Compositions for and methods of expanding γδ t cells

Abstract

Disclosed herein are methods of making and using an expanded population of γδ T cells. Disclosed herein is an allogeneic γδ CAR T-cell therapy.

Description

COMPOSITIONS FOR AND METHODS OF EXPANDING γδ T CELLS I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 642,849 filed 5 May 2024 and to U.S. Provisional Application No.63 / 472,896 filed 14 June 2023, each of which is incorporated herein in its entirety. II. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under CA124515 and CA278907 awarded by the National Institutes of Health. The government has certain rights in the invention. III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted 14 June 2024 as an XML file named "24-3014-WO_ Sequence Listing", created on 14 June 2024 and having a size of 19 kB is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). IV. BACKGROUND

[0004] γδ T-cells directly recognize and kill transformed cells independently of HLA-antigen presentation, which makes them a highly promising effector cell compartment for cancer immunotherapy. The clinical implementation of novel γδ T-cell-based immunotherapies rests on the ability to generate populations of purified γδ T cell populations.

[0005] Thus, there remains a need for the rapid expansion of γδ T cells into purified populations of specific subsets of γδ T cells. The compositions and methods disclosed herein meet this need. V. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG.1A – FIG.1B show the prevalence of γδ T cells in peripheral blood and umbilical cord blood. FIG. 1A shows that in human peripheral blood from adults, γδ T cells are predominantly expressing the delta-2 TCR chain (Vδ2) while the delta-3 TCR chain (Vδ3) (Vd1negVd2neg) cells are almost absent, and the delta-1 TCR chain (Vδ1) is in low proportions. FIG.1B shows that in contrast, in umbilical cord blood, γδ T cells are predominantly expressing the delta-1 and delta-3 TCR chains (Vδ1; Vδ3) while Vδ2 T cells are at very low percent.

[0007] FIG.2A – FIG.2B show the analysis by iRepertoire of FACS-Sorted Vd1 cell subsets. FIG.2A shows that hPBL had a reduced usage of TCR gamma chains. FIG.2B shows that in contrast, UCB samples showed a wide usage of TCR gamma chains that recapitulates γδ T cells at tumor beds, which indicates UCB is a good supplier for Vd1 and Vd3 γδ T cells for clinical trials.

[0008] FIG.3 provides a schematic of the optimized protocol for expansion and transduction of human Vd1 / Vd3 γδ T cells from umbilical cord blood (UCB). This optimized expansion protocol recapitulated proportions of γδ lymphocyte subsets found at tumor beds with features of effector activity and stemness.

[0009] FIG. 4 shows γδ T cells expanded from UCB after two weeks were enriched in T cells expressing the delta-1 and delta-3 chains (Vδ1 / Vδ3).

[0010] FIG. 5 shows γδ T cells expanded from UCB after two weeks by a modified rapid expansion protocol did not show significant changes in the gamma chain TCR repertoire usage and kept a wide repertoire of gamma TCR chains.

[0011] FIG.6A shows that γδ T cells expanded from UCB were enriched in T cells expressing the TCR delta-3 chain (Vd1negVd2neg), while FIG.6B shows that the total fractions of expanded γδ T cells had efficient cytotoxic activity of the ovarian tumor cell line A2780 (killing up to 100% of the cancer cells).

[0012] FIG.7A shows that γδ T cells from UCB, in contrast to hPBL, showed a high proportion of Vd1 and Vd3 T cells subsets, while FIG.7B shows that γδ T cells expanded from UCB showed high proportions of transduced cells measured by the GFP reporter.

[0013] FIG.8 shows that γδ T cells expanded from UCB had expression of markers for effector tumor activity, thereby making these cells suitable candidates for CAR T-cell anti-tumor therapies. This expression was observed in the (Vd1Vg) expressing the δ1 chain and the Vd1negVD2neghighly enriched in the δ3 chain.

[0014] FIG. 9A - FIG. 9B shows that γδ T cells expanded from UCB had high expression of markers for stemness (FIG. 9A) and low T cell exhaustion (FIG.9B). This expression was observed in the (Vd1Vg) expressing the δ1 chain and the Vd1negVD2neghighly enriched in the δ3 chain.

[0015] FIG.10 shows that γδ T cells expanded from UCB showed high expression of NK markers associated with anti-tumor cytotoxic activity. This expression was observed in the (Vd1Vg) expressing the δ1 chain and the Vd1negVD2neghighly enriched in the δ3 chain.

[0016] FIG. 11 shows γδ CAR-T cells targeting TAS2R13 show superior cytotoxic activity compared with αß CAR-T cells. Here, the γδ CAR-T cells were generated using the disclosed rapid expansion protocol. VI. BRIEF SUMMARY

[0017] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T cells expanded by a disclosed method. Disclosed herein is a method of treating cancer,the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of CAR γδ T-cells expanded by a disclosed method.

[0018] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 CAR γδ T- cells and / or Vδ3 CAR γδ T-cells.

[0019] Disclosed herein is a method of treating cancer, the method comprising administering to the subject having cancer a therapeutically effective amount of a population of γδ T-cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising administering to a subject having cancer a therapeutically effective amount of a population of CAR γδ T-cells expanded by a disclosed method.

[0020] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T-cells expanded by a disclosed method, wherein the expanded population of CAR γδ T- cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of CAR γδ T-cells expanded by a disclosed method, wherein the expanded population of CAR γδ T-cells predominantly comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0021] Disclosed herein is a method of treating cancer, the method comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a population of γδ T cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method.

[0022] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising treating a subjectin need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method.

[0023] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0024] Disclosed herein is a method of treating cancer, the method comprising administering to a subject having cancer a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T-cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising administering to a subject having cancer a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method.

[0025] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T-cells expanded by a disclosed method, wherein the expanded population of γδ T-cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method, wherein the expanded population of CAR γδ T-cells predominantly comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0026] Disclosed herein is a population γδ T cells expanded by a disclosed method. Disclosed herein is a population of CAR γδ T-cells expanded by a disclosed method. Disclosed herein is a population of Vδ1 γδ T cells and / or Vδ3 γδ T-cells expanded by a disclosed method. Disclosed herein is a population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells expanded by a disclosed method. Disclosed herein is a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells.VII. DETAILED DESCRIPTION

[0027] The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and / or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0028] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. A. Definitions

[0029] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0030] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0031] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0032] The phrase "consisting essentially of" limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase "consisting of" excludes any component, step, or element that is not recited in the claim. The phrase "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended. "Comprising" does not exclude additional, unrecited components or steps.

[0033] As used herein, when referring to any numerical value, the term "about" means a value falling within a range that is ± 10% of the stated value.

[0034] Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0035] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0036] As used herein, the terms "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0037] As used herein, the term "subject" refers to the target of administration, e.g., a human being. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have cancer, be suspected of having cancer, or be at risk of developing cancer.

[0038] In an aspect, the term "diagnosed" means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed expanded populations of Vδ1 γδ T-cells and / or Vδ3 γδ T- cells (expanded by a disclosed method) and / or the disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, "diagnosed with a disease or disorder" means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as cancer) that can be treated by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, the disclosed expanded populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, "suspected of having a disease or disorder" can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as cancer) that can likely be treated by one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, the disclosed expanded populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.), scans (e.g., CT scans, PET scans, etc.), and assays (e.g., enzymatic assay), or a combination thereof.

[0039] A "patient" refers to a subject afflicted with a disease or disorder (e.g., cancer). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder such as cancer. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder (such as a cancer).

[0040] As used herein, the phrase "identified to be in need of treatment for a disease or disorder," or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., cancer) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the cancer. In an aspect, the identification can be performed by a person differentfrom the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0041] As used herein, "activated" and "activation" can refer 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" can refer to T cells that are proliferating. Signals generated through the TCR alone may be insufficient for full activation of the T cell and one or more secondary or costimulatory signals may also be required. Thus, T cell activation comprises a primary stimulation signal through the TCR / CD3 complex and one or more secondary costimulatory signals. Co-stimulation can be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0042] As used herein, "inhibit," "inhibiting", and "inhibition" mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not receiving the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, or 90-100% as compared to a native or control level (e.g., a subject not receiving the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed expanded populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof). In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level (such as a pre-cancer state).

[0043] The words "treat" or "treating" or "treatment" include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathologicalcondition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having cancer). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as cancer). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having cancer). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder (such as cancer).

[0044] As used herein, the term "prevent" or "preventing" or "prevention" refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and / or chromatin dysregulation is intended. The words "prevent", "preventing", and "prevention" also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as cancer) or related complication from progressing to that complication. In an aspect, preventing metastasis is intended.

[0045] As used herein, the terms "administering" and "administration" refer to any method of providing one or more of the disclosed interfering molecules, the disclosed anti-PD1 molecules, the disclosed pharmaceutical formulations, or a combination thereof to a subject. Such methodsare well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intratumoral administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra- arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed composition, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, a disclosed RNA therapeutic, or any combination thereof can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more routes.

[0046] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), disclosed pharmaceutical formulations, or a combination thereof to treat or prevent a disease or disorder (such as cancer). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof.

[0047] By "determining the amount" is meant both an absolute quantification of a particular analyte (e.g., biomarker for cancer, for example) or a determination of the relative abundance of a particular analyte (e.g., a cancer biomarker). The phrase includes both direct or indirect measurements of abundance or both.

[0048] As used herein, "modifying the method" can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. In an aspect, a method can be altered by changing the amount of the disclosed isolated nucleic acid molecules, disclosed vectors,disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof to a subject, by changing the duration of time that the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof is administered to a subject, or by substituting for one or more of the disclosed components and / or reagents with a similar or equivalent component and / or reagent. The same applies to all disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or combinations thereof.

[0049] A "transposon" is a mobile genetic element that efficiently moves between vectors and chromosomes using the "cut and paste" or "copy and paste" mechanism. During transposase transposition (for example, PB transposase in the PiggyBac transposon system) recognizes transposon-specific sequences of inverted terminal repeats (ITRs) located at both ends of the transposon (there are 5'- and 3'-ITRs in any transposon system), it moves the contents from source sites and embeds them in chromosomal sites, such as TTAA chromosomal sites. In an aspect, the powerful activity of the PiggyBac transposon system makes it easy to transfer genes of interest located between two ITRs to target genomes. The transposon can be divided into Class I transposon (retrotransposon) and Class II transposon (DNA transposon). In Class I transposon, after RNA is transcribed from nucleic acid in a cell or from transposon DNA on the animal genome, the DNA reverse-transcribed from the RNA is transferred to another location on the animal genome. It works by inserting it. Class II transposon cuts nucleic acid in cells or transposon DNA on the animal genome, and then inserts the cut transposon DNA into another location on the animal genome. The Class II transposon may include a first polynucleotide at a 5' end, a second polynucleotide at a 3' end, and a third polynucleotide. The first polynucleotide and the second polynucleotide may include an inverted terminal repeat (ITR) sequence. The third polynucleotidemay be located between the first polynucleotide and the second polynucleotide. The third polynucleotide may include an exo-polynucleotide. The third polynucleotide may include a polynucleotide encoding a transposase. Unless otherwise specified below, the term "transposon" assumes the case of Class II transposon, but even if the term "transposon" is interpreted as Class I transposon, it is technically If there is no problem, it will not be necessary to limit the interpretation to Class II transposon.

[0050] An "enriched" cell population or preparation refers to a cell population derived from a starting mixed cell population that contains a greater percentage of a specific cell type than the percentage of that cell type in the starting population. For example, a starting mixed cell population can be enriched for a specific γδ T cell population. In an aspect, the enriched γδ T cell population contains a greater percentage of Vδ1 T cells and / or Vδ3 T cells than the percentage of that cell type in the starting population. For example, an enriched γδ T cell population can contain a greater percentage of both Vδ1 T cells and / or a greater percentage of Vδ3 T cells than the percentage of that cell type in the starting population. For example, an enriched γδ T cell population can contain a greater percentage of both Vδ1 T cells and / or a greater percentage of Vδ3 T cells than the percentage of that cell type in the starting population. In an aspect, the enriched γδ CAR T-cell population contains a greater percentage of Vδ1 CAR T-cells and / or Vδ3 CAR T-cells than the percentage of that cell type in the starting population. For example, an enriched γδ CAR T-cell population can contain a greater percentage of both Vδ1 CAR T-cells and / or a greater percentage of Vδ3 CAR T-cells than the percentage of that cell type in the starting population. For example, an enriched γδ CAR T-cell population can contain a greater percentage of both Vδ1 CAR T-cells and / or a greater percentage of Vδ3 CAR T-cells than the percentage of that cell type in the starting population. In an aspect, the enriched γδ T-cell population can contain a lesser percentage of αβ T-cell populations.

[0051] In an aspect, "expanded" refers to the number of the desired or target cell type (e.g., Vδ1 T cells and / or Vδ3 T cells or Vδ1 CAR T-cells and / or Vδ3 CAR T-cells) in the enriched preparation is higher than the number in the initial or starting cell population. In an aspect, "selectively expand" can mean that the target cell type or cell types (e.g., Vδ1 T cells and / or Vδ3 T cells and / or Vδ3 T-cells or or Vδ1 CAR T-cells and / or Vδ3 CAR T-cells) are preferentially expanded over other non-target cell types (e.g., αβ T-cells or NK cells). In an aspect, the activating agents of the invention selectively expand, e.g., engineered or non-engineered, Vδ1 T-cells and / or Vδ3 T-cells or Vδ1 CAR T-cells and / or Vδ3 CAR T-cell without significant expansion of Vδ2 T- cells or Vδ2 CAR T-cells. In an aspect, "without significant expansion of" means that thepreferentially expanded cell population are expanded at least 10-fold, preferably 100-fold, and more preferably 1,000-fold more than the reference cell population.

[0052] As used herein, "concurrently" means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0053] The term "contacting" as used herein refers to bringing one or more of the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed populations of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or a combination thereof together with a target area or intended target area in such a manner that the disclosed populations of Vδ1 γδ T- cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed populations of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or a combination thereof can exert an effect on the intended target or targeted area either directly or indirectly. A target area or intended target area can be one or more of a subject's organs (e.g., lungs, heart, liver, kidney, brain, etc.) hosting cancerous cells. In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as cancer). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as cancer).

[0054] As used herein, "determining" can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, cancer. Methods and techniques used to determine the presence and / or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and / or diagnose the presence, severity, or both of a disease or disorder (such as, for example, cancer).

[0055] As used herein, "effective amount" and "amount effective" can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and / or prevention of a disease or disorder (e.g., a cancer) or a suspected disease or disorder. As used herein, the terms "effective amount" and "amount effective" can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a cancer). For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.

[0056] In an aspect, "therapeutically effective amount" means an amount of a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed cell, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a cancer), (ii)attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., cancer), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., cancer). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T- cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can befound in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a "prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and / or mutant protein or enzyme.

[0057] The term "antibody" (Ab) includes, without limitation, a glycoprotein immunoglobulin that binds specifically to an antigen. An antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain can comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise three constant domains, CH1, CH2 and CH3. Each light chain can comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region can comprise one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL can comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains can contain a binding domain that interacts with an antigen. The constant regions of the Abs can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies can be approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. The heavy and light chains can be linked or connected to one another by a single disulfide bond and two other disulfide bonds can connect the heavy chain hinge regions to one another, so that the dimers can be connected to one another and the tetramer can be formed. Naturally produced antibodies are also glycosylated, e.g., on the CH2 domain. The term "antibody" is used to mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing etc., through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1. IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to- other molecules such as toxins, radioisotopes, etc.

[0058] The term "variable region" or "variable domain" is used interchangeably. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). In an aspect, the variable region is a human variable region. In an aspect, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In an aspect, the variable region is a primate (e.g., non-human primate) variable region. In an aspect, the variable region comprises rodent or murine CDRs and primate framework regions (FRs).

[0059] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof. The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.

[0060] The terms "constant region" and "constant domain" are interchangeable and have a meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0061] The term "heavy chain" when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (6), epsilon (E), gamma (y) and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3and IgG4.

[0062] The term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (A) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In an aspect, the light chain is a human light chain.

[0063] "Endogenous" with reference to a gene, protein, and / or nucleic acid refers to the natural presence of that gene, protein, and / or nucleic acid in a cell, such as an immune cell.

[0064] "Exogenous" refers to an introduced agent, such as a nucleic acid, gene, or protein, into a cell, for example from an outside source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein which is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase the expression of the protein over the level that would naturally be found in the cell under similar conditions, e.g., without introduction of the exogenous nucleic acid.

[0065] A "T cell receptor" or "TCR" refers to antigen-recognition molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes, a, 0., y, and 6, may rearrange leading to highly diverse TCR proteins.

[0080] As used herein, "effector function" can refer to a biological result of interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions comprise, without limitation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement mediated cytotoxicity (CMC). An effector function may be antigen binding dependent, antigen binding independent, or both. ADCC refers to lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve Fc receptor (FcR)-bearing effector cells recognizing and subsequently killing antibody-coated target cells (e.g., cells that express on their surface antigens to which an antibody is bound). Effector cells that mediate ADCC may comprise immune cells, comprising yet not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, eosinophils.

[0099] The term "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 an immune response. Examples of immunotherapy can include, but are not limited to, NK cells and T cell therapies. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACTTM), and allogeneic T cell transplantation. However, one of skill in the art would recognize that the conditioning methods disclosed herein would enhance the effectiveness of any transplanted T cell therapy.

[0066] The T cells or NK cells of the immunotherapy can come from any source known in the art. For example, T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population (for example iPSCs) or can be obtained from a subject. T cells and NK cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan.

[0067] As used herein, 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, single chain antibodies, and multi-specific antibodies formed from antibody fragments.

[0068] A "monoclonal antibody" as used herein refers to homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of manners including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0069] As used herein, the term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and capability. In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residue either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domainscontaining all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.

[0070] That an antibody "selectively binds" or "specifically binds" to an epitope or receptor means that the antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope or receptor than with alternative substances, including unrelated proteins. "Selectively binds" or "specifically binds" means, for instance, that an antibody binds to a protein with a KD of about 0.1 mM or less, more usually about 1 μM or less. "Selectively binds" or "specifically binds" means at times that an antibody binds to a protein with a KD of about 0.1 mM or less, at times about 1 μM or less, at times about 0.1 μM or less, at times about 0.01 μM or less, and at times about 1 nM or less. It is understood that, in an aspect, an antibody or binding moiety that specifically binds to a first target may or may not specifically bind to a second target. As such, "specific binding" does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, an antibody may, in an aspect, specifically bind to more than one target (e.g., any cancer- specific antigen and / or cancer-related antigen).

[0071] An "antigen" refers to a compound, composition, or substance that may stimulate the production of antibodies or a T cell response in a human or animal, including compositions (such as one that includes a tumor-specific protein) that are injected or absorbed into a human or animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is not substantially found on the surface of other normal (desired) cells and to which a binding domain of a TCR or CAR contemplated herein, is designed to bind.

[0072] A "target" is any molecule bound by a binding motif, CAR, TCR or antigen binding agent, e.g., an antibody. In an aspect, a target can be a cancer-specific antigens and cancer-related antigens including, but are not limited to, the following: HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mud, Muc16, NCAM, NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof.). In an aspect, the multiple targets may be bound by the same antigen-binding site on the antibody.

[0073] "Antigen-specific targeting region" (ASTR) refers to the region of the CAR or TCR which targets specific antigens. The targeting regions on the CAR or TCR are extracellular. In an aspect, the antigen-specific targeting regions comprise an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof and each of the targeting regions target a different antigen. The targeting regions may comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen.

[0074] In an aspect, the term "autologous" refers to any material derived from the same individual to which it is later to be re-introduced. For example, a subject's own cells can be obtained, made to express one or more disclosed CARs, and then administered to the same subject.

[0075] In an aspect, the term "allogeneic" refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In an aspect, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0076] In an aspect, the term "antibody production" can have both general and specific meanings. In the broad sense, it can refer to the entire process of creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. In the more restricted sense, antibody production refers to the steps leading up to antibody generation but does not include various forms of purifying and labeling the antibody for particular uses. Antibody production involves preparation of antigen samples and their safe injection into laboratory or farm animals to evoke high expression levels of antigen-specific antibodies in the serum, which can then be recovered from the animal. Polyclonal antibodies are recovered directly from serum (bleeds). Monoclonal antibodies are produced by fusing antibody-secreting spleen cells from immunized mice with immortal myeloma cell to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant. Successful antibody production depends upon careful planning and implementation with respect to several important steps and considerations: (i) synthesize or purify the target antigen (e.g., peptide or hapten); (ii) choose an appropriate immunogenic carrier protein; (iii) conjugate the antigen and carrier protein to createthe immunogen; immunize animals using appropriate schedule and adjuvant formula; and screen serum (or hybridoma) for antibody titer and isotype (also called antibody characterization).

