Identification of suitable donors of γδ t cells

Abstract

The present disclosure provides methods for identifying a human donor or a donor blood sample that is suitable for obtaining a population of Vδ1+ gamma delta (γδ) T cells, the method comprising assaying for the presence of naïve Vδ1+ γδ T cells in the donor blood sample by detecting the presence of cells that express CD27, CD127, CD73, or any combination thereof. Further provided are populations of γδ T cells obtained from the suitable donor blood sample and methods of using the same.

Description

IDENTIFICATION OF SUITABLE DONORS OF y5 T CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No.63 / 679,552, filed on August 5, 2024, which is incorporated by reference herein in its entirety.FIELD OF DISCLOSURE

[0002] The present disclosure relates to methods of immunotherapy. In particular, the disclosure provides methods of identifying a human donor or a donor blood sample that is suitable for obtaining a population of V51+gamma delta (y5) T cells for an allogeneic immunotherapy.BACKGROUND

[0003] The growing interest in T cell immunotherapy for cancer has focused on the evident capacity of engineered T cells as therapeutic moieties. Gamma delta (y5) T cells represent a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 T cells can be broadly classified as one or two types: peripheral blood-resident y5 T cells and tissue-resident y5 T cells. Most blood-resident y5 T cells express a V52 TCR, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 and / or other V5 chains.

[0004] Fully developed Vbl+yb T cells (Vbl cells) exist in their naive state until an activating stimulus is applied. Once activated, Vbl cells undergo functional and phenotypic (and presumably metabolic) differentiation, to give rise to effector and memory Vbl cells. Like their naive ocp TCR-expressing counterparts, naive Vbl cells possess the highest proliferation potential among all hierarchical differentiation states of Vbl cells.

[0005] In the human peripheral blood, Vbl cells constitute a rare compartment. This highlights the challenge of obtaining a yield of Vbl -derived drug substances that meetsthe quality control and dosing requirements. It is imperative that the manufacturing process be started with batches of material possessing relatively high numbers of highly proliferative V81 cells.

[0006] Traditionally, naive V81 cells are identified as those co-expressing CD45RAand CD27 (CD45RA+CD27+V81+y8 T cells). V81 cells have been understood to undergo activation associated with clonal expansion in response to CMV infection. In CMV seropositive adults, a sizable proportion of clonally diverse (indicating a lack of activation) V81 cells do not express CD45RA, indicating that CD45RA may not be a reliable marker of naive V81 cells.

[0007] Similarly, CD27 shows different levels of expression in V81 cells, and the repertoire can be classified into three groups: CD27", CD2710and CD27111. The CD27111subgroup possesses the highest clonal diversity and retains the expression of markers that are identifiers of naive ocp T cells: CD127, CD62L, CCR7 and CD28 among others. Interestingly, CD27" and CD2710V8 l cells downregulate the naive markers, while expressing those markers that are associated with differentiation and cytotoxicity such as CX3CR1, granzyme A and perforin.

[0008] Assays based solely on CD45RA and / or CD27 thus may be insufficient to accurately and reproducibly identify naive V81+y5 T cells in a donor sample.SUMMARY OF THE DISCLOSURE

[0009] To address the deficiencies in current selection assays based primarily on CD45RA and / or CD27 detection, the inventors have developed improved methods to more efficiently identify and select naive V81+y5 T cells in a donor sample.

[0010] Some aspects of the present disclosure are directed to methods of identifying a donor blood sample suitable for obtaining a population of V51+gamma delta (y5) T cells, the method comprising assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express (i) CD27, (ii) CD127, (iii) CD73, or any combination thereof.

[0011] Some aspects of the present disclosure are directed to methods of identifying a human donor that is suitable for obtaining a population of V51+gamma delta (y5) T cells, the method comprising obtaining a donor blood sample from the human donor, and assaying for the presence of naive V51+y5 T cells in the donor blood sample by detectingthe presence of cells that express (i) CD27, (ii) CD127, (iii) CD73, or any combination thereof.

[0012] In some aspects, the naive V51+y5 T cells are (i) CD27 and (ii) CD127, CD73, or both. In some aspects, the naive V51+y5 T cells are CD27+CD127+. In some aspects, the naive V51+y5 T cells are CD27+CD73+. In some aspects, the naive V51+y5 T cells are CD73+CD127+. In some aspects, the naive V51+y5 T cells are CD27+CD127+CD73+. In some aspects, the naive V51+y5 T cells do not express CX3CR1. In some aspects, the naive V51+y5 T cells do not express HLA-DR. In some aspects, the naive V51+y5 T cells do not express CX3CR1 and do not express HLA-DR.

[0013] In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined by detecting CD27, CD127, and / or CD73 protein; CD27, CD127, and / or CD73 mRNA; or any combination thereof. In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined by using an immunohistochemical assay. In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined using flow cytometry.

[0014] In some aspects, the method comprises: (i) sorting cells obtained from the donor blood sample based on expression of CD27; (ii) sorting the CD27+cells from (i) based on expression of CD127; and (iii) sorting CD27+CD127+cells from (ii) based on expression of CD73.

[0015] In some aspects, the method comprises: (i) sorting cells obtained from the donor blood sample based on expression of CD127; (ii) sorting the CD127+cells from (i) based on expression of CD27; and (iii) sorting CD27+CD127+cells from (ii) based on expression of CD73.

[0016] In some aspects, the method further comprses calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+triple-positive.

[0017] In some aspects, the method further comprises: (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of CX3CR1; and (v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR1 ’.

[0018] In some aspects, the method further comprises: (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of HLA-DR; and (v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+HLA-DR'.

[0019] In some aspects, the method further comprises: (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of CX3CR1; (v) sorting the CD27+CD127+CD73+CX3CR1" cells from (iv) based on the expression of HLA-DR; and (vi) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR1 "HLA-DR".

[0020] In some aspects, the method further comprises: (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of HLA-DR; (v) sorting the CD27+CD127+CD73+HLA-DR" cells from (iv) based on the expression of CX3CR1; and (v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR1 "HLA-DR".

[0021] In some aspects, the donor blood sample is identified as suitable if at least about 0.05%, at least about 0.08%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of immune cells in the donor blood sample are naive V51+y5 T cells.

[0022] In some aspects, the donor blood sample is obtained from a human donor. In some aspects, the human donor is at least about 18 years old to about 55 years old. In some aspects, the human donor has a BMI of at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35. In some aspects, the human donor has a BMI of at least about 24. In some aspects, the human donor has a body weight of at least about 150 lb., at least about 155 lb., at least about 160 lb., at least about 165 lb., at least about 170 lb., at least about 175 lb., at least about 180 lb., at least about 185 lb., at least about 190 lb., at least about 195 lb., or at least about 200 lb. In some aspects, the human donor has a body weight of at least about 180 lb.

[0023] In some aspects, the human donor is a healthy human. In some aspects, the human donor has not been diagnosed with a cancer, an infectious disease, an autoimmune disease, a metabolic disease, or any combination thereof.

