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WO2024259371A2 - COMPOSITIONS ET PROCÉDÉS D'EXPANSION DE LYMPHOCYTES T γδ - Google Patents

COMPOSITIONS ET PROCÉDÉS D'EXPANSION DE LYMPHOCYTES T γδ Download PDF

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Publication number
WO2024259371A2
WO2024259371A2 PCT/US2024/034197 US2024034197W WO2024259371A2 WO 2024259371 A2 WO2024259371 A2 WO 2024259371A2 US 2024034197 W US2024034197 W US 2024034197W WO 2024259371 A2 WO2024259371 A2 WO 2024259371A2
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Prior art keywords
cells
disclosed
population
car
cancer
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WO2024259371A3 (fr
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Jose R. Conejo Garcia
Ricardo CHAURIO GONZALEZ
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Duke University
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Duke University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • FIG.1A – FIG.1B show the prevalence of ⁇ T cells in peripheral blood and umbilical cord blood.
  • FIG. 1A shows that in human peripheral blood from adults, ⁇ T cells are predominantly expressing the delta-2 TCR chain (V ⁇ 2) while the delta-3 TCR chain (V ⁇ 3) (Vd1 neg Vd2 neg ) cells are almost absent, and the delta-1 TCR chain (V ⁇ 1) is in low proportions.
  • FIG.1B shows that in contrast, in umbilical cord blood, ⁇ T cells are predominantly expressing the delta-1 and delta-3 TCR chains (V ⁇ 1; V ⁇ 3) while V ⁇ 2 T cells are at very low percent.
  • 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 ⁇ T cells at tumor beds, which indicates UCB is a good supplier for Vd1 and Vd3 ⁇ T cells for clinical trials.
  • FIG.3 provides a schematic of the optimized protocol for expansion and transduction of human Vd1/Vd3 ⁇ T cells from umbilical cord blood (UCB).
  • This optimized expansion protocol recapitulated proportions of ⁇ lymphocyte subsets found at tumor beds with features of effector activity and stemness.
  • FIG. 4 shows ⁇ T cells expanded from UCB after two weeks were enriched in T cells expressing the delta-1 and delta-3 chains (V ⁇ 1/V ⁇ 3).
  • FIG. 5 shows ⁇ 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.
  • FIG.8 shows that ⁇ 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 ⁇ 1 chain and the Vd1 neg VD2 neg highly enriched in the ⁇ 3 chain.
  • FIG. 9A - FIG. 9B shows that ⁇ 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 ⁇ 1 chain and the Vd1 neg VD2 neg highly enriched in the ⁇ 3 chain.
  • FIG.10 shows that ⁇ 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 ⁇ 1 chain and the Vd1 neg VD2 neg highly enriched in the ⁇ 3 chain.
  • FIG. 11 shows ⁇ CAR-T cells targeting TAS2R13 show superior cytotoxic activity compared with ⁇ ß CAR-T cells. Here, the ⁇ CAR-T cells were generated using the disclosed rapid expansion protocol. VI.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T cells expanded by a disclosed method.
  • 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 ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 CAR ⁇ T- cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • 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 ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer the method comprising administering to a subject having cancer a therapeutically effective amount of a population of CAR ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T- cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of CAR ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T-cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a method of treating cancer comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a population of ⁇ 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 ⁇ T-cells expanded by a disclosed method.
  • 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 ⁇ T cells expanded by a disclosed method.
  • 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 ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of ⁇ T cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer 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 ⁇ T-cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a method of treating cancer comprising administering to a subject having cancer a therapeutically effective amount of a pharmaceutical formulation comprising a population of ⁇ 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 ⁇ T-cells expanded by a disclosed method.
  • 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 ⁇ T-cells expanded by a disclosed method, wherein the expanded population of ⁇ T-cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ 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 ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T-cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a population ⁇ T cells expanded by a disclosed method Disclosed herein is a population of CAR ⁇ T-cells expanded by a disclosed method.
  • Disclosed herein is a population of V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T-cells expanded by a disclosed method.
  • Disclosed herein is a population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells expanded by a disclosed method. Disclosed herein is a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ 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.
  • 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.
  • 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.
  • a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.
  • the term "subject” refers to the target of administration, e.g., a human being.
  • 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.).
  • livestock e.g., cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.
  • the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • 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.
  • a subject can be a human patient.