[0077] As used herein, "RNA therapeutics" can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2'-O-Me-RNA, 2'-MEO-RNA, 2'-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non- coding RNA (lncRNA), such as, for example, a long intergenic non-coding RNA (lincRNA), pre- transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre- miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post- transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0078] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.

[0079] The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation such as cancer.

[0080] "Tumor" and "neoplasm" as used herein refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions. "Metastasis" as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A "metastatic" or "metastasizing" cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[0081] The terms "cancer cell" or "tumor cell" and grammatical equivalents refer to the total population of cells derived from a tumor including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).

[0082] As used herein "tumorigenic" refers to the functional features of a solid tumor stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non- tumorigenic tumor cells) that allow solid tumor stem cells to form a tumor.

[0083] As used herein, "lipid nanoparticles" or "LNPs" can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be use interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.

[0084] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical" or "essentially similar" when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequencescan have "substantial sequence identity" if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as "variants" herein, e.g., other variants of a missing, deficient, and / or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3'- and / or 5'-side are 100% identical.

[0085] As used herein, "immune-modulating" refers to the ability of a disclosed isolated nucleic acid molecules, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0086] In an aspect, a disclosed pharmaceutically acceptable carrier can refer to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. In an aspect, examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluiditycan be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0087] As used herein, the term "excipient" refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0088] As used herein, the term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0089] As used herein, the term "in combination" in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration "in combination with" one or more further therapeutic agents includessimultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or the disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof) can be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy to a subject having or diagnosed with cancer.

[0090] Disclosed are the components to be used to prepare the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additionalsteps can be performed with any specific aspect or combination aspects of the methods of the invention.

[0091] In an aspect, "Vδ3 T cells" can be used interchangeably with "Vδ1- Vδ2- T cells".

[0092] In this application, the following abbreviations apply: ACT (adoptive cell therapy); ADCC (antibody-dependent cell-mediated cytotoxicity); APCs (antigen-presenting cells); BAFF (B-cell activating factor); BCMA (B cell maturation antigen); CARs (chimeric antigen receptors); CAR- T cells (chimeric antigen receptor redirected T cells); CART-19 (CD19-targeted CAR T-cell therapies); Chemistry, Manufacturing, and Controls (CMC); CMs (costimulatory molecules); CRS (cytokine release syndrome); DNT (double-negative T cell); FCR (Fc receptor); HLA (human leukocyte antigen); IL-2 (Interleukin-2); ITAMs (immunoreceptor tyrosine-based activation motifs); JAK (Janus kinase); MCL (mantle cell lymphoma); NFAT (nuclear factor of the activated T-cell); NKG2D (natural killer group 2D); NSCLC (non-small cell lung cancer); OS (overall survival); PB (peripheral blood); PBMCs (peripheral blood mononuclear cells); scFv (single-chain fragment variant); STAT (signal transducer and activator of transcription); TAM (tumor-associated macrophage); TCR (T cell receptor); TIL (tumor-infiltrating lymphocyte); TMD (transmembrane domain); TRAIL (TNF-related apoptosis-inducing ligand); and TRUCKs (T cell redirected for universal cytokine-mediated killing). B. T Cells 1. γδ T cells vs. αβ T cells

[0093] Th dichotomy between γδ and αβ T cells extends across numerous dimensions, encompassing TCR structural variances, thymic developmental trajectories, mechanisms of antigen identification and presentation, activation cues, their roles in pathological conditions, and their applications in immunotherapy. The structural divergence of the γδ TCR and αβ TCR serves as the bedrock for distinguishing the functional roles these two T cell subsets assume within immune responses. This distinction becomes particularly evident when delving into their respective thymic development, as elaborated upon in forthcoming sections. Specifically, while the developmental journey of αβ T cells entails stages of double-negative (DN), double-positive (DP), and single-positive (SP) prior to dissemination into the circulation, γδ T cells follow a distinct course. The latter either exit the thymus during the DN (DN2-DN3) phase or progress through DN and DP or DN, DP, and SP stages before embarking into circulation. This unique developmental pattern equips γδ T cells with a greater spectrum of immune functions, spanning both innate and adaptive roles, along with diversified capacities in antigen recognition and presentation.

[0094] Distinctly stratified in their antigen recognition, αβ T cells operate within the confines of MHC-dependent recognition, whereas γδ T cells extend their sensing capabilities to include stress- induced antigens, phosphoantigens, and other non-peptidic molecules, all while circumventing the need for MHC mediation. While activation of αβ T cells necessitates a dual input of signals - antigen recognition and co-stimulation - to orchestrate immune responses, γδ T cells can be activated by a singular signal, such as a phosphoantigen. Notably, within the realm of antigen presentation, a specific subset of human γδ T cells, the Vδ2 T cells, takes on the role of professional antigen-presenting cells (APCs), a function beyond the purview of αβ T cells. This unique attribute situates γδ T cells as key regulators of immune functions within the broader immune cell landscape.

[0095] In terms of disease, αβ T cells are well-known for their adaptive immune responses and are critical in combating infections and mounting antigen-specific immune responses. They are highly specialized and undergo clonal expansion upon encountering specific antigens, however, the frequency of cells among αβ T cells which can recognize a given peptide antigen is extremely low. In contrast, γδ T cells exhibit characteristics of both innate and adaptive immunity. They can rapidly respond to various pathogens through their innate-like receptors, allowing for early immune defense even in the absence of prior antigen exposure. Moreover, γδ T cells are actively involved in tissue surveillance at barrier sites and contribute to the maintenance of tissue homeostasis. In disease settings, γδ T cells have been implicated in both protective and pathogenic roles. Their ability to respond rapidly to infections and produce cytokines enables them to contribute to pathogen clearance. However, dysregulation of γδ T cell activation and function has been associated with the development of autoimmune diseases, where these cells can recognize self-antigens and contribute to tissue damage. 2. Vδ1, Vδ2, and Vδ3 T cells

[0096] In the realm of cellular immunity, γδ T cells stand apart from their αβ T cell counterparts due to their distinct attributes, which intricately shape their roles in both pathogenesis of diseases and the field of immunotherapy. αβ T cells, forming the predominant subset of CD3+T cells within the immune repertoire, predominantly recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. In contrast, γδ T cells adopt an alternative T cell receptor (TCR) architecture, consisting of γ and δ chains, granting them the ability to perceive a wider array of antigens in MHC-independent manner. αβTCR recognize an antigen presented by an MHC molecule, whereas γδ TCR recognize an antigen independently of MHC presentation.

[0097] Human γδ T cells are classified into three subtypes based on the expression of δ chains. First, Vδ1 T cells are enriched in the thymus and mucosal epithelial tissues. Vδ1 T cells arepredominantly associated with the VγI gene family (Vγ2 / 3 / 4 / 5 / 8). They produce a variety of cytokines like TNF-α and IFN-γ, and lyse infected or transformed target cells by cytotoxicity. Second, Vδ2 T cells, which exert cytotoxicity in tumor immunoregulation and viral infection, are mainly distributed in peripheral blood. Vδ2 T cells co-express VγII (Vγ9). T cell receptors (TCRs) expressed on the surface of Vδ2 T cells are able to recognize phosphoantigens produced by malignant cells, thereby activating Vδ2 T cells to release perforins, granzymes, and IFN-γ. MDSCs may inhibit the production of IFN-γ and degranulation in phosphoantigens-activated Vδ2T cells. Third, Vδ3 T cells are mainly distributed in the liver tissue and rarely in the peripheral blood, which can directly kill the target cells and secrete cytokines.

[0098] Vδ1 T cells are players of adaptive immunity. Vδ1+ T cell relies on γδTCR- and natural killer receptors (NKR)-mediated recognition of tumor antigens or stress signals, similar to Vδ2 T cell. However, there are significant differences between the two subtypes. Specifically, Vδ1 TCR recognizes MHC-like proteins of the CD1 family, such as CD1c and CD1d, Annexin A2, and MHC class I chain-related protein A and B (MICA / B), which are mostly upregulated in transformed cells and virus infected cells. Evidence indicates δ1TCR has a much higher affinity toward CD1d than MICA / B. The drastic difference in their TCR ligand recognition patterns further implies non-redundant roles of Vδ1 and Vδ2 in establishing immune surveillance. Based on published studies, we can conclude that Vδ1 T cells play a significant role in adaptive immunity among γδ T cell subsets. Compared with Vδ2 T cells, Vδ1 T cells account for a small proportion of γδ T cells. They are mainly distributed on the surface of the mucosa. Although a large number of studies about the antitumor effects of γδ T cells mainly focus on Vγ9Vδ2 T cell subset, it is becoming more evident that Vδ1 T cells also play a critical role in progression of hematological malignancies and some epithelial-derived solid tumors (including CRC).

[0099] Like Vδ2 T cells, Vδ1 T cells also highly express natural killer group 2 member D (NKG2D), which is a stress-sensing molecule that recognizes its cognate ligand MICA / B on the surface of the cancer cells. However, Vδ1+ TCR and NKG2D do not share binding sites on MICA / B, and the strength of NKG2D-MICA / B binding is 1000-fold stronger than that of Vδ1+TCR-MICA / B. Despite discrepancies in their antigen recognition, both Vδ2 and Vδ1 T cells rely on secretion of the perforin / granzyme-B mediated secretory and death receptor (TRAIL / TRAIL-R, Fas / FasL) pathways to execute their anti-tumor cytotoxic activity.

[0100] Vδ2 T cells are the most abundant subset of γδ T cells, accounting for 50 to 90% of the total number of γδ T cells. Among Vδ2 T cells, Vγ9Vδ2 T cells that co-express Vδ2 and Vγ9 chains are the most abundant subtype. Vγ9Vδ2 T cells elicit robust inhibitory effects on the tumorigenesis and growth of different tumors.

[0101] To this end, extensive infiltration of human tumors by γδ T cells, including in ovarian cancer has been reported in the range of 3%-15% of total tumor-infiltrating T lymphocytes (TILs). These γδ T lymphocytes produce a variety of cytokines; have the capacity to cross-present antigens; and can re-direct antibodies against target cells through antibody-dependent cellular cytotoxicity. Unlike their conventional αβ T cell counterparts, which recognize peptides presented by major histocompatibility complex (MHC) molecules, γδ T cells possess a broader and incompletely understood recognition repertoire. Various γδ T-cell receptors (TCRs) can identify diverse antigens and butyrophilins (BTN) or BTN dimers, while their cytotoxic activity is primarily mediated through NKG2D signaling, or other natural killer cell receptors (NKRs), prompting the release of cytotoxic molecules such as perforin and granzymes. Consistent with all these observations, most studies in other human cancers have also identified γδ T cell infiltration with superior outcomes. This includes lung, breast, hepatocellular, renal, gastric, head and neck, and bladder cancers, in addition to melanoma. C. Chimeric Antigen Receptors

[0102] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to comprise a binding domain and a means of activating immune cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells, NK cells or combination thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. In an aspect, a CAR comprises a binding domain, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. A T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell. Similarly, an NK cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR NK cell.

[0103] Chimeric antigen receptors (CARs) are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. CARs comprise an extracellular ligand-binding domain, most commonly a single chain variable fragment (scFv), a spacer domain, a transmembrane domain, and one or more cytoplasmic domains.

[0104] In an aspect, a disclosed CAR can be characterized by its generation.

[0105] Generally, 1G CAR contains only a CD3ζ in its signaling domain with no CM. Generally, 2G CAR harbors CD3ζ and one CM, allowing dual signaling pathway. Generally, a 3G CAR combines CD3ζ and several CMs that deliver multiple signaling. Generally, a 4G CAR resembles 2G CAR with the incorporation of an additional NFAT-responsive cassette that expresses cytokines playing anti-cancer activities. Thus, 4G CAR has triple signals from primary CD3ζ, CM, and expressed transgenic proteins. Generally, 5G CAR is based on 2G CAR by addingmembrane receptors IL-2Rβ that provides a binding site for STAT3 and activates the JAK-STAT signaling domain. 5G CAR has also a synergistic activation of triple signals from CD3ζ, CMs, and cytokine-inducing JAK-STAT3 / 5 pathway. Abbreviations: IL-2R, interleukin receptor; Jak, Janus kinase; NFAT, nuclear factor of the activated T-cell; STAT, signal transducer and activator of transcription.

[0106] First generation (1G) CAR T-cells contain only CD3ζ without any CM. Typically, 1G CAR T-cells have only one signaling and can be capable of T cell activation only. The use of 1G CAR T-cells can be limited by inadequate T-cell proliferation, cytokine release, T-cell persistence, and anti-tumor activity. Second generation (2G) CAR T-cells contain CD3ζ and CMs (CD28, CD134 / OX-40, or CD137 / 4-1BB). Typically, 2G CAR T-cells provide two signals via CD3ζ and CM and can offer better T-cell activation, proliferation, and persistence than 1G CAR T-cells. The use of 2G CAR T-cells can be limited by issues concerns persistence and relapse. Third generation (3G) CAR T-cells combine CD3ζ and multiple CMs (CD28, CD137 / 41BB, CD134 / OX-40, NKG2D, CD27, TLR2 or ICOS). Typically, 3G CAR T-cells can be characterized by multiple signaling via CD3ζ and two CMs. 3G CAR T-cells can provide better safety profiles, proliferation, perseverance, and anti-tumor functions than do 2G CAR T-cells. The use of 3G CAR T-cells can be limited by a higher incidence of severe side-effects such as CRS and faster T-cell exhaustion. Fourth generation (4G) CAR T-Cells harbor CD3ζ, NFAT-responsive cassette containing cytokines (IL-2, IL-5, IL-12, IFNγ) and CMs (CD28, OX-40 or 4-1BB). Typically, 4G CAR T-cells can be characterized by primary CD3ζ, costimulatory signals, and the expression of transgenic proteins. 4G CAR T-cells can potentiate T-cell expansion, persistence, and anti- tumor capacity by overcoming tumor antigen loss by cytokine activation at the tumor site and modulating the tumor milieu. 4G CAR T-cells can also help a patient regain its immune system and can reduce toxicity to more than 3G. The use of 4G CAR T-cells can be limited by poor cancer-killing potential in solid tumors, side effects such as on-target off-tumor activation of TRUCK T-cells and release of the transgenic cytokine in healthy tissues, and the double modification of T-cells. Fifth generation (5G) CAR T-cells incorporates CD3ζ and additional membrane receptors: IL-2Rβ and STAT3. Typically, 5G CAR T-cells can provide better T-cell activation, proliferation, solid tumor infiltration, and persistence by cytokine-inducing JAK / STAT signaling. 5G CAR T-cells can also have a better safety profile and a wider therapeutic window. 5G CAR T-cells can create a more favorable tumor milieu and reestablish the immune system after infusion. The use of 5G CAR T-cells can be limited by r limitations by continuous development and some side effects.

[0107] The design of each module of the CAR structure can contribute to CAR-T–cell signaling mechanisms, effector functions, and its eventual efficacy and toxicity. It is evident that modules such as the scFv and intracellular cytoplasmic domains play a key role in ligand recognition and signaling. It has recently become clear that non-signaling domains such as the spacer and transmembrane domains can also influence CAR functions.

[0108] scFvs are the most used ligand-binding domains in CAR structures. scFv affinity is a key parameter that has been modulated to improve specificity of the CAR and reduce "on-target, off- tumor" side effects, which is of particular importance when the target antigen is ubiquitously expressed on healthy tissue.

[0109] Spacer domains that connect the scFv to the transmembrane domain lend flexibility to the scFv and help improve efficacy. Appropriate spacer domain engineering can enable recognition of target epitopes that are otherwise sterically inaccessible. Spacer domain modulation can also be used to regulate synaptic cleft distances and hence signaling phenomena such as kinetic segregation. To maintain optimal synapse distance, membrane-distal epitopes usually require shorter spacers whereas membrane-proximal epitopes require longer spacers. Apart from limiting exclusion of inhibitory phosphatases, increasing epitope-paratope distance can also result in impaired delivery of granzymes and perforins to the target cell, thus reducing lytic efficiency. In a physiological T-cell setting, the highly dense immune synapse hinders diffusion of lytic granules, which enhances pore formation by perforins and granzyme delivery [

[0110] Transmembrane domains in CAR structures serve as a fulcrum for transducing ligand recognition signals to the intracellular cytoplasmic domain.

[0111] In T cells, the intracellular signaling domain of the TCR-CD3 complex transduces the necessary "signal 1" to kick-start the signaling cascade. Co-stimulatory receptors, especially CD28, convey "signal 2" which is important for sustained signaling, prevention of anergy, and proliferation. 4-1BB, ICOS, and OX40 are other co-stimulatory receptors that affect T-cell differentiation pathways, metabolic cycles, as well as apoptosis and activation-induced cell death.

[0112] In CAR-T cells, co-stimulatory signals are usually included in-cis with the CD3ζ cytoplasmic domain. The number and type of co-stimulatory signals, as well as their order and proximity to the membrane, are critical parameters that have been addressed in literature (Fig.5). Immunoreceptor Tyrosine Activation Motifs (ITAMs) present on cytoplasmic domains of TCR- CD3 complexes are the phosphorylation sites, which recruit ZAP70, critical for signaling cascades. In T cells, ITAM diversity and number of functional CD3ζ ITAMs are important for optimal signaling. In CAR-T cells, the number of functional ITAMs is gaining attention as an important design strategy to ensure efficacy.D. Compositions for Use in the Disclosed Methods 1. Expanded Population of γδ T cells

[0113] Disclosed herein is a population γδ T cells expanded by a disclosed method. Disclosed herein is a population of CAR γδ T-cells expanded by a disclosed method. Disclosed herein is a population of Vδ1 γδ T cells and / or Vδ3 γδ T-cells expanded by a disclosed method. Disclosed herein is a population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells expanded by a disclosed method. Disclosed herein is a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells.

[0114] Disclosed herein is a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells made according to the rapid expansion protocol set forth in Example 1, infra.

[0115] Disclosed herein is a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells made according to the rapid expansion protocol set forth in Example 2, infra.

[0116] In an aspect, predominantly can mean that the number of Vδ1 γδ T cells in a disclosed expanded population of γδ T cells significantly exceeds the number of Vδ2 γδ T cells in the same expanded population. In an aspect, predominantly can mean that the number of V3 γδ T cells in a disclosed expanded population of γδ T cells significantly exceeds the number of Vδ2 γδ T cells in the same expanded population. In an aspect, predominantly can mean that the number of Vδ1 γδ T cells and the number of Vδ3 γδ T cells in a disclosed expanded population of γδ T cells significantly exceeds the number of Vδ2 γδ T cells in the same expanded population.

[0117] Disclosed herein is a population of expanded CAR γδ T-cells comprising predominantly Vδ1 γδ CAR T-cells and / or V3 γδ CAR T-cells. In an aspect, predominantly can mean that the number of Vδ1 γδ CAR T-cells in a disclosed expanded population of CAR γδ T-cells significantly exceeds the number of V2 γδ T cells in the same expanded population. In an aspect, predominantly can mean that the number of V3 γδ CAR T-cells in a disclosed expanded population of CAR γδ T-cells significantly exceeds the number of V2 CAR γδ T-cells in the same expanded population. In an aspect, predominantly can mean that the number of Vδ1 γδ CAR T- cells and the number of Vδ3 γδ CAR T-cells in a disclosed expanded population of CAR γδ T- cells significantly exceeds the number of Vδ2 CAR γδ T-cells in the same expanded population.

[0118] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells made by a disclosed method can be cryopreserved for future use and / or analysis. In an aspect, apopulation of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells made by a disclosed method can be cryopreserved for future use and / or analysis.

[0119] In an aspect, expanded γδ T cells and / or expanded CAR γδ T-cells can be added to a freezing media (90% FBS + 10% DMSO) to achieve a final concentration of about 2e7 cryopreserved cells / mL to about 5e7 cryopreserved cells / mL.

[0120] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells remains can be administered to one or more subjects having cancer. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered to one or more subjects having cancer.