[0024] In some aspects, the method further comprises isolating V51+y5 T cells from a donor sample identified as suitable. In some aspects, the V51+y5 T cells comprise naive V51+ y5 T cells.

[0025] In some aspects, the isolated V51+y5 T cells are expanded ex vivo. In some aspects, the expanded V51+y5 T cells are administered to a subject.

[0026] Some aspects of the disclosure are directed to a method of expanding a population of y5 T cells for an allogenic y5 T cell-derived therapy, comprising (i) identifying a donor sample suitable for obtaining a population of V51+y5 T cells according to a method disclosed herein, (ii) isolating y5 T cells from the donor sample identified as suitable, and (iii) expanding the y5 T cells. In some aspects, the isolated V51+y5 T cells comprise naive V51+ y5 T cells.

[0027] Some aspects of the present disclosure are directed to a population of allogenic y5 T cells obtained by a method disclosed herein.

[0028] Some aspects of the present disclosure are directed to a method of treating a subject in need thereof, comprising administering a population of allogenic y5 T cells disclosed herein to the subject.

[0029] Some aspects of the disclosure are directed to a method of treating a subject in need thereof, comprising (i) identifying a human donor sample suitable for obtaining a population of V51+y5 T cells according to a method disclosed herein, (ii) isolating y5 T cells from the donor sample identified as suitable, (iii) expanding the y5 T cells, and (iii) administering the y5 T cells to the subject. In some aspects, the isolated V51+y5 T cells comprise naive V51+ y5 T cells.

[0030] In some aspects, the y5 T cells are modified prior to administration to the subject. In some aspects, the y5 T cells are genetically modified. In some aspects, the y5 T cells are modified to express a chimeric antigen receptor (CAR). In some aspects, the CAR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the CAR comprises an antigen-binding domain that specifically binds mesothelin.

[0031] In some aspects, the y5 T cells are modified to reduce the immunogenicity of the y5 T cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGs. 1 A-1I are flow cytometry plots illustrating the enumeration of naive V51 cells using a single marker: CD27. Controls are shown in FIGs. 1 A (percent of lymphocytes), IB (viable cells as measured by eFluor780 and FSC-A expression), and 1C (single cells). Cells were stained for CD45 (FIG. ID), y5 TCR (FIG. IE), ocp TCR, V51 TCR (FIG. 1G), and CD27 (FIG. 1H). FIG. II shows an overlay of cells and rainbow beads to distinguish CD2710from CD27111cells.

[0033] FIGs. 2A-2B are graphical representations of the comprehensive surface phenotyping V51 cells. FIG. 2A shows expression of CX3CR1 and HLA-DR on V51 cells from donors with high or low frequencies of CD27hiV51 cells. FIG. 2B shows expression of CD27, CD127, and CD73 on CD27hiV51 as compared to CD27loV51 cells.

[0034] FIGs. 3A-3J illustrate polarized expression of CD73, CD127, CX3CR1 and HLA- DR relative to CD27. FIGs. 3 A-3I are flow cytometry plots illustrating the gating strategy for selecting CD27111and CD27lo / " V51 cells. First cells were gated based on CD27 high or low expression (FIG. 3A), then on CD127 expression (FIGs. 3B-3C), then on CD73 expression (FIGs. 3D-3E), then on CX3CR1 expression (FIGs. 3F-3G), and then on HLA-DR expression (FIGs. 3H-3I), as indicated. CD27111gate placement was user subjective. The expression of the indicated markers in CD27hi (right column) and CD271o / - (left column) populations of V51 cells, using a single donor. FIG. 3 J is a heatmap showing the expression of indicated markers in CD27hi and CD271o / - V51 cells in a larger cohort of screened donors (n=47, wherein each row is a unique donor).

[0035] FIGs. 4A-4D are flow cytometry plots illustrating enumeration of naive V51 cells by utilizing a combination of multiple naive and effector markers of V51 cells. Sequential gating is shown by first sorting on expression of CD27 (FIG. 4A), then on expression of CD127 and CD27 (FIG. 4B), then on expression of CD73 and CD27 (FIG. 4C), and finally on lack of expression of both HLA-DR and CX3CR1 (FIG. 4D).

[0036] FIGs. 5A-5I show a comparison of the populations selected by the Donor Screening Protocol (DSP) (FIGs. 5B, 5D, 5F, and 5H) and a naiveness determining screening protocol (NDSP) (FIGs. 5C, 5E, 5G, and 51), disclosed herein. The cells identified as naive by each method are in red (FIGs. 5F-5I; darker data points, within the dashed boxes). FIG. 5 A shows the correlation between the frequencies of naive V51 cells calculated using relative internal measures: DSP (x-axis) and NDSP (y-axis). FIGs. 5B-5E provide an example demonstrating fold expansion (FOE) of DSP as compared with NDSP using transduced (TD) and un-transduced (UTD) cells. FIGs. 5F-5I provide an example demonstrating the rigidity of DSP as compared with NDSP. Naive cells identified by the indicated method are in red (darker data points, within the dashed boxes).

[0037] FIGs. 6A-6D are graphical representations of the fold expansion (FOE) in transduced (TD) and un-transduced (UTD) cells relative to DSP or NDSP values.DETAILED DESCRIPTION

[0038] Some aspects of the present disclosure are directed to methods of identifying a donor blood sample suitable for obtaining a population of V51+gamma delta (y5) T cells. Some aspects of the present disclosure are directed to methods of identifying a human donor that is suitable for obtaining a population of V51+gamma delta (y5) T cells. In some aspects, the method comprises obtaining a donor blood sample from the human donor. In some aspects, the method comprises assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express CD27, CD127, CD73, or any combination thereof. In some aspects, the method comprises assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express (i) CD27 and (ii) CD127, CD73, or both. In some aspects, the naive V51+y5 T cells are CD27+CD127+. In some aspects, the naive V51+y5 T cells are CD27+CD73+. In some aspects, the naive V51+y5 T cells are CD73+CD127+. In some aspects, the naive V51+y5 T cells are CD27+CD127+CD73+. In some aspects, the naive V51+y5 T cells do not express CX3CR1 and / or HLA-DR. In some aspects, the donor blood sample is identified as suitable if at least about 0.05% of all immune cells (e.g., all CD45+ cells) in the donor blood sample are naive V51+y5 T cells.

[0039] Some aspects of the present disclosure are directed to methods of expanding a population of y5 T cells for an allogenic y5 T cell-derived therapy, comprising (i) identifying a donor sample suitable for obtaining a population of V51+y5 T cells according to a method disclosed herein, (ii) isolating y5 T cells from the donor sample identified as suitable, and (iii) expanding the y5 T cells.

[0040] Some aspects of the present disclosure are directed to methods of treating a subject in need thereof, comprising administering a population of allogenic y5 T cells prepare according to the methods disclosed herein.I. Definitions

[0041] It is to be understood that aspects and aspects of the disclosure described herein include "comprising," "consisting," and "consisting essentially of aspects and aspects. As used herein, the singular form "a," "an," and "the" includes plural references unless indicated otherwise.