  • a subject can have cancer, be suspected of having cancer, or be at risk of developing cancer.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T- cells (expanded by a disclosed method) and/or the disclosed expanded population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • diagnosis 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed expanded population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods.
  • a condition such as cancer
  • "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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed expanded population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method), disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods.
  • a condition such as cancer
  • 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.
  • a "patient” refers to a subject afflicted with a disease or disorder (e.g., cancer).
  • a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder such as cancer.
  • 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).
  • 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.
  • 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.
  • the identification can be performed by a person different from the person making the diagnosis.
  • the administration can be performed by one who performed the diagnosis.
  • 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.
  • 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.
  • inhibitor means 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.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (ex
  • 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.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed expanded population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof).
  • a native or control level e.g., a subject not receiving the disclosed isolated nucleic acid molecules, disclosed vector
  • the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% as compared to native or control levels.
  • a native or control level can be a pre-disease or pre-disorder level (such as a pre-cancer state).
  • 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, pathological condition, 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.
  • 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.
  • a mammal e.g., a human
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • preventing metastasis is intended.
  • administering refers 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 methods are 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.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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).
  • a disease or disorder such as cancer
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof.
  • disclosed isolated nucleic acid molecules disclosed vectors, disclosed cells (e.g., the disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof.
  • 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.
  • modifying the method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method)), disclosed pharmaceutical formulations, or a combination thereof to a subject
  • a “transposon” is a mobile genetic element that efficiently moves between vectors and chromosomes using the "cut and paste” or "copy and paste” mechanism.
  • transposase transposition for example, PB transposase in the PiggyBac transposon system
  • ITRs inverted terminal repeats
  • 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).
  • 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.
  • ITR inverted terminal repeat
  • the third polynucleotide may 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.
  • 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.
  • 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.
  • a starting mixed cell population can be enriched for a specific ⁇ T cell population.
  • the enriched ⁇ T cell population contains a greater percentage of V ⁇ 1 T cells and/or V ⁇ 3 T cells than the percentage of that cell type in the starting population.
  • an enriched ⁇ T cell population can contain a greater percentage of both V ⁇ 1 T cells and/or a greater percentage of V ⁇ 3 T cells than the percentage of that cell type in the starting population.
  • an enriched ⁇ T cell population can contain a greater percentage of both V ⁇ 1 T cells and/or a greater percentage of V ⁇ 3 T cells than the percentage of that cell type in the starting population.
  • the enriched ⁇ CAR T-cell population contains a greater percentage of V ⁇ 1 CAR T-cells and/or V ⁇ 3 CAR T-cells than the percentage of that cell type in the starting population.
  • an enriched ⁇ CAR T-cell population can contain a greater percentage of both V ⁇ 1 CAR T-cells and/or a greater percentage of V ⁇ 3 CAR T-cells than the percentage of that cell type in the starting population.
  • an enriched ⁇ CAR T-cell population can contain a greater percentage of both V ⁇ 1 CAR T-cells and/or a greater percentage of V ⁇ 3 CAR T-cells than the percentage of that cell type in the starting population.
  • the enriched ⁇ T-cell population can contain a lesser percentage of ⁇ T-cell populations.
  • “expanded” refers to the number of the desired or target cell type (e.g., V ⁇ 1 T cells and/or V ⁇ 3 T cells or V ⁇ 1 CAR T-cells and/or V ⁇ 3 CAR T-cells) in the enriched preparation is higher than the number in the initial or starting cell population.
  • "selectively expand” can mean that the target cell type or cell types (e.g., V ⁇ 1 T cells and/or V ⁇ 3 T cells and/or V ⁇ 3 T-cells or or V ⁇ 1 CAR T-cells and/or V ⁇ 3 CAR T-cells) are preferentially expanded over other non-target cell types (e.g., ⁇ T-cells or NK cells).
  • the activating agents of the invention selectively expand, e.g., engineered or non-engineered, V ⁇ 1 T-cells and/or V ⁇ 3 T-cells or V ⁇ 1 CAR T-cells and/or V ⁇ 3 CAR T-cell without significant expansion of V ⁇ 2 T- cells or V ⁇ 2 CAR T-cells.
  • "without significant expansion of” means that the preferentially expanded cell population are expanded at least 10-fold, preferably 100-fold, and more preferably 1,000-fold more than the reference cell population.
  • “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.
  • the term "contacting" as used herein refers to bringing one or more of the disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed populations of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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 V ⁇ 1 ⁇ T- cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed populations of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method), the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed
  • 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.