[0121] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells remains can be administered to one or more subjects having one or more αβ T cell refractory cancers. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered to one or more subjects having one or more αβ CAR T-cell refractory cancers.

[0122] In an aspect, an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered as an allogeneic cell therapy to a subject having cancer. In an aspect, an expanded population of predominantly population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a subject having cancer. In an aspect, an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancer. In an aspect, an expanded population of predominantly purified Vδ1 CAR γδ T- cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancers.

[0123] In an aspect, an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered to a subject having an αβ T cell refractory cancer. In an aspect, an expanded population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a subject having an αβ CAR T-cell refractory cancer.

[0124] In an aspect, an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered to a plurality of subjects having an αβ T cell refractory cancer. In an aspect, an expanded population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a plurality subjects having an αβ CAR T-cell refractory cancer.

[0125] In an aspect, an expanded population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can comprise a CAR for any cancer-specific antigen and / or cancer- related antigen. In an aspect, an expanded population of predominantly purified Vδ1 CAR γδ T- cells and / or Vδ3 CAR γδ T-cells can comprise one or more CARs for any cancer-specific antigen and / or cancer-related antigen. In an aspect, cancer-specific antigens and cancer-related antigens include, but are not limited to, the following: HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-A1+NY-ES0-1, HLA-A2+NY- ES0-1, HLA-A3+NYES0-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mud, Muc16, NCAM, NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof.

[0126] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be used in one or more disclosed methods. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be used in one or more disclosed methods. In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be used in one or more disclosed methods of treating a subject. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be used in one or more disclosed methods of treating a subject.

[0127] In an aspect, a disclosed population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be validated and / or characterized by several protocols known to the skilled person in the art. For example, a disclosed population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be subjected to sorting using FACS or a similar type of flow cytometry. In an aspect, a disclosed population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be evaluated in a cytotoxicity assay.

[0128] In an aspect, a disclosed population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to kill stressed cells (e.g., tumor or virally infected cells) and can be combined with IgG targeting molecules on the tumor cell surface, thereby addressing the problem of antigen heterogeneity.

[0129] In an aspect, a disclosed expanded population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to avoid T cell exhaustion.

[0130] In an aspect, using UCB as a biological sample for a use in a disclosed method ensures that an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can comprise a broader repertoire of gamma and delta chains. In an aspect, using PB as a biological sample yields a narrower repertoire of gamma and delta chains. 2. Isolated Nucleic Acid Molecules

[0131] Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a CAR for any cancer-specific antigen and / or cancer-related antigen. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any generation of CAR.

[0132] Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more disclosed CARs. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more disclosed CARs for any cancer-specific antigen and / or cancer-related antigen. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more CARs of any generation.

[0133] In an aspect, a disclosed nucleic acid sequence can encode a CAR comprising from N- terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and / or one or more co-stimulatory domains.

[0134] In an aspect, a disclosed nucleic acid sequence can encode a CAR comprising from N- terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; an intracellular domain, a cleavage signal; a signal peptide; and a tumor- specific antigen or fragment thereof.

[0135] In an aspect, a disclosed nucleic acid sequence can encode a CAR comprising an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.

[0136] In an aspect, a disclosed nucleic acid sequence can encode a CAR comprising from N- terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; and an intracellular domain comprising one or more immunoreceptor tyrosine-based activation domains (ITAMs) and / or one or more co-stimulatory domains.

[0137] In an aspect, a disclosed nucleic acid sequence can encode a CAR comprising from N- terminus to C-terminus a signal peptide; an antigen binding domain; a hinge domain; a transmembrane domain; an intracellular domain, a cleavage signal; a signal peptide; and a tumor specific antigen or fragment thereof.

[0138] In an aspect, a disclosed antigen binding domain can be a scFV and wherein the scFV can comprise a linker. In an aspect, a disclosed encoded linker can join the VHand VLregions of the ScFv. In an aspect of a disclosed isolated nucleic acid molecule encoding a cancer-specific antigen and / or cancer-related antigen can comprise an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a part or all an immunoglobulin constant region, or any combination thereof.

[0139] In an aspect, a disclosed nucleic acid sequence can encode one or more CARs specific for a cancer-specific antigen and / or cancer-related antigen. In an aspect, cancer-specific antigens and cancer-related antigens include, but are not limited to, the following: HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4- 1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA- A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0-1, IL-11Ra, IL-13Ra2, Lambda, Lewis- Y, Kappa, Mesothelin, Mud, Muc16, NCAM, NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof. 3. Vectors

[0140] Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a vector comprising a nucleic acid sequence encoding a disclosed CAR. Disclosed herein a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any disclosed CAR. Disclosed herein a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a CAR for expression by a population of predominantly purified γδ T cells expanded by a disclosed method. Disclosed herein a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a CAR for expression by a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells expanded by a disclosed method. Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a vector comprising a nucleic acid sequence encoding one or more disclosed CARs. Disclosed herein a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more disclosed CARs. Disclosed herein a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more CARs for expression by a population of predominantly purified γδ T cells expanded by a disclosed method. Disclosed herein a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more CARs for expression by apopulation of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells expanded by a disclosed method.

[0141] In an aspect, a disclosed vector can be used to introduce a disclosed isolated nucleic acid molecule encoding a disclosed CAR or disclosed isolated nucleic acid sequence encoding a disclosed CAR to one or more populations of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells expanded by a disclosed method.

[0142] In an aspect, a disclosed vector can be an integrating vector or a non-integrating vector. In an aspect, integration can mean that the nucleotides of nucleic acid sequence can be stably inserted into the cellular genome (e.g., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA).

[0143] In an aspect, a disclosed vector can be a viral vector. In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picornavirus vector. In an aspect, a disclosed viral vector can be an adeno-associated virus (AAV) vector In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and / or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV- HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein. In an aspect, a disclosed viral vector can be a lentiviral vector or a retroviral vector.

[0144] As used herein, "operably linked" refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. For example, a control element "operably linked" to a functional element is associated in such a way that expressionand / or activity of the functional element is achieved under conditions compatible with the control element. In an aspect, a promotor can be operably linked to nucleic acids.

[0145] In an aspect, a disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter. In an aspect, a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed CAR or one or more disclosed CARs. In an aspect, a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed tumor antigen. Promoters are known to the art. In an aspect, a disclosed promoter can be a promoter / enhancer. Promoter / enhancers are known to the art. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, a disclosed promoter or a disclosed promoter / enhancer can be used for constitutive and efficient expression of a disclosed CAR or one or more disclosed CARs. In an aspect, a disclosed promoter or a disclosed promoter / enhancer can be used for constitutive and efficient expression of a disclosed tumor antigen. In an aspect, a disclosed vector can be used to engineer cells to express a disclosed CAR or one or more disclosed CARs.

[0146] In an aspect, a disclosed vector can be a non-viral vector. In an aspect, a disclosed non- viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector.

[0147] In an aspect, a disclosed non-viral vector can be a transposon-based vector such as Sleeping Beauty and PiggyBac, both of which are known in the art. In an aspect, a first plasmid can be loaded with a disclosed nucleic acid sequence encoding a disclosed CAR or one or more disclosed CARs, named transposon, surrounded by inverted repeats (IRs) that contain short direct repeats (DRs), while a second plasmid encodes the enzyme (transposase) that can recognize the sequences from the first plasmid and cut the transposon out of the first plasmid. Then the sequence for a disclosed CAR or one or more disclosed CARs can be successfully delivered into the targeted cell (e.g., a T cell or a NK cell or a macrophage) cytoplasm and inserted randomly into TA dinucleotide base pairs of the recipient DNA sequence. In an aspect, using this vector, stable integration and reliable long-term expression of the sequence of a disclosed CAR or one or more disclosed CARs can be achieved.

[0148] In an aspect, a disclosed non-viral vector can comprise lipid and / or polymer-based nanoparticles loaded with mRNA encoding one or more disclosed CARs. In an aspect, a disclosed non-viral vector can comprise lipid and / or polymer-based nanoparticles loaded with transposon- based plasmids such as, for example, transposon-based plasmids comprising a sequence encoding one or more disclosed CARs. In an aspect, a disclosed non-viral vector can comprise mRNAencoding one or more disclosed CARs. In an aspect, a disclosed non-viral vector can comprise lipid and / or polymer-based nanoparticles loaded with mRNA encoding one or more disclosed CARs. In an aspect, a disclosed non-viral vector can be a plasmid. In an aspect, a disclosed plasmid can comprise a disclosed nucleic acid sequence. Recombinant plasmids are known to the art.

[0149] In an aspect, a disclosed viral vector or a disclosed non-viral can comprise a nucleic acid one or more CARs for any cancer-specific antigen and / or cancer-related antigen. In an aspect, cancer-specific antigens and cancer-related antigens include, but are not limited to, the following: HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mud, Muc16, NCAM, NKG2D Ligands, NYE- S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof. 4. Pharmaceutical Formulations

[0150] Disclosed herein is a pharmaceutical formulation comprising a disclosed population of expanded γδ T cells; and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein is a pharmaceutical formulation comprising a disclosed population of CAR γδ T-cells; and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein is a pharmaceutical formulation comprising a population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells expanded by a disclosed method; and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein is a pharmaceutical formulation comprising Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells expanded by a disclosed method a disclosed isolated nucleic acid molecule; and one or more pharmaceutically acceptable carriers and / or excipients.

[0151] Disclosed herein is a pharmaceutical formulation comprising a purified population of expanded γδ T cells; and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein is a pharmaceutical formulation comprising a purified population of expanded CAR γδ T-cells; and one or more pharmaceutically acceptable carriers and / or excipients.

[0152] Disclosed herein is a pharmaceutical formulation comprising a purified population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells; and one or more pharmaceutically acceptable carriers and / or excipients. Disclosed herein is a pharmaceutical formulation comprising a purified population ofVδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells; and one or more pharmaceutically acceptable carriers and / or excipients.

[0153] Disclosed herein is a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule; and one or more pharmaceutically acceptable carriers. Disclosed herein is a pharmaceutical formulation comprising a disclosed viral vector; and one or more pharmaceutically acceptable carriers. Disclosed herein is a pharmaceutical formulation comprising a disclosed non-viral vector; and one or more pharmaceutically acceptable carriers. Disclosed herein is a pharmaceutical formulation comprising a disclosed plasmid; and one or more pharmaceutically acceptable carriers. Disclosed herein is a pharmaceutical formulation comprising a disclosed cell; and one or more pharmaceutically acceptable carriers.

[0154] In an aspect, a disclosed pharmaceutical formulation can further comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.

[0155] In an aspect, a disclosed pharmaceutical formulation can further comprise one or more anti-inflammatory agents. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

[0156] In an aspect, NSAIDs can comprise ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as rofecoxib and celecoxib, sialylates, or any combination thereof. In an aspect, analgesics can comprise acetaminophen, oxycodone, tramadol, proporxyphene hydrochloride, or any combination thereof. In an aspect, glucocorticoids can comprise cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or any combination thereof. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g., etanercept, adalimumab, and infliximab, chemokine inhibitors and adhesion molecule inhibitors. In an aspect, biological response modifiers can comprise monoclonal antibodies as well as recombinant forms of molecules. In an aspect, exemplary disease-modifying anti-rheumatic drugs (DMARDs) can comprise include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), minocycline, or any combination thereof.

[0157] In an aspect, a disclosed chemotherapeutic agent in a disclosed pharmaceutical formulation can comprise an anthracycline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof. In an aspect, a disclosed chemotherapeutic agent can comprise 5-fluorouracil (Adrucil, Efudex), 6-mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), bicalutamide (Casodex), bleomycin sulfate (Blenoxane), bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC- Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), doxorubicin hydrochloride (Adriamycin, Rubex), etoposide (Vepesid), fludarabine phosphate (Fludara), flutamide (Eulexin), folic acid antagonists, gemcitabine (difluorodeoxycitidine), gemtuzumab, gliotoxin, hydroxyurea (Hydrea), Idarubicin (Idamycin), ifosfamide (IFEX), ifosfamide, irinotecan (Camptosar), L-asparaginase (ELSPAR), lenalidomide), leucovorin calcium, melphalan (Alkeran), melphalan, methotrexate (Folex), mitoxantrone (Novantrone), mylotarg, N4-pentoxycarbonyl-5 deoxy-5-fluorocytidine, nab-paclitaxel (Abraxane), paclitaxel (Taxol), pentostatin, phoenix (Yttrium90 / MX-DTPA), polifeprosan 20 with carmustine implant (Gliadel), purine analogs and adenosine deaminase inhibitors (fludarabine), pyrimidine analogs, rituximab, tamoxifen citrate (Nolvadex), temozolomide), teniposide (Vumon), tezacitibine, thalidomide or a thalidomide derivative, thiotepa, tirapazamine (Tirazone), topotecan hydrochloride for injection (Hycamptin), tositumomab), vinblastine (Velban), vinblastine, vincristine (Oncovin), vindesine, vinorelbine (Navelbine), or any combination thereof.

[0158] In an aspect, a disclosed pharmaceutical formulation can comprise an anti-chemokine therapy that enhances the resident memory T cell formations in tumor-free tissues. In an aspect, a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCR2, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof.

[0159] In an aspect, a disclosed pharmaceutical formulation can further comprise abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomabm bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab,brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, namatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, 3F8, or any combination thereof.

[0160] In an aspect, a disclosed pharmaceutical formulation can be prepared for systemic or direct administration. In an aspect, a disclosed pharmaceutical formulation can be prepared for oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof. In an aspect, a disclosed pharmaceutical formulation can be prepared for any method of administration disclosed herein. In an aspect, a disclosed pharmaceutical formulation can be prepared for administration via multiple routes either concurrently or sequentially. For example, in an aspect, a disclosed pharmaceutical formulation can be first administered intratumorally and then be administered intravenously. In an aspect, a disclosed pharmaceutical formulation can be first administered intratumorally and then be administered orally. A skilled clinical can determine the best route of administration for a subject at a given time.

[0161] In an aspect, a disclosed pharmaceutical formulation can comprise one or more immune modulators. In an aspect, a disclosed pharmaceutical formulation can comprise one or more proteasome inhibitors. In an aspect, a disclosed pharmaceutical formulation can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed pharmaceutical formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).

[0162] In an aspect, a disclosed pharmaceutical formulation can comprise an RNA therapeutic. An RNA therapeutic can comprise RNA-mediated interference (RNAi) and / or antisenseoligonucleotides (ASO). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzyme that is overexpressed or is overactive due to a missing, deficient, and / or mutant protein or enzyme (such as, for example, a missing, deficient, and / or mutant protein or enzyme related to cancer and / or associated with cancerous cells). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzyme that is overexpressed or is overactive and related to cancer and / or associated with cancerous cells.

[0163] In an aspect, a disclosed pharmaceutically acceptable carrier can comprise any disclosed carrier and / or any disclosed excipient.

[0164] In an aspect, a disclosed pharmaceutical formulation comprising an expanded population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to avoid T cell exhaustion. 5. Animals

[0165] Disclosed herein are animals used to validate the efficacy and / or safety of one or more disclosed populations of expanded γδ T cells, one or more disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method), or one or more Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), one or more disclosed CARs, one or more disclosed cells, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, a disclosed animal can be treated with one or more disclosed populations of expanded γδ T cells, one or more disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method), or one or more Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), one or more disclosed CARs, one or more disclosed cells, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, animals can be assessed and / or monitored for one or more biological and / or chemical functions prior to treatment, during treatment, after treatment, or any combination thereof. In an aspect, a disclosed treated subject can be a mouse or a rat. In an aspect, a disclosed treated subject can be a transgenic mouse or a transgenic rat. In an aspect, a disclosed treated subject can have one or more types of cancers and / or tumors. E. Methods of Expanding a Desired T Cell Population

[0166] Disclosed herein is a method of expanding a desired T cell population, the method comprising isolating a population of T cells from a biological sample; and expanding the selected T cell population.

[0167] Disclosed herein is a method of expanding a desired T cell population, the method comprising isolating a population of T cells from a biological sample and expanding the selected T cell population according to the rapid expansion protocol set forth in Example 1, infra.

[0168] Disclosed herein is a method of expanding a desired T cell population, the method comprising isolating a population of T cells from a biological sample and expanding the selected T cell population according to the rapid expansion protocol set forth in Example 2, infra.

[0169] Disclosed herein is a method of expanding Vd1 γδ T cells and Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells; and depleting residual δβ T cells from the expanded γδ T cells.

[0170] Disclosed herein is a method of generating CAR Vd1 γδ T cells and CAR Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells; depleting residual δβ T cells from the expanded γδ T cells; and transducing the expanded γδ T cells with a vector encoding a chimeric antigen receptor.

[0171] Disclosed herein is a method of expanding Vd1 γδ T cells and Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells, wherein purifying and expanding the isolated γδ T cells comprises preparing the autologous feeder cells and co-incubating the isolated γδ T cells with the autologous feeder cells; and depleting residual δβ T cells from the expanded γδ T cells.

[0172] Disclosed herein is a method of generating CAR Vd1 γδ T cells and CAR Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells; wherein purifying and expanding the isolated γδ T cells comprises preparing the autologous feeder cells and co- incubating the isolated γδ T cells with the autologous feeder cells; depleting residual δβ T cells from the expanded γδ T cells; and transducing the expanded γδ T cells with a vector encoding a chimeric antigen receptor.

[0173] Disclosed herein is a method of expanding Vd1 γδ T cells and Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells; depleting residual δβ T cells from the expanded γδ T cells; and characterizing the expanded γδ T cells.

[0174] Disclosed herein is a method of generating CAR Vd1 γδ T cells and CAR Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood;

[0175] isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells; depleting residual δβ T cells from the expanded γδ T cells; characterizing the expanded γδT cells; and transducing the expanded γδ T cells with a vector encoding a chimeric antigen receptor.

[0176] Disclosed herein is a method of expanding Vd1 γδ T cells and Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells, wherein purifying and expanding the isolated γδ T cells comprises preparing the autologous feeder cells and co-incubating the isolated γδ T cells with the autologous feeder cells; depleting residual δβ T cells from the expanded γδ T cells; and characterizing the expanded γδ T cells.

[0177] Disclosed herein is a method of generating CAR Vd1 γδ T cells and CAR Vd3 γδ T cells, the method comprising isolating total leukocytes from umbilical cord blood; isolating γδ T cells from the total leukocytes; purifying and expanding the isolated γδ T cells; wherein purifying and expanding the isolated γδ T cells comprises preparing the autologous feeder cells and co- incubating the isolated γδ T cells with the autologous feeder cells; depleting residual δβ T cells from the expanded γδ T cells; characterizing the expanded γδ T cells; and transducing the expanded γδ T cells with a vector encoding a chimeric antigen receptor.

[0178] In an aspect, a disclosed method of expanding a desired T cell population can be performed ex vivo. In an aspect, a disclosed desired T cell population can be expanded γδ T-cells or certain types of expanded γδ T-cells. In an aspect, a disclosed desired expanded T cell population can comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. In an aspect, a disclosed desired expanded T cell population can comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0179] In an aspect, a disclosed biological sample can be umbilical cord blood (UCB), PBMCs, induced pluripotent stem cells (iPSCs), or any combination thereof. In an aspect, using UCB as a biological sample ensures that an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can comprise a broader repertoire of gamma and delta chains. In an aspect, using PB as a biological sample yields a narrower repertoire of gamma and delta chains.

[0180] In an aspect, UCB can serve as a more tolerable source material than other sources such as, for example, PBMCs. In an aspect, UCB can allow for less stringent HLA matching between a donor and a recipient. In an aspect, UCB can serve as an enriched source of hematopoietic stem cells (HSCs). HSCs can self-renew and can be used to differentiate into T-cells. In an aspect, T- cells isolated from UCB can have a unique antigen-naive status, which can be linked to thedecreased alloreactivity observed. In an aspect, UCB T-cells can be characterized by impaired nuclear factor of activated T-cells (NFAT) signaling and reduced activity, which can further contribute to the reduced GvHD.

[0181] In an aspect, a disclosed biological sample can be obtained from PBMCs. In an aspect, T- cells can be isolated from PBMCs. In an aspect, stem cells and / or hematopoietic progenitor cells can be isolated from PBMCs, and can be activated and transduced into CAR T-cells. In an aspect, a disclosed biological sample can be obtained from PBMCs. In an aspect, T-cells can be derived from iPSCs. In an aspect, iPSCs can have an unlimited capacity for self-renewal and can be banked. In an aspect, T-cells derived from iPSCs can be transduced into CAR T-cells. For example, in an aspect, isolating a population of T cells from a biological sample can comprise isolating leukocytes from UCB, PBMCs, iPSCs, or any combination thereof. For example, in an aspect, isolating a population of T cells from a biological sample can comprise isolating leukocytes from UCB. In an aspect, isolating a population of T cells from a biological sample such as cord blood can comprise any process known to the art.