[0042] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. In some instances, "about" encompass variations of +20%, in some instances +10%, in some instances +5%, in some instances +1%, or in some instances +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. When the term "about" is used to modify a value of measurement, e.g., mM, or a quantity, e.g., percent of cells that are naive V51+y5 T cells, the term refers to a range of plus or minus 10% of the value or quantity. As such, "about 20%" refers to a range of 18% to 22%.

[0043] As used herein, the term "engineered y5 T cell" refers to a y5 T cell that expresses a transgene (i.e., a gene that has been transduced into the engineered y5 T cell or a parental cell thereof).

[0044] As used herein, the term "donor blood sample" refers to a blood sample taken from a blood donor suitable for obtaining a population of V51+gamma delta (y5) T cells.

[0045] As used herein, the term "suitable" refers to a donor blood sample that is at least about 0.05%, at least about 0.08%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of immune cells (e.g., all CD45+ cells) in the donor blood sample are naive V51+y5 T cells.

[0046] As used herein, the term "immune cell" refers to a cell of the immune system. In some aspects, the immune cell is selected from a T lymphocyte ("T cell"), B lymphocyte ("B cell"), natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell orneutrophil). In some aspects, the immune cell is a tumor-infiltrating cell (TIL). As used herein, the term "Gamma delta T cell" or "y5 T cell" refers to a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 T cells can be broadly classified as one or two types: peripheral blood-resident y5 T cells and tissue-resident y5 T cells. Most blood-resident y5 T cells express a V52 TCR, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 and / or other V5 chains.

[0047] As used herein, the term "naive V51+y5 T cells" refers to precursors for effector and memory y5 T cell subsets. Phenotypically, naive T cells are small cells with little cytoplasm; they express surface markers, such as CD45RA, CCR7, CD62L, CD 127, and CD 132. They lack expression of markers of previous activation, such as CD25, CD44, CD69, CD45RO, or HLA-DR.

[0048] As used herein, the term "sorting" refers the process of flow cytometry, for example, through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as extracellular and / or intracellular protein expression, size, morphological parameters, viability, or a combination thereof. "Detecting" refers to identifying presence of cells with the surface expression of interest.

[0049] As used herein, the term "human donor" refers to any healthy subject, from which a biological sample, e.g., blood, can be obtained. In some aspects, the human donor is not afflicted with a cancer, infectious disease, or both.

[0050] As used herein, the term "BMI" refers to Body Mass Index and is a calculation that estimates body fat percentage and is used to screen for weight categories and disease risk. BMI is calculated by dividing a person's weight in kilograms by their height in meters squared. The formula is BMI = kg / m2. BMI categories are underweight, healthy weight, overweight, obese, and severely obese. For adults, a healthy BMI is between 18.5 and 24.9, while a BMI of 25-29.9 is considered overweight and a BMI of 30 or higher is considered obese.

[0051] As used herein, "CD27" (UniProt P26842) refers to a receptor specifically expressed at the surface of T cells, which binds and is activated by its ligand CD70 / CD27L, which is expressed by activating cells, such as B cells and dendritic cells. The CD70-CD27 signaling pathway mediates antigen-specific T cell activation and expansion, which in turn provides immune surveillance of B cells.

[0052] As used herein, "CD127" (UniProt P16871) refers to a receptor for interleukin-7, which may also act as a receptor for thymic stromal lymphopoietin (TSLP).

[0053] As used herein, "CD73" (UniProt P21589) refers to an antigen that catalyzes the hydrolysis of nucleotide monophosphates, releasing inorganic phosphate and the corresponding nucleoside, with AMP being the preferred substrate. CD73 shows a preference for ribonucleotide monophosphates over their equivalent deoxyribose forms. Other substrates include IMP, UMP, GMP, CMP, dAMP, dCMP, dTMP, NAD and NMN.

[0054] As used herein, "CX3CR1 " (UniProt P49238) refers to a receptor for the C-X3-C chemokine fractalkine (CX3CL1), which is present on many early leukocyte cells. CX3CR1-CX3CL1 signaling exerts distinct functions in different tissue compartments, such as immune response, inflammation, cell adhesion and chemotaxis. CX3CR1- CX3CL1 signaling mediates cell migratory functions and is responsible for the recruitment of natural killer (NK) cells to inflamed tissues.

[0055] As used herein, "HLA-DR" (UniProt Q29900) refers to an alpha chain of antigen- presenting major histocompatibility complex class II (MHCII) molecule. In complex with the beta chain HLA-DRB, HLA-DR displays antigenic peptides on professional antigen presenting cells (APCs) for recognition by alpha-beta T cell receptor (TCR) on HLA-DR- restricted CD4-positive T cells. This guides antigen-specific T-helper effector functions, both antibody-mediated immune response and macrophage activation, to ultimately eliminate the infectious agents and transformed cells. Typically presents extracellular peptide antigens of 10 to 30 amino acids that arise from proteolysis of endocytosed antigens in lysosomes.

[0056] As used herein, a "population" of y5 T cells refers to a group of three or more y5 T cells (e.g., at least 10, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, at least 1012, or at least 1013) y5 T cells (e.g., naive y5 T cells). A population of a particular cell type (e.g., a population of naive y5 T cells) refers to the cells of that type and not to cells of a different type within a broader population. For example, if 10% of the cells of a starting population of 108T cells are y5 T cells, the starting population of y5 T cells is 107.

[0057] As used herein, the phrase "in an amount effective to" refers to an amount that induces a detectable result (e.g., a number of cells having a statistically significant increased number relative to its starting population, e.g., at a p < 0.05).

[0058] The term "culturing" as used herein refers to the controlled growth of cells ex vivo and / or in vitro. As used herein, "culturing" includes the growth of cells, e.g., y5 T cells, during cell expansion. In some aspects, the cultured cells are obtained from a subject, e.g., a human subject. In some aspects, the cultured cells comprise y5 T cells obtained from a human subject. In some aspects, the y5 T cells are isolated or purified prior to the culture. In some aspects, the cell culturing is intended to expand the number of cultured cells, e.g., to increase proliferation of the cells.

[0059] "Expand" or "expansion," as used herein in reference to y5 T cells refers to the process of culturing the cells to allow for and / or encourage proliferation of the cells. The expansion process can lead to an increase in the proportion or the total number of desired cells, e.g., an increase in the proportion or total number of y5 T cells, in a population of cultured cells, after the cells cultured. Expansion does not require that all cell types in a population of cultured cells are increased in number. Rather, in some aspects, only a subset of cells in a population of cultured cells are increased in number during expansion, while the number of other cell types may not change or may decrease.

[0060] As used herein, the term "yield" refers to the total number of cells following a culture method or a portion thereof. In some aspects, the term "yield" refers to a particular population of cells, e.g., y5 T cells or, in particular, naive y5 T cells. The yield can be determined using any methods, including, but not limited to, estimating the yield based on a representative sample.

