the isolated nucleic acid, or an, e.g., contiguous, portion thereof containing the binding domain transmembrane domain and one or more signaling and/or costimulation endodomains is integrated into the genome of a host cell, such as a host immune cell. In an exemplary embodiment, the isolated nucleic acid is retroviral vector.

Immune cells

[00174] In embodiments, the immune cells have in vitro or in vivo cytotoxic activity against a cell (e.g., a tumor cell such as a hematological tumor cell) that exhibits cell surface expression of a desired disease associated antigen. In some cases, the cytotoxic activity is innate activity. In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds a disease associated antigen expressed on the surface of a cell. In some cases, the immune cells exhibit killing activity of a cell expressing a disease associated antigen greater than an innate level of in vitro and/or in vivo killing activity in a control immune cell. In some cases, the control immune cell does not comprise a CAR construct. In some cases, the control immune cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, a signaling domain described herein, and/or a costimulation endodomain described herein.

[00175] In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds disease-associated antigen or an epitope within the disease-associated antigen. In some cases, the immune cells functionally express a disease-associated antigen specific CAR encoded by an isolated nucleic acid described herein. [00176] In embodiments, immune cells described herein can exhibit HLA-restricted (e.g., HLA class I restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all of the cytotoxic activity is not HLA-restricted (e.g., HLA class I restricted). HLA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against an HLA (e.g., HLA class I) (null) tumor cell line versus in vitro cytotoxicity against an HLA+ (e.g., HLA class I⁺) tumor cell line. In embodiments, the HLA-restricted cytotoxic activity is at least in part, significantly (>25%), or entirely, provided by the use of a T cell Receptor-like binding domain. T cell receptor like binding domains are binding domains that specifically recognize the antigen when presented on the surface of a cell in complex with an MHC molecule. T cell Receptor-like binding domains are further described, e.g., in WO 2016/199141.

[00177] In embodiments, immune cells described herein can exhibit robust and/or persistent tumor cell killing activity. In some cases, the tumor cell killing activity can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a tumor cell. In some cases, the tumor cell killing activity of a immune cell described herein, or a progeny thereof, can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a tumor cell, or from administration of the immune cell described herein. This persistent tumor cell killing activity can be exhibited in vitro, in vivo, or both in vitro and in vivo.

[00178] In embodiments, the immune cells proliferate in response to contact with cells that exhibit cell surface expression, or overexpression, of the disease associated antigen. The cells that exhibit cell surface expression of the disease associated antigen can be normal hematological cells, such as normal B cells. The cells that exhibit cell surface expression, or overexpression, of the disease associated antigen can be tumor cells. In some cases, the proliferation is an innate activity. In some cases, the proliferation is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds the disease associated antigen expressed on the surface of the cell or tumor cell. In some cases, the immune cells exhibit a greater level of in vitro and/or in vivo proliferation as compared to a control immune cell. In some cases, the control immune cell does not comprise a CAR construct. In some cases, the control immune cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, a signaling domain described herein, and/or a costimulation endodomain described herein. [00179] In some cases, the proliferation is at least in part, significantly (> about 20 or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds a disease associated antigen or an epitope within a disease associated antigen . In some cases, immune cells exhibiting proliferation in response to contact with a cell or tumor cell that exhibits cell surface expression of a disease associated antigen functionally express a disease associated antigen specific CAR encoded by an isolated nucleic acid described herein.

[00180J In embodiments, immune cells can exhibit robust and/or persistent proliferation in a host organism that comprises the disease cell or tumor cell that exhibits cell surface expression, or overexpression, of the disease associated antigen. In some cases, the proliferation can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a tumor cell or from a date of administration of the immune cell to the host organism. In some cases, the proliferation of a immune cell described herein, or a progeny thereof, in the host organism that comprises the cell or tumor cell that exhibits cell surface expression, or overexpression, of the disease associated antigen can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a cell or tumor cell or from the date of first administration of the immune cell to the host organism. In some cases, the proliferation in the host organism is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds a disease associated antigen or an epitope within the disease associated antigen. In some cases, immune cells exhibiting proliferation in the host organism comprising a cell or tumor cell that exhibits cell surface expression of disease associated antigen functionally express a disease associated antigen specific CAR encoded by an isolated nucleic acid described herein.

[00181] In embodiments, the immune cells described herein express, or persistently express, pro-inflammatory cytokines such as, but not limited to, tumor necrosis factor alpha or interferon gamma after contact with the cell or tumor cell. In some embodiments, the immune cells described herein, or progeny thereof, express, or persistently express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with the cell or tumor cell, e.g., in a host organism comprising the cell or tumor cell.

[00182] In embodiments, the immune cell, or a pharmaceutical composition containing the immune cell, exhibits essentially no, or no graft versus host response when introduced into an allogeneic host. Tn embodiments, the immune cell, or a pharmaceutical composition containing the immune cell, exhibits a clinically acceptable level of graft versus host response when introduced into an allogeneic host. In embodiments, a clinically acceptable level is an amount of graft versus host response that does not require cessation of a immune cell treatment to achieve a therapeutically effective treatment. In embodiments, a clinically acceptable level of graft versus host response (GvHD) is an acute response that is less severe than Grade C according to an applicable IBMTR grading scale. The severity of acute graft versus host response is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined to produce an overall grade, which has prognostic significance. Grade 1(A) GvHD is characterized as mild disease, grade 11(B) GvHD as moderate, grade III(C) as severe, and grade IV(D) life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings etal., Br J Haematol 1997; 97:855):

•Grade A - Stage 1 skin involvement alone (maculopapular rash over <25 percent of the body) with no liver or gastrointestinal involvement

•Grade B - Stage 2 skin involvement; Stage 1 to 2 gut or liver involvement

•Grade C - Stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg/dL; diarrhea 1500 to 2000 mL/day)

•Grade D - Stage 4 involvement of any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg/dL; diarrhea >2000 mL/day OR pain OR ileus).

See also, Schoemans et al., Bone Marrow Transplantation volume 53, pagesl401-1415 (2018), e.g., at Tables 1 and 2, which discloses criteria for assessing and grading acute GvHD.

[00183] In embodiments, the y3 T cell, or a pharmaceutical composition containing the y8 T cell, exhibits reduced or substantially reduced graft versus host response when introduced into an allogeneic host as compared to a graft versus host response exhibited by control 0 T cells, or a control pharmaceutical composition comprising the control a0 T cells, administered to an allogeneic host. In some cases, the control aP T cell is an allogeneic non-engineered control aP T cell. In some cases, the control aP T cell does not comprise a CAR or does not comprise the same CAR as a reference y8 T cell. [00184] The y8 T cells described herein can be 81, 82, 83, or 84 y8 T cells, or combinations thereof. In some cases, the y8 T cells are mostly (>50%), substantially (>90%), essentially all, or entirely 82’ yd T cells. In some cases, the y8 T cells are mostly (>50%), substantially (>90%), essentially all, or entirely 81 yd T cells.

[00185] The immune cells can be obtained from an allogeneic or an autologous donor. The immune cells can be, partially or entirely purified, or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in WO 2017/197347. The expansion may be performed before or after, or before and after, a CAR construct is introduced into the immune cell(s).

[00186] The immune cells described herein can be stored, e.g., cryopreserved, for use in adoptive cell transfer.

[00187] In another aspect, the present disclosure provides immune cells expressing (e.g., stably expressing) at least one c-kit agonist (e.g., a recombinant or synthetic stem cell factor described herein). In embodiments, the immune cells further express (e.g., stably express) one or more antigen recognition moieties, such as CAR, and at least one c-kit agonist (e.g., a recombinant or synthetic stem cell factor described herein).

[00188] Engineered immune cells may be generated with various methods known in the art, including those described in WO2021113558, which is incorporated by reference in its entirety.

[00189] In embodiments, the engineered immune cells may further comprise one or more modifications, e.g., one or more of disruption of CBL-B gene, disruption of REGNASE-1 gene, disruption of roquin gene, disruption of TGF-0 receptor type 2 (TGF0R2) gene, expression of a dominant negative TGF-P receptor type 2 (dnTGFpR2), expression of a membrane-bound IL-12, disruption of CISH gene, expression of a cytokine switch receptor, disruption of ICAM-1 gene, disruption of CD58 gene, disruption of Fas gene, expression of a dominant-negative Fas receptor (dnFas), expression of a chimeric antigen receptor (CAR) binding to CD70, disruption of RASA2 gene, disruption of MED12 gene, expression of a heterologous cytokine receptor (e.g., dnTGFpR2), or any combination thereof. Further examples of such modifications include those described in WO2021113558, W02024073111, U.S. provisional application 63/627,746, each of which is incorporated by reference herein in its entirety. Biomarkers, Methods of Detection, and Use Thereof

[00190] Biomarkers are biological indicators of disease or therapeutic effects that can be measured in vivo by biomedical/molecular imaging, as well as other in vitro or laboratory methodologies. As disclosed herein, one or more biomarkers can advantageously be relied upon to inform cell activation, treatment efficacy and/or follow-on treatment regimens. With respect to administration of immune cells to a subject in need thereof as herein described, one or more biomarkers can be relied upon as indicator(s) of effectiveness, potential for effectiveness, or lack thereof in terms of, e.g., promoting an anti -turn or effect in the subject. In embodiments, one or more biomarkers can be relied upon to determine, for example, whether to administer one or more additional dosing regimens, and if so, whether to adjust a dosage level (e.g., increase, decrease, or maintain the same dosage of immune cells), to include one or more additional or alternative therapies, to adjust a previously planned dosing schedule, to administer immune cells derived from a same or a different donor, or whether to halt/postpone treatment or discontinue treatment altogether, and the like.