[0182] For example, in an aspect, to isolate specific cell populations from cord blood, mononuclear cell preparation can be obtained by (i) using a density gradient medium (e.g., Lymphoprep by StemCell Technologies) and centrifugation, or (ii) by depleting RBCs. In an aspect, depleting RBCs from cord blood can require special considerations. Standard ammonium chloride lysis can be too harsh for the hematopoietic or immune progenitor cells contained in cord blood. In an aspect, a disclosed method can employ a commercially available product (e.g., EasySep or HetaSep by StemCell Technologies). In an aspect, a disclosed cord blood sample can have high platelet counts, which can facilitate the formation of clots. In an aspect, a disclosed method can further comprise using suitable anticoagulants (e.g., acid citrate dextrose (ACD) or EDTA).

[0183] In an aspect, following the manufacturer's instructions, ~250 million of total leukocytes were isolated using SepMate PBMC Isolation 50 mL tubes (StemCell Technologies, Cat #85450) and Lymphoprep (StemCell Technologies, Cat. # 07801).

[0184] In an aspect, use of Lymphoprep (StemCell Technologies) can comprise the following manufacturer's instructions: (1) warm Lymphoprep to room temperature (15 °C – 25 °C) before use: (2) remove the bottle cap and mix thoroughly by inverting the bottle several times; (3) withdraw Lymphoprep using either a syringe or a pipettor as follows (w / Syringe - with the bottle in the inverted position, insert the syringe needle through the rubber septum and withdraw desired volume; and w / Pipettor - lift the aluminum ring and pull off the metal seal. Remove the silver ring. Using aseptic technique, remove the rubber septum and withdraw the required volume. Usingaseptic technique, reinsert the rubber septum; (4) add Lymphoprep™ to tube; (5) dilute blood with an equal amount of Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS; Catalog #07905), or other suitable culture medium; (6) layer blood on top of Lymphoprep taking care to minimize mixing of blood with Lymphoprep; (7) centrifuge at 800 x g for 20 minutes at room temperature (15 °C – 25 °C) with brake off. If the blood has been stored for more than 2 hours, then increase the centrifugation time to 30 minutes; (8) remove and discard upper plasma layer without disturbing the plasma:Lymphoprep interface; (9) remove and retain the MNC layer at the plasma:Lymphoprep interface without disturbing the erythrocyte / granulocyte pellet; and (10) wash MNCs once with medium.

[0185] In an aspect, disclosed isolated leukocytes can be aliquoted. In an aspect, an aliquot of disclosed isolated leukocytes can comprise about 5 million cells per aliquot to about 15 million cells per aliquot. In an aspect, an aliquot of disclosed isolated leukocytes can comprise about 15 million cells per aliquot to about 25 million cells per aliquot. In an aspect, an aliquot of disclosed isolated leukocytes can comprise 5 million cells per aliquot, about 6 million cells per aliquot, about 7 million cells per aliquot, about 8 million cells per aliquot, about 9 million cells per aliquot, about 10 million cells per aliquot, about 11 million cells per aliquot, about 12 million cells per aliquot, about 13 million cells per aliquot, about 14 million cells per aliquot, about 15 million cells per aliquot, about 16 million cells per aliquot, about 17 million cells per aliquot, about 18 million cells per aliquot, about 19 million cells per aliquot, about 20 million cells per aliquot, about 21 million cells per aliquot, about 22 million cells per aliquot, about 23 million cells per aliquot, about 24 million cells per aliquot, about 25 million cells per aliquot, or more than 25 millions cells per aliquot.

[0186] In an aspect, disclosed isolated leukocytes can be cryopreserved. In an aspect, disclosed isolated leukocytes can be aliquoted and can be cryopreserved. In an aspect, a disclosed method can further comprise cryopreserving disclosed isolated leukocytes.

[0187] In an aspect, cryopreserving can comprise storing one or more disclosed biological samples (such as, for example, UCB, PBMCs, iPSCs, or any combination thereof) at about -20 °C or below -20 °C. In an aspect, cryopreserving can comprise storing the one or more disclosed biological samples (such as, for example, UCB, PBMCs, iPSCs, or any combination thereof) at about -80 °C or below -80 °C. In an aspect, cryopreserving can comprise storing one or more disclosed aliquots of isolated leukocytes at about -20 °C or below -20 °C. In an aspect, cryopreserving can comprise storing the disclosed aliquots of isolated leukocytes at about -80 °C or below -80 °C.

[0188] In an aspect, cryopreserving can comprise about 1 day to about 10 days, about 10 days to about 20 days, about 20 days to about 30 days, about 30 days to about 40 days, about 40 days to about 50 days, about 50 days to about 60 days, about 60 days to about 70 days, about 70 days to about 80 days, about 80 days to about 90 days, or about 90 days to about 100 days. In an aspect, cryopreserving can comprise about 1 month, about 1 month to about 2 months, about 2 months to about 3 months, about 3 months to about 4 months, about 4 months to about 5 months, about 5 months to about 6 months, about 6 months to about 7 months, about 7 months to about 8 months, about 8 months to about 9 months, about 9 months to about 10 months, about 10 months to about 11 months, or about 11 months to 12 months. In an aspect, cryopreserving can comprise about 1 year, about 1 year to about 2 years, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, or more than about 5 years. In an aspect, cryopreserving can comprise more than 5 years, more than 6 years, more than 7 years, more than 8 years, more than 9 years, or more than 10 years. In an aspect, cryopreserving can comprise an indefinite period.

[0189] In an aspect, a disclosed method can further comprise isolating γδ T cells from a disclosed population of isolated leukocytes. In an aspect, a population of γδ T cells can be immunopurified from a disclosed aliquot of isolated leukocytes. In an aspect, a population of γδ T cells can be immunopurified from a disclosed aliquot of isolated leukocytes using immunomagnetic positive selection. In an aspect, a population of γδ T cells can be immunopurified from a disclosed aliquot of isolated leukocytes using immunomagnetic negative selection.

[0190] In an aspect, immunopurification of γδ T cells can be achieved using a commercially available kit (e.g., EasySep™ Human Gamma / Delta T Cell Isolation Kit by StemCell Technologies). In an aspect, a disclosed immunopurification kit can employ one or more monoclonal and / or polyclonal antibodies that target specific antigens on the cell surface of unwanted cells. In an aspect, for example, unwanted cells (cells other than γδ T cells) can be labeled with antibodies and magnetic particles, and then separated using a magnet. In an aspect, immunopurification and / or sorting of γδ T cells can be performed without a column. In an aspect, immunopurification and / or sorting of γδ T cells can be performed with a column.

[0191] In an aspect, following immunopurification, a disclosed separated fraction can be a γδ T cell fraction. In an aspect, following immunopurification, a disclosed separated fraction can be a non-γδ T cell fraction.

[0192] In an aspect, a magnet can be used for at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or more than 10 minutes. In an aspect, a magnet can be used for aslong as recommended by the manufacturer of a commercial kit. In an aspect, a magnet can be used for as long as necessary to ensure the success of the immunopurification.

[0193] In an aspect, using a commercial γδ T cells isolation kit and immunomagnetic negative selection can allow the desired cells (i.e., γδ T cells) to be poured into one or more new tubes. In an aspect, once separated into one or more new tubes, the disclosed isolated γδ T cells can now be employed in one or more applications including, but not limited to flow cytometry, cell culture, DNA / RNA extraction, or any combination thereof.

[0194] In an aspect, a population of disclosed immunopurified and / or sorted γδ T cells can be centrifuged and re-suspended in media. In an aspect, the disclosed immunopurified and / or sorted γδ T cells can be re-suspended in a commercially available medium. In an aspect, a disclosed medium can be AIM V (ThermoFisher, Cat. # 12055083). In an aspect, AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 μg / mL), and gentamicin sulfate (e.g., 10 μg / mL). In an aspect, a disclosed medium can further comprise one or more antibodies and / or one or more transcription factors. For example, in an aspect, a disclosed medium can comprise OKT3 (orthoclone-muromonab-CD3). In an aspect, OKT3 can be added to a disclosed medium to achieve a final concentration of about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, or about 80 ng / mL.

[0195] For example, in an aspect, a disclosed medium can comprise IL-2. In an aspect, about 4000 IU / mL, about 5000 IU / mL, about 6000 IU / mL, about 7000 IU / mL, or about 8000 IU / mL of IL-2 can be added to a disclosed medium. In an aspect, AIM V medium can comprise about 6000 IU / mL of IL-2 and about 60 ng / mL OKT3.

[0196] In an aspect, immunopurified and / or sorted γδ T cells can be centrifuged at about 300 x g, about 400 x g, at about 500 x g, about 600 x g, or at about 700 x g. In an aspect, immunopurified and / or sorted γδ T cells can be centrifuged for about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, or about 7 minutes. In an aspect, immunopurified and / or sorted γδ T cells can be centrifuged at about 500 x g for about 5 minutes. In an aspect, centrifuging the disclosed immunopurified and / or sorted γδ T cells can be done at a temperature other than room temperature. In an aspect, centrifuging the disclosed immunopurified and / or sorted γδ T cells can be done at about 4 °C. In an aspect, immunopurified and / or sorted γδ T cells can be centrifuged at about 500 x g and at 4 °C for about 5 minutes.

[0197] In an aspect, a disclosed method can further comprise using a disclosed separated fraction comprising non-γδ T cells. For example, in an aspect, a disclosed separated fraction comprising non-γδ T cells (that is, cells other than γδ T cells) can be used as feeder cells. In an aspect, disclosed feeder cells can be used for the immunopurified and / or sorted γδ T cells. In an aspect,a disclosed method can further comprise transferring disclosed feeder cells (such as, for example, a disclosed separated fraction comprising non-γδ T cells) to a new medium. In an aspect, a disclosed method can further comprise transferring a population of disclosed feeder cells (such as, for example, non-γδ T cells) to a new medium. In an aspect, a disclosed medium can be a commercially available medium with or without modifications.

[0198] In an aspect, a population of disclosed feeder cells can be transferred and / or added to a 10% FBS RPMI medium. In an aspect, a disclosed medium comprising disclosed feeder cells be centrifuged. In an aspect, centrifugation of a disclosed medium comprising disclosed feeder cells can at about 300 x g, about 400 x g, at about 500 x g, about 600 x g, or at about 700 x g. In an aspect, a disclosed medium comprising disclosed feeder cells can be centrifuged for about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, or about 7 minutes. In an aspect, a disclosed medium comprising disclosed feeder cells can be centrifuged at about 500 x g for about 5 minutes. In an aspect, centrifuging a disclosed medium comprising disclosed feeder cells can be done at a temperature other than room temperature. In an aspect, centrifuging a disclosed medium comprising disclosed feeder cells can be done at about 4 °C. In an aspect, a disclosed medium comprising disclosed feeder cells can be centrifuged at about 500 x g and at 4 °C for about 5 minutes.

[0199] In an aspect, a disclosed method can further comprise re-suspending disclosed feeder cells in an appropriate medium. In an aspect, the disclosed feeder cells previously centrifuged can be re-suspended in an appropriate medium such as, for example, a RPMI media supplemented with 10% FBS with 1X DNAse I. In an aspect, a disclosed method can further comprise incubating the re-suspended disclosed feeder cells at room temperature. In an aspect, incubating the re- suspended disclosed feeder cells can last for at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, or more than 15 minutes. In an aspect, incubating the re-suspended disclosed feeder cells can last for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 1 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, or more than 15 minutes.

[0200] In an aspect, a disclosed method can further comprise re-suspending disclosed feeder cells in a RPMI media supplemented with 10% FBS with 1X DNAse I, and incubating the re-suspended disclosed feeder cells at room temperature for about 10 minutes. In an aspect, a disclosed method can further comprise centrifuging a disclosed medium comprising re-suspended feeder cells. In an aspect, a disclosed medium having DNAse I and comprising re-suspended feeder cells can have been incubated. For example, a disclosed incubation can comprise any disclosed incubation.

[0201] In an aspect, a disclosed method can further comprise centrifuging the disclosed feeder cells re-suspended in a RPMI media supplemented with 10% FBS with 1X DNAse I and incubated at room temperature for about 10 minutes. In an aspect, centrifugation of the disclosed incubated medium comprising re-suspended feeder cells can be at about 300 x g, about 400 x g, at about 500 x g, about 600 x g, or at about 700 x g. In an aspect, a disclosed incubated medium comprising re-suspended feeder cells can be centrifuged for about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, or about 7 minutes. In an aspect, a disclosed incubated medium comprising re-suspended feeder cells can be centrifuged at about 500 x g for about 5 minutes.

[0202] In an aspect, centrifuging a disclosed incubated medium comprising re-suspended feeder cells can be done at a temperature other than room temperature. In an aspect, centrifuging a disclosed incubated medium comprising re-suspended feeder cells can be done at about 4 °C. In an aspect, a disclosed incubated medium comprising re-suspended feeder cells can be centrifuged at about 500 x g and at 4 °C for about 5 minutes.

[0203] In an aspect, a disclosed method can further comprise re-suspending disclosed feeder cells following a centrifugation step. In an aspect, re-suspending disclosed feeder cells can be done in a commercially available medium with or without modifications. In an aspect, AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 μg / mL), and gentamicin sulfate (e.g., 10 μg / mL). In an aspect, a disclosed medium can further comprise one or more antibodies and / or one or more transcription factors. For example, in an aspect, a disclosed medium can comprise OKT3 (orthoclone-muromonab-CD3). In an aspect, OKT3 can be added to a disclosed medium to achieve a final concentration of about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, or about 80 ng / mL. For example, in an aspect, a disclosed medium can comprise IL-2. In an aspect, about 4000 IU / mL, about 5000 IU / mL, about 6000 IU / mL, about 7000 IU / mL, or about 8000 IU / mL of IL-2 can be added to a disclosed medium. In an aspect, AIM V medium can comprise about 6000 IU / mL of IL-2 and about 60 ng / mL OKT3.

[0204] In an aspect, a disclosed method can further comprise (i) re-suspending disclosed feeder cells in a RPMI media supplemented with 10% FBS with 1X DNAse I, (ii) incubating the re- suspended disclosed feeder cells at room temperature for about 10 minutes, (iii) centrifuging the incubated re-suspended disclosed feeder cells at about 500 x g for about 5 minutes at about 4 °C, and (iv) re-suspended in a medium (e.g., AIM V) at about 3e6 / mL.

[0205] In an aspect, a disclosed method can further comprise irradiating a population of disclosed feeder cells. In an aspect, irradiating feeder cells can comprise any irradiation protocol known to the skilled person in the art. In an aspect, irradiating the disclosed feeder cells can comprise usingan X-ray irradiator. In an aspect, use of an X-ray irradiator can comprise a protocol comprising 5000 rad | 160kV | 19.0 mA | 714 seconds | 30 cm.

[0206] In an aspect, irradiating a disclosed population of feeder cells can induced expression of NKG2D ligands in autologous cells. In an aspect, inducing expression of NKG2D ligands in autologous cells can cause preferential activation of γδ T cells.

[0207] In an aspect, a disclosed method can further comprise adding together a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells. In an aspect, a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be added to a container for incubation (such as, e.g., a flask). In an aspect, a disclosed flask can be an upright 25 cm2flask. In an aspect, a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be added to a flask.

[0208] In an aspect, adding together a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be at a ratio of about 1:150 (T cells:feeder cells), at a ratio of about 1:200 (T cells:feeder cells), at a ratio of about 1:250 (T cells:feeder cells), or at some other ratio. In an aspect, adding together a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be at a ratio of about 1:200 (T cells:feeder cells), at a ratio of about 1:250 (T cells:feeder cells).

[0209] In an aspect, a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells. In an aspect of a disclosed method, changing the medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be repeated one or more times. For example, in an aspect, changing the medium can occur on day 4 of incubation and on day 7 of incubation.

[0210] In an aspect, a disclosed method can further comprise removing from a disclosed flask some or all the spent medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells. In an aspect, changing the medium can comprise removing spent medium from the flask containing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells, and adding fresh medium to the flask.

[0211] In an aspect, spent medium can be collected and subjected to immediate analysis. In an aspect, spent medium can be collected and cryopreserved. In an aspect, cryopreserved spent medium can be thawed and subjected to analysis.

[0212] In an aspect, changing the medium can be done without disrupting the cells.

[0213] In an aspect, fresh medium can be added slowly to the flask to avoid disruption of the cells. In an aspect, fresh medium can be added to the flask by placing the tip of a pipette against the non-tissue culture treated side of the flask.

[0214] In an aspect of a disclosed method, fresh medium can comprise a pre-warmed medium. In an aspect, fresh medium can comprise AIM V medium. In an aspect, fresh AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 μg / mL), and gentamicin sulfate (e.g., 10 μg / mL). In an aspect, a disclosed fresh medium can further comprise one or more antibodies and / or one or more transcription factors. For example, in an aspect, a disclosed fresh medium can comprise IL-2. In an aspect, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, or about 4000 IU / mL of IL-2 can be added to a fresh disclosed medium. In an aspect, fresh AIM V medium can comprise about 3000 IU / mL of IL-2 and can be pre-warmed.

[0215] In an aspect, a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells after 3 days of incubating, after 4 days of incubating, after 5 days of incubating, or more than 5 days of incubating.

[0216] In an aspect, on day 4 of incubation, a disclosed method can further comprise removing from a disclosed flask some or all the spent medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells, and adding fresh pre-warmed medium to the flask.

[0217] In an aspect of a disclosed method, after the spent medium is removed and fresh medium is added, a disclosed flask containing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be placed upright.

[0218] In an aspect, on day 7 of incubation, a disclosed method can further comprise repeating the removing of spent medium and the adding of fresh medium. For example, on day 7 of incubation, a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells.

[0219] In an aspect, on day 7 of incubation, a disclosed method can further comprise removing from a disclosed flask some or all the spent medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells. In an aspect, on day 7 of incubation, changing the medium can comprise removing spent medium from the flask containing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells, and adding fresh medium to the flask.

[0220] In an aspect, spent medium can be collected and subjected to immediate analysis. In an aspect, spent medium can be collected and cryopreserved. In an aspect, cryopreserved spent medium can be thawed and subjected to analysis.

[0221] In an aspect, on day 7 of incubation, changing the medium can be done without disrupting the cells. In an aspect, fresh medium can be added slowly to the flask to avoid disruption of the cells. In an aspect, fresh medium can be added to the flask by placing the tip of a pipette against the non-tissue culture treated side of the flask.

[0222] In an aspect of a disclosed method, fresh medium can comprise a pre-warmed medium. In an aspect, fresh medium can comprise AIM V medium. In an aspect, fresh AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 μg / mL), and gentamicin sulfate (e.g., 10 μg / mL). In an aspect, a disclosed fresh medium can further comprise one or more antibodies and / or one or more transcription factors. For example, in an aspect, a disclosed fresh medium can comprise IL-2. In an aspect, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, or about 4000 IU / mL of IL-2 can be added to a fresh disclosed medium. In an aspect, fresh AIM V medium can comprise about 3000 IU / mL of IL-2 and can be pre-warmed.

[0223] In an aspect, on day 7 of incubation, a disclosed method can further comprise removing from a disclosed flask some or all the spent medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells, and adding fresh pre-warmed medium to the flask.

[0224] In an aspect of a disclosed method, after the spent medium is removed and fresh medium is added for a second time, a disclosed flask containing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be placed on its side or place on the flat surface.

[0225] In an aspect, changing the medium for a second time can comprise removing spent medium from the flask containing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells, and adding fresh medium to the flask.

[0226] In an aspect, spent medium can be collected for a second time and subjected to immediate analysis. In an aspect, spent medium can be collected and cryopreserved. In an aspect, cryopreserved spent medium can be thawed and subjected to analysis.

[0227] In an aspect, changing the medium for a second time can be done without disrupting the cells. In an aspect, fresh medium can be added slowly to the flask to avoid disruption of the cells. In an aspect, fresh medium can be added to the flask by placing the tip of a pipette against the non-tissue culture treated side of the flask.