[0061] As used herein, the term "chimeric antigen receptor" or alternatively a "CAR" refers to a recombinant polypeptide construct including an extracellular antigen binding domain, a transmembrane domain, and, optionally, an intracellular domain that propagates an activation signal that activates the cell and / or a costimulatory signal. In some aspects, the CAR includes an optional leader sequence at the N-terminus of the CAR fusion protein. In some aspects, the CAR lacks an intracellular (e.g., signaling) domain.

[0062] "Immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0063] As used herein, the terms "treat," "treatment," or "treatment of' when used in the context of treating a disease or condition in a subject, e.g., a cancer, refer to reducingdisease pathology, reducing or eliminating disease symptoms, promoting increased survival rates, and / or reducing discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce one or more disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing one or more clinical symptom and / or inhibition or delay in the progression of the condition and / or prevention or delay of the onset of a disease or illness.

[0064] As used herein, "cancer" refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can result in the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0065] As used herein, the terms "subject," "individual," or "patient," refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0066] Various aspects described herein are described in further detail in the following subsections.II. Methods of the DisclosureILA. Methods of Identifying Suitable Donor Samples

[0067] Some aspects of the present disclosure are directed to methods of identifying a donor blood sample suitable for obtaining a population of V51+gamma delta (y5) T cells. Some aspects of the present disclosure are directed to methods of identifying a human donor that is suitable for obtaining a population of V51+gamma delta (y5) T cells. In some aspects, the method comprises obtaining a donor blood sample from the human donor. In some aspects, the method comprises assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express CD27, CD127, CD73, or any combination thereof. In some aspects, the method comprises assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express (i) CD27 and (ii) CD127, CD73, or both. In some aspects, the method comprises assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express (i) CD73 and (ii) CD127, CD27, or both. In some aspects, the naive V51+y5 T cells are CD27+CD127+. In some aspects, the naive V51+y5 T cells are CD27+CD73+. In some aspects, the naiveV51+y5 T cells are CD73+CD127+. In some aspects, the naive V51+y5 T cells are CD27+CD127+CD73+.

[0068] In some aspects, the naive V51+y5 T cells do not express CX3CR1. In some aspects, the naive V51+y5 T cells are CD27+CD127+and CXCRL. In some aspects, the naive V51+y5 T cells are CD27+CD73+and CXCR1-. In some aspects, the naive V51+y5 T cells are CD73+CD127+and CXCR1-. In some aspects, the naive V51+y5 T cells are CD27+CD127+CD73+and CXCRl’.

[0069] In some aspects, the naive V51+y5 T cells do not express HLA-DR. In some aspects, the naive V51+y5 T cells are CD27+CD127+and HLA-DR’. In some aspects, the naive V51+y5 T cells are CD27+CD73+and HLA-DR’. In some aspects, the naive V51+y5 T cells are CD73+CD127+and HLA-DR’. In some aspects, the naive V51+y5 T cells are CD27+CD127+CD73+and HLA-DR’.

[0070] In some aspects, the naive V51+y5 T cells do not express CX3CR1 and do not express HLA-DR. In some aspects, the naive V51+y5 T cells are CD27+CD127+and CXCRL HLA-DR’. In some aspects, the naive V51+y5 T cells are CD27+CD73+and CXCR1’HLA-DR’. In some aspects, the naive V51+y5 T cells are CD73+CD127+and CXCRl’HLA-DR’. In some aspects, the naive V51+y5 T cells are CD27+CD127+CD73+and CXCRl’HLA-DR’.

[0071] The expression of one or more of CD27, CD127, CD73, CXCRl, and HLA-DR can be detected using any methods. In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined by detecting CD27, CD127, and / or CD73 protein. In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined by detecting CD27, CD127, and / or CD73 mRNA. In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined by using an immunohistochemical assay. Any immunohistochemical assay can be used. In some aspects, the presence of naive V51+y5 T cells in the donor blood sample is determined using flow cytometry.

[0072] In some aspects, the donor blood sample is identified as suitable if at least about 1% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, (i) sorting cells obtained from the donor blood sample based on expression of CD27; (ii) sorting the CD27+cells from (i) based on expression of CD 127; and (iii) sorting CD27+CD127+cells from (ii) based on expression of CD73.

[0073] In some aspects, the method comprises (i) sorting cells obtained from the donor blood sample based on expression of CD127; (ii) sorting the CD127+cells from (i) based on expression of CD27; and (iii) sorting CD27+CD127+cells from (ii) based on expression of CD73.

[0074] In some aspects, the method comprises further calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+triple-positive.

[0075] In some aspects, the method further comprises (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of CX3CR1; and (v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR1 '.

[0076] In some aspects, the method further comprises (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of HLA-DR; and (v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+HLA-DR".

[0077] In some aspects, the method further comprises (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of CX3CR1; (v) sorting the CD27+CD127+CD73+CX3CR1‘ cells from (iv) based on the expression of HLA-DR; and (vi) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR1 'HLA-DR-.

[0078] In some aspects, the method further comprises (iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of HLA-DR; (v) sorting the CD27+CD127+CD73+HLA-DR’ cells from (iv) based on the expression of CX3CR1; and (v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR1 'HLA-DR-.

[0079] In some aspects, the relative abundance is equivalent to the percent of the immune cells that are selected (z.e., naive V51+y5 T cells), out of the total number of immune cells present in the sample pre-sort.

[0080] In some aspects, the donor blood sample is identified as suitable if at least about 0.05% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 0.05%, at least about 0.08%, at least about 0.1%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 0.5% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 0.8% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 0.1% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 1% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 2% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 3% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 4% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 5% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 6% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 7% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 8% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 9% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 10% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 11% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 12% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 13% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 14% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable ifat least about 15% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 20% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 25% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 30% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 35% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 40% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 45% of immune cells in the donor blood sample are naive V51+y5 T cells. In some aspects, the donor blood sample is identified as suitable if at least about 50% of immune cells in the donor blood sample are naive V51+y5 T cells.

[0081] In some aspects, the human donor is screened for one or more additional aspects that are can improve the likelihood that the donor is suitable. In some aspects, the human donor is at least about 18 years old. In some aspects, the human donor is less than about 55 years old. In some aspects, the human donor is at least about 18 years old to about 55 years old. In some aspects, the human donor is about 18 years old to about 50 years old. In some aspects, the human donor is about 18 years old to about 49 years old. In some aspects, the human donor is about 18 years old to about 48 years old. In some aspects, the human donor is about 18 years old to about 47 years old. In some aspects, the human donor is about 18 years old to about 46 years old. In some aspects, the human donor is about 18 years old to about 45 years old. In some aspects, the human donor is about 18 years old to about 44 years old. In some aspects, the human donor is about 18 years old to about 43 years old. In some aspects, the human donor is about 18 years old to about 42 years old. In some aspects, the human donor is about 18 years old to about 41 years old. In some aspects, the human donor is about 18 years old to about 40 years old.