[00191] In embodiments, activation and/or expansion of an administered immune cells can be monitored by way of flow cytometry detection of CAR+ immune cells and/or via quantitative polymerase chain reaction (qPCR) detection of a transgene in the immune cells (e.g., a CAR such as anti-CD20 CAR). Preferably, such methodology(s) are conducted at a number of time points following administration of the immune cells, for example daily, every other day, every 3 days, every 4 days, etc., up to e.g., 14 days, 28 days, 2 months, 3 months, or more, as it is well known that the presence and the status of CAR-T cells in peripheral blood can vary over time (Shah et al. (2020) Nat Med., 26: 1569-75). In the art, PCR results have been reported to correlate with CAR surface expression as monitored by flow cytometry, however flow cytometry has advantages in that it allows for identification and characterization of CAR expressing immune cell subpopulations and of a patient’s immune cells in a fast way and at a single cell level. In addition, flow cytometry detects the CAR at the proteomic level and thus can provide information regarding CAR cell functionality. The experimental manner in how to rely on one or more of flow cytometry, qPCR and/or other detection methodologies to monitor activation and/or expansion of the immune cells is readily determined by the skilled artisan, as shown and described, e.g., as described e.g., by Hu and Huang (2020) Front. Immunol., 11(1770). [00192] In embodiments, the immune cells administered to a subject can induce release of one or more cytokines. In embodiments, the one or more cytokines are secreted from the disease antigen-binding CAR immune cells. In additional or alternative embodiments, the one or more cytokines are secreted from cells other than the immune cells including, e.g., T cells, NK cells, dendritic cells, and macrophages. In embodiments, induction of one or more inflammatory cytokines mitigates immunosuppression caused by a tumor microenvironment, and can in turn improve clinical response to the immune cell therapy.

[00193] In embodiments, one or more cytokines are biomarkers of cell activation, cell expansion and/or therapeutic efficacy of an engineered immune cell therapy as herein disclosed. Relevant cytokines can include but are not limited to SCF, INFy, GM-CSF, IL-2, IL-7, IL-15, TNFa, IL-ip, IL-6, IL-8, IL-10, MIPla, MIPip, CRP, ferritin, monocyte chemotactic protein-1 (MCP-1), CXCL9, CXCL10, CXCL11, CCL5, IL-5, IL-IRA, IL-18, soluble MICA, IL-10, IL-4, IL-13, IL-17, CCL2, CXCL12, CCL17, and CCL22. In embodiments, the one or more cytokines comprise or consist of IL-2 and IL-8.

[00194] In embodiments, the one or more cytokines comprise or consist of SCF. The level of SCF (e.g., the level of endogenous SCF in a subject) may be detected using an affinity-based assay such as ELISA with an anti-SCF antibody, e.g., AB 1498 (by Sigma-Aldrich) and abl76109 (by Abeam). An exemplary assay for testing the SCF level in human patient sample is demonstrated in Example 1 .

[00195] In embodiments, induction of cytokines for use as biomarkers of therapeutic efficacy occurs within a timeframe between one day or less and 28 days following administration of CAR immune cells. In embodiments, said timeframe is between one day or less and 21 days, or 18 days, or 14 days, or 10 days following administration of immune cells. In embodiments, a cytokine biomarker comprises IL-8 and induction of IL-8 occurs between one day or less and 28 days, for example between one day or less and 21 days, for example between one day or less and 14 days following administration of immune cells. In some additional or alternative embodiments, a cytokine biomarker comprises IL -2 and induction of IL-2 occurs between one day or less and 28 days, for example between one day or less and 21 days, for example between one day or less and 14 days following administration of immune cells. In some additional or alternative embodiments, induction of cytokines for use as biomarkers occurs within a timeframe between one day or less following LD and 28 days following administration of immune cells. In embodiments, such a timeframe is between one day or less following LD and 21 days, or 18 days, or 14 days, or 10 days following administration of immune cells. In some embodiments, a cytokine biomarker comprises IL-8 and induction of IL-8 occurs between one day or less following LD and 28 days following administration of immune cells, for example between one day or less following LD and 21 days following administration of immune cells, for example between one day or less following LD and 14 days following administration of immune cells. In some additional or alternative embodiments, a cytokine biomarker comprises IL-2 and induction of IL -2 occurs between one day or less following LD and 28 days following administration of immune cells, for example between one day or less following LD and 21 days following administration of immune cells, for example between one day or less following LD and 14 days following administration of immune cells.

[00196] Measurement of serum levels of single cytokines are commonly performed using enzyme-linked immunosorbent assay (ELISA) and/or chemiluminesent assays, and multiplex bead-based assays can be used to determine serum levels of a plurality of cytokines in a single test (Knight et al. (2020) Archives of Pathology & Laboratory Medicine, 144(10)). In embodiments, serum levels of one or more cytokines are measured before administration of immune cells, e.g., before lymphodepletion and/or following/during lymphodepletion but prior to immune cells. In additional or alternative embodiments, serum levels of one or more cytokines are measured following administration of immune cells. In embodiments, serum levels of one or more cytokines for use as biomarkers are measured before administration of immune cells (e.g., between 1 and 7 days prior to administration), and/or are measured one or more times following administration of immune cells up to about 28 days. In embodiments, a plurality of measurements of serum levels of one or more cytokines encompassing a timeframe before and/or following administration of immune cells provides a time course of induction of the one or more cytokines. Such a time course can be used to establish peak serum levels of said one or more cytokines and/or the time course can be used to establish approximate total levels of cytokine induction during the time course. It is within the scope of this disclosure that peak levels of one or more cytokines are used as a biomarker metric. Additionally or alternatively, it is within the scope of this disclosure that total levels of release of one or more cytokines are used as a biomarker metric.

[00197] In embodiments, a presence of a biomarker (e.g., a cytokine) is confirmed in response to said biomarker being measured above some predetermined threshold, for example following administration of immune cells. In embodiments, a biomarker is IL-8, and the presence of the biomarker is confirmed responsive to serum levels of IL-8 reaching or exceeding about 100 pg/mL, or about 125 pg/mL, or about 150 pg/mL, or about 175 pg/mL, or about 200 pg/mL within a predetermined timeframe (e.g., 21 days or less) following administration of immune cells. In additional or alternative embodiments, a biomarker is IL-2 and the presence of the biomarker is confirmed responsive to serum levels of IL-2 reaching or exceeding about 75 pg/mL, or about 80 pg/mL, or about 85 pg/mL within a predetermined timeframe (e.g., 21 days or less) following administration of immune cells.

[00198] In embodiments, the biomarker is SCF, and the presence of the biomarker is confirmed responsive to serum levels of SCF reaching or exceeding about 100 pg/mL, about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 300 pg/mL, about 350 pg/mL, about 400 pg/mL, about 450 pg/mL, about 500 pg/mL, about 550 pg/mL, about 600 pg/mL, about 650 pg/mL, about 700 pg/mL, about 750 pg/mL, about 800 pg/mL, about 850 pg/mL, about 900 pg/mL, about 950 pg/mL, or about 1000 pg/mL within a predetermined timeframe (e.g., 21 days or less) following administration of immune cells.

[00199J In embodiments, confirmation of the presence of one or more cytokine biomarkers as herein described is used to inform follow-on treatments. For example, in response to cytokine biomarker confirmation following administration of a first dose of immune cells, a second dose may be optional, or a dosage of the corresponding second dose may be adjusted accordingly (e.g., maintained the same as the first dose or decreased). In additional or alternative embodiments, a lack of cytokine biomarker confirmation following administration of a first dose of immune cells may indicate a need for a second dose (e.g., with or without another lymphodepletion step), that a cell dosage amount be increased for said second dose, and/or that the second dose comprise immune cells derived from a different donor as compared to the first dose. Similar logic additionally or alternatively applies to an indication of presence or absence of biomarkers indicative of in vivo activation and/or expansion of administered immune cells as described above.