  • a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as cancer).
  • 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).
  • "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.
  • a disease or disorder such as, for example, cancer
  • 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.
  • 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).
  • 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.
  • “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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR
  • disclosed isolated nucleic acid molecules disclosed vectors, disclosed cells (e.g., the disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • a single dose of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells e.g., the disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • 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.
  • a progen 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).
  • CDRs complementarity determining regions
  • FR framework regions
  • 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.
  • 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.
  • 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.
  • 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 antigen recognition 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.
  • the term "variable region” or "variable domain” is used interchangeably.
  • 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.
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable region is a human variable region.
  • the variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • the variable region is a primate (e.g., non-human primate) variable region.
  • variable region comprises rodent or murine CDRs and primate framework regions (FRs).
  • VL and VL domain are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof.
  • VH and VH domain are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.
  • 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.
  • 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., IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • 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.
  • “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.
  • “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.
  • 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.
  • 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).
  • FcR Fc receptor
  • 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.
  • NK natural killer
  • 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.
  • TIL tumor-infiltrating lymphocyte
  • eACTTM engineered autologous cell therapy
  • allogeneic T cell transplantation can enhance the effectiveness of any transplanted T cell therapy.
  • the T cells or NK cells of the immunotherapy can come from any source known in the art.
  • 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.
  • iPSCs hematopoietic stem cell population
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • the term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CDR complementary determining region
  • FR Fv framework region
  • 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.
  • the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing 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.
  • Fc immunoglobulin constant region or domain
  • 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 ⁇ M 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 ⁇ M or less, at times about 0.1 ⁇ M or less, at times about 0.01 ⁇ M 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.
  • an antibody may, in an aspect, specifically bind to more than one target (e.g., any cancer- specific antigen and/or cancer-related antigen).
  • 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.
  • a “target” is any molecule bound by a binding motif, CAR, TCR or antigen binding agent, e.g., an antibody.
  • 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
  • the multiple targets may be bound by the same antigen-binding site on the antibody.
  • ASTR Antigen-specific targeting region
  • the targeting regions on the CAR or TCR are extracellular.
  • 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.
  • the term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced.
  • a subject's own cells can be obtained, made to express one or more disclosed CARs, and then administered to the same subject.
  • 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.
  • allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • 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 create the immunogen; immunize animals using appropriate schedule and adjuvant formula; and screen serum (or hybridoma) for antibody titer and isotype (also called antibody characterization).
  • target antigen e.g., peptide or hapten
  • 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.
  • a therapeutic RNA can comprise one or more expression sequences.
  • 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.
  • 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.
  • 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.
  • lncRNA long non- coding RNA
  • lincRNA long intergenic non-coding RNA
  • pre- transcript pre-miRNA
  • pre-mRNA pre-mRNA
  • ceRNA competing endogenous RNA
  • snRNA small nuclear RNA
  • snoRNA small nucleolar RNA
  • pseudo-gene rRNA
  • tRNA tRNA
  • ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre- miRNA).
  • 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.
  • RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation.
  • 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.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • cancer examples 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.
  • proliferative disorder and “proliferative disease” refer to disorders associated with abnormal cell proliferation such as cancer.
  • Tuor and neoplasm refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.
  • Methodastasis 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.
  • 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).
  • 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.
  • lipid nanoparticles 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.
  • 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.
  • 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 sequences can 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.
  • 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.
  • examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water.
  • examples of gaseous carriers can include carbon dioxide and nitrogen.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • Proper fluidity can 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.
  • biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that
  • 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.
  • 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.
  • proteins e.
  • 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.
  • the term "in combination” in the context of the administration of other therapies includes the use of more than one therapy (e.g., drug therapy).
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order.
  • a first therapy e.g., the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g., the disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or the disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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), concurrent
  • a first therapy e.g., the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells (e.g
  • 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.
  • V ⁇ 3 T cells can be used interchangeably with "V ⁇ 1- V ⁇ 2- T cells”.
  • ACT advanced 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
  • CMC Chemistry, Manufacturing, and Controls
  • CMs costimulatory molecules
  • CRS cytokine release syndrome
  • DNT double-negative T cell
  • FCR Fc receptor
  • HLA human leukocyte antigen
  • IL-2 Interleukin-2
  • ITAMs immunoimmunoreceptor tyrosine-based activation motifs
  • JAK Janus kinase
  • MCL mantle cell lymphoma
  • NFAT nuclear factor of the activated T-cell
  • NKG2D natural killer group 2D
  • T Cells 1. ⁇ T cells vs. ⁇ T cells [0093] Th dichotomy between ⁇ and ⁇ 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 ⁇ TCR and ⁇ 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.