[0082] In some aspects, the human donor has a BMI of at least about 18 to about 35. In some aspects, the human donor has a BMI of at least about 18 to about 34, at least about 18 to about 33, at least about 18 to about 32, at least about 18 to about 31, at least about 18 to about 30, at least about 18 to about 29, at least about 18 to about 28, at least about 18 to about 27, at least about 18 to about 26, at least about 18 to about 25, at least about18 to about 24, at least about 18 to about 23, at least about 18 to about 22, at least about 18 to about 21, or at least about 18 to about 20. In some aspects, the human donor has a BMI of at least about 18 to about 35, at least about 19 to about 35, at least about 20 to about 35, at least about 21 to about 35 c, at least about 22 to about 35, at least about 23 to about 35, at least about 24 to about 35, at least about 25 to about 35, at least about 26 to about 35, at least about 27 to about 35, at least about 28 to about 35, at least about 29 to about 35, at least about 30 to about 35.

[0083] In some aspects, the human donor has a BMI of at least about 18. In some aspects, the human donor has a BMI of at least about 19. In some aspects, the human donor has a BMI of at least about 20. In some aspects, the human donor has a BMI of at least about 21. In some aspects, the human donor has a BMI of at least about 22. In some aspects, the human donor has a BMI of at least about 23. In some aspects, the human donor has a BMI of at least about 24. In some aspects, the human donor has a BMI of at least about 25. In some aspects, the human donor has a BMI of at least about 26. In some aspects, the human donor has a BMI of at least about 27. In some aspects, the human donor has a BMI of at least about 28. In some aspects, the human donor has a BMI of at least about 29. In some aspects, the human donor has a BMI of at least about 30. In some aspects, the human donor has a BMI of at least about 31. In some aspects, the human donor has a BMI of at least about 32. In some aspects, the human donor has a BMI of at least about 33. In some aspects, the human donor has a BMI of at least about 34. In some aspects, the human donor has a BMI of at least about 35.

[0084] In some aspects, the human donor has a body weight of at least about 125 lb. to about 200 lb. In some aspects, the human donor has a body weight of at least about 130 lb. to about 200 lb., at least about 135 lb. to about 200 lb., at least about 140 lb. to about 200 lb., at least about 145 lb. to about 200 lb., at least about 150 lb. to about 200 lb., at least about 155 lb. to about 200 lb., at least about 160 lb. to about 200 lb., at least about 165 lb. to about 200 lb., at least about 170 lb. to about 200 lb., at least about 175 lb. to about 200 lb., at least about 180 lb. to about 200 lb., at least about 180 lb. to about 200 lb., at least about 185 lb. to about 200 lb., or at least about 190 lb. to about 200 lb. In some aspects, the human donor has a body weight of at least about 125 lb. to about 195 lb., at least about 125 lb. to about 190 lb., at least about 125 lb. to about 185 lb., at least about 125 lb. to about 180 lb., at least about 125 lb. to about 175 lb., at least about 125 lb. to about 170 lb., at least about 125 lb. to about 165 lb., at least about 125 lb. to about 160lb., at least about 125 lb. to about 155 lb., at least about 125 lb. to about 150 lb., at least about 125 lb. to about 145 lb., at least about 125 lb. to about 140 lb., at least about 125 lb. to about 135 lb., at least about 125 lb. to about 130 lb.

[0085] In some aspects, the human donor has a body weight of at least about 125 lb. In some aspects, the human donor has a body weight of at least about 130 lb. In some aspects, the human donor has a body weight of at least about 135 lb. In some aspects, the human donor has a body weight of at least about 140 lb. In some aspects, the human donor has a body weight of at least about 145 lb. In some aspects, the human donor has a body weight of at least about 150 lb. In some aspects, the human donor has a body weight of at least about 155 lb. In some aspects, the human donor has a body weight of at least about 160 lb. In some aspects, the human donor has a body weight of at least about 165 lb. In some aspects, the human donor has a body weight of at least about 170 lb. In some aspects, the human donor has a body weight of at least about 175 lb. In some aspects, the human donor has a body weight of at least about 180 lb. In some aspects, the human donor has a body weight of at least about 185 lb. In some aspects, the human donor has a body weight of at least about 190 lb. In some aspects, the human donor has a body weight of at least about 195 lb. In some aspects, the human donor has a body weight of at least about 200 lb.

[0086] In some aspects, the suitable human donor is a healthy human. In some aspects, the human donor has not been diagnosed with a cancer. In some aspects, the human donor has not been diagnosed with an infectious disease. In some aspects, the human donor has not been diagnosed with an autoimmune disease. In some aspects, the human donor has not been diagnosed with a metabolic disease.II.B. Harvesting and Expansion y6 T Cells

[0087] In some aspects, y5 T cells, e.g., naive V51+y5 T cells, are obtained from the subject that is identified as being a suitable donor, according to the methods disclosed herein. As such, some aspects of the present disclosure are directed to methods of expanding a population of y5 T cells for an allogenic y5 T cell-derived therapy, comprising (i) identifying a donor sample suitable for obtaining a population of V51+y5 T cells according to a method disclosed herein, (ii) isolating y5 T cells from the donor sample identified as suitable, and (iii) expanding the y5 T cells.

[0088] In some aspects, the y5 T cells, e.g., naive V51+y5 T cells, are then expanded in the presence of a cytokine, such as IL- 15, IL-2, or in the absence of exogenous IL-2 or IL-15. In some aspects, the y5 T cells, e.g., naive V51+y5 T cells, are expanded in the presence of exogenous IL-15. In some aspect, the y5 T cells, e.g., naive V51+y5 T cells, express secreted IL- 15 and do not require exogenous IL- 15 for expansion. In some aspects, the method includes expanding the population of y5 T cells, e.g., naive V51+y5 T cells, in the presence of exogenous IL-2.

[0089] In some aspects, the methods further comprise separating and expanding V51 cells from blood. Methods of isolating and expanding V51 cells from blood include those described, for example, in U.S. Patent No. 9,499,788, International Patent Publication No. WO 2016 / 198480, U.S. Patent No. 11,166,983, and U.K. Patent Application No. 2204926.6, each of which is incorporated herein by reference in its entirety.

[0090] In some aspects, peripheral blood mononuclear cells (PBMCs) can be obtained from a donor subject according to any suitable method. PBMCs can be cultured in the presence of aminobisphosphonates (e.g., zoledronic acid), synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1PP, or 2-methyl-3-butenyl-l- pyrophosphate in the presence of IL-2 for one-to-two weeks to generate an enriched population of V51+cells. Alternatively, immobilized anti-TCRyS (e.g., pan TCRyS) can induce preferential expansion of V52 cells from a population of PBMCs in the presence of IL-2, e.g., for approximately 14 days. In some aspects, preferential expansion of V51+cells from PBMCs can be achieved upon culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2 and IL-4. In some aspects, the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, IL-2, and IL-4. Alternatively, artificial antigen presenting cells can be used to promote preferential expansion of y5 T cells, such as V51+cells. For example, PBMC-derived y5 T cells cultured in the presence of irradiated aAPC, IL-2, and / or IL-21 can expand to generate a population of y5 T cells including a high proportion of V51+cells, moderate proportion of V51 cells, and some double negative cells. In some aspects of the aforementioned methods, PBMCs can be pre-enriched or post-enriched (e.g., through positive selection with TCRyS-specific agents or negative selection of TCRaP-specific agents). Such methods and other suitable methods for expansion of y5 T cells are described in detail, for example, by Deniger et al., Frontiers in Immunology 5, 636: 1-10, 2014, which is incorporated herein by reference in its entirety.