[00200] In additional or alternative embodiments, indicators of minimal residual disease (MRD) can serve as a biomarker for informing follow-on treatment regimens. Discussed herein, MRD refers to some amount of cancer cells remaining in the body of a subject following a course of treatment (e.g., administration of one or more doses of immune cells). In embodiments, MRD analysis is conducted some predetermined time duration following a last administration of immune cells. In embodiments, said time duration is at least 20 days, for example at least 25 days, for example at least 28 days, for example at least 30 days following a last administration of immune cells. In embodiments, an MRD positive test is indicative of disease continuing to be detected following treatment, whereas an MRD negative test is indicative of disease not being detected following treatment. In embodiments, an MRD positive test can indicate a need for an additional treatment regimen, for example a second course of treatment comprising administration of another round of immune cells, preferably at a higher cell dosage, preferably including an additional lymphodepletion step. In some embodiments, a first course of treatment comprising administration of immune cells may follow a standard course of lymphodepletion (e.g., comprising or consisting of fludarabine at 30 mg/m²/day plus cyclosporamide at 500 mg/m²/day for three days), and responsive to an MRD positive test, a second course of treatment may comprise an enhanced lymphodepletion step (e.g., comprising or consisting of fludarabine at 30 mg/m²/day for four days plus cyclosporamide at 1000 mg/m²/day for three days).

[00201] In embodiments, MRD analysis is conducted via one or more of multiparametric flow cytometry and immunosequencing as known in the art (see, e.g., Wood et al. (2018) Blood, 131(12): 1350-1359).

Methods of monitoring and prognosing adoptive cell therapy and treating diseases

[00202J In another aspect, the present disclosure provides methods for monitoring and/or prognosing an adoptive cell therapy in a subject. In embodiments, the methods comprise determining a level (e.g., detection of the presence, absence, and/or amount) of endogenous stem cell factor (SCF) in a biological sample from the subject. The determined level of endogenous SCF may be is informative of i) the in vivo activation of the adoptive immune cells in the subject; ii) the in vivo expansion of the adoptive immune cells in the subject; iii) the anti-tumor effect of the adoptive cell therapy; iv) the anti-autoimmune effect of the adoptive cell therapy, v) the antipathogen effect of the adoptive cell therapy; vi) the need for a treatment modification; vii) the need for a treatment extension; or viii) any combination of i)-vii).

[00203] In embodiments, the treatment modification comprises adjusting a dosage level (e.g., increasing or decreasing the dosage of the adoptive cell therapy). Additionally or alternatively, the treatment modification may comprise adjusting a previously planned dosing schedule. [00204] Additionally or alternatively, the treatment modification may comprise administering one or more adjunctive therapies. In embodiments, the one or more adjunctive therapies comprises administering at least one c-kit agonist as described herein. For example, the administration of the at least one c-kit agonist may be in conjunction with the administration of the adoptive cell therapy. In some examples, the administration of the at least one c-kit agonist may be simultaneous with the administration of the adoptive cell therapy. In some examples, the administration of the at least one c-kit agonist may be after the administration of the adoptive cell therapy.

[00205] Additionally or alternatively, the treatment modification may comprise administering one or more alternative therapies. For example, the one or more alternative therapies may be administration of an adoptive cell therapy different from the adoptive cell therapy of whose activation and/or expansion the level of endogenous SCF is informative.

[00206] In embodiments, the treatment extension comprises increasing the number of doses and/or the frequency of doses of the adoptive cell therapy given to the subject. For example, the treatment extension may comprise administering a second dose of the adoptive cell therapy to the subject at least 1 day, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, at least 1 month, at least 2 months, or at least 3 months after the first dose. The second dose may be administered with an additional LD regimen. Alternatively, the second dose may be administered without an additional LD regimen.

[00207] In embodiments, the biological sample is a bodily liquid sample. In one example, the biological sample is blood. In another example, the biological sample is plasma. In another example, the biological sample is serum.

[00208] The biological sample may be obtained from the subject at a suitable time point to provide informative SCF level. In embodiments, the biological sample is obtained from the subject after administering a lymphodepletion (LD) regimen to the subject and prior to administering a dose of the adoptive cell therapy to the subject. In some examples, the biological sample may be obtained at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, or at least 168 hours after the administration of the LD regimen. In some examples, the biological sample may be obtained from 6 hours to 24 hours, from 12 hours to 48 hours, from 24 hours to 72 hours, from 48 hours to 96 hours, from 72 hours to 120 hours, from 96 hours to 144 hours, or from 120 hours to 168 hours after the administration of the LD regimen.

[00209] In some examples, the biological sample may be obtained from the subject pre-infusion

10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day before the first dose of the adoptive cell therapy. In some examples, the biological sample may be obtained from the subject pre-infusion on the same day as the first dose of the adoptive cell therapy.

[00210] In embodiments, the methods comprise determining the level of endogenous SCF in at least one additional biological sample. The additional biological sample(s) may be obtained from one or more time points different from the first biological sample. For example, at least one additional biological sample may be obtained from the subject after the administration of the adoptive cell therapy. In some examples, at least one additional biological sample may be obtained from the subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, or 16 weeks after the administration of the adoptive cell therapy. In some examples, at least one additional biological sample may be obtained from the subject from 1 day to 7 days, from 1 week to 2 weeks, from 2 weeks to 3 weeks, from 3 weeks to 4 weeks, from 4 weeks to 5 weeks, from 6 weeks to 7 weeks, from 7 weeks to 8 weeks, from 8 weeks to 9 weeks, from 9 weeks to 10 weeks, from 10 weeks to

11 weeks, from 11 weeks to 12 weeks, from 12 weeks to 13 weeks, from 13 weeks to 14 weeks, from 14 weeks to 15 weeks, or from 15 weeks to 16 weeks after the administration of the adoptive cell therapy.

[00211] In embodiments, the methods further comprise detecting the endogenous SCF level before a LD regimen in a subject, e g., to provide a baseline for the subject. For example, the SCF level may be detected in a biological sample obtained from the subject prior to a LD regimen. In some examples, the biological sample may be obtained from the subject on the same day as the LD regimen and before the LD regimen is started. In some examples, the biological sample, or a series of biological samples, may be obtained from the subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, or 16 weeks before the LD regimen. In some examples, the biological sample, or a series of biological samples, may be obtained from the subject from 1 day to 7 days, from 1 week to 2 weeks, from 2 weeks to 3 weeks, from 3 weeks to 4 weeks, from 4 weeks to 5 weeks, from 6 weeks to 7 weeks, from 7 weeks to 8 weeks, from 8 weeks to 9 weeks, from 9 weeks to 10 weeks, from 10 weeks to 11 weeks, from 11 weeks to 12 weeks, from 12 weeks to 13 weeks, from 13 weeks to 14 weeks, from 14 weeks to 15 weeks, or from 15 weeks to 16 weeks before the LD regimen.

[00212] In embodiments, the level of SCF is used to determine the approach to facilitate the inducement of a subject’ response to the adoptive cell therapy. In some examples, the level of SCF may be used to determine whether to use LD, one or more c-kit agonists (e.g., SCF), or a combination thereof to induce a subject’s response to the adoptive cell therapy.

[00213] Alternatively or additionally, at least one additional biological sample may be obtained from the subject after administration of an alternative therapy. Alternatively or additionally, at least one additional biological sample may be obtained from the subject after administration of an adjunctive therapy.

[00214] In embodiments, the level (e.g., the presence, absence and/or amount) of endogenous SCF in the first biological sample and/or the at least one additional biological sample may be used to determine the dose, or the continuing dose, of the at least one c-kit agonist to be administered to the subject. In embodiments, the level (the presence, absence and/or amount) of endogenous SCF in the at least one additional sample may be used to determine the dose or continuing dose of the at least one alternative therapy to be administered to the subj ect. In embodiments, the level (the presence, absence and/or amount) of endogenous SCF in the at least one additional sample may be used to determine the need to administer one or more additional doses of the adoptive cell therapy to the subject, and the dosage of the one or more additional doses of the adoptive immune cell therapy.

[00215] In embodiments, detecting an amount of endogenous SCF greater than 100 pg/ml, greater than 150 pg/ml, greater than 200 pg/ml, greater than 300 pg/ml, greater than 350 pg/ml, greater than 400 pg/ml, greater than 450 pg/ml, greater than 500 pg/ml, greater than 550 pg/ml, greater than 600 pg/ml, greater than 650 pg/ml, greater than 700 pg/ml, greater than 750 pg/ml, greater than 800 pg/ml, greater than 850 pg/ml, greater than 900 pg/ml, greater than 950 pg/ml, or greater than 1000 pg/ml in a serum sample from the subject is supportive of a positive prognosis. In one example, detecting an amount of endogenous SCF greater than 500 pg/ml in a serum sample from the subject is supportive of a positive prognosis.

[00216] In embodiments, detecting an amount of endogenous SCF greater than 100 pg/ml, greater than 150 pg/ml, greater than 200 pg/ml, greater than 300 pg/ml, greater than 350 pg/ml, greater than 400 pg/ml, greater than 450 pg/ml, greater than 500 pg/ml, greater than 550 pg/ml, greater than 600 pg/ml, greater than 650 pg/ml, greater than 700 pg/ml, greater than 750 pg/ml, greater than 800 pg/ml, greater than 850 pg/ml, greater than 900 pg/ml, greater than 950 pg/ml, or greater than 1000 pg/ml in a plasma sample from the subject is supportive of a positive prognosis. In one example, detecting an amount of endogenous SCF greater than 500 pg/ml in a plasma sample from the subject is supportive of a positive prognosis.