  • ⁇ T cells While the developmental journey of ⁇ T cells entails stages of double-negative (DN), double-positive (DP), and single-positive (SP) prior to dissemination into the circulation, ⁇ 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.
  • DN2-DN3 double-negative
  • SP single-positive
  • ⁇ T cells Distinctly stratified in their antigen recognition, ⁇ T cells operate within the confines of MHC-dependent recognition, whereas ⁇ 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 ⁇ T cells necessitates a dual input of signals - antigen recognition and co-stimulation - to orchestrate immune responses, ⁇ T cells can be activated by a singular signal, such as a phosphoantigen.
  • ⁇ 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 ⁇ T cells which can recognize a given peptide antigen is extremely low. In contrast, ⁇ T cells exhibit characteristics of both innate and adaptive immunity.
  • ⁇ T cells are actively involved in tissue surveillance at barrier sites and contribute to the maintenance of tissue homeostasis. In disease settings, ⁇ 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 ⁇ 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.
  • V ⁇ 1, V ⁇ 2, and V ⁇ 3 T cells In the realm of cellular immunity, ⁇ T cells stand apart from their ⁇ T cell counterparts due to their distinct attributes, which intricately shape their roles in both pathogenesis of diseases and the field of immunotherapy. ⁇ 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, ⁇ T cells adopt an alternative T cell receptor (TCR) architecture, consisting of ⁇ and ⁇ chains, granting them the ability to perceive a wider array of antigens in MHC-independent manner.
  • TCR T cell receptor
  • V ⁇ 1 T cells are enriched in the thymus and mucosal epithelial tissues. V ⁇ 1 T cells are predominantly associated with the V ⁇ I gene family (V ⁇ 2/3/4/5/8). They produce a variety of cytokines like TNF- ⁇ and IFN- ⁇ , and lyse infected or transformed target cells by cytotoxicity. Second, V ⁇ 2 T cells, which exert cytotoxicity in tumor immunoregulation and viral infection, are mainly distributed in peripheral blood.
  • V ⁇ 2 T cells co-express V ⁇ II (V ⁇ 9).
  • T cell receptors (TCRs) expressed on the surface of V ⁇ 2 T cells are able to recognize phosphoantigens produced by malignant cells, thereby activating V ⁇ 2 T cells to release perforins, granzymes, and IFN- ⁇ .
  • MDSCs may inhibit the production of IFN- ⁇ and degranulation in phosphoantigens-activated V ⁇ 2T cells.
  • V ⁇ 3 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]
  • V ⁇ 1 T cells are players of adaptive immunity.
  • V ⁇ 1+ T cell relies on ⁇ TCR- and natural killer receptors (NKR)-mediated recognition of tumor antigens or stress signals, similar to V ⁇ 2 T cell.
  • NCR natural killer receptors
  • V ⁇ 1 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.
  • MICA/B MHC class I chain-related protein A and B
  • Evidence indicates ⁇ 1TCR 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 V ⁇ 1 and V ⁇ 2 in establishing immune surveillance.
  • V ⁇ 1 T cells play a significant role in adaptive immunity among ⁇ T cell subsets. Compared with V ⁇ 2 T cells, V ⁇ 1 T cells account for a small proportion of ⁇ T cells. They are mainly distributed on the surface of the mucosa. Although a large number of studies about the antitumor effects of ⁇ T cells mainly focus on V ⁇ 9V ⁇ 2 T cell subset, it is becoming more evident that V ⁇ 1 T cells also play a critical role in progression of hematological malignancies and some epithelial-derived solid tumors (including CRC).
  • V ⁇ 1 T cells Like V ⁇ 2 T cells, V ⁇ 1 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.
  • NKG2D natural killer group 2 member D
  • V ⁇ 1+ 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 V ⁇ 1+TCR-MICA/B.
  • V ⁇ 2 and V ⁇ 1 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.
  • TRAIL/TRAIL-R perforin/granzyme-B mediated secretory and death receptor
  • Fas/FasL perforin/granzyme-B mediated secretory and death receptor
  • TILs tumor-infiltrating T lymphocytes
  • MHC major histocompatibility complex
  • 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.