[0091] In some aspects, V51 T cells can be engineered to express a transgene e.g., a heterologous targeting construct). Any suitable method of obtaining a population of V51 T cells can be used. For example, Almeida et al. (Clinical Cancer Research, 22, 23; 5795- 5805, 2016), incorporated herein by reference in its entirety, provides suitable methods of obtaining a population of V51 T cells that can be engineered to express a heterologous targeting construct described herein. For example, in some aspects, PBMCs are preenriched using magnetic bead sorting, which can yield greater than 90% y5 T cells. These cells can be cultured in the presence of one or more factors (e.g., TCR agonists, coreceptor agonists, and / or cytokines, e.g., IL-4, IL-15, and / or IFN-y) in gas-permeable bioreactor bags for up to 21 days or more. Variations of this method, and other methods of obtaining V51 T cells are suitable as part of the present disclosure. For example, blood derived V51 T cells can alternatively be obtained using methods described, for example, in U.S. Patent No. 9,499,788, U.S. Patent No. 11,166,983, and International Patent Publication No. WO 2016 / 198480, each of which is incorporated herein by reference in its entirety.II.C. yd T cells

[0092] The method disclosed herein provide a means of identifying human subject and / or blood samples obtained from a human subject that are suitable for obtaining y5 T cells for an allogeneic immunotherapy. As such, some aspects of the present disclosure are directed to a population of y5 T cells obtained by a method disclosed herein.

[0093] y5 T cells represent a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 T cells can be broadly classified as one or two types — peripheral blood-resident y5 T cells and tissue-resident y5 T cells. Most blood-resident y5 T cells express a V52 TCR, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 and / or other V5 chains.

[0094] The y5 T cells described herein may lack a vesicular stomatitis virus G glycoprotein (VSV-G) entry receptor (e.g., LDL). The y5 T cell (e.g., endogenous y5 T cells or primed y5 T cells) may express ASCT-1 and / or ASCT-2. The expression of ASCT-1 and / or ASCT-2 may permit transduction with a betaretroviral pseudotyped vector (e.g., BaEV and RD114). The lack of expression of VSV-G may prevent transduction with a VSV-G pseudotyped vector.

[0095] In some aspects, the y5 T cell is transduced with a viral vector encoding a transgene (e.g, a CAR). In some aspects, the viral vector is a retroviral vector. In some aspects, the viral vector is a lentiviral vector. In some such aspects, the cell may stably express the transgene. In some aspects, the cell may transiently express the transgene.

[0096] In some aspects, the y5 T cells are modified to reduce the immunogenicity of the y5 T cells.ILD. Methods of Treatment

[0097] In some aspects of the present disclosure, y5 T cells, e.g, naive V51+y5 T cells, are obtained from a suitable human donor (identified according to the methods disclosed herein), expanded, and administered to a subject in need thereof, wherein the patient is not the suitable human donor. In some aspects, the subject is afflicted with a disease or condition. As such, some aspects of the present disclosure are directed to methods of treating a subject in need thereof, comprising (i) identifying a human donor sample suitable for obtaining a population of V51+y5 T cells according to a method disclosed herein, (ii) isolating y5 T cells from the donor sample identified as suitable, (iii) expanding the y5 T cells, and (iii) administering the y5 T cells to the subject.

[0098] In some aspects, the disease or condition comprises a cancer, e.g., the subject is afflicted with a cancer. Generally, the growth and / or life span of a cancer cell exceeds, and is not coordinated with, that of the normal cells and tissues around it. Cancers may be benign, pre-malignant or malignant. Cancer occurs in a variety of cells and tissues, including the oral cavity (e.g. mouth, tongue, pharynx, etc.), digestive system (e.g. esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gall bladder, pancreas, etc.), respiratory system (e.g. larynx, lung, bronchus, etc.), bones, joints, skin (e.g. basal cell, squamous cell, meningioma, etc.), breast, genital system, (e.g. uterus, ovary, prostate, testis, etc.), urinary system (e.g. bladder, kidney, ureter, etc.), eye, nervous system (e.g. brain, etc.), endocrine system (e.g. thyroid, etc.), and haematopoietic system (e.g. lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.). In some aspects, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML) (e.g., relapsed or refractory AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia (ALL), chronic myelogenous leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is relapsed. In some aspects, the cancer is refractory or relapsed following one or more prior anti-cancer therapy. In some aspects, the one or more prior anti-cancer therapies comprise a standard of care therapy.

[0099] In some aspects, the y5 T cells, e.g., naive V51+y5 T cells, are administered in combination with an additional anti-cancer therapy. In some aspects, the additional anticancer therapy comprises a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof. In some aspects, the additional anti-cancer therapy comprises a chemotherapy. In some aspects, the additional anti-cancer therapy comprises an immune-checkpoint inhibitor. In some aspects, the additional anti-cancer therapy comprises a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR antagonist, or any combination thereof. In some aspects, the anticancer therapy comprises an antibody or antigen-binding portion thereof the specifically binds and inhibits PD-1 (e.g., pembrolizumab or nivolumab). In some aspects, the anticancer therapy comprises an antibody or antigen-binding portion thereof the specifically binds and inhibits PD-L1 (e.g., atezolizumab, avelumab, or durvalumab).

[0100] In some aspects, the method further comprises pretreating the subject prior to administering the population of immune cells. In some aspects, the subject is administered a chemotherapy prior to administering the population of immune cells. In some aspects, the subject is administered an immuno-depleting chemotherapy prior toadministering the population of immune cells. In some aspects, the immuno-depleting chemotherapy comprises cyclophosphamide, fludarabine, or both.

[0101] In some aspects, the method comprises administering to the subject (i) y5 T cells, e.g., naive V51+y5 T cells, disclosed herein and (ii) a cytokine. In some aspects, the cytokine comprises IL-2, an analog thereof, a variant thereof, or a fragment thereof.II.E. Chimeric Antigen Receptors

[0102] In some aspects, the y5 T cells obtained from the human donor blood sample are genetically modified prior to administration to a subject. In some aspects, the y5 T cells are modified to express a chimeric antigen receptor (CAR). In some aspects, the CAR comprises an antigen-binding domain that specifically binds a tumor antigen. As such, some aspects of the present disclosure are directed to a population of y5 T cells (e.g., a population of V51+ y5 cells derived from blood) comprising y5 T cells comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigenbinding domain that specifically binds a tumor antigen.

[0103] In some aspects, the tumor antigen comprises CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-llRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY- ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE2, claudin (e.g., CLDN18.2), B7H3 or any combination thereof.

[0104] In some aspects, the CAR comprises an antigen-binding domain that specifically binds mesothelin, e.g, human mesothelin. In some aspects, the population of y5 T cells (e.g, a population of V51+ y5 cells derived from blood) comprises y5 T cells comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigenbinding domain that specifically binds human mesothelin.