[00217] The level of SCF may be detected using methods known in the art. In embodiments, the level of SCF is measured using methods and reagents described in Smith KA et al., Measurement of human and murine stem cell factor (c-kit ligand), Curr Protoc Immunol. 2001 May:Chapter 6:6.17.1-6.17.11; Carsons SE et al., Detection and quantitation of stem cell factor (kit ligand) in the synovial fluid of patients with rheumatic disease, J Rheumatol. 2000 Dec;27(12):2798-800, each of which is incorporated by reference herein in its entirety. Additional reagents for measuring the level of SCF include SCF Monoclonal Antibody, PeproTech (Invitrogen Catalog # 500-M44-500UG) and Human SCF ELISA Kit - Quantikine (R&D Systems, Inc. Catalog #: DCK00).

[00218] In another aspect, the present disclosure provides methods for treating a disease (e.g., a cancer, autoimmune disease, or a pathogen infection), comprising administering to the subject a lymphodepletion (LD) regimen, administering to the subject a dose of an adoptive cell therapy, and simultaneously or sequentially administering a therapeutically effective amount of at least one c-kit agonist to the subject to enhance the in vivo expansion and/or activation of the adoptive immune cells. In embodiments, the at least one c-kit agonist is administered simultaneously with the administration of the adoptivecell therapy. In embodiments, the at least one c-kit agonist is administered after the administration of the adoptive cell therapy. [00219] In embodiments, the adoptive cell therapy comprises non-engineered immune cells such as, e.g., LAKs, TILs, virus-specific T cells (VSTs), and the like. In embodiments, the adoptive cell therapy comprises engineered immune cells such as, e.g., TCR-T cells, CAR-T cells, CAR-NK cells, CAR-M cells, CAR-yST cells, CAR-NKT cells, and the like.

[00220] In embodiments, a LD regimen comprises administration of fludarabine at about 30 mg/m2/day plus cyclophosphamide at about 500 mg/m2/day for three days. In embodiments, aLD regimen comprises administration of fludarabine at about 20 mg/m2/day plus cyclophosphamide at about 300 mg/m2/day for three days. In some examples, in these LD regimens, both fludarabine and cyclophosphamide are administered on Days -5, -4, and -3 prior to administration of an immune cell therapy.

[00221] In embodiments, a LD regimen comprises administration of fludarabine at about 30 mg/m2/day for four days, plus cyclophosphamide at about 1000 mg/m2/day for three days. In some examples, in the LD regimen, the fludarabine is administered on Days -6, -5, -4, and -3, and the cyclophosphamide is administered Days -5, -4, and -3 prior to administration of an immune cell therapy.

[00222] In embodiments, the LD regimens further comprise administration of an anti-CD52 antibody and/or an anti-CD19 antibody.

[00223] In embodiments, a dose of the adoptive cell therapy is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least

14 days, or at least 15 days after the administration of a LD regimen. In embodiments, a dose of the adoptive cell therapy is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days after the administration of the LD regimen.

[00224] In embodiments, the method of treating a disease herein further comprise administering IL-15 (e.g., as an adjunctive therapy). In some examples, the IL-15 is administered prior to the administration of the c-kit agonist (e.g., SCF). In some examples, the IL-15 is administered simultaneously with the administration of the c-kit agonist (e.g., SCF). In some examples, the IL-

15 is administered after the administration of the c-kit agonist (e.g., SCF). [00225] In embodiments, the method of treating a disease herein further comprise modified and/or extended treatment, e.g., one or more additional doses, one or more alternative therapies, and/or one or more adjunctive therapies, described herein.

[00226] One or multiple non-engineered, immune cell populations, engineered, immune cell populations, and/or admixtures thereof, having cytotoxic activity against a cell (e.g., tumor cell) can be administered to a subject in any order or simultaneously. If simultaneously, the multiple non-engineered, immune cell population, engineered, immune cell population, and/or admixtures thereof, of the invention can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, s.c, injections or pills. The non-engineered, immune cell population, engineered, immune cell population, and/or admixtures thereof, of the invention can be packed together or separately, in a single package or in a plurality of packages. One or all of the non-engineered immune cell population, engineered immune cell population, and/or admixtures thereof, of the invention can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some cases, a non-engineered, enriched immune cell population, an engineered, enriched immune cell population, and/or admixtures thereof, of the invention can proliferate within a subject's body, in vivo, after administration to a subject. One or more non-engineered immune cell populations, one or more engineered immune cell populations, and/or admixtures thereof, can be frozen to provide cells for multiple treatments with the same cell preparation. One or more non-engineered immune cell populations, one or more engineered immune cell populations, and/or admixtures thereof, of the disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit may include instructions (e.g., written instructions) on the use of the non-engineered immune cell population, the engineered immune cell population, and/or admixtures thereof, and compositions comprising the same.

[00227] In some cases, a method of treating a disease comprises administering to a subject a therapeutically-effective amount of a non-engineered immune cell population, an engineered immune cell population, and/or admixtures thereof, wherein the administration treats the disease. In some embodiments the therapeutically-effective amount of the non-engineered, immune cell population, the engineered immune cell population, and/or admixtures thereof, is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 5 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 8 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In embodiments the therapeutically-effective amount of the non-engineered immune cell population, the engineered immune cell population, and/or admixtures thereof, is administered for at least one week. In embodiments the therapeutically-effective amount of the non-engineered immune cell population, the engineered immune cell population, and/or admixtures thereof, is administered for at least two weeks.

[00228] A non-engineered immune cell population, an engineered immune cell population, and/or admixtures thereof, described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering a pharmaceutical composition containing the immune cell population can vary. For example, the immune cell population can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein. In some examples, the administration of a immune cell population of the disclosure is an intravenous administration. One or multiple dosages of the immune cell population can be administered as soon as is practicable after the onset of a disease and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. In embodiments, one or multiple dosages of the immune cell population can be administered years after onset of the cancer and before or after other treatments.

[00229] In embodiments, the immune cell population is administered simultaneously or sequentially with at least one c-kit agonist as described herein. For example, the administration of the at least one c-kit agonist may be in conjunction with the administration of the immune cell population. In some examples, the administration of the at least one c-kit agonist may be simultaneous with the administration of the immune cell population. In some examples, the administration of the at least one c-kit agonist may be after the administration of the immune cell population. [0018] In embodiments, the c-kit agonist comprises recombinant SCF, a nucleic acid encoding SCF, or an immune cell engineered to stably express SCF, wherein the immune cell engineered to stably express SCF may be the same or different than the adoptive cell therapy, and included in the immune cell population.

[00230] In embodiments, the immune cell population is administered simultaneously or sequentially with one or more methods to elevate common gamma chain cytokine(s). As used herein, “one or more methods to elevate common gamma chain cytokine(s): refers to a method, or combination of methods, that alters the physiological state of a subject, such that at least one common gamma chain cytokine level is elevated in the subject. In embodiments, the method elevates the level of one or more common gamma chain cytokine(s) selected from the group consisting of IL-2, IL-7, and IL-15, SCF. In one example, the method elevates the level of SCF in the subject. In one example, the method elevates the level of IL-15 in the subject. In one example, the method elevates the levels of both IL-15 and SCF in the subject. In some embodiments, the method comprises lymphodepletion. In some embodiments, the method comprises administering one or more common gamma chain cytokine(s) to the subject. In some cases, SCF, IL-2, IL-7, and/or IL-15, preferably IL-15, SCF, or both SCF and IL-15, are administered. In some embodiments, the method comprises secreting common gamma chain cytokine(s) from an administered, e.g., immune cell. In some cases, IL-2, IL-7, and/or IL-15, preferably IL-15, SCF, or both IL-15 and SCF are secreted.

[00231] In embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before introducing the immune cell(s). In embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the immune cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced immune cell(s), preferably wherein the method comprises administering IL-2 or one or more mimetics thereof, more preferably wherein the method comprises administering IL-15 or one or more mimetics thereof, more preferably wherein the method comprises administering SCF or one or more mimetics thereof, more preferably wherein the method comprises administering IL-15 and SCF or one or more mimetics thereof. The amount of administered common gamma chain cytokine(s) can be an amount effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced immune cell (s) before and/or after introducing the immune cell(s). Exemplary amounts of IL-15 include, without limitation between 0.01 - 10 pg/kg/dose every 24 hours for IL-15. Exemplary amounts of IL-2 include, without limitation, between about 3xl0⁶ and about 22x10⁶ units every 8 - 48 hours. For example, the dosing regimen for IL-2 in RCC is 600,000 International Units/kg (0.037 mg/kg) IV q8hr infused over 15 minutes for a maximum 14 doses. Exemplary amounts and dose regimens of SCF include those described in Clinical trial NCT01016795 (Stem Cell Factor (SCF) Priming of Haematopoietic Stem Cell Grafts in Malignant Lymphoma (SCF980266)”, Drug: r-metHuSCF and Filgrastim); Clinical Trial NCT00001398 (Stem Cell Factor Medication for Aplastic Anemia); Clinical Trial NCT00005783 (A Phase Eli Trial of Recombinant-Methionyl Human Stem Cell Factor (SCF) in Adult Patients With Sickling Disorders); Clinical Trials NCT00001398 (Stem Cell Factor Medication for Aplastic Anemia); Clinical Trial NCT02501811 (Combination of Mesenchymal and C-kit+ Cardiac Stem Cells as Regenerative Therapy for Heart Failure (CONCERT-HF)); Tricot G et al., Superior mobilization of peripheral blood progenitor cells (PBPC) with r-metHuSCF (SCF) and r- metHuG-CSF (filgrastim) in heavily pretreated multiple myeloma (MM) patients, Blood, 88 (1996), p. 388a; E J Shpall et al., A randomized phase 3 study of peripheral blood progenitor cell mobilization with stem cell factor and filgrastim in high-risk breast cancer patients, Blood. 1999 Apr 15;93(8):2491-501; Kurzrock R et al., Trilineage responses seen with stem cell factor (STEMGEN, SCF) and filgrastim (G-CSF) treatment in aplastic anemia (AA) patients (Pts) Br J Haematol. 1998.