  • BTN butyrophilins
  • NRRs natural killer cell receptors
  • Chimeric Antigen receptor 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.
  • a CAR comprises a binding domain, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain.
  • CAR T cell A T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell.
  • an NK cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR NK cell.
  • CARs Chimeric antigen receptors
  • mAb monoclonal antibody
  • 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.
  • a disclosed CAR can be characterized by its generation.
  • 1G CAR contains only a CD3 ⁇ in its signaling domain with no CM.
  • 2G CAR harbors CD3 ⁇ and one CM, allowing dual signaling pathway.
  • a 3G CAR combines CD3 ⁇ and several CMs that deliver multiple signaling.
  • a 4G CAR resembles 2G CAR with the incorporation of an additional NFAT-responsive cassette that expresses cytokines playing anti-cancer activities.
  • 4G CAR has triple signals from primary CD3 ⁇ , CM, and expressed transgenic proteins.
  • 5G CAR is based on 2G CAR by adding membrane receptors IL-2R ⁇ 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 CD3 ⁇ , CMs, and cytokine-inducing JAK-STAT3/5 pathway.
  • First generation (1G) CAR T-cells contain only CD3 ⁇ without any CM.
  • 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 CD3 ⁇ and CMs (CD28, CD134/OX-40, or CD137/4-1BB).
  • 2G CAR T-cells provide two signals via CD3 ⁇ 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 CD3 ⁇ and multiple CMs (CD28, CD137/41BB, CD134/OX-40, NKG2D, CD27, TLR2 or ICOS).
  • 3G CAR T-cells can be characterized by multiple signaling via CD3 ⁇ 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.
  • 4G CAR T-Cells harbor CD3 ⁇ , NFAT-responsive cassette containing cytokines (IL-2, IL-5, IL-12, IFN ⁇ ) and CMs (CD28, OX-40 or 4-1BB).
  • 4G CAR T-cells can be characterized by primary CD3 ⁇ , 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.
  • 5G CAR T-cells incorporates CD3 ⁇ and additional membrane receptors: IL-2R ⁇ and STAT3.
  • 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.
  • 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.
  • 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.
  • 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.
  • membrane-distal epitopes usually require shorter spacers whereas membrane-proximal epitopes require longer spacers.
  • increasing epitope-paratope distance can also result in impaired delivery of granzymes and perforins to the target cell, thus reducing lytic efficiency.
  • the highly dense immune synapse hinders diffusion of lytic granules, which enhances pore formation by perforins and granzyme delivery
  • Transmembrane domains in CAR structures serve as a fulcrum for transducing ligand recognition signals to the intracellular cytoplasmic domain.
  • 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.
  • co-stimulatory signals are usually included in-cis with the CD3 ⁇ cytoplasmic domain.
  • Expanded Population of ⁇ T cells [0113] Disclosed herein is a population ⁇ T cells expanded by a disclosed method. Disclosed herein is a population of CAR ⁇ T-cells expanded by a disclosed method. Disclosed herein is a population of V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T-cells expanded by a disclosed method. Disclosed herein is a population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells expanded by a disclosed method. Disclosed herein is a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells.
  • a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells made according to the rapid expansion protocol set forth in Example 1, infra.
  • a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells made according to the rapid expansion protocol set forth in Example 2, infra.
  • predominantly can mean that the number of V ⁇ 1 ⁇ T cells in a disclosed expanded population of ⁇ T cells significantly exceeds the number of V ⁇ 2 ⁇ T cells in the same expanded population.
  • predominantly can mean that the number of V3 ⁇ T cells in a disclosed expanded population of ⁇ T cells significantly exceeds the number of V ⁇ 2 ⁇ T cells in the same expanded population. In an aspect, predominantly can mean that the number of V ⁇ 1 ⁇ T cells and the number of V ⁇ 3 ⁇ T cells in a disclosed expanded population of ⁇ T cells significantly exceeds the number of V ⁇ 2 ⁇ T cells in the same expanded population. [0117] Disclosed herein is a population of expanded CAR ⁇ T-cells comprising predominantly V ⁇ 1 ⁇ CAR T-cells and/or V3 ⁇ CAR T-cells.