[0105] In some aspects, the CAR further comprises a 4- IBB and CD3 zeta domain. In some aspects, the y5 T cells comprise a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds a tumor antigen, e.g., human mesothelin, and further comprise a 4- IBB and CD3 zeta domain. In some aspects, the CAR further comprises a 4- IBB and CD3 zeta domain.

[0106] In some aspects, the CAR further comprises a hinge region between the antigenbinding domain and the transmembrane domain. In some aspects, the hinge is derived from an immunoglobulin (e.g., derived from hinge regions or loop regions). In certain aspects, these hinges comprise, e.g., IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge regions, fragments thereof (alone or capped by additional sequences, e.g., CHI or CH2 regions sequences), or combinations of fragments from IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge regions. In some aspects, the hinges comprise, e.g., IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM constant domain loop regions, fragments thereof (alone or capped by additional sequences, e.g., from adjacent P- strands), or combinations of fragments from IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM loop regions. In some aspects, the hinge of the present disclosure comprises hinge region derived sequences, loop region derived sequences, or combinations thereof.

[0107] In some aspects, the hinge region is selected from a CD8 hinge, a CD28 hinge, and an immunoglobulin hinge. In some aspects, the hinge comprises a CD28 hinge.

[0108] In some aspects, the CAR further comprises a transmembrane domain. The transmembrane domain can be derived either from a natural or from a recombinant source. Where the source is natural, the domain can be derived from any membranebound or transmembrane protein. In some aspects, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR of the present disclosure has bound to a target.

[0109] In some aspects, a transmembrane domain can include at least the transmembrane region(s) of, e.g., CD8, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55),PSGL1, CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, NKG2C, or CD 19.

[0110] In some aspects, the TM domain is derived from CD8, CD2, CD4, CD28, CD45, PD1, CD 152, or any combination thereof.[OHl] In some aspects, the CAR further comprises a costimulatory domain. In some aspects, the costimulatory domain comprises a costimulatory domain of 4-1BB / CD137, interleukin-2 receptor (IL-2R), interleukin- 12 receptor (IL-12R), IL-7, IL-21, IL-23, IL- 15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP10, B7-H3, CD28 deleted for Lek binding (ICA), BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor 8 chain, a ligand that specifically binds with CD83, or any combination thereof. In some aspects, the CAR comprises a 4- IBB costimulatory domain.

[0112] In some aspects, the CAR further comprises an intracellular signaling domain. In some aspects, the intracellular signaling domain comprises a CD3 C, activating domain, a CD35 activating domain, a CD3s activating domain, a CD3r| activating domain, a CD79A activating domain, a DAP 12 activating domain, a FCER1G activating domain, a DAP10 / CD28 activating domain, a ZAP70 activating domain, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 C, activating domain.

[0113] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-FV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986));Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2ndEd. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0114] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0115] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1

[0116] To test the utility of a fluorescence standard based solely on CD27 expression, calibration beads (Rainbow Beads; Biolegend #422903) were used to demarcate CD27hiV81 cells from the rest of the population (FIG. 1 A). Fifth peak of the beads in the FITC channel serves as the threshold for the identification of naive V81 cells. Allogenic cellular drug substances must be substantially depleted of ocp T cells, which are depleted at the start of the manufacturing process. The bead-based method estimates the frequency of naive V81 cells in the starting material that remains after ocp T cell depletion. This beadbased method was termed "Donor Screening Protocol (DSP)."

[0117] DSP may have challenges with respect to its use as a method for identifying the target population. First, the method uses a non-biological standard to measure the fluorescence of cells. Second, the method is based on a single parameter: the expression level of CD27. As such, this method assumes the CD27hiV8 l population to be homogenous. Finally, this method applies a "one size fits all" approach by assuming that every naive V81 cell in every donor is at least as bright as the fifth peak of the calibration beads in the FITC channel.

[0118] An independent method that simultaneously assesses multiple naive and activation markers was designed to address the potential shortcomings of DSP. A comprehensive surface phenotyping was performed of V81 cells to encompass known surface markers. First, a comparison was performed between V81 cells from donors containing high (good donors) or low (bad donors) frequencies of CD27111V81 cells in their blood. When the phenotype of pan V81 cells was interrogated, CX3CR1 and HLA-DR were observed to be enriched in bad donors as compared with good donors (FIG. 2A). When the focus was narrowed to compare CD27111V81 cells with CD2710V81 cells, CD 127 and CD73 were found to be enriched in the CD27111subset of V81 cells (FIG. 2B).

[0119] These data were used to develop a naiveness determining screening protocol (NDSP), which utilizes a novel expanded definition of naive V81 cells: CD27+CD127+CD73+CX3CR1'HLA'DR".

[0120] All markers of the NDSP signature show polar expression in relation to CD27.CD127 and CD73 that are enriched in CD27hiV81 cells, and CX3CR1 and HLA-DR are enriched in CD27lo / " subsets (FIG. 3 A). This expression pattern was broadly true in a larger cohort of donors screened (FIG. 3B).

[0121] An example of the NDSP strategy is depicted in FIGs. 4A-4D. First, V81 cells are resolved based on the expression of CD27 (FIG. 4A), which is the foundational naive marker. Next, cells that are double positive for CD127 and CD27 are identified (FIG. 4B). The next step resolved the cells based on the expression of CD73 within the CD27+CD127+subset (FIG. 4C). The expression of two markers, CX3CR1 and HLA-DR, within the triple positive naive subset (CD27+CD127+CD73+) cannot be ruled out. The final step identifies true naive V81 cells by eliminating those with the expression of the two activation markers (FIG. 4D).

[0122] The rigid DSP and more flexible NDSP mostly concur with each other when estimating the frequency of naive V81 cells in the starting material (FIG. 5A) and when measuring the expansion of the transduced (TD) or un-transduced (UTD) cells (FIGs. 5B- 5E). However, the addition of more markers to identify V81 cells can be beneficial. DSP assumes that naive V81 cells in every donor are universally at least as bright as the fifth bead peak. That may not always be the case (FIG. 5F-5G). As is evident, V81 cells can display a naive phenotype while being dimmer than the fifth bead peak in the CD27 channel. Conversely, DSP considers the CD27111population to be homogeneous. Thispresumption may be incorrect, as CD27111Vbl cells can express CX3CR1 and HLA-DR (FIGs. 5H-5I), which indicates that such cells are effector cells (z.e., activated) and not naive cells.

[0123] Further analysis of the cells revealed that the NDSP had a lower false negative rate than the DSP; however, both the DSP and the NDSP were observed to have both false negative and false positive results (FIGs. 6A-6D).

[0124] These data illustrate the flexibility of the NDSP method described herein, which results from the multi -marker qualitative approach (as opposed to DSP’s rigid singlemarker, fixed MFI approach). The methods disclosed herein thus provide an improved, consistent, and more accurate means of evaluating whether a human donor blood sample is a suitable source of V51+y5 T cells that can be used, for example, in various cell therapies.OTHER ASPECTS

[0125] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0126] While the disclosure has been described in connection with specific aspects thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0127] Other aspects are within the claims.