[00232] In embodiments, the administering one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before administering the immune cell(s) and administering simultaneously with introducing the immune cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced immune cell(s).

[00233] It should be understood that other suitable lymphodepletion methods can be utilized in the methods of the present disclosure. Exemplary lymphodepletion methods are disclosed in, for example, Amini, et al., “Preparing for CAR T cell therapy: patient selection, bridging therapies and lymphodepletion,” Nat. Rev. Clin. Oncol. 19(5):342-355 (May 2022) and Bechman, N. and Maher, J., “Lymphodepletion strategies to potentiate adoptive T-cell immunotherapy - what are we doing; where are we going,” Expert Opin. Biol. Ther. 21(5):627-637 (May 2021), each of which is incorporated herein by reference in its entirety. EXAMPLES

[00234] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 - Expansion, persistence and pharmacodynamic profile of adi-001, a first-inclass allogeneic CD20-targeted CAR gamma delta T cell therapy, in patients with relapsed/refractory aggressive B-cell Non-Hodgkin’s Lymphoma

[00235] ADI-001 is a first-in-class allogeneic gamma delta (y5) CAR T cell therapy targeting the B cell antigen, CD20. Expansion and persistence of cell therapy products and release of functional cytokines have correlated with patient outcomes. This example shows report cellular kinetic and pharmacodynamic correlates from a phase 1, multicenter, open-label, dose escalation study to evaluate ADI-001 in R/R B cell NHL.

[00236] ADI-0001 is CAR+ V81 78 T cells expressing an anti-CD20 CAR (SEQ ID NO: 41), which are described in Nishimoto KP et al., Allogeneic CD20-targeted y6 T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models, Clin. Transl.

Immunology. 2022; 11(2): el373, which is incorporated by reference herein in its entirety.

[00237] A summary of the study is shown in FIG. 1. In brief, of the 24 DLT-evaluable patients, 3 received ADI-001 at dose level 1 (DL1) (30 million CAR+ cells), 3 received ADI-001 at dose level 2 (DL2) (100 million CAR+ cells), 6 received ADI-001 at dose level 3 (DL3) (300 million CAR+ cells), 4 received two infusions of ADI-001 at DL3 (two doses of 300 million CAR+ cells, one on day 1 and the second dose on day 7 following a single lymphodepletion), and 8 received ADI-001 at dose level 4 (DL4) (1 billion CAR+ cells).

[00238] Cellular kinetics of ADI-001 were measured using three orthogonal methods, including quantitative SNP profiling of cell product (AlloCell), flow-cytometry for CAR+ V81 78 T cells, and droplet digital PCR (ddPCR) quantification of CAR transgene copies. Using these methods, expansion of ADI-001 was assessed in the peripheral blood for 24 DLT evaluable patients across four dose levels in association with a phase 1 dose-escalation trial (NCT04735471). Relationships between ADI-001 cellular kinetics and radiographic clinical responses were also examined. Overall, ADI-001 was well tolerated throughout the dose escalation including in patients dosed at DL4, particularly in comparison with the safety profile of approved autologous CD 19 CAR T. Serum biomarkers related to host immune cell recovery during lymphodepletion, and cytokine release were monitored for pharmacodynamic purposes. Other correlative characteristics were also evaluated, including degree of shared HLA alleles between patient and ADI-001 product in relation to response and/or ADI-001 expansion and persistence.

[00239] ADI-001 was detected at all dose levels tested using ddPCR and flow cytometry to quantify CAR transgene copies and CAR+ V81 yS T cells, respectively. Treatment at the highest dose level (1E9, DL4), achieved a mean Cmax of 201,666 copies/pg or approximately 364 cells/pL, a mean day 28 persistent exposure of 16,553 copies/pg or approximately 27 CAR+ cells/pL, and a mean time to peak (Tmax) of 8.1 days. Additional measures of ADI-001 exposure (AlloCell) further demonstrated a robust dose-dependent expansion profile of ADI-001 in the peripheral blood. Subj ects with a BOR of CR or PR were observed to have a mean peak of 180, 107 CAR copies/pg versus a mean peak of 20,950 CAR copies/pg in subjects with a BOR of SD or PD. The results are shown in FIGS. 2A-2E and 3A-3D.

[00240] Additionally, following lymphodepletion, a transient increase in the endogenous SCF and homeostatic cytokine IL-15, coinciding with ADI-001 expansion and host-mediated immune cell recovery, was observed. A HMPCore2 assay was used to measure SCF and IL-15 levels. The HMPCore2 assay is microsphere-based and uses antigen-specific antibodies optimized in a capture-sandwich format. All incubations took place at room temperature. In brief, 5 pL of a diluted mixture of capture-antibody microspheres were mixed with 5 pL blocker and 10 pL standard, pre-diluted sample, or control in a hard-bottom microtiter plate. Plasma and serum samples were diluted to the appropriate dilution. The plate was incubated for 1 hour. 10 pL biotinylated detection antibody was added to each well, thoroughly mixed, and incubated for 1 hour. 10 pL diluted Streptavidin-phycoerythrin was added to each well, thoroughly mixed, and incubated for 60 minutes. A filter-membrane microtiter plate was pre-wetted by adding 100 pL wash buffer followed by aspiration via a vacuum manifold device. The reaction contents of the hard-bottom plate were then transferred to the respective wells of the filter plate. All wells were vacuum aspirated, and the contents were washed twice with 100 pL wash buffer. After the last wash, 100 pL wash buffer was added to each well, and the washed microspheres were resuspended with thorough mixing. The plate was then analyzed on the Luminex platform. The results are shown in FIGS. 4A-4B. A total of 34 subjects were assessed for SCF levels at D-l pre-infusion. Table 1 shows the proportion of response rates of subjects with less than or greater than a SCF level of 500 pg/mL.

Table 1

[00241] ddPCR (for results in FIG. 2A and FIGS. 3A-3D): Genomic DNA (gDNA) was isolated from whole blood and assessed by droplet digital PCR (ddPCR). The ddPCR data was analyzed by Bio-Rad QuantaSoft™ Software (vl .7.4). The data was reported as Anti-CD20 copies per ug and Vector Copy Number (Anti-CD20 copies per cell). AUCO-28 was calculated using a model-based cellular kinetics analysis for CAR T cells for the first 28 days after infusion (units of days*CAR copies/ug DNA).

[00242] Flow Cytometry (for results in FIG. 2D and 4B): A Combination of 2 panels (i.e. CAR T Cell (for FIG. 2D) and TBNK Panels (for FIG. 4B) were used in order to enumerate CAR T cells. Sample staining with the CAR T cell Panel was performed in a plate-based format using a lyse/wash methodology, and relative frequencies for multiple cell subsets were determined. In parallel, sample staining with the TBNK Panel was performed in a tube format (i.e. Trucount tubes) using a lyse/no wash methodology. Cell counts (i.e. cells/pL of blood) generated via the TBNK Panel was used to mathematically enumerate CAR T cells using relative frequencies derived from the CAR T Cell Panel. The TBNK panel has been previously validated and can be used on clinical samples without any additional development or set up steps. The CAR T cell Panel has been validated.

Example 2 -

[00243] This example describes an in vivo study using ADI-001 and SCF in a subcutaneous model of human B cell lymphoma in NSG mice to evaluate tumor growth inhibition (TGI). It describes the methodology for model establishment, animal dosing, data collection and analyses for the TGI method. The experiments herein assess the TGI of ADI-001 test articles in vivo in a human Burkitt’s lymphoma Raji xenograft tumor model (expressing the target of ADI-001, CD20), with exogenous human SCF and/or c-KIT agonist supplement (e.g., amino acid or small molecule).

[00244] Overview

[00245] Human lymphoma Raji cells are subcutaneously implanted in female NSG mice and tumors allowed to grow for 6 days. On day 6 post implantation, when tumor volumes across all animals reach a mean of approximately 180 mm3 ± 40 mm³, mice are assigned to the following groups (n=8 per group): a vehicle-treated group, group(s) receiving a single dose level of ADI-001 test article(s), and a positive control group. In the case of evaluating multiple lots (and in the interest of minimizing the use of animals) multiple lots may be individually tested alongside shared vehicle-treated control and positive control groups in the same experiment. At least five extra mice are implanted with Raji cells so that mice with similar tumor volumes can be randomized and assigned to each group. Mice are excluded from the study if a tumor demonstrates a >20% difference in tumor volume as compared to the mean or if tumors are multi-lobed at the time of randomization.