[0228] In an aspect of a disclosed method, fresh medium can comprise a pre-warmed medium. In an aspect, fresh medium can comprise AIM V medium. In an aspect, fresh AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 μg / mL), and gentamicin sulfate (e.g., 10 μg / mL). In an aspect, a disclosed fresh medium can further comprise one or more antibodies and / or one or more transcription factors. For example, in an aspect, a disclosed fresh medium can comprise IL-2. In an aspect, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, or about 4000 IU / mL of IL-2 can be added to a fresh disclosed medium. In an aspect, fresh AIM V medium can comprise about 3000 IU / mL of IL-2 and can be pre-warmed.

[0229] In an aspect, a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells after 3 days of incubating, after 4 days of incubating, after 5 days of incubating, or more than 5 days of incubating. In an aspect, on day 4 of incubation, a disclosed method can further comprise removing from a disclosed flask some or all the spent medium bathing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells, and adding fresh pre-warmed medium to the flask.

[0230] In an aspect of a disclosed method, after the spent medium is removed and fresh medium is added, a disclosed flask containing a disclosed population of immunopurified and / or sorted γδ T cells and a disclosed population of irradiated feeder cells can be placed upright. In an aspect of a disclosed method, by day 10 of incubation, a disclosed population of irradiated feeder cells can be dead. In an aspect of a disclosed method, by day 10 of incubation, the expanded population of disclosed population of immunopurified and / or sorted γδ T cells can be counted.

[0231] In an aspect, a disclosed expanded population of γδ T cells can comprise at least 200 million γδ T cells, at least 250 million γδ T cells, at least 300 million γδ T cells, or more than at least 300 million γδ T cells. In an aspect, a disclosed expanded population of γδ T cells can comprise predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells. In an aspect, a disclosed expanded population of γδ T cells can comprise predominantly Vδ1 γδ T cells. In an aspect, a disclosed expanded population of γδ T cells can comprise predominantly Vδ3 γδ T cells. In an aspect, a disclosed expanded population can comprise clinically relevant amounts of Vδ1 γδ T cells and / or Vδ3 γδ T cells.

[0232] In an aspect, a disclosed method can further comprise transducing the expanded population of γδ T cells with one or more chimeric antigen receptors (CARs). CARs are known to the art and discussed supra. Generally, CARs include an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or moreimmunostimulatory domains. In an aspect, a disclosed CAR can be a 1stgeneration CAR, a 2ndgeneration CAR, a 3rdgeneration CAR, a 4thgeneration CAR, or a 5thgeneration CAR.

[0233] In an aspect, a disclosed method can further comprise transducing the expanded population of γδ T cells with one or more chimeric antigen receptors (CARs) using a transduction protocol known to the skilled person in the art. In an aspect, a disclosed method can further comprise transducing the expanded population of γδ T cells with one or more chimeric antigen receptors (CARs) using a transduction protocol employing a viral vector (e.g., a lentiviral vector or a retroviral vector) or a non-viral vector (e.g., transposons and mRNA electroporation).

[0234] In an aspect, a lentiviral vector can be used to integrate the genetic material for one or more CARs into the host cell's genome. In an aspect, the primary advantage of lentiviral vectors is their high transduction efficiency, even in non-dividing cells, which can make them highly effective for T lymphocyte modification. In an aspect, a retroviral vector can be used to integrate the genetic material for one or more CARs into the host cell genome. In an aspect, the advantage of retroviral vectors lies in their robustness and established methodologies. In an aspect, a transposon system (e.g., nonviral approaches like the Sleeping Beauty system) can utilize transposase enzymes to integrate the CAR gene into the T-cell genome. Transposons are DNA elements that can move from one genomic location to another. In CAR-T therapy, a transposon carrying the CAR gene is introduced into T-cells along with a transposase enzyme. The transposase mediates the integration of the CAR gene into the T-cell genome, enabling CAR expression. In an aspect, mRNA electroporation is another non-viral technique involving CAR gene transient expression. Electroporating mRNA encoding the chimeric antigen receptor construct into T lymphocytes enables the temporary expression of CAR proteins on the cell surface.

[0235] For example, in an aspect, a disclosed expanded population of γδ T cells can be transduced using lentiviral or retroviral transduction, transfection, electroporation, or any combination thereof. In an aspect, a disclosed expanded population of γδ T cells can be transduced using spin- infection. Spin-infection protocols are generally known in the art. In an aspect, a disclosed expanded population of γδ T cells can be transduced with one or more desired CARs and then re- suspended in AIM media. In an aspect, AIM V medium can be serum-free and can contain L- glutamine, streptomycin sulfate (e.g., 50 μg / mL), and gentamicin sulfate (e.g., 10 μg / mL). In an aspect, a disclosed AIM V medium can further comprise one or more antibodies and / or one or more transcription factors. For example, in an aspect, a disclosed AIM V medium can comprise IL-2. In an aspect, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, or about 4000 IU / mL of IL-2 can be added to a fresh disclosed medium. In an aspect, AIM Vmedium can comprise about 3000 IU / mL of IL-2. In an aspect, a disclosed transduced expanded population of γδ T cells can be incubated while laying flat in a flask or other container.

[0236] In an aspect of an expanded population of Vδ1 γδ T cells and / or Vδ3 γδ T cells, a disclosed antigen binding domain can comprise a binding domain for a known cancer-specific antigen or cancer-related antigen. In an aspect, cancer-specific antigens and cancer-related antigens are known to the art and include, but are not limited to, the following: HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4- 1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA- A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0-1, IL-11Ra, IL-13Ra2, Lambda, Lewis- Y, Kappa, Mesothelin, Mud, Muc16, NCAM, NKG2D Ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof.

[0237] In an aspect, a disclosed antigen binding domain can be a scFV and wherein the scFV can comprise a linker. In an aspect, a disclosed linker can join the VHand VLregions of the ScFv.

[0238] In an aspect, a disclosed the extracellular domain can further comprise a signal peptide. In an aspect, a disclosed signal peptide can comprise a CD8 signal peptide or a fragment thereof.

[0239] In an aspect, a disclosed chimeric antigen receptor can further comprise a spacer domain between the extracellular domain and the transmembrane domain. In an aspect, a disclosed spacer domain can comprise an immunoglobulin hinge region, an extracellular region of a type 1 membrane proteins, a part or all of an immunoglobulin constant region, or any combination thereof. In an aspect, a disclosed spacer domain can comprise a hinge region. In an aspect, a disclosed hinge region can comprise a hinge region of CD8α, CD28, IgG1, IgG2, IgG3, IgG4, IgA, IgD, or any combination thereof. In an aspect, a disclosed hinge region can be from or can be derived from CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD813, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D(KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK- p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, Toll ligand receptor, or any combination thereof, or any fragment / truncation thereof, or any combination of fragments / truncations thereof.

[0240] In an aspect, a disclosed chimeric antigen receptor can further comprise a transmembrane domain. In an aspect, a disclosed transmembrane can further comprise a transmembrane domain of CD2, CD3γ, CD3ε, CD3δ, CD3ξ, CD4, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD79B, CD80, CD86, CD95 (FAS), CD134 (OX40), CD137 (4-1BB), CD154, CD278(ICOS), TCRα, TCRβ, NKG2D, 2B4, or any combination thereof.

[0241] In an aspect, a disclosed transmembrane domain can be from or can be derived from the alpha, beta or zeta chain of a T-cell receptor, NKG2D, 2B4, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DAP- 12, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, a ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / 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; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a combination thereof, or afragment / truncation thereof, or a combination of fragments and / or truncations thereof. In an aspect, a disclosed transmembrane domain can be from or can be derived from NKG2D,

[0242] In an aspect, a disclosed intracellular domain can comprise the intracellular domain of CD28. In an aspect, a disclosed intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (ITAMs). In an aspect, a disclosed ITAM can comprise the signaling domain of DAP10, DAP12, TCRζ, FcRγ, FcRβ, CD3γ, CD3ξ, CD3ε, CD3δ, CD3ξ, CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), or any combination thereof.

[0243] In an aspect, a disclosed intracellular domain can further comprise one or more co- stimulatory domains. In an aspect, a disclosed co-stimulatory domain can comprise the signaling domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7 LIGHT, NKG2C, B7-H3, or any combination thereof. In an aspect, a disclosed costimulatory molecule can comprise 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1- 1a, CD1-1b, CD1-1c, CD1-1d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO- 3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0244] In an aspect of a disclosed chimeric antigen receptor, a disclosed intracellular domain can comprise an ITAM and one or more co-stimulatory domains, wherein the ITAM can comprise the signaling domain of CD3γ, CD3ξ, CD3ε, CD3δ, CD3ξ, CD5, CD22, CD79a, CD278 (ICOS), or any combination thereof, and wherein the co-stimulatory domains can compris the signaling domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7 LIGHT, NKG2C, B7-H3, or any combination thereof.

[0245] In an aspect of a disclosed chimeric antigen receptor, a disclosed intracellular domain can comprise one or more ITAMs and one or more co-stimulatory domains, wherein the ITAMs can comprise the signaling domain of CD3γ, CD3ξ, CD3ε, CD3δ, CD3ξ, CD5, CD22, CD79a, CD278 (ICOS), or any combination thereof, and wherein the co-stimulatory domains can comprise the signaling domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7 LIGHT, NKG2C, B7-H3, or any combination thereof.

[0246] In an aspect, the disclosed two co-stimulatory domains can comprise two of the same signaling domain. In an aspect, the disclosed two co-stimulatory domains can comprise two different signaling domains. In an aspect, a disclosed co-stimulatory domain can comprise CD28. In an aspect, a disclosed CD28 co-stimulatory domain can demonstrate one or more functional aspects: (i) lower persistence and differentiation towards effector memory phenotype compared to 41BB second-generation CARs, (ii) more prone to tonic signaling and causes early exhaustion, (iii) imparts resistance to Tregs in-vitro, in-vivo models however suggested that CD28 co- stimulation causes increased infiltration of Tregs and were less effective against tumors in presence of Tregs, (iv) resistant to CTLA4 inhibition, (v) faster and higher signaling intensity, (vi) does not alter scFv "affinity ceiling" - affinity beyond which IFNγ, IL2 secretion and cytotoxicity do not increase, or (vii) any combination thereof. In an aspect, a disclosed co-stimulatory domain can comprise 4-1BB. In an aspect, a disclosed 4-1BB co-stimulatory domain can demonstrate one or more functional aspects: (i) greater persistence and differentiation towards central memory phenotype compared to CD28 second- generation CARs, (ii) can reduce tonic signaling at optimal expression levels and decrease exhaustion, (iii) slower and less intense signaling, or (iv) any combination thereof. In an aspect of a chimeric antigen receptor, a disclosed intracellular domain can further comprise a self-cleaving peptide. In an aspect, a disclosed self-cleaving peptide can comprise a T2A, a P2A, a E2A, or F2A peptide. In an aspect, a GSG linker can be added to the N-terminus of a disclosed 2A peptide. In an aspect of a chimeric antigen receptor, a disclosed intracellular domain can further comprise a sequence encoding an EGFR domain or a truncated EGFR domain.

[0247] In an aspect, a disclosed CAR can comprise a first-generation CAR, a second-generation CAR, a third-generation CAR, a fourth-generation CAR, or a fifth-generation CAR. CARs are discussed supra.

[0248] In an aspect, a disclosed method can further comprise removing and / or depleting residual αβ T cells from a disclosed expanded population of γδ T cells. In an aspect, removing and / or depleting residual αβ T cells from a disclosed expanded population of γδ T cells can be performed using a commercially available kit (e.g., CliniMACS TCRα / β product line (Miltenyi Biotec, Cat.# 200-070-409)), or can be performed using another technique or protocol known to the skilled person in the art.

[0249] In an aspect, removing and / or depleting residual αβ T cells from a disclosed expanded population of γδ T cells can be performed at day 8, day 9, day 10, day 11, or day 12 of incubation. In an aspect, removing and / or depleting residual αβ T cells from a disclosed expanded population of γδ T cells can be performed at day 10 of incubation.

[0250] In an aspect, if a disclosed expanded population of γδ T cells is transduced with one or more CARs, then removing and / or depleting αβ T cells from a disclosed expanded population of γδ T cells can be performed at day 12, day 13, day 14, day 15, or day 16 of incubation. In an aspect, if a disclosed expanded population of γδ T cells is transduced with one or more CARs, then removing and / or depleting αβ T cells from a disclosed expanded population of γδ T cells can be performed at day 14 of incubation.

[0251] In an aspect of a disclosed method, following the removing and / or depleting of αβ T cells from a disclosed expanded population of γδ T cells, the remaining population of γδ T cells can comprise predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells. In an aspect of a disclosed method, following the removing and / or depleting of αβ T cells from a disclosed expanded population of transduced γδ T cells, the remaining population of CAR γδ T-cells can comprise predominantly Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0252] In an aspect, predominantly can mean that the number of Vδ1 γδ T cells and / or V3 γδ T cells in a disclosed expanded population of γδ T cells significantly exceeds the number of V2 γδ T cells in the same expanded population.

[0253] In an aspect, predominantly can mean that the number of Vδ1 γδ T cells in a disclosed expanded population of γδ T cells significantly exceeds the number of V2 γδ T cells in the same expanded population. In an aspect, predominantly can mean that the number of V3 γδ T cells in a disclosed expanded population of γδ T cells significantly exceeds the number of V2 γδ T cells in the same expanded population.

[0254] In an aspect, when all steps of a disclosed method have been performed, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells remains. In an aspect, when all steps of a disclosed method have been performed, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells remains. In an aspect, a disclosed method can generate a disclosed expanded population of γδ T cells that comprises predominantly Vδ1 γδ T cells and / or V3 γδ T cells (rather than V2 γδ T cells). In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells remains can be cryopreserved for future use and / or analysis.In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T- cells can be cryopreserved for future use and / or analysis.

[0255] In an aspect, expanded γδ T cells and / or expanded CAR γδ T-cells can be added to a freezing media (90% FBS + 10% DMSO) to achieve a final concentration of about 2e7 cryopreserved cells / mL to about 5e7 cryopreserved cells / mL.

[0256] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells remains can be administered to one or more subjects having cancer. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered to one or more subjects having cancer.

[0257] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered as an allogeneic cell therapy to a subject having cancer. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a subject having cancers. In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancer. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancers.

[0258] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered to a subject having an αβ T cell refractory cancer. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a subject having an αβ CAR T-cell refractory cancer.

[0259] In an aspect, a population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can be administered to a plurality of subjects having an αβ T cell refractory cancer. In an aspect, a population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells can be administered as an allogeneic cell therapy to a plurality subjects having an αβ CAR T-cell refractory cancer.

[0260] In an aspect, a disclosed method of expanding a desired T cell population can further comprise measuring the purity of a population of γδ T cells following expansion by a disclosed method and / or the purity of a population of CAR γδ T-cells following expansion by a disclosed method. In an aspect, a disclosed method can increase of purity of a population of Vδ1 T-cells. In an aspect, a disclosed method can increase of purity of a population of Vδ1- Vδ2- T-cells. In an aspect, a disclosed method can increase of purity of a population of Vδ3 T-cells. In an aspect, a disclosed method can increase of purity of a population of Vδ1 T-cells and / or Vδ1- Vδ2- T-cells and / or Vδ3 T-cells.

[0261] In an aspect, a disclosed method of expanding a desired T cell population can further comprise validating the efficacy of one or more of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method). In an aspect, validating the efficacy of one or more populations of expanded T cells can comprise administering the expanded T cells to cancer cells in culture and / or to a laboratory animal having cancer.

[0262] In an aspect, a disclosed method of expanding a desired T cell population can further comprise subjecting one or more disclosed population of expanded γδ T cells to a delta chain analysis using, for example, iRepertoire. In an aspect, a disclosed method of expanding a desired T cell population can further comprise sorting one or more disclosed population of expanded γδ T cells using FACS or a similar type of flow cytometry. In an aspect, a disclosed method of expanding a desired T cell population can further comprise evaluating one or more disclosed population of expanded γδ T cells in a cytotoxicity assay.

[0263] For example, in an aspect, a disclosed flow panel can consider one or more of the following: (1) BD Horizon™ BV786 Mouse Anti-Human CD45. Clone: HI30; Cat No:563716; (2) BD Horizon™ BUV395 Mouse Anti-Human CD3. Clone: SK7. Cat No: 564001; (3) BD Pharmingen™ Alexa Fluor® 700 Mouse Anti-Human CD14. Clone M5E2. Cat No:557923; (4) BD Pharmingen™ PE Mouse Anti-Human TCRαβ. Clone: IP26 (also known as IP26A). Cat Number: 564728; (4) BD Horizon™ BV711 Mouse Anti-Human γδ TCR. Clone: 11F2. Cat No: 568490; (6) Invitrogen. TCR V delta 1 Monoclonal Antibody (TS8.2), PE-Cyanine7, eBioscience™; (7) Biolegend. APC anti-human TCR Vδ2 Antibody. Clone B6. Cat: 331417; (8) Biolegend. Zombie NIR™ Fixable Viability Kit. Cat# 423106; (9) Biolegend. Brilliant Violet 605™ anti-human IFN-γ Antibody. Clone B27. Cat 506542; (10) BD Horizon™ BV421 Mouse Anti-Human Perforin. Clone δG9. Cat# 563393; (11) BD Horizon™ R718 Mouse Anti-Human Granzyme B. Clone GB11. Cat# 566964; (12) BD Pharmingen™ PerCP-Cy5.5 Mouse Anti- Human CD27. Cat# 567181; (13) Cell Signaling Technology. TCF1 / TCF7 (C63D9) Rabbit mAb (Alexa Fluor® 647 Conjugate) #6709; (14) Biolegend. Brilliant Violet 421™ anti-human CD366 (Tim-3) Antibody. Clone F38-2E2. Cat# 345008; (15) Biolegend. FITC anti-human CD223 (LAG-3) Antibody. Clone 11C3C65. Cat# 369308; (16) Biolegend. Brilliant Violet 605™ anti- human CD337 (NKp30) Antibody. Cat# 325234; and (17) Biolegend. PE / Dazzle™ 594 anti- human CD314 (NKG2D) Antibody. Clone 1D11. Cat# 320828.

[0264] In an aspect, a disclosed gating strategy can comprise viable CD45+CD14negCD3+TCRαβnegTCRγδ+Vd1+Vd2+. In an aspect, expression for specific markers (IFN-γ, Perforin, Granzyme B, CD27, TCF1 / TCF7, Tim-3, NKG2D, LAG-3 and NKp30 can beevaluated separately for the Vδ1 and Vδ2 T cells subsets. In an aspect, expression for expecific markers (IFN-γ, Perforin, Granzyme B, and TCF1 / TCF7 (an intracellular staining)) can be performed following the manufacturer's instructions (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set Cat. #00-5523-00; ThermoFisher). Samples can be run either on a BD LSRII / BD LSRFortessa X-20 for flow analysis and / or can besorted using a BD FACS Aria / Beckman Coulter MoFlo Astrios. All sorted populations can be confirmed to be >98% pure through post-sort analysis.

[0265] In an aspect, a disclosed method can further comprise cryopreserving a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method). In an aspect, a disclosed method can further comprise thawing a disclosed cryopreserved population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed cryopreserved population of Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell s(expanded by a disclosed method) or a pharmaceutical formulation thereof.

[0266] In an aspect, a disclosed method can further comprise administering a subject having cancer a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method) or a pharmaceutical formulation thereof.

[0267] In an aspect, a disclosed method can further comprise administering to a plurality of subjects having cancer an allogeneic cell therapy comprising a disclosed population of Vδ1 γδ T- cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method) of a pharmaceutical formulation thereof.

[0268] In an aspect, a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T cells (expanded by a disclosed method) of a pharmaceutical formulation thereof can be fresh when administered to a subject (e.g. recently generated) or can be previously cryopreserved and thawed when administered to a subject having cancer.

[0269] Disclosed herein is a method of expanding a desired T cell population, the method comprising isolating leukocytes from a sample comprising cord blood, immunopurifying γδ T cells from the isolated leukocytes; sorting the isolated leukocytes into γδ+T cells and γδ−T cells; using the γδ−T cells as feeder cells; irradiating the feeder cells to create preferential activation of γδ+T cells; incubating the irradiated feeder cells and the activated γδ T cells at a ratio of about 1:200 (γδ T cells: feeder cells) in an upright flask; on day 4, removing spent media from the topof the flask and adding fresh media to the flask; on day 7, removing spent media from the top of the flash and adding fresh media to the flask and placing the flask on its side; and on day 10, depleting the αβ T cells and removing and counting the γδ T cells. In an aspect, the method can further comprise transducing the γδ T cells with a vector comprising a nucleic acid sequence encoding one or more chimeric antigen receptors on day 14.