  • predominantly can mean that the number of V ⁇ 1 ⁇ CAR T-cells in a disclosed expanded population of CAR ⁇ T-cells significantly exceeds the number of V2 ⁇ T cells in the same expanded population. In an aspect, predominantly can mean that the number of V3 ⁇ CAR T-cells in a disclosed expanded population of CAR ⁇ T-cells significantly exceeds the number of V2 CAR ⁇ T-cells in the same expanded population. In an aspect, predominantly can mean that the number of V ⁇ 1 ⁇ CAR T- cells and the number of V ⁇ 3 ⁇ CAR T-cells in a disclosed expanded population of CAR ⁇ T- cells significantly exceeds the number of V ⁇ 2 CAR ⁇ T-cells in the same expanded population.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells made by a disclosed method can be cryopreserved for future use and/or analysis.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells made by a disclosed method can be cryopreserved for future use and/or analysis.
  • expanded ⁇ T cells and/or expanded CAR ⁇ 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.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells remains can be administered to one or more subjects having cancer.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered to one or more subjects having cancer.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells remains can be administered to one or more subjects having one or more ⁇ T cell refractory cancers.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered to one or more subjects having one or more ⁇ CAR T-cell refractory cancers.
  • an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered as an allogeneic cell therapy to a subject having cancer.
  • an expanded population of predominantly population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a subject having cancer.
  • an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancer.
  • an expanded population of predominantly purified V ⁇ 1 CAR ⁇ T- cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancers.
  • an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered to a subject having an ⁇ T cell refractory cancer.
  • an expanded population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a subject having an ⁇ CAR T-cell refractory cancer.
  • an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered to a plurality of subjects having an ⁇ T cell refractory cancer.
  • an expanded population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a plurality subjects having an ⁇ CAR T-cell refractory cancer.
  • an expanded population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can comprise a CAR for any cancer-specific antigen and/or cancer- related antigen.
  • an expanded population of predominantly purified V ⁇ 1 CAR ⁇ T- cells and/or V ⁇ 3 CAR ⁇ T-cells can comprise one or more CARs for any cancer-specific antigen and/or cancer-related antigen.
  • 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-ES
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be used in one or more disclosed methods.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be used in one or more disclosed methods.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be used in one or more disclosed methods of treating a subject.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be used in one or more disclosed methods of treating a subject.
  • a disclosed population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be validated and/or characterized by several protocols known to the skilled person in the art.
  • a disclosed population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be subjected to sorting using FACS or a similar type of flow cytometry.
  • a disclosed population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be evaluated in a cytotoxicity assay.
  • a disclosed population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ 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.
  • stressed cells e.g., tumor or virally infected cells
  • IgG targeting molecules on the tumor cell surface, thereby addressing the problem of antigen heterogeneity.
  • a disclosed expanded population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be used to avoid T cell exhaustion.
  • using UCB as a biological sample for a use in a disclosed method ensures that an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can comprise a broader repertoire of gamma and delta chains.
  • using PB as a biological sample yields a narrower repertoire of gamma and delta chains.
  • 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.
  • 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.
  • ITAMs immunoreceptor tyrosine-based activation domains
  • 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.
  • 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.
  • 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.
  • ITAMs immunoreceptor tyrosine-based activation domains
  • 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.
  • a disclosed antigen binding domain can be a scFV and wherein the scFV can comprise a linker.
  • a disclosed encoded linker can join the V H and V L regions of the ScFv.
  • 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.
  • a disclosed nucleic acid sequence can encode one or more CARs specific for a cancer-specific antigen and/or cancer-related antigen.
  • 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-
  • Vectors 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 ⁇ T cells expanded by a disclosed method.
  • a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a CAR for expression by a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells expanded by a disclosed method.
  • a vector comprising a disclosed isolated nucleic acid molecule.
  • a vector comprising a nucleic acid sequence encoding one or more disclosed CARs.
  • a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding one or more disclosed CARs.
  • 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 ⁇ T cells expanded by a disclosed method.
  • 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 V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells expanded by a disclosed method.
  • 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 V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells expanded by a disclosed method.
  • a disclosed vector can be an integrating vector or a non-integrating vector.
  • 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).
  • a disclosed vector can be a viral vector.
  • 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.
  • a disclosed viral vector can be an adeno-associated virus (AAV) vector
  • 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.
  • ICTV International Committee on Taxonomy of Viruses
  • 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.
  • a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1).
  • a disclosed AAV vector can be a self-complementary AAV as disclosed herein.