Claims

CLAIMS1. A method of identifying a donor blood sample suitable for obtaining a population of V51+gamma delta (y5) T cells, the method comprising assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express (i) CD27, (ii) CD 127, (iii) CD73, or any combination thereof.

2. A method of identifying a human donor that is suitable for obtaining a population of V51+gamma delta (y5) T cells, the method comprising obtaining a donor blood sample from the human donor, and assaying for the presence of naive V51+y5 T cells in the donor blood sample by detecting the presence of cells that express (i) CD27, (ii) CD127, (iii) CD73, or any combination thereof.

3. The method of claim 1 or 2, wherein the naive V51+y5 T cells are (i) CD27 and (ii) CD 127, CD73, or both.

4. The method of claim 1 or 2, wherein the naive V51+y5 T cells are CD27+CD127+.

5. The method of claim 1 or 2, wherein the naive V51+y5 T cells are CD27+CD73+.

6. The method of claim 1 or 2, wherein the naive V51+y5 T cells are CD73+CD127+.

7. The method of any one of claims 1 to 6, wherein the naive V51+y5 T cells areCD27+CD127+CD73+.

8. The method of any one of claims 1 to 7, wherein the naive V51+y5 T cells do not express CX3CR1.

9. The method of any one of claims 1 to 8, wherein the naive V51+y5 T cells do not express HLA-DR.

10. The method of any one of claims 1 to 9, wherein the naive V51+y5 T cells do not express CX3CR1 and do not express HLA-DR.

11. The method of any one of claims 1 to 10, wherein the presence of naive V51+y5 T cells in the donor blood sample is determined by detecting CD27, CD127, and / or CD73 protein; CD27, CD127, and / or CD73 mRNA; or any combination thereof.

12. The method of any one of claims 1 to 11, wherein the presence of naive V51+y5 T cells in the donor blood sample is determined by using an immunohistochemical assay.

13. The method of any one of claims 1 to 12, wherein the presence of naive V51+y5 T cells in the donor blood sample is determined using flow cytometry.

14. The method of any one of claims 1 to 13, comprising:(i) sorting cells obtained from the donor blood sample based on expression of CD27;(ii) sorting the CD27+cells from (i) based on expression of CD127; and(iii) sorting CD27+CD127+cells from (ii) based on expression of CD73.

15. The method of any one of claims 1 to 13, comprising:(i) sorting cells obtained from the donor blood sample based on expression of CD127;(ii) sorting the CD127+cells from (i) based on expression of CD27; and(iii) sorting CD27+CD127+cells from (ii) based on expression of CD73.

16. The method of claim 14 or 15, further comprising calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+triple-positive.

17. The method of any one of claims 14 to 16, further comprising:(iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of CX3CR1; and(v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+CX3CR1’ .

18. The method of any one of claims 14 to 16, further comprising:(iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of HLA-DR; and(v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD127+CD73+HLA-DR'.

19. The method of any one of claims 14 to 16, further comprising:(iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of CX3CRl;(v) sorting the CD27+CD127+CD73+CX3CRL cells from (iv) based on the expression of HLA-DR; and(vi) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR 1 'HLA-DR'.

20. The method of any one of claims 14 to 16, further comprising:(iv) sorting the CD27+CD127+CD73+cells from (iii) based on the expression of HLA-DR;(v) sorting the CD27+CD127+CD73+HLA-DR' cells from (iv) based on the expression of CX3CR1; and(v) calculating the relative abundance of cells in the donor blood sample that are CD27+CD 127+CD73+CX3 CR 1 'HLA-DR'.

21. The method of any one of claims 1 to 20, wherein the donor blood sample is identified as suitable if at least about 0.05%, at least about 0.08%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of immune cells in the donor blood sample are naive V51+y5 T cells.

22. The method of any one of claims 1 and 3 to 21, wherein the donor blood sample is obtained from a human donor.

23. The method of any one of claims 2 to 22, wherein the human donor is at least about 18 years old to about 55 years old.

24. The method of any one of claims 2 to 23, wherein the human donor has a BMI of at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at leastabout 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35.

25. The method of any one of claims 2 to 24, wherein the human donor has a BMI of at least about 24.

26. The method of any one of claims 2 to 25, wherein the human donor has a body weight of at least about 150 lb., at least about 155 lb., at least about 160 lb., at least about 165 lb., at least about 170 lb., at least about 175 lb., at least about 180 lb., at least about 185 lb., at least about 190 lb., at least about 195 lb., or at least about 200 lb.

27. The method of any one of claims 2 to 26, wherein the human donor has a body weight of at least about 180 lb.

28. The method of any one of claims 2 to 27, wherein the human donor is a healthy human.

29. The method of any one of claims 2 to 27, wherein the human donor has not been diagnosed with a cancer, an infectious disease, an autoimmune disease, a metabolic disease, or any combination thereof.

30. The method of any one of claims 1 to 29, further comprising isolating V51+y5 T cells from a donor sample identified as suitable.

31. The method of claim 30, wherein the V51+y5 T cells comprise naive V51+ y5 T cells.

32. The method of claim 30 or 31, wherein the isolated V51+y5 T cells are expanded ex vivo.

33. The method of claim 32, wherein the expanded V51+y5 T cells are administered to a subject.

34. A method of expanding a population of y5 T cells for an allogenic y5 T cell-derived therapy, comprising (i) identifying a donor sample suitable for obtaining a population of V51+y5 T cells according to the method of any one of claims 1 to 29, (ii) isolating y5 T cells from the donor sample identified as suitable, and (iii) expanding the y5 T cells.

35. The method of claim 34, wherein the isolated V51+y5 T cells comprise naive V51+ y5 T cells.

36. A population of allogenic y5 T cells obtained by the method of any one of claims 30 to 35.

37. A method of treating a subject in need thereof, comprising administering the population of allogenic y5 T cells of claim 36 to the subject.

38. A method of treating a subject in need thereof, comprising (i) identifying a human donor sample suitable for obtaining a population of V51+y5 T cells according to the method of any one of claims 1 to 29, (ii) isolating y5 T cells from the donor sample identified as suitable, (iii) expanding the y5 T cells, and (iii) administering the y5 T cells to the subject.

39. The method of claim 38, wherein the isolated V51+y5 T cells comprise naive V51+ y5 T cells.

40. The method of any one of claims 37 to 39, wherein the y5 T cells are modified prior to administration to the subject.

41. The method of claim 40, wherein the y5 T cells are genetically modified.

42. The method of claim 40 or 41, wherein the y5 T cells are modified to express a chimeric antigen receptor (CAR).

43. The method of claim 42, wherein the CAR comprises an antigen-binding domain that specifically binds a tumor antigen.

44. The method of claim 43, wherein the CAR comprises an antigen-binding domain that specifically binds mesothelin.

45. The method of any one of claims 42 to 44, wherein the y5 T cells are modified to reduce the immunogenicity of the y5 T cells.

Images