[00246] Treatment groups receive 5E6 viable V81 CAR+ cells from the test article or positive control per mouse as a single bolus dose by intravenous (i.v.) tail vein injection on Day 0 (7 days post implantation). Appropriate adjustments to target 5E6 viable V81 CAR+ cells per dose are performed based on the viable cell density as determined by the NC-200 counts after the product is thawed and washed and the %V81 CAR+ population determined during released testing. Human recombinant stem cell factor (SCF) and/or c-KIT agonist supplement is given to each mouse in the treatment groups, three times a week (3X/week) by intra-peritoneal (i.p.) injection for the duration of the study. The first SCF and/or c-KIT agonist dose is given to each mouse within one hour prior to dosing of test article and positive control.

[00247] Tumor growth is monitored by digital caliper measurement twice a week up to day 18, then every day following day 18, for the duration of the study. Body weight data for each mouse are collected twice a week using an electronic balance for the duration of the study. When the average tumor volume reaches approximately 4000 mm3 ± 250 mm3 in any study group, the study is terminated. Modifications to the schedule of tumor measurements and the criteria for study termination can be made.

[00248] Materials and equipment

[00249] Immunodeficient mice. The NSG (NOD SCID gamma) mice are purchased from Jackson Laboratory. Female mice at the age of 6-8 weeks old at the time of xenograft tumor implantation are used in the study. Mice are acclimated within the study vivarium for a minimum of 3 days prior to study handling. From the time of arrival to the end of the study, the health status of all animals is monitored. All mice are group housed with environmental enrichment. Mice are provided with irradiated feed and sterilized water, ad libitum.

[00250] Human B cell lymphoma cell line. The Raji cell line obtained from American Cell Type Culture Collection (ATCC® CCL-86™) is a human B lymphoblastoid cell line originally derived from a patient with Burkitt’s lymphoma and is known to express CD20. RediFect™ Red-shifted Firefly Luciferase (Luciola italica), referred to as RFLuc, is introduced into the Raji cells via stable viral integration which enables the cells, when viable, to emit light in the presence of luciferase substrate. Raji-RFLuc cell clones are single cell sorted and expanded to derive the clone Raji- RFLuc/B4, which are used in the study. Raji cell lines are cultured in RPMI 1640 (ATCC modified) medium (Gibco™, A 10491) containing 10% HyClone™ characterized FBS (Cytiva, SH30071.03)

[00251] Human recombinant SCF. A human recombinant SCF and/or c-kit agonist, is used for exogenous SCF and/or c-KIT agonist supplementation in the study.

[00252] Matrigel (Basement Membrane Matrix). Coming® Matrigel® Matrix Phenol Red-free (Coming Inc., 356237) is used in the study to co-inject with Raji cells to establish xenograft tumors.

[00253] Preparation of SCF and/or c-KIT agonist stock solution

[00254] Prepare vials containing desired concentration(s). Reconstitute one vial of lyophilized SCF and/or c-kit ligand containing 22E6 IU in 1.2mL of WFI, aseptically. Gently swirl the vial without shaking, to fully homogenize the lyophilized pellet.

[00255] Aliquot 20 pL of reconstituted SCF and/or c-KIT agonist into individual 1.5 mL Eppendorf tubes labeled “18E6 lU/mL SCF and/or c-KIT agonist, along with the lot #, initials of analyst and date of preparation. Store reconstituted aliquots in a -80°C freezer for the duration of the dosing period.

[00256] A 20 pL aliquot of SCF and/or c-KIT agonist contains sufficient dosing for three study groups containing eight animals per group. For example, a study containing three study groups (24 mice total) would use one SCF and/or c-KIT agonist aliquot per dosing period.

[00257] Culture of Raji-RFLuc/B4 cell line. An appropriate number of vials of Raji-RFLuc/B4 cells are thawed. A single vial is typically sufficient for experiments containing up to three study groups with a lead time of three-days prior to Raji-RFLuc/B4 implantation. Thaw an additional vial for each additional set of three groups if the lead time is less than four days prior to initiation of the study. If multiple vials are thawed, pool them to generate one homogeneous starting pool. In this assay, Raji-RFLuc/B4 cells are cultured for a limited period of 3 to 10 days post thaw, prior to implantation in animals. Raji-FLuc/B4 cells cultured for more than 20 days are not used in this assay.

[00258] Prepare RPMI 1640 + 10% FBS cell growth media.

[00259] Combine 900 mb of RPMI 1640 growth media with 100 mL of FBS and filter through a 1 L sterile vacuum filter/storage system. Label the 1 L bottle of filtered FBS containing medium “RPMI 1640 + 10% FBS” and include initials and expiry date (four weeks from the date of preparation). Cap the bottle tightly and store in 2° - 8°C refrigerator for the duration of the cell culture process.

[00260] Remove RPMI 1640 + 10% FBS growth media and warm in a water bath set at 37°C for at least 30 minutes. Remove frozen vial(s) of Raji-RFLuc/B4 cells from liquid nitrogen storage and immediately thaw the vial(s) in the water bath set at 37°C until only a small ice crystal remains. Immediately transfer thawed cells to a 50 mL conical tube containing 40 mL of pre-warmed growth media and spin down in a centrifuge set at 300 g for 5 min at room temperature.

[00261] Aspirate the supernatant being careful to not disturb the cell pellet and resuspend in 20 mL growth media. In triplicate, measure and record the % viability and cell concentration via Countess II (1 : 1 v/v cell suspension to Trypan blue). For optimal cell concentration measurements using Countess II, perform cell counts within the range of 1E5 to 4E6 cells/mL. [00262] Adjust the concentration of the Raji-RFLuc/B4 cells to achieve a seeding density of 2E5 viable cells/mL (± 0.5E5 viable cells/mL) per tissue culture flask and label the flasks with the cell line name, date, and initials. Incubated at 37°C with 5% CO2 in a TC incubator. Replace culture medium every 2-4 days.

[00263] Perform subculture when cell density reaches 70-80% confluency. To perform subculturing, collect spent media containing cells in suspension and spin down in a centrifuge set at 300 g for 5 min at room temperature. Each mouse is injected subcutaneously with 1E6 viable cells. When the number of viable cells needed to implant the study animals are available, cells are harvested and prepared for implantation into study mice.

[00264] The following parameters are under evaluation. If one or more of the following parameters is not met, then determination of culture termination is made at the discretion of the Nonclinical Development Study Director. If a decision is made to terminate a culture, then a new culture using a separate thaw of Raji-RFLuc/B4 cells are initiated for the study.

• Average post-thaw viability of > 60% and average viability during the tissue culture duration of > 80%.

• Normal Raji-RFLuc/B4 cell morphology for tissue culture duration. An average growth rate of 0.025 cells/hour (± 0.015 cells/hour) per 72-to-96-hour culture period following Raji-RFLuc/B4 seeding at 2E5 viable cells/mL (± 0.5E5 viable cells/mL) per tissue culture flask. cells concentration ( — j- ) i_n _ mL ^J cells cells seeding density -j-)

Growth Rate (- - ) = - - - - - hour hours in culture

• On the day of formulation for tumor implantation, Raji-RFLuc/B4 cells have an average viability of > 85% to be used in animals.

[00265] Harvest Raji-RFLuc/B4 cells for implantation into NSG mice

[00266] Pre-warm RPMI 1640+ 10% FBS growth media in a warm water bath set at 37°C for at least 30 minutes. Harvest and spin down Raji-RFLuc/B4 cells. Aspirate the supernatant being careful to not disturb cell pellet and resuspend in 50 mL PBS. In triplicate, measure and record the pre-formulation % viability and cell concentration via Countess II (1 :1 v/v cell suspension to Trypan blue). Determine the final total volume to achieve the desired final concentration of 10E6 viable cells/mL for implantation in mice. Once the final total volume has been determined, spin down the cell pellet in a centrifuge set at 300 g for 5 min at room temperature. Add the appropriate amount of PBS to achieve the desired final concentration of 10E6 viable cells/mL to dose all mice in the study (ensure the final total volume accounts for the cell pellet volume). Transport prepared Raji-RFLuc/B4 cells in a tightly capped 50 mL conical tube on ice for implantation in NSG mice in the vivarium.

[00267] Subcutaneous implantation of Raji-RFLuc/B4 cells in NSG mice

[00268] Shave the right hind flank of each NSG mouse to prepare for implantation. Remove the Matrigel Membrane Matrix from -80°C freezer and thaw overnight in a 2° - 8°C refrigerator. Matrigel Membrane Matrix must be kept on ice (to prevent solidification) before and after mixing with Raji-RFLuc/B4 cells and immediately prior to implantation in NSG mice.

[00269] One vial of Matrigel Membrane Matrix contains sufficient material (10 mL) to dose approximately 83 animals. If a study contains more than 10 study groups (containing 8 animals per group), than thaw an additional vial of Matrigel Membrane Matrix.