[0270] In an aspect of a disclosed method, a disclosed population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to kill stressed cells (e.g., tumor or virally infected cells) and can be combined with IgG targeting molecules on the tumor cell surface, thereby addressing the problem of antigen heterogeneity.

[0271] In an aspect of a disclosed method, a disclosed expanded population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to avoid T cell exhaustion. F. Methods of Treating Cancer

[0272] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of CAR γδ T-cells expanded by a disclosed method.

[0273] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 CAR γδ T- cells and / or Vδ3 CAR γδ T-cells.

[0274] Disclosed herein is a method of treating cancer, the method comprising administering to the subject having cancer a therapeutically effective amount of a population of γδ T-cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising administering to a subject having cancer a therapeutically effective amount of a population of CAR γδ T-cells expanded by a disclosed method.

[0275] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of γδ T-cells expanded by a disclosed method, wherein the expanded population of CAR γδ T-cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of CAR γδ T-cells expanded by a disclosed method, wherein the expanded population of CAR γδ T-cells predominantly comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0276] Disclosed herein is a method of treating cancer, the method comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a population of γδ T cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method.

[0277] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method.

[0278] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method, wherein the expanded population of γδ T cells predominantly comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0279] Disclosed herein is a method of treating cancer, the method comprising administering to a subject having cancer a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T-cells expanded by a disclosed method. Disclosed herein is a method of treating cancer, the method comprising administering to a subject having cancer a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method.

[0280] Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of γδ T-cells expanded by a disclosed method, wherein the expanded population of γδ T-cells predominantly comprise Vδ1 γδ T-cells and / or Vδ3 γδ T-cells. Disclosed herein is a method of treating cancer, the method comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of CAR γδ T-cells expanded by a disclosed method, wherein the expanded population of CAR γδ T-cells predominantly comprise Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells.

[0281] Use of a population γδ T cells expanded by a disclosed method to treat a subject having cancer. Use of a population of CAR γδ T-cells expanded by a disclosed method to treat a subject having cancer. Use of a population of Vδ1 γδ T cells and / or Vδ3 γδ T-cells expanded by a disclosed method to treat a subject having cancer. Use of a population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells expanded by a disclosed method to treat a subject having cancer. Use of a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells to treat a subject having cancer.

[0282] Use of a population γδ T cells expanded by a disclosed method in the manufacture of a medicament to treat a subject having cancer. Use of a population of CAR γδ T-cells expanded by a disclosed method in the manufacture of a medicament to treat a subject having cancer. Use of a population of Vδ1 γδ T cells and / or Vδ3 γδ T-cells expanded by a disclosed method in the manufacture of a medicament to treat a subject having cancer. Use of a population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells expanded by a disclosed method in the manufacture of a medicament to treat a subject having cancer. Use of a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells in the manufacture of a medicament to treat a subject having cancer.

[0283] In an aspect, a disclosed γδ CAR T-cell can target one or more antigens on the surface of a cancer cell. In an aspect, a disclosed γδ CAR T-cell can target a cancer cell in a solid tumor. In an aspect, a disclosed γδ CAR T-cell can target a blood borne cancer cell. Cancer-specific and / or cancer-related antigens are known in the art and can include, but are not limited, to the following: HPV-16 E6 and HPV-16 E7, alpha folate receptor, 5T4, avp6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIll, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLAA1+MAGEI, HLA-A2+MAGE1,HLAA3+MAGE1, HLA-A1+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NYES0-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mud, Muc16, NCAM, NKG2D Ligands, NYE- S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEMs, VEGFRII, or any combination thereof. In an aspect, a disclosed population of expanded γδ T-cells or a disclosed population of expanded CAR γδ T-cells can target an antigen on a αβ T-cell refractory tumor or an αβ CAR T-cell refractory cancer cell or tumor cell.

[0284] In an aspect, a disclosed method of treating cancer can comprise stimulating an effector cell mediated immune modulator response to a cancer cell targeted by a disclosed γδ T-cell or a disclosed γδ CAR T-cell . In an aspect, a disclosed method of treating cancer can induce a tumor reducing immune response. In an aspect, a disclosed method of treating cancer can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed method of treating cancer can cross-prime an anti-tumor T cell response. In an aspect, a disclosed method of treating cancer can induce a tumor eliminating immune response. In an aspect, a disclosed method of treating cancer can effect tumor cell death.

[0285] In an aspect, a disclosed γδ T cell and / or a disclosed γδ CAR T-cell can be allogeneic to a subject having cancer and / or a subject in need thereof.

[0286] In an aspect, the disclosed Vδ1 γδ T cells and / or Vδ3 γδ T cells (expanded by a disclosed method) and / or the disclosed Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) can be used against ovarian cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, malignant pleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bile duct cancer, bladder cancer, transitional cell carcinoma, papillary carcinoma, kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, testicular seminoma, testicular teratoma, embryonic testicular cancer, uterine cancer, teratocarcinoma, embryonal carcinoma, or any combination thereof. In an aspect, a disclosed subject can have an αβ T-cell refractory tumor or an αβ CAR T-cell refractory cancer. In an aspect, a disclosed subject can one or more αβ T-cell refractory tumors or one or more αβ CAR T-cell refractory tumors.

[0287] In an aspect, a disclosed method of treating cancer can further comprise collecting one or more blood and / or biological samples from a subject at the same time or at different times. Forexample, in an aspect, a blood sample and / or a biological sample can be collected from a subject at a pre-determined interval. In an aspect, a pre-determined interval can be once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, or at a longer interval. In an aspect, a pre-determined interval can be once a month, once every 2 months, once every 3 months, once every 5 months, once every 5 months, once every 6 months, or at a longer interval. In an aspect, a blood sample and / or a biological sample can be collected from a subject prior to treatment, during treatment, after treatment, or any combination thereof. In an aspect, a blood and / or a biological sample can be collected from a subject at any time deemed medically and / or clinically appropriate by the skilled clinician.

[0288] In an aspect, a therapeutically effective amount of a disclosed expanded population of γδ T cells predominantly comprising Vδ1 γδ T-cells and / or Vδ3 γδ T-cell and / or a disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell or a pharmaceutical formulation can be about 1 x 103cells to about 1 x 109cells per subject. In an aspect, a therapeutically effective amount of a disclosed expanded population of γδ T cells predominantly comprising Vδ1 γδ T-cells and / or Vδ3 γδ T-cell and / or a disclosed expanded population of Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell or a pharmaceutical formulation can be about 1 x 104cells / kg per subject, about 1 x 105cells / kg per subject, about 1 x 106cells / kg per subject, about 1 x 107cells / kg per subject, about 1 x 108cells / kg per subject, or about 1 x 109cells / kg per subject.

[0289] In an aspect, a disclosed expanded population of γδ T cells predominantly comprising Vδ1 γδ T-cells and / or Vδ3 γδ T-cell can be delivered a dosing regimen. For example, in an aspect, a first dose of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered. If the subject does not show signs of CRS, then a second dose of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered to the subject. If the subject does not show signs of CRS, then a third dose Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered to the subject.

[0290] In an aspect, a disclosed expanded population of γδ T cells predominantly comprising Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell can be delivered a dosing regimen. For example, in an aspect, a first dose of Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered. If the subject does not show signs of CRS, then a second dose of Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered to the subject. If thesubject does not show signs of CRS, then a third dose Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) can be administered to the subject.

[0291] In an aspect, several factors including target antigen, intracellular signaling domain, and product characteristics such as cell viability and number of CAR molecules per cell, can impact the efficacy and toxicity of the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method). All of these factors should be considered in determining the total dose of CAR-T cells. The dose that achieves an optimal effector (CAR-T cell)-to-tumor target cell (E:T) ratio can result in the maximal eradication of the tumor cells and achieve the most durable responses for a given product. Lower doses may result in sub-optimal E:T ratio and exhaustion of CAR-T cells whereas very high doses can precipitate intolerable toxicity.

[0292] In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of the disclosed Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can protect a subject against the onset of a disease and / or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0293] In an aspect, administering Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can comprise systemic or direct administration. In an aspect, administering can comprise oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof. In an aspect, Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered by any method of administration disclosed herein. In an aspect, Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered via multiple routes either concurrently or sequentially.

[0294] For example, in an aspect, Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof can be first administered intratumorally and thenbe administered intravenously. In an aspect, Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof can be first administered intravenously and then be administered intratumorally. A skilled clinician can determine the best route of administration for a subject at a given time.

[0295] In an aspect, local administration of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof can comprise delivery to one or more of the subject's body systems having cancerous cells or tumorous growth. In an aspect, the subject's one or more body systems having cancerous cells or tumorous growth can comprise the subject's cardiovascular system, the subject's digestive system, the subject's endocrine system, the subject lymphatic system, the subject's muscular system, the subject's nervous system, the subject's reproductive system, the subject's respiratory system, the subject's skeletal system, the subject's urinary system, the subject's integumentary system, or any combination thereof.

[0296] In an aspect, a disclosed method of treating cancer can further comprise repeating the administering of the Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) a pharmaceutical formulation thereof.

[0297] In an aspect, a disclosed method of treating cancer can comprise protecting the subject from metastasis. In an aspect, a disclosed method of treating cancer can comprise reducing the risk of developing metastasis. In an aspect, a disclosed method of treating cancer can comprise preventing or inhibiting metastasis.

[0298] In an aspect, a disclosed method can comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, a disclosed method can comprise continuing to treat the subject. In an aspect, continuing to treat the subject can comprise continuing to administer Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof. In an aspect, in the presence of adverse effects, a disclosed method can comprise modifying one or more steps of the method. In an aspect, modifying one or more steps of a disclosed method can comprise modifying the administering step. In an aspect, modifying the administering step can comprise changing the amount of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof administered to the subject, changing the frequency of administration of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell(expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, changing the duration of administration of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, changing the route of administration of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, or any combination thereof.

[0299] In an aspect, a disclosed method of treating cancer can comprise administering to the subject one or more additional anti-cancer therapies. Anti-cancer therapies are known to the art. In an aspect, a disclosed anti-cancer therapy can comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof. In an aspect, a disclosed anti-cancer therapy can comprise one or more chemotherapeutic agents. In an aspect, a disclosed chemotherapeutic agent can comprise an anthracycline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof. In an aspect, a disclosed chemotherapeutic agent can comprise 5- fluorouracil (Adrucil, Efudex), 6-mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), axitinib (Inlyta), bevacizumab (Avastin), bicalutamide (Casodex), bleomycin sulfate (Blenoxane), bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC- Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), doxorubicin hydrochloride (Adriamycin, Rubex), etoposide (Vepesid), fludarabine phosphate (Fludara), flutamide (Eulexin), folic acid antagonists, gemcitabine (difluorodeoxycitidine), gemtuzumab, gliotoxin, hydroxyurea (Hydrea), Idarubicin (Idamycin), ifosfamide (IFEX), ifosfamide, irinotecan (Camptosar), L-asparaginase (ELSPAR), lenalidomide), leucovorin calcium, melphalan (Alkeran), melphalan, methotrexate (Folex), mitoxantrone (Novantrone), mylotarg, N4-pentoxycarbonyl-5 deoxy-5-fluorocytidine, nab-paclitaxel (Abraxane), paclitaxel (Taxol), pentostatin, phoenix (Yttrium90 / MX-DTPA), polifeprosan 20 with carmustine implant (Gliadel), purine analogs and adenosine deaminase inhibitors (fludarabine), pyrimidine analogs, rituximab,tamoxifen citrate (Nolvadex), temozolomide), teniposide (Vumon), tezacitibine, thalidomide or a thalidomide derivative, thiotepa, tirapazamine (Tirazone), topotecan hydrochloride for injection (Hycamptin), tositumomab), vinblastine (Velban), vinblastine, vincristine (Oncovin), vindesine, vinorelbine (Navelbine), or any combination thereof.

[0300] In an aspect, a disclosed method of treating cancer can comprise administering to the subject an anti-chemokine therapy. In an aspect, a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCR2, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof.

[0301] In an aspect, a disclosed method of treating cancer can comprise administering to the subject one or more immune modulators. In an aspect, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-Rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGF1R antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration.

[0302] In an aspect, a disclosed method of treating cancer can comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. CRISPR / Cas9 systems and methods are known to the art.

[0303] In an aspect, a disclosed method of treating cancer can further comprise preventing or inhibiting metastasis of cancer cells. In an aspect, a disclosed method can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease or reduction in the risk of and / or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T- cell (expanded by a disclosed method) or a pharmaceutical formulation thereof). In an aspect, preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or any amount of decrease or reduction in the risk of and / or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof).

[0304] In an aspect, a disclosed method of treating cancer can comprise surgically resecting the tumor and / or cancer cells from the subject. In an aspect, following resecting the tumor and / or cancer cells from the subject, a disclosed method of treating cancer can comprise continuing to administer to the subject a therapeutically effective amount of a disclosed anti-cancer agent, a disclosed anti-chemokine therapy, a disclosed chemotherapeutic agent, any disclosed therapeutic agent, or any combination thereof.

[0305] In an aspect, a disclosed method of treating cancer can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0306] In an aspect, a disclosed method of treating cancer can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation in a subject, such as, for example, a subject having cancer or cancerous cells. In an aspect, a disclosed interfering molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation in a subject having cancer. In an aspect, metabolic dysregulation can be associated with cancer or cancerous cells. In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and / or ameliorating autophagy);(iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of the multi-systemic manifestations of a cancer; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a cancer, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and / or preserving one or more aspects of cellular structural and / or functional integrity. In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and / or functionality is similar to that of a wild-type or control level. In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise preventing or inhibiting metastasis of cancer cells in the subject.

[0307] In an aspect, following the administering step, (i) the risk of developing metastases can be prevented and / or decreased; (ii) the survival of the subject can be prolonged; (iii) the subject's quality of life can be enhanced and / or improved; (iv) the likelihood of surgical intervention can be reduced and / or minimized; (v) preventing and / or delaying recurrence of the cancer; (vi) the size of one or more tumors in the subject can be reduced and / or decreased; (vii) one or more tumors in the subject can be eliminated; (viii) extending and / or prolonging disease-free or tumor- free survival time; (ix) increasing and / or lengthening overall survival time; (x) reducing and / or minimizing the frequency of treatment; (xi) relieving and / or ameliorating one or more symptoms of the cancer; (xii) reducing and / or decreasing tumor burden, (ix) preventing and / or facilitating surgical intervention; (xiii) normal metabolism of one or more organ systems in the subject can be improved and / or restored, (xiv) one or more aspects of cellular homeostasis and / or cellular functionality, and / or metabolic dysregulation can be restored and / or improved, or (xv) any combination thereof.

[0308] In an aspect of a disclosed method of treating cancer, techniques to monitor, measure, and / or assess the restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided below.

[0309] In an aspect, a disclosed method of treating cancer can comprise titrating the dose of a disclosed targeted therapy, a disclosed composition, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, a disclosed RNA therapeutic, or any combination thereof to identify an effective dose and / or to identify an effective dose eliciting only mild adverse and / or side effects.

[0310] In an aspect, a disclosed method of treating cancer can comprise titrating the dose of a disclosed treatment regimen in a specific or disclosed subject. In an aspect, a disclosed method of treating and / or preventing cancer can comprise titrating the dose of a disclosed composition, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, a disclosed RNA therapeutic, or any combination thereof to identify an effective dose and / or to identify an effective dose eliciting only mild adverse and / or side effects for a specific or disclosed subject.

[0311] In an aspect, administering to a subject Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof can be done prior to, concurrent with, or after the administering of any other therapeutic agent and / or targeted therapy can change during a treatment regimen. In an aspect, the order of administering the one or more disclosed populations of expanded γδ T cells, one or more disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method), or the one or more disclosed populations of Vδ1 CAR γδ T- cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), or a pharmaceutical formulation thereof and the administering of any other therapeutic agent and / or targeted therapy can change during a treatment regimen.

[0312] In an aspect, a disclosed method of treating and / or preventing cancer can further comprise obtaining a biological sample from the subject prior to administering a disclosed treatment regimen. In an aspect, a disclosed method of treating and / or preventing cancer can further comprise obtaining a biological sample from the subject after administering a disclosed treatment regimen. In an aspect, a disclosed method of treating and / or preventing cancer can further comprise subjecting the biological sample to a cell-free DNA (cfDNA) analysis. cfDNA analyses are known to the skilled person in the art. In an aspect, a disclosed cfDNA analysis can be repeated one or more times. In an aspect, a disclosed obtaining step can be repeated one or more times.

[0313] In an aspect of a disclosed method of treating and / or preventing cancer, a disclosed cfDNA analysis can comprise next generation sequencing. In an aspect, next generation sequencing (NGS) can comprise using one or more commercially available platforms. Commercially available NGS sequencing platforms can comprise, for example, Guardant360 CDx (Guardant Health, Inc.), FoundationOne CDx (F1CDx) (Foundation Medicine, Inc.), or Tempus xT (Tempus).

[0314] In an aspect of a disclosed method of treating cancer, next generation sequencing can comprise sequencing one or more cancer related genes. In an aspect of a disclosed method of treating and / or preventing cancer, sequencing one or more cancer related genes can comprise identifying one or more genomic aberrations. In an aspect, one or more genomic aberrations can comprise somatic genomic aberrations. In an aspect, the disclosed one or more somatic genomic aberrations can comprise mutations, insertions, deletions, chromosomal rearrangements, copy number aberrations, or any combination thereof.

[0315] In an aspect of a disclosed method, one or more genomic aberrations in the biological sample can be detected. In an aspect, a disclosed method can further comprise detecting one or more genomic aberrations in the biological sample. In an aspect, a disclosed method can further comprise identifying one or more genomic aberrations in a panel of genes. In an aspect, disclosed genomic aberrations can be identified in a panel of genes. In an aspect, a disclosed panel of genes can comprise at least one gene, at least two genes, 3 or more genes, 5 or more genes, 7 or more genes, or 10 or more genes. In an aspect, a disclosed panel of genes can comprise 2 or more genes, 3 or more genes, 4 or more genes, 5 or more genes, 6 or more genes, 7 or more genes, 8 or more genes, 9 or more genes, or 10 or more genes. In an aspect, a disclosed panel of genes can comprise any combination of disclosed genes or disclosed cancer-related genes.

[0316] In an aspect, a disclosed method can comprise detecting the expression of one or more disclosed genes and / or one or more disclosed gene aberrations. In an aspect, a disclosed method can comprise generating a control sample and / or a pooled control sample. In an aspect, a disclosedmethod can comprise generating a reference sample and / or a pooled reference sample. In an aspect, a disclosed method can comprise generating a range of a control samples and / or a pooled control samples. In an aspect, a disclosed method can comprise generating a range of reference samples and / or a pooled reference samples.

[0317] In an aspect, if the expression and / or amount and / or presence of the disclosed one or more genomic aberrations in the post-treatment biological sample is lower than the expression and / or amount and / or presence of the same one or more genomic aberrations in a pre-treatment sample, then a disclosed method of treating cancer can comprise continuing to administer to the subject a disclosed treatment regimen comprising of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof. In an aspect, if the expression and / or amount and / or presence of the disclosed one or more genomic aberrations in the post-treatment biological sample is lower than the expression and / or amount and / or presence of the same one or more genomic aberrations in a prior post-treatment sample, then a disclosed method of treating and / or preventing cancer of treating and / or preventing cancer can comprise continuing to administer to the subject a disclosed treatment regimen comprising of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.

[0318] In an aspect, a disclosed method of treating cancer can further comprise measuring the subject's tumor response to the administering of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof. In an aspect, a subject's tumor response can comprise a partial response or a complete response. In an aspect, a disclosed partial response can comprise a decrease in the size of a tumor or a decrease in one or more tumors by 25% or more when compared to the size of the same tumor or the same one or more tumors prior to the treatment with of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof. In an aspect, a disclosed partial response can comprise a decrease in the size of a tumor or a decrease in one or more tumors by 50% or more when compared to the size of the same tumor or the same one or more tumors prior to treatment with of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof. In an aspect, a disclosed partial response can comprise a decrease in the sizeof a tumor or a decrease in one more tumors by about 100% or more when compared to the size of the same tumor or the same one or more tumors prior to treatment.