[00270] Raji-RFLuc/B4 cells are implanted in mice within 1.5 hours following formulation. In the vivarium, aliquot 600 pL of formulated cells into individual chilled sterile 1.5 mL Eppendorf tubes.

[00271] Anesthetize mice according to standard practice: Turn on the anesthesia machine, place mice in the induction chamber. As mice are undergoing anesthesia, dilute one chilled 1.5 mL Eppendorf tubes of Raji-RFLuc/B4 cells with 600 pL of Matrigel (1 : 1 ratio) and carefully resuspend with a P1000 pipette until homogenous.

[00272] While mice undergo anesthesia, load a 25G tuberculin syringe with 1 mL of formulated Raji-RFLuc/B4 cells (10E6 viable cells/mL) per five mice to be implanted. Swab the injection site with 70% IPA and inject 200 pL containing 1E6 viable cells into the right hind flank (superior to the femoral head) of each animal under anesthesia.

[00273] Following tumor cell injection, remove the needles by rotation on axis to close the site and prevent leakage. In the event of technical failure, remove the mouse from the study and replace with a new mouse. Monitor mice for recovery following anesthesia. [00274] Randomization of tumor-bearing mice into study groups

[00275] A minimum of five extra mice are implanted with tumor cells so that enough animals with similar tumor sizes are available to be randomized into each study group.

[00276] Implanted tumors are allowed to grow seven days prior to treatment with ADI-001. On the day prior to treatment with ADI-001 (day 6 post Raji cell implantation, or study day -1), when tumors reach approximately 180 mm³ ± 40 mm³ in average tumor volume, animals are randomized into groups required for the study. Mice are randomized based on tumor volume and body weight using the “Multi-Task” randomization option in Studylog software which factors in both tumor volume and body weight and uses hierarchical clustering in conjunction with leaf-order optimization to assign experimental units in multivariate space into groups with similar means and standard deviations. Mice may be excluded from the study if a tumor demonstrates a > 20% difference in tumor volume as compared to the mean or if tumors are multi-lobed at the time of randomization. Each mouse is assigned a permanent ID number (ear notch or tail marking) following randomization.

[00277] Preparation and administration of SCF and/or c-KIT agonist in NSG mice from frozen stock solution.

[00278] Remove one vial (per 3 study groups) of reconstituted SCF and/or c-KIT agonist labeled “18E6 HJ/mL SCF and/or c-KIT agonist from the -80°C freezer and thaw on ice. If 6 study groups are being tested, thaw 2 vials. If more than 1 vial of SCF and/or c-KIT agonist is used, prepare the 130,000 lU/mL final concentration for each vial individually. Then pool the final solutions to make one preparation of 130,000 lU/mL. Aliquot 1.749 mL of PBS into a centrifuge tube. Add 500 pL of PBS to the thawed vial labeled “18E6 lU/mL SCF and/or c-KIT agonist. Transfer the contents to the centrifuge tube. Rinse the vial labeled “18E6 lU/mL SCF and/or c- KIT agonist, with an additional 500 pL of PBS and transfer the rinsate to the centrifuge tube and mix well. The final concentration of formulated SCF and/or c-KIT agonist solution should be sufficient for dosing 24 animals.

[00279] Withdraw 500 pL of the final SCF and/or c-KIT agonist solution into a 27G tuberculin syringe. Load the appropriate number of syringes to dose all SCF and/or c-KIT agonist supplement cohorts. Dose each mouse with 100 pL SCF and/or c-KIT agonist solution intra-peritoneally via the lower quadrant of the abdomen. Prepare fresh SCF and/or c-KIT agonist dosing solution from a new frozen aliquot on each given dosing day and discard any remaining SCF and/or c-KIT agonist solution.

[00280] Administer the first dose of SCF and/or c-KIT agonist within one hour prior to treatment with the test articles and subsequently three (3) times a week until study termination on a Monday-Wednesday-Friday schedule.

[00281] Preparation and dosing of test article and positive control cells in NSG mice

[00282] Administration of the test article and positive control cells is initiated on Day 0. Cell dosage in the study is based on the number of viable V51 CAR⁺ T cells (all references to “cells/mL” are intended to be “viable cells/mL”).

[00283] Each treatment group animal receive 200 pL of the formulated cell suspension. Animals in the vehicle-treated control group receive 200 pL of vehicle only. Load cells into 27G tuberculin syringes with one syringe per animal dosed.

[00284] Briefly, animals are placed under a heat lamp in a holding cage under supervision. Immediately prior to injection of test article(s), mice are briefly restrained and the injection site is disinfected with a 70% IPA swab. Cells are dosed intravenously via the lateral tail vein and the dosing timestamp is recorded using Studylog software. Following, injection animals are returned to their home cage and monitored by the study operator(s) for acute signs of toxicity.

[00285] Dosing is completed within one hour of cell formulation. Once dosing is completed, the remaining ADI-001 product is transferred to staff to verify cell count and viability post-dosing.

[00286] Data collection

[00287] Tumor size measurement. Tumor size is obtained by external caliper measurements two times per week throughout the study until day 18 post dosing, after which tumor size is measured daily until study termination. Tumor volume (mm³) is calculated as follows, where length is the largest dimension and width is the smallest.

Tumor volume (mm³) = length x width² x 0.5

[00288] If the formation of multiple subcutaneous tumors more than 3 mm apart is observed post-randomization in any study animal, the affected study animal is excluded from the study. Tumor size data are automatically captured and recorded in the Studylog system via an electronic caliper with audit trails. [00289] Body weight measurements. Animal body weight data are obtained two times per week using an electronic balance for the duration of the study until study termination.

[00290] Study endpoint. The study is terminated when the mean tumor volume in any of the groups reaches approximately 4000 mm³ +/- 250 mm³ or when animal health issues warrant group euthanasia according to standard husbandry practices (such as body weight loss of over 20% or paralysis due to tumor burden). If a non-treatment related health event for a given animal occurs and warrants euthanasia, the study is allowed to continue to the endpoint, but the related animal is excluded from the study data analysis.

[00291] Data analysis

[00292] Statistical analysis of tumor volume data is performed using GraphPad Prism. This protocol is designed to achieve at least 90% statistical power to detect a minimum effect size of 20% tumor growth inhibition (a one-way 20% directional reduction in mean tumor volume in test group versus tumor only control group).

[00293] As designed, n=7 mice per arm achieves this with an actual designed power of 93%. An 8^th animal per group is included to enable an outlier analysis while maintaining this level of designed power. If no outlier is removed, the actual designed power exceeds 93% power to detect a minimum effect size of 20% tumor growth inhibition.

[00294] The outlier analysis is conducted using Grubbs’ method with Alpha of 0.2 to remove a maximum of one outlier in each group, if identified by the statistical method (performed using GraphPad Prism).

[00295] The final mean tumor volume data for each group (after outlier analysis) are plotted over time with SEM bars until the last time point when all animals in any given group are still alive or until the study endpoint. Additionally, individual tumor volume data at the last time point are plotted along with mean and SEM bars to examine the distribution of the data. The mean tumor volume, SEM, and SD for each study group on the last day of the study is reported in tabular format. Tumor growth inhibition (TGI) is calculated using the following formula:

%TGI = (1 — (MTV of test article group - MTV of vehicle group')') x 100 [00296] Additional data analysis may be performed as needed, and additional statistical analyses may be applied. [00297] Table 2. Exemplary sequences

[00298] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims.

Claims

CLAIMS: What is claimed is:

1. A method for monitoring and/or prognosing an adoptive cell therapy in a subject, comprising determining a level of endogenous stem cell factor (SCF) in a biological sample from the subject; wherein detection of a presence, absence, and/or amount of endogenous SCF is informative of the in vivo activation and/or expansion of the adoptive immune cells, the antitumor effect of the adoptive cell therapy, and/or the need for a treatment modification and/or extension.

2. The method of claim 1, wherein the adoptive cell therapy comprises non-engineered immune cells; preferably wherein the adoptive cell therapy comprises lymphokine-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TILs), virus-specific T cells (VSTs), and the like.

3. The method of claim 1, wherein the adoptive cell therapy comprises engineered immune cells; preferably wherein the adoptive cell therapy comprises engineered T cell receptor (TCR)-T cells, chimeric antigen receptor (CAR)-T cells, CAR-natural killer (NK) cells, CAR-macrophage (M) cells, C AR-y3 T cells, CAR-NKT cells, and the like.

4. The method of any preceding claim, wherein said biological sample is obtained from the subject after administering a lymphodepletion (LD) regimen to the subject and prior to administering a first dose of the adoptive cell therapy to the subject; preferably at least 24, 48, 72, 96, or 120 hours after administration of the LD regimen.

5. The method of claim 4, wherein said biological sample is obtained from the subject preinfusion on the same day as the first dose of the adoptive cell therapy.

6. The method of any preceding claim, wherein the treatment modification comprises adjusting a dosage level (e.g., increasing or decreasing the dosage of the adoptive cell therapy), adjusting a previously planned dosing schedule, and/or administering one or more adjunctive or alternative therapies.

7. The method of claim 6, wherein the one or more alternative therapies comprises administering a different adoptive cell therapy.

8. The method of claim 6, wherein the one or more adjunctive therapies comprises administering at least one c-kit agonist to said subject in conjunction with, administering the adoptive cell therapy.

9. The method of claim 8, wherein the c-kit agonist comprises recombinant SCF, a nucleic acid encoding SCF, or an immune cell engineered to stably express SCF, and wherein the immune cell engineered to stably express SCF may be the same or different than the adoptive cell therapy.

10. The method of claim 8 or 9, further comprising determining a level of endogenous SCF in at least one additional biological sample obtained from the subject after administration of the adoptive cell therapy, and determining a dose, or a continuing dose, of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in said at least one additional sample.

11. The method of any preceding claim, wherein the adoptive cell therapy is an engineered immune cell therapy comprising T cells, NK cells and/or macrophages that are engineered to stably express one or more antigen recognition moi eties; preferably wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen.

12. The method of claim 11, wherein the antigen recognition moiety is selected from the group consisting of a TCR, a TCR, y8 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).

13. The method of claim 11 or 12, wherein the T cells, NK cells and/or macrophages are engineered to express two or more antigen recognition moieties, preferably wherein the two or more antigen recognition moieties are different, and wherein each different antigen recognition moiety is engineered to recognize different epitopes of the same antigen or to recognize different epitopes of different antigens.

14. The method of claim 11, wherein the T cells, NK cells and/or macrophages are further engineered to stably express at least one c-kit agonist; preferably wherein said at least one c-kit agonist comprises human SCF (hSCF).

15. The method of any preceding claim, wherein detecting an amount of endogenous SCF greater than 500 pg/ml in a serum sample from the subject is supportive of a positive prognosis.

16. The method of claim 1, wherein the treatment extension comprises administering a second dose of the adoptive cell therapy to the subject at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, or at least one month after the first dose, with or without administering an additional LD regimen.

17. A method of treating a disease in a subject in need thereof, the method comprising a) administering to the subject a lymphodepletion (LD) regimen; b) administering to the subject a first dose of an adoptive cell therapy at least 5 days after administering said LD regimen; and c) simultaneously or sequentially administering a therapeutically effective amount of at least one c-kit agonist to the subject to enhance in vivo expansion of the adoptive immune cells.

18. The method of claim 17, wherein the at least one c-kit agonist is administered after the administration of the adoptive cell therapy.

19. The method of claim 17 or 18, wherein the c-kit agonist comprises recombinant SCF, a nucleic acid encoding SCF, or an immune cell engineered to stably express SCF, wherein the immune cell engineered to stably express SCF may be the same or different than the adoptive cell therapy.

20. The method of claim 17, further comprising determining a level of endogenous SCF in a biological sample obtained from the subject after administration of the LD regimen and before administration of the engineered immune cell therapy, and determining a dose of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in said sample.

21. The method of claim 20, wherein the biological sample is obtained from the subject at least 24, 48, 72, 96, or 120 hours after administration of the LD regimen; preferably wherein the biological sample is obtained from the subject prior to infusion on the same day as the first dose of the adoptive cell therapy.

22. The method of claim 17, further comprising determining a level of endogenous SCF in at least one biological sample obtained from the subject after administration of the adoptive cell therapy, and determining a dose of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in said sample.

23. The method of claim 18, further comprising determining a level of endogenous SCF in at least one additional biological sample obtained from the subject after administration of the adoptive cell therapy, and determining a dose, or a continuing dose, of the c-kit agonist based on the presence, absence and/or amount of endogenous SCF in said at least one additional sample.

24. The method of any one of claims 17-23, wherein the adoptive cell therapy is an engineered immune cell therapy comprising T cells, NK cells and/or macrophages that are engineered to stably express one or more antigen recognition moi eties; preferably wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen

25. The method of claim 24, wherein the antigen recognition moiety is selected from the group consisting of a TCR, a TCR, yd TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).

26. The method of claim 24 or 25, wherein the T cells, NK cells and/or are engineered to express two or more antigen recognition moieties, preferably wherein the two or more antigen recognition moieties are different, and wherein each different antigen recognition moiety is engineered to recognize different epitopes of the same antigen or to recognize different epitopes of different antigens.

27. The method of claim 24, wherein the T cells, NK cells and/or macrophages are further engineered to stably express at least one c-kit agonist; preferably wherein said at least one c-kit agonist comprises hSCF.

28. The method of any one of claims 15-24, further comprising administering a second dose of the engineered immune cell therapy to the subject at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, or at least one month after the first dose, with or without administering an additional LD regimen.

29. The method of any one of claims 2-28, wherein the LD regimen comprises administration of fludarabine at about 30 mg/m2/day plus cyclophosphamide at about 500 mg/m2/day for three days.

30. The method of any one of claims 2-28, wherein the LD regimen comprises administration of fludarabine at about 30 mg/m2/day for four days, plus cyclophosphamide at about 1000 mg/m2/day for three days.

31. A composition comprising T cells, NK cells and/or macrophages that are engineered to stably express one or more antigen recognition moi eties and at least one c-kit agonist; preferably wherein the antigen recognition moiety recognizes a tumor antigen, an antigen associated with an autoimmune disease, or a pathogenic antigen, and the c-kit agonist is hSCF.

32. The composition of claim 31, wherein the antigen recognition moiety is selected from the group consisting of a TCR, a.p TCR, y8 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).

33. The composition of claim 31 or 32, wherein the T cells, NK cells and/or macrophages are engineered to express two or more antigen recognition moieties, preferably wherein the two or more antigen recognition moieties are different, and wherein each different antigen recognition moiety is engineered to recognize different epitopes of the same antigen or to recognize different epitopes of different antigens.

34. An isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding at least one c-kit agonist.

35. The isolated polynucleotide of claim 34, wherein the c-kit agonist is hSCF.

36. The isolated polynucleotide of claim 34, wherein the CAR comprises an affinity binding entity comprising an antigen binding domain that specifically binds to (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a complex with MHC (peptide-MHC complex).

37. The isolated polynucleotide of claim 36, wherein said CAR further comprises a hinge domain; optionally wherein said hinge domain comprises a glycine polymer, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, immunoglobulin heavy chain hinge, or receptor-derived hinge.

38. The isolated polynucleotide of claim 37, wherein said receptor-derived hinge is a CD8 alpha hinge domain.

39. The isolated polynucleotide of any one of claims 34-38, wherein said CAR further comprises a transmembrane (TM) domain; optionally wherein said TM domain comprises a TM region of 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7- H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CD 11 a, CD1 lb, CD11c, CD1 Id, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD 162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof.

40. The isolated polynucleotide of claim 39, wherein said TM domain comprises a TM domain of CD8, preferably wherein said CD8 TM domain is a TM domain of CD8 alpha.

41. The isolated polynucleotide of any one of claims 34-40, wherein said CAR further comprises at least one costimulatory domain; optionally wherein said costimulatory domain comprises a costimulatory domain of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8a, CD8p, CDl la, CDl lb, CDl lc, CDl ld, IL2RP, IL2y, IL7Ra, IL4R, IL7R, IL15R, IL21R, CD18,

CD 19, CD 19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD 100 (SEMA4D), CD103, CD134 (0X40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CDl la/CD18), FcR, FcyRI, FcyRII, FcyRIII, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG/Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, J AML, CD150, PSGL1, TSLP, TNFR2, or TRANCE/RANKL, or a portion thereof, or combinations thereof.

42. The isolated polynucleotide of claim 41, wherein said costimulatory domain is a 4- IBB costimulatory domain

43. The isolated polynucleotide of any one of claims 34-42, wherein said CAR and/or said c- kit agonist further comprises a signal peptide.

44. The isolated polynucleotide of any one of claims 34-43, further comprising a nucleic acid sequence encoding at least one multicistronic linker region; optionally wherein the multi ci str onic region encodes a cleavage sequence and/or an internal ribosomal entry site (IRES).

45. The isolated polynucleotide of any one of claims 34-44, wherein the cleavage sequence is selected from T2A, F2A, P2A, E2A, furin, and furin-P2A (FP2A).

46. The isolated polynucleotide of any one of claims 34-45, wherein the c-kit agonist is operably linked to a nucleic acid sequence encoding a signal peptide.

47. An expression vector comprising the isolated polynucleotide of any one of claims 34-46, operably linked to a cis-acting regulatory element.

48. A cell comprising the isolated polynucleotide of any one of claims 34-46, and/or the expression vector of claim 47.

49. A modified immune cell, comprising the isolated polynucleotide of any one of claims 34- 46, and/or the expression vector of claim 47.

50. The modified immune cell of claim 49, wherein said modified immune cell is a y8 T cell, a y8 NKT cell, an a.p T cell, a NK cell, a NKT cell, or a macrophage.

51. The modified immune cell of claim 50, wherein said modified immune cell is a y6 T cell; optionally wherein said y8 T cell is a 81, a 82, a S3, or a 84 y8 T cell, preferably a 82' y8 T cell, more preferably a 81 y8 T cell.