[0319] In an aspect, a complete response can be defined as complete disappearance of all tumors with no recurrence of tumors for at least four weeks. In an aspect, a partial response can be defined as a 50% reduction in total tumor size with such reduction lasting at least four weeks. In an aspect, stable disease can be defined as less than 50% reduction in size but no more than 25% increase in size of the tumor mass lasting for at least twelve weeks.

[0320] In an aspect, a disclosed method of treating cancer can further comprise measuring the subject's molecular response to a disclosed treatment regimen. In an aspect, a disclosed molecular response can comprise a decrease in the number of somatic genomic aberrations in a disclosed biological sample obtained from the subject. In an aspect, disclosed somatic genomic aberrations can comprise mutations, insertions, deletions, chromosomal rearrangements, copy number aberrations, fusions, or any combination thereof.

[0321] In an aspect, a subject can be a human patient. In an aspect a subject can be any age (e.g., geriatric, adult, young adult, teenager, tween, adolescent, child, toddler, baby, or infant), can be male or female, can be any nationality, can be of any ethnicity, and / or can be of any race. In an aspect, a subject can have a terminal cancer. In an aspect, a disclosed subject can an αβ T-cell refractory tumor or an αβ CAR T-cell refractory cancer. In an aspect, a disclosed subject can one or more αβ T-cell refractory tumors or one or more αβ CAR T-cell refractory tumors.

[0322] In an aspect, a disclosed targeted therapy can comprise one or more monoclonal antibodies. In an aspect, a disclosed monoclonal antibody can comprise an angiogenesis inhibitor (e.g., bevacizumab), a HER-2 targeted agent (e.g., trastuzumab, pertuzumab, etc.), an anti-CD20 monoclonal antibody (e.g., rituximab, obinutuzumab, etc.), or any combination thereof. In an aspect, a disclosed targeted therapy can comprise one or more small molecule inhibitors. In an aspect, a disclosed small molecule inhibitor can comprise a tyrosine kinase inhibitor (e.g., erlotinib, sunitinib, imatinib, dasatinib, etc.), a mTOR inhibitor (e.g., everolimus), a PARP inhibitor (e.g., olaparib), a CDK inhibitor (e.g., palbociclib, ribociclib, abermaciclib, etc.), a CD4 and / or CD6 inhibitor, or any combination thereof.

[0323] In an aspect, a disclosed targeted therapy can comprise abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab (tocilizumab), atorolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab,benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, blinatumomab, blosozumab, bococizumab, brentuxim abvedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cbr96-doxorubicin immunoconjugate, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, crenezumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, derlotuximab biotin, detumomab, dinutuximab, diridavumab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etanercept, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirvetuximab soravtansine, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab,oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotumumab, rinucumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarextumab, tefibazumab, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, ticilimumab, tigatuzumab, tildrakizumab, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, or any combination thereof.

[0324] In an aspect, a disclosed targeted therapy can comprise abemaciclib, ado-trastuzumab emtansine, afatinib, alectinib, alemtuzumab, alpelisib, atezolizumab, avelumab, axitinib, bevacizumab, binimetinib, blinatumomab, bosutinib, brentuximab, brigatinib, cabozantinib, carfilzomib, cemiplimab, ceritinib, cetuximab, gilteritinib, cobimetinib, copanlisib, crizotinib, dabrafenib, dacomitinib, daratumumab, dasatinib, denosumab, dinutuximab, durvalumab, duvelisib, elotuzumab, encorafenib, entrectinib, erdafitinib, erlotinib, fam-trastuzumab deruxtecan-nxki, gefitinib, gemtuzumab, ibritumomab tiuxetan, ibrutinib, imatinib, inotuzumab, ipilumumab, ivosidenib, lapatinib, larotrectinib, Lenvatinib, lorlatinib, margetuximab-cmkb, necitumumab, neratinib, nilotinib, niraparib, nivolumab, obinutuzumab, ofatumumab, olaparib, olaratumab, osimertinib, palbociclib, panitumumab, pazopanib, pembrolizumab, pertuzumab, ponatinib, ramucirumab, regorafenib, ribociclib, rituximab, rucaparib, sorafenib, sunitinib, talazoparib, tivozanib, tositumomab, trametinib, trastuzumab, tucatinib, vandetanib, vemurafenib, vismodegib, or any combination thereof.

[0325] In an aspect, a disclosed targeted therapy for oral administration can comprise abemaciclib, afatinib, alectinib, alpelisib, axitinib, binimetinib, bosutinib, brigatinib, cabozantinib, cobimetinib, crizotinib, dabrafenib, dacomitinib, dasatinib, duvelisib, encorafenib, entrectinib, erdafitinib, erlotinib, gefitinib, gilteritinib, ibrutinib, imatinib mesylate, ivosidenib, lapatinib, larotrectinib, lorlatinib, lenvatinb, neratinib, nilotinib, niraparib, olaparib, osimertinib palbociclib, pazopanib, ponatinib, regorafenib, ribociclib, rucaparib, sorafenib, sunitinib malate, talazoparib, tivozanib, trametinib, vandetanib, vemurafenib, vismodegib, or any combination thereof.

[0326] In an aspect, a disclosed targeted therapy for intravenous administration can comprise ado- trastuzumab emtansine, alemtuzumab, atezolizumab, avelumab, bevacizumab, blinatumomab, brentuximab vedotin, carfilzomib, cetuximab, cemiplimab-rwlc, certinib, copanlisib, daratumumab, dinutuximab, durvalumab, elotuzumab, fam-trastuzumab deruxtecan-nxki, gemtuzumab ozogamicin, ibritumomab tiuxetan, inotuzumab ozogamicin, ipilimumab, margetuximab-cmkb, necitumumab, nivolumab, obinutuzumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, tositumomab and iodine I131 tositumomab, trastuzumab, ublituximab-xiiy, or any combination thereof.

[0327] In an aspect, a disclosed targeted therapy for subcutaneous administration can comprise denosumab, ofatumumab, or any combination thereof.

[0328] In an aspect, a disclosed method of treating cancer can comprise administering to the subject a therapeutically effective amount of a targeted immunotherapy agent (e.g., fam- trastuzumab-deruxtecan-nxki; trastuzumab; Herceptin Hylecta (injectable Herceptin); Herceptin biosimilars (e.g., Herzuma, Kanjinti, Ogivri, Ontruzant, and Trazimera); ado-trastuzumab emtansine; margetuximab-cmkb; pertuzumab, trastuzumab, and hyaluronidase-zzxf; pertuzumab; sacituzumab govitecan-hziy; or any combination thereof).

[0329] In an aspect, a disclosed method can be modified or one or more steps of a disclosed method can be modified. In an aspect, a disclosed method of treating cancer and / or metastatic cancer can comprise modifying or changing one or more features or aspects of one or more steps. In an aspect, a method can be altered by changing the amount of a disclosed treatment regimen, the disclosed Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the disclosed Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, a disclosed targeted therapy, a disclosed pharmaceutical formulation, a disclosed anti-chemokine, a disclosed anti-cancer agent, a disclosed chemotherapeutic agent, or a combination thereof administered to a subject, or by changing the frequency of administration of disclosed treatment regimen, the disclosed of Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the disclosed Vδ1 CAR γδ T-cells and / orVδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, a disclosed targeted therapy, a disclosed pharmaceutical formulation, a disclosed anti-chemokine, a disclosed anti-cancer agent, a disclosed chemotherapeutic agents, or a combination thereof to a subject, by changing the duration of time that disclosed treatment regimen, the disclosed Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the disclosed Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, a disclosed targeted therapy, a disclosed pharmaceutical formulation, a disclosed anti-chemokine, a disclosed anti-cancer agent, a disclosed chemotherapeutic agent, or a combination thereof is administered to a subject, or by substituting for one or more of the disclosed components and / or reagents with a similar or equivalent component and / or reagent. The same applies to all disclosed treatment regimens, the disclosed Vδ1 γδ T-cells and / or Vδ3 γδ T-cell (expanded by a disclosed method) and / or the disclosed Vδ1 CAR γδ T-cells and / or Vδ3 γδ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof , disclosed targeted therapies, disclosed pharmaceutical formulations, disclosed anti-chemokines, disclosed anti-cancer agents, disclosed chemotherapeutic agents, or combinations thereof.

[0330] In an aspect of a disclosed method of treating cancer, a disclosed population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to kill stressed cells (e.g., tumor or virally infected cells) and can be combined with IgG targeting molecules on the tumor cell surface, thereby addressing the problem of antigen heterogeneity.

[0331] In an aspect of a disclosed method, a disclosed expanded population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to avoid T cell exhaustion.

[0332] In an aspect, using UCB as a biological sample for a disclosed method ensures that an expanded population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells can comprise a broader repertoire of gamma and delta chains. In an aspect, using PB as a biological sample yields a narrower repertoire of gamma and delta chains.

[0333] In an aspect, a disclose method can comprise monitoring the subject for develoment of cytokine release syndrome (CRS). G. Kits

[0334] Disclosed herein is a kit comprising one or more disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or disclosed populations of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed populations of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / ordisclosed populations of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed cells, or any combination thereof.

[0335] Disclosed herein is a kit comprising a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells made according to the rapid expansion protocol set forth in Example 1, infra.

[0336] Disclosed herein is a kit comprising a population of expanded γδ T cells comprising predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells made according to the rapid expansion protocol set forth in Example 2, infra.

[0337] Disclosed herein is a kit for use in a method of expanding a desired T cell population, the method comprising isolating a population of T cells from a biological sample and expanding the selected T cell population according to the rapid expansion protocol set forth in Example 1, infra.

[0338] Disclosed herein is a kit for use in a method of expanding a desired T cell population, the method comprising isolating a population of T cells from a biological sample and expanding the selected T cell population according to the rapid expansion protocol set forth in Example 2, infra.

[0339] In an aspect, a disclosed kit can comprise one or more additional active agents and / or therapeutic agents (such as, for example, a disclosed anti-cancer agent). In an aspect, the one or more agents can treat, prevent, inhibit, and / or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and / or ameliorate cellular and / or metabolic complications related to cancer or cancer cells or cancerous cells.

[0340] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a disease or disorder such as cancer). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on a computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof, and a label orpackage insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed populations of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof can be used for treating, preventing, inhibiting, and / or ameliorating a disease or disorder or complications and / or symptoms associated with a disease or disorder such as cancer or metastatic cancer. A kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes.

[0341] In an aspect, a disclosed kit can be used in a disclosed method of expanding a diesred T cell population, such as for example, γδ T cells or CAR γδ T-cells. In an aspect, a disclosed kit can be used to treat cancer. In an aspect, a disclosed kit can be used to preventing or inhibiting metastasis of cancer cells. In an aspect, a disclosed kit can be used to risk of developing metastases. In an aspect, a disclosed kit can be used to validate the efficacy and / or toxicity of a disclosed population of Vδ1 γδ T-cells and / or Vδ3 γδ T-cells (expanded by a disclosed method) and / or a disclosed population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells (expanded by a disclosed method), a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed host cell, or any combination thereof.

[0342] In an aspect, a disclosed kit comprising an expanded population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to generate an allogeneic CAR T-cell therapy.

[0343] In an aspect, a disclosed kit comprising an expanded population of predominantly purified Vd1 γδ T cells and / or Vδ3 γδ T cells can be used to avoid T cell exhaustion. H. Miscellaneous

[0344] Disclosed herein is a method for the isolation and expansion of polyclonal Vδ1 and Vδ1- Vδ2- γδ T cells from a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) isolating leukocytes from the sample; (c) immunopurifying γδ T cells from the isolated leukocytes; (d) sorting the isolated leukocytes into γδ+T cells and γδ−T cells; (e) using the γδ−Tcells as feeder cells; (f) irradiating the feeder cells to created preferential activation of γδ T cells; (g) incubating the feeder cells and activated γδ T cells at a ratio of about 1:200 (γδ T cells: Feeder cells) in an upright flask for at least 10 days; (h) adding IL-2 to the flask and removing 10 ml of media from the top of the flask so as not to disturb the cells at the bottom at day 4; (i) adding 10 ml of new media to the flask such that the cells at the bottom are not disturbed; (j) repeating steps (g)-(i) on day 7 followed by placing the flask on its side; (k) removing the γδ T cells and counting on day 10; (l) optionally transducing the γδ T cells with chimeric antigen receptors; and (m) depleting the αβ T cells at day 14. Disclosed herein is a therapeutic composition comprising isolated Vδ1 and Vδ1-Vδ2- γδ T cells and / or γδ CAR-T cells and a pharmaceutically acceptable carrier, diluent and / or excipient. Disclosed herein is method of preventing and / or treating a subject suffering from a disease, the method comprising administering to the subject a therapeutically effective amount of an isolated Vδ1 and Vδ1-Vδ2- γδ T cells and / or γδ CAR-T cells and / or a therapeutic composition thereof as in any of the preceding claims such that the disease is prevented and / or treated in the subject. In an aspect, a disease can comprise cancer. In an aspect, a method can further comprise administering one or more additional therapeutic agents. In an aspect, one or more additional therapeutic agents can be administered prior to the Vδ1 and Vδ1-Vδ2- γδ T cells and / or γδ CAR-T cells and / or therapeutic composition thereof. In an aspect, one or more additional therapeutic agents can be administered concurrently with the Vδ1 and Vδ1- Vδ2- γδ T cells and / or γδ CAR-T cells and / or therapeutic composition thereof. In an aspect, the one or more additional therapeutic agents can be administered after the Vδ1 and Vδ1-Vδ2- γδ T cells and / or γδ CAR-T cells and / or therapeutic composition thereof. VIII. EXAMPLES

[0345] Little is known about the role of heterogeneous γδ T cells infiltrating human tumors. Vd2 γδ T cells are found in tiny numbers in solid tumors, indicating that Vd2 likely circulate in blood. And while, Vd2 γδ T cells can be important to treat hematological tumors or bone marrow metastases, Vd2 γδ T cells do not appear to significant treat a solid tumor. Therefore, those γδ T cells that found in the tumor microenvironment are Vd1 and Vd3 γδ T cells.

[0346] To gain more insight into the phenotypes of γδ tumor-infiltrating lymphocytes (TILs), T cells from dissociated human high-grade serous ovarian cancer (HGSOCs) cells and lung cancer cells and malignant pleural effusions, were FACS-sorted. FACS analysis confirmed that ~6 % of TILs were γδ T cells and that δ1 T cells outnumbered δ2 lymphocytes ~4:1. Next, encapsulation and running with 10X (single-cell) analyses, with sorted, bar-coded, δ1 plus δ2 TILs were performed. In both diseases (HGSOC and lung cancer), δ1 TIL subsets showed distinctive clustersof (TCF7+CD27+IL7Rdim) stem-like cells (δ1stem); and effector T cells expressing high levels of granzymes, IFNG and perforin, plus NK receptors (δ1Eff). Importantly, low expression of PD-1 was found by RNA-seq and FACS, with only a small cluster of δ1 TILs with TOX expression in NSCLC.

[0347] Based on the phenotypes found in human cancer, it was reasoned that Vδ1 and Vδ1-Vδ2- T cells could be effective as an allogeneic CAR T-cell platform against αβ CAR T-cell-refractory tumors. To pave the way for this clinical expansion, effective and clinically relevant methods for selective expansion of Vδ2- γδ T cells from human umbilical cord blood were optimized.

[0348] The majority of γδ T cells in umbilical cord blood are naive Vδ2- T cells expressing all Vγ chains, including Vγ2 / Vγ5. From 40 mL of cord blood, > 250 million viable cells were purified. Using 18 million cells from 2 different donors for optimization, bead-purified γδ T cells (initially > 8 x 104) were incubated with γ-irradiated autologous feeder PBMCs (γδ T cell-negative fraction expressing NKG2D ligands; 1:200, T cell:irradiated feeder cells), plus 50 ng / mL of OKT3, plus 3000 U / mL of hIL-2 ("modified rapid expansion").

[0349] This resulted in an expanded >79% pure γδ T cell population in 10 days, which contained ~96% Vδ2- (tumor homing γδ) T cells, with > 3,300-fold increase in Vδ2- T cell numbers (256 million). Expanded Vδ1 T cells showed an effector phenotype.

[0350] The Examples that follow are illustrative of specific aspects of the invention, and various uses thereof. They set forth for explanatory purposes only and are not to be taken as limiting the invention. Example 1 Development of an Isolation and Expansion Protocol for Vδ1 and Vδ3 T Cells

[0351] Described herein is a rapid expansion protocol detailing the isolation and expansion of polyclonal Vδ1 γδ T cells and Vδ3 γδ T cells and from cord blood. Here, "Vδ1-Vδ2-γδ T cells" can be used interchangeably with "Vδ3 γδ T cells" and vice versa.

[0352] Step 1. Umbilical cord blood samples were obtained and provided in 40 mL bags. Total leucocytes were isolated from blood by using in according to the manufacturer's instructions.

[0353] Step 2. Second, by following the manufacturer's instructions (Cat #85450), ~250 million of total leukocytes were isolated using SepMate™- PBMC Isolation 50 mL tubes and lymphoprepTM (StemCEll Technologies; Cat.# 07801). Aliquots were then cryopreserved at ~18 million cells / aliquot.

[0354] Step 3. Again, following manufacturer's instructions for using the EasySep™ Human Gamma / Delta T Cell Isolation Kit and ~5 minutes of magnetic separation, ~80,000 γ-δ T cells were immunopurified from each 18 million cell aliquot, , .

[0355] Step 4. The sorted cells were centrifuged at ~500 x g for 5 minutes at 4 °C and resuspended in 10 mL AIM-V media containing 6000 IU / mL of IL-2 and 60 ng / mL OKT3 (final concentration). AIM-V media is serum-free and contains L-glutamine, streptomycin sulfate at 50 μg / mL, and gentamicin sulfate at 10 μg / mL.

[0356] Step 5. The γδ T cell negative fraction was used as feeder cells, by transferring the cells into 10% FBS RPMI media, spinning the cells at 500 x g for 5 minutes at 4 °C and resuspending the cells in 10% FBS RPMI media with 1X DNAse I. This process was followed by incubation at room temperature for 10 min, centrifugation at 500 x g for 5 minutes at 4 °C and resuspension in AIM-V media at 3e6 / mL.

[0357] Step 6. The feeder cells were irradiated using X-ray irradiator (using 5000 rad | 160kV | 19.0 mA | 714 seconds | 30 cm). This induced the expression of NKG2D ligands in autologous cells, resulting in preferential activation of γδ T cells.

[0358] Step 7. The γδ T cells and feeder cells were added to upright 25 cm2flasks at a 1:200 ratio (T cell:feeder cell).

[0359] Step 8. On day 4, AIM-V media containing 3000 IU / mL of IL-2 was pre-warmed. Using a 10 mL pipette, 10 mL of media was removed from the top, without disrupting the cells lying on the bottom of the flask.

[0360] Step 9. 10 mL of new media was added slowly to the flask by placing the tip of the pipette against the non-tissue culture treated side of the flask.

[0361] Step 10. The 25 cm2flask were kept upright in the incubator.

[0362] Step 11. At day 7, Steps 8-10 were repeated, but the 25 cm2flask were now placed lying flat.

[0363] Step 12. At day 10, feeder cells were dead and T cells could be counted. Approximately 256 million γδ T cells were expanded at this point, with ~63% of them representing Vδ1 T cells and ~34% representing Vδ3 γδ lymphocytes.

[0364] Step 13. T cells are optionally transduced with chimeric antigen receptors using spin- infection, then resuspended into 20 mL of AIM-V containing 3000 IU / mL of IL-2, and transferred to 75 cm2flasks, which are deposited laying flat into the incubator.

[0365] Step 14. Following the manufacturer's instructions, residual human αβ T cells are depleted by using the CliniMACS TCRα / β product line (Miltenyi Biotec; Cat#.200-070-409). This stepis performed at day 10, or if chimeric antigen receptors are transduced, then it is performed at day 14.

[0366] Step 15. The isolated expanded Vδ1 γδ T cells and Vδ3 γδ T cells and / or the expanded Vγ1 γδ CAR T cells and the Vγ3 γδ CAR T cells are used for allogeneic administration into cancer patients. Example 2 Optimization of an Isolation and Expansion Protocol for Vδ1 and Vδ3 T Cells

[0367] Building on the protocol set forth in Example 1, a protocol for expanding human Vδ1 and Vδ3 γδ T cells from umbilical cord blood (UCB) was further optimized. Notably, Vδ3+T cells are almost absent from peripheral blood. This expansion protocol recapitulated proportions of γδ lymphocyte subsets found at tumor beds, with features of effector activity and stemness.

[0368] Isolation of total human leukocytes. Total human leucocytes were isolated from either Peripheral Blood (hPBL) or Umbilical Cord Blood (UCB) by using lymphoprepTM(StemCEll Technologies; Cat. #07801) in according to the manufacturer's instructions. SepMate™-50 tubes were used for the cell's separation in according to the manufacturer's instructions (Cat. #85450). Isolation of gamma delta human (γδ) T cells from total leukocytes. Human γδ T cells were purified by negative selection from a fraction of total leukocytes by using the EasySep kit (Stem Cell Technologies; Cat. #19255) in according to the manufacturer's instructions.

[0369] Purified (γδ) T cells were expanded by a modified Rapid Expansion Protocol (REP) following the protocol below:

[0370] On day 0, the autologous feeder cells were prepared. A 1X DNase solution I in R10 media was prepared from a stock at 100X. R10 medium is defined as RPMI-1640 (Sigma), supplemented with L-glutamine (2 mM, Gen Clone), penicillin (100 U / mL, Lonza), streptomycin (100 μg / mL Lonza), sodium pyruvate (1 mM) (ThermoScientific) and 10% Fetal Bovine Serum (FBS) (Sigma). Feeder cells were obtained from the same donor. A ratio 1:200 ratio of T cells:Feeder cells was used. For example, for 1.5e5 T cells, 30e6 irradiated feeder cells were needed. Here, to avoid TCR alpha-beta (αβ) T cells expansion, autologous feeder cells were used. If feeder cells were frozen in Cryo Tubes, each vial was thawed in a water bath at 37 °C. Immediately thereafter, feeder cells were transferred into to a 15 mL conical tube with R10 media (without DNase I) from each vial of cells. Feeder cells were then spun at 520 x g for 5 minutes at 4 °C. The supernatant was decanted. About 15 mL of R10 media with 1X DNase I was added to each cell pellet. Pipetting was used to thorough mix. The feeder cells were then incubated at room temperature for 10 min. The feeder cells were then spun again at 520 x g for 5 minutes at 4 °C. The spent medium was decanted, and the pellet was re-suspended in fresh R10 medium. The cells werecounted. The feeder cells were then spun again at 520 x g for 5 minutes at 4 °C and the resulting supernatant was discarded. The feeder cells were re-suspended in a mix of mixture of R10 medium and AIM-V medium (1:1 ratio) at cell density ranging between 2e6 / mL to 3e6 / mL (Feeder Media). While FBS was employed here, human serum can supplement the R10 medium for clinical use. The feeder cells were then irradiated by using an X-ray irradiator (X-RAD 160) with the following settings: 6000 rad; 160 kV; 19.0 mA. The cells were collected, and the concentration was adjusted. This protocol was optimized to expand T cells starting from a range of 5e4 T cells to 2e5 T cells. The lowest number of T cells required for this optimized protocol is 5e4 T cells.

[0371] On day 0, the γδ T cells were co-incubated with the autologous feeder cells. After the isolation of gamma-delta T cells by the EasySepTMkit, the cells were counted. Here, an aliquot containing a range of 5e4 cells to 2e5a cells was re-suspended in 10 mL of feeder cell media containing 6000 IU / mL of human IL-2 (Peprotech), 100 ng / mL of In Vivo Ready™ Anti-Human CD3, clone UCHT1 (TonboTM), and 1 µg / mL of In Vivo Ready™ Anti-Human CD28 (clone CD28.2) (TonboTM). The T cells were added to a 25 cm2flask and was kept upright. The, 10mL of the feeder cells were added for a final volume of 20 mL. The addition of the feeder cells diluted the media yielding a final concentration of IL-2 (3000 IU / mL), UCHT1 / OKT3 (50 ng / mL), and CD28 (0.5 µg / mL). The flask was kept upright and stored in an incubator at 37 °C and 5% CO2.

[0372] From this point forward, only AIM V medium was used.

[0373] On day 4, AIM V medium containing 3000 IU / mL of hIL-2 was warmed. The flask was held upright and carefully placed in the biological cabinet. The flask rested for about 5 to 10 minutes. This allowed cells that were disrupted to settle down to the bottom of the flask. Using a 10 mL pipette, 10 mL of spent medium was carefully removed from the top. Care was taken not to disrupt the cells lying on the bottom of the flask. Then, 10 mL of fresh medium very slowly added to the flask either drop by drop or by placing the tip of the pipette against the non-tissue culture treated side of the flask. The flask was kept upright,

[0374] On day 7, work in 25 cm2flask continued. First, about 10 mL of AIM-V medium containing 3000 IU / mL of hIL-2 was warmed. Second, about 10 mL of spent medium was removed from the the top and about 10 mL of fresh medium was added. Third, the media and cells were re-suspended by pipetting in the flask to remove TIL from the bottom of the flask and the flask was laid flat. Finally, the inclination angle of the flask was changed from 90C to 180C to ensure that the flask was now lying flat.

[0375] On day 10, the cells were transferred to a 75 cm2flask. First, 20 mL of AIM-V medium containing 3000 IU / mL of hIL-2 was prepared. Second, the cell suspension was removed from the original 25 cm2flask and transferred to the 75 cm2flask. Third, the bottom of the original 25cm2flask was rinsed a few times (3x-5x) with the new medium, then the cells were transferred to the 75 cm2flask. Fourth, the new 75 cm2flask was placed back into the incubator and laid flat. By this time, the feeder cells had died.

[0376] On day 12, the cells were transferred to a 150 cm2flask. First, 40 mL of AIM-V medium containing 3000 IU / mL of hIL-2 was prepared. Second, the contents of the 75 cm2flask were transferred to a 150 cm2flask. Third, the 75 cm2flask was carefully rinsed with some of the new medium, then the cells were transferred to the 175 cm2flask. The flask was kept lying flat and was placed back into incubator.

[0377] On day 14, the cells were mixed, collected, and placed into 50 mL conical tubes. Then, the 175 cm2flask was rinsed with 20 mL PBS and the cells were transferred. This rinsing step was repeated 2-3 times to optimize cell collection. The tubes were spun at 520 x g for 5 minutes at 4 °C. The medium was decanted, and the cells were re-suspended and counted. The cells were now ready for either experimental use or for cryopreservation.

[0378] Although the depletion of possible residual αβ T cells can be performed either before or after cell expansion, residual αβ T cells were removed from the expanded T cells was effected using the CliniMACS TCRα / β product line (Miltenyi Biotec; Cat. # 200-070-409).

[0379] The expanded γδ T cells were subsequently characterized using flow cytometry. Specifically, the following flow panel was employed: (1) BD Horizon™ BV786 Mouse Anti- Human CD45. Clone: HI30; Cat No:563716; (2) BD Horizon™ BUV395 Mouse Anti-Human CD3. Clone: SK7. Cat No: 564001; (3) BD Pharmingen™ Alexa Fluor® 700 Mouse Anti-Human CD14. Clone M5E2. Cat No:557923; (4) BD Pharmingen™ PE Mouse Anti-Human TCRαβ. Clone: IP26 (also known as IP26A). Cat Number: 564728; (4) BD Horizon™ BV711 Mouse Anti- Human γδ TCR. Clone: 11F2. Cat No: 568490; (6) Invitrogen. TCR V delta 1 Monoclonal Antibody (TS8.2), PE-Cyanine7, eBioscience™; (7) Biolegend. APC anti-human TCR Vδ2 Antibody. Clone B6. Cat: 331417; (8) Biolegend. Zombie NIR™ Fixable Viability Kit. Cat# 423106; (9) Biolegend. Brilliant Violet 605™ anti-human IFN-γ Antibody. Clone B27. Cat 506542; (10) BD Horizon™ BV421 Mouse Anti-Human Perforin. Clone δG9. Cat# 563393; (11) BD Horizon™ R718 Mouse Anti-Human Granzyme B. Clone GB11. Cat# 566964; (12) BD Pharmingen™ PerCP-Cy5.5 Mouse Anti-Human CD27. Cat# 567181; (13) Cell Signaling Technology. TCF1 / TCF7 (C63D9) Rabbit mAb (Alexa Fluor® 647 Conjugate) #6709; (14) Biolegend. Brilliant Violet 421™ anti-human CD366 (Tim-3) Antibody. Clone F38-2E2. Cat# 345008; (15) Biolegend. FITC anti-human CD223 (LAG-3) Antibody. Clone 11C3C65. Cat# 369308; (16) Biolegend. Brilliant Violet 605™ anti-human CD337 (NKp30) Antibody. Cat#325234; and (17) Biolegend. PE / Dazzle™ 594 anti-human CD314 (NKG2D) Antibody. Clone 1D11. Cat# 320828.

[0380] The following gating strategy was used - Viable CD45+CD14negCD3+TCRαβnegTCRγδ+Vd1+Vd2+. Expression for specific markers (IFN-γ, Perforin, Granzyme B, CD27, TCF1 / TCF7, Tim-3, NKG2D, LAG-3 and NKp30 was evaluated separately for the Vδ1 and Vδ2 T cells subsets. For IFN-γ, Perforin, Granzyme B, and TCF1 / TCF7 (an intracellular staining) was performed following the manufacturer's instructions (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set Cat. #00-5523-00; ThermoFisher). Samples were run either on a BD LSRII / BD LSRFortessa X-20 for flow analysis or sorted using a BD FACS Aria / Beckman Coulter MoFlo Astrios. All sorted populations were confirmed to be >98% pure through post-sort analysis.

[0381] After expansion of gamma-delta T cells, the δ-1 and δ-3 chains were confirmed from sorted fractions. Briefly, TCRγδ+Vd1+and TCRγδ+Vd1negVd2negexpanded fractions were FACS-sorted. Immediately after sorting, RNA was isolated (RNeasy PlusMicroKit, QIAGEN, Cat#74034) and samples were sent for iRepertoire analysis of the delta chains.

[0382] Fluorescence-activated cell sorting (FACS) was performed on the transduced cells. Sorting of antibody heavy chain-transduced HEK293T cells (GFP expressing) were performed by staining with DAPI (Thermo Scientific) viability dye and gated for DAPI-GFP+cells. Similarly, PIGR-transduced HCT116 cells were sorted by gating DAPI-GFP+cells. Samples were subsequently fluorescence-activated cell sorting (FACS) sorted using BD FACS ARIA. Here, one hundred thousand KRASG12Dtransduced OVCAR3 cells were placed in 6-well plates. After 12 hr, the wells were washed, fresh medium was added, and the cells were treated with anti-human KRASG12D-IgA1 or KRASG12D-IgG4 or non-specific human IgA (Abcam, ab91025) at 0.5 μg / mL final concentration or vehicle (PBS) and incubated for 16 hr in a 37 °C incubator. The total apoptotic cells (annexin V+propidium iodide+ / −) were analyzed by flow cytometry.

[0383] For transduction, retrovirus was generated by transfecting 293GP cells with either pBMN- I-GFP or pBMNI-GFP-TAS2R13-CD28 CAR. The CAR targeted an extracellular domain in TAS2R13 (i.e., CD28-co-stimulated CAR targeting an ECD in TAS2R13 (SEQ ID NO:01, SEQ ID NO:09), and contained the following components: CD28 TM domain (SEQ ID NO:02, SEQ ID NO:10), CD28 IC domain (SEQ ID NO:03, SEQ ID NO:11), CD3zeta (SEQ ID NO:04, SEQ ID NO:12), Heavy Chain (SEQ ID NO:05, SEQ ID NO:13), Light Chain (SEQ ID NO:06, SEQ ID NO:14), CD28 hinge domain (SEQ ID NO:07, SEQ ID NO:15), and Signal Peptide (SEQ ID NO:07, SEQ ID NO:16). The γδ CAR T cells, which were expanded using a disclosed rapid expansion protocol, were tested in a reduction to practice against cancer cells (FIG.11).

[0384] Briefly, the cells were in a 10 cm culture dish. When the cells reached 70-80% confluency, the cells were transfected with a mix of our DNA and the RD114 envelope vector using Lipofectamine 3000 (Invitrogen). The supernatant containing the retroviral particles was collected at 48 hr, 72 hr, and 96 hr after transfection.

[0385] To prepare conventional αβ T cells for transduction, red blood cells lysis on healthy human apheresis samples was performed. This was followed by the activation of T cells in AIM-V medium with 5% FBS, 1% penicillin / streptomycin, 1% L-glutamine, 300 U / mL IL-2, and 0.05 µg / mL anti-CD3 at a concentration of 106cells / mL.

[0386] Umbilical cord blood samples were obtained. γδ T cells were expanded from umbilical cord blood and transduced as detailed in the enclosed protocol. Here, 3 spin infections were performed on Retronectin coated plates (Takara) – at 48 hr, 72 hr, and 96 hr. Spent medium was removed and new medium containing 300 U / mL of IL-2 was added to maintain a concentration of 106cells / mL. Transduction efficiencies and T cell populations were measured with a BD LSRII flow cytometer.

[0387] For the cytotoxicity assays, about 105target cells per well were plated in a flat bottom 96 well plate with 100 µL culture media. The following day, the target cells were transfected with a luciferase expressing vector (Promega pGL4.51[luc2 / CMV / Neo]) (RRID:Addgene_132962) using the JetPrime system (Polyplus Cat# 101000015) for tumor cells and the JetOptimus system (Polyplus Cat# 101000051) for primary healthy cells. About 6-8 hours after transfection, the media was removed and replaced with fresh culture media. The appropriate number of either TAS2R13-CD28 CAR, or mock transduced T cells were added the following day and co-cultured for 8 hours. Cytotoxicity was measured via Luciferase Assay (Promega Cat# E1501). Cytotoxicity was calculated as (maximum viability control - individual well) / (maximum viability control - maximum death control) *100 as a percentage.

[0388] To establish a tumor model, flank tumors were initiated by injecting 1 × 107KRASG12DA427 NCSCL cells (ATCC) in mice. Tumor volume was calculated as: 0.5 × (L × W2), where L is length and W is width. Intraperitoneal injections of CAR T-cells were done at day 7. Summary of Examples

[0389] Described herein is a protocol for expanding human Vδ1 / 3 γδ T cells from umbilical cord blood (UCB). This expansion protocol recapitulated proportions of ^ ^ lymphocyte subsets found at tumor beds, with features of effector activity and stemness.

[0390] The data provided herein show (i) that γδ CAR-T outperformed αβ CAR-T cells, demonstrating enhanced cytotoxic activity, (ii) that γδ CAR-T cells exhibited heightenedresponsiveness against solid tumors, and that the integration of NKG2D domains enhanced the functionality of γδ CAR-T.

[0391] While expanding Vd1 γδ T cells from aphaeresis is technically feasible, it is ineffective. Vd3 γδ T cells are not found in sufficient amounts in peripheral blood to provide a meaningful means for expansion. However, using the compositions and methods disclosed herein, the simultaneous expansion of Vd1 and Vd3 γδ T cells from umbilical cord blood (UCB) was possible. The disclosed expansion protocol yielded high numbers of expanded γδ T cells. Moreover, the proportion of Vd1 and Vd3 γδ T cells in that expanded population mimicked that seen in the microenvironment of different human solid cancers.

[0392] One of the significant advantages provided by the disclosed compositions and methods concerns an allogeneic CAR T-cell therapy. This allogeneic CAR T-cell therapy can be used to treat those patients having solid tumors that (i) are rapidly deteriorating and cannot wait for the generation of an autologous infusion product, and / or (ii) do not have the appropriate T cells for the generation of an autologous infusion product due to extension prior treatment.

[0393] Another significant advantage provided by the disclosed compositions and methods concerns the ability of the disclosed compositions and methods to generate expanded populations of γδ T cells, especially Vd1 γδ T cells and Vd3 γδ T cells, from UCB, which is an easily accessible and nearly inexhaustible source of material.

[0394] Still another significant advantage provided by the disclosed compositions and methods concerns cost. The estimated cost for autologous CAR-T therapy is about $500,000 to $1 million dollars, which is cost-prohibitive for many desperate patients. But, when expanded according to the disclosed rapid expansion protocol, CAR γδ T-cells can be provided as an effective, allogenic "off-the-self" and "ready to use" product for a large population of patients having cancer. This is a financial game-changer for healthcare.

Claims

IX. CLAIMS What is claimed is:

1. A method of expanding a desired T cell population, the method comprising: isolating a population of T cells from a biological sample; and expanding the selected T cell population.

2. The method of Claim 1, wherein the biological sample comprises umbilical cord blood (UCB).

3. The method of Claim 1, wherein isolating a population of T cells from a biological sample comprises isolating leukocytes from the UCB.

4. The method of Claim 3, further comprising immunopurifying a population of γδ T cells from the isolated leukocytes.

5. The method of Claim 1, wherein immunopurifying a population of γδ T cells comprises using immunomagnetic negative selection.

6. The method of Claim 1, immunopurifying a population of γδ T cells generates a fraction comprising non-γδ T cells and a fraction comprising γδ T cells.

7. The method of Claim 6, further comprising centrifuging and re-suspending in a medium the fraction comprising the γδ T cells.

8. The method of Claim 1, wherein the medium comprises AIM V medium, and wherein the AIM V medium comprises IL-2 and OKT3.

9. The method of Claim 8, further comprising centrifuging the γδ T cells at about 500 x g at about 4 °C for about 5 minutes.

10. The method of Claim 6, further comprising using the fraction comprising non-γδ T cells as feeder cells.

11. The method of Claim 10, further comprising preparing the fraction comprising non-γδ T cells to serve as feeder cells.

12. The method of Claim 11, wherein preparing the fraction comprising non-γδ T cells to serve as feeder cells comprises irradiating the non-γδ T cells.

13. The method of Claim 12, wherein irradiating the feeder cells induces expression of NKG2D ligands, and wherein the expression of NKG2D ligands in the feeder cells preferentially activates γδ T cells.

14. The method of Claim 12, further comprising adding together the γδ T cells and the irradiated feeder cells.

15. The method of Claim 14, wherein the ratio of γδ T cells and irradiated feeder cells is about 1:200 (T cells:feeder cells).

16. The method of Claim 14, further comprising incubating the mixture of the γδ T cells and the irradiated feeder cells.

17. The method of Claim 16, further comprising changing the medium one or more times during the incubating step.

18. The method of Claim 14, wherein on day 10 of the incubating step, the method generates a population of expanded γδ T cells.

19. The method of Claim 18, wherein the population of expanded γδ T cells comprises predominantly Vδ1 γδ T cells and / or Vδ3 γδ T cells.

20. The method of Claim 18, wherein method generates a clinically relevant amount of Vδ1 γδ T cells and / or Vδ3 γδ T cells.

21. The method of any one of Claims 19 - 21, further comprising removing and / or depleting residual αβ T cells from the expanded population of γδ T cells.

22. The method of any one of Claims 19 - 21, further comprising transducing the expanded population of γδ T cells with one or more chimeric antigen receptors (CARs).

23. A population of predominantly purified Vδ1 γδ T cells and / or Vδ3 γδ T cells made by the method of any one of Claims 1 - 21.

24. A population of predominantly purified Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells made by the method of Claim 22.

25. A pharmaceutical formulation comprising the population of Vδ1 γδ T cells and / or Vδ3 γδ T cells of Claim 23 or the population of Vδ1 CAR γδ T-cells and / or Vδ3 CAR γδ T-cells of Claim 24, and one or more pharmaceutically acceptable carriers.

26. A method of treating cancer, the method comprising: treating a subject having cancer by administering to the subject a therapeutically effective amount of the pharmaceutical formulation of Claim 25.

27. The method of Claim 26, wherein the subject has ovarian cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous cell lung carcinoma, adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, malignant pleomorphic adenoma, sarcoma, synovial sarcoma, carcinosarcoma, bile duct cancer, bladder cancer, transitional cell carcinoma, papillary carcinoma, kidney cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, colon cancer, small bowel cancer, small bowel adenocarcinoma, adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer,testicular seminoma, testicular teratoma, embryonic testicular cancer, uterine cancer, teratocarcinoma, embryonal carcinoma, or any combination thereof.

28. The method of Claim 26, wherein the subject has an αβ T-cell refractory tumor or an αβ CAR T-cell refractory cancer.

29. The method of any one of Claims 26 - 28, further comprising administering to the subject one or more additional anti-cancer therapies.

30. The method of Claim 29, wherein the one or more anti-cancer therapies comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof.

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