  • a disclosed viral vector can be a lentiviral vector or a retroviral vector.
  • a promotor can be operably linked to nucleic acids.
  • a disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter.
  • a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed CAR or one or more disclosed CARs.
  • a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed tumor antigen. Promoters are known to the art.
  • a disclosed promoter can be a promoter/enhancer. Promoter/enhancers are known to the art.
  • a disclosed promoter can be an endogenous promoter.
  • 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.
  • 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.
  • 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.
  • the targeted cell e.g., a T cell or a NK cell or a macrophage
  • a disclosed non-viral vector can comprise lipid and/or polymer-based nanoparticles loaded with mRNA encoding one or more disclosed CARs.
  • a disclosed non-viral vector can be a plasmid.
  • a disclosed plasmid can comprise a disclosed nucleic acid sequence. Recombinant plasmids are known to the art.
  • 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.
  • 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-ES
  • compositions comprising a disclosed population of expanded ⁇ T cells; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a disclosed population of CAR ⁇ T-cells; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells expanded by a disclosed method; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells expanded by a disclosed method a disclosed isolated nucleic acid molecule; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a purified population of expanded ⁇ T cells; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a purified population of expanded CAR ⁇ T-cells; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a purified population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a purified population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells; and one or more pharmaceutically acceptable carriers and/or excipients.
  • a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule; and one or more pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a disclosed viral vector; and one or more pharmaceutically acceptable carriers.
  • a disclosed pharmaceutical formulation comprising a disclosed non-viral vector; and one or more pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a disclosed plasmid; and one or more pharmaceutically acceptable carriers.
  • a pharmaceutical formulation comprising a disclosed cell; and one or more pharmaceutically acceptable carriers.
  • 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.
  • 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.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone
  • NSAIDS nonsteroidal anti-inflammatory drugs
  • NSAIDs can comprise ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as rofecoxib and celecoxib, sialylates, or any combination thereof.
  • analgesics can comprise acetaminophen, oxycodone, tramadol, proporxyphene hydrochloride, or any combination thereof.
  • glucocorticoids can comprise cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or any combination thereof.
  • 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.
  • cytokine inhibitors such as the TNF antagonists (e.g., etanercept, adalimumab, and infliximab, chemokine inhibitors and adhesion molecule inhibitors.
  • TNF antagonists e.g., etanercept, adalimumab, and infliximab
  • chemokine inhibitors e.g., chemokine inhibitors and adhesion molecule inhibitors.
  • biological response modifiers can comprise monoclonal antibodies as well as recombinant forms of molecules.
  • exemplary disease-modifying anti-rheumatic drugs can comprise include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), minocycline, or any combination thereof.
  • 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.
  • GITR TNFR glucocorticoid induced TNFR related protein
  • 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
  • a disclosed pharmaceutical formulation can comprise an anti-chemokine therapy that enhances the resident memory T cell formations in tumor-free tissues.
  • 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.
  • 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, ensit
  • a disclosed pharmaceutical formulation can be prepared for systemic or direct administration.
  • a disclosed pharmaceutical formulation can be prepared for oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof.
  • a disclosed pharmaceutical formulation can be prepared for any method of administration disclosed herein.
  • a disclosed pharmaceutical formulation can be prepared for administration via multiple routes either concurrently or sequentially.
  • a disclosed pharmaceutical formulation can be first administered intratumorally and then be administered intravenously.
  • 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.
  • a disclosed pharmaceutical formulation can comprise one or more immune modulators.
  • a disclosed pharmaceutical formulation can comprise one or more proteasome inhibitors.
  • a disclosed pharmaceutical formulation can comprise one or more immunosuppressives or immunosuppressive agents.
  • an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof.
  • a disclosed pharmaceutical formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).
  • a disclosed pharmaceutical formulation can comprise an RNA therapeutic.
  • RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO).
  • 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).
  • 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.
  • a disclosed pharmaceutically acceptable carrier can comprise any disclosed carrier and/or any disclosed excipient.
  • a disclosed pharmaceutical formulation comprising an expanded population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ 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 ⁇ T cells, one or more disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method), or one or more V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • a disclosed animal can be treated with one or more disclosed populations of expanded ⁇ T cells, one or more disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method), or one or more V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • 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.
  • a disclosed treated subject can be a mouse or a rat.
  • a disclosed treated subject can be a transgenic mouse or a transgenic rat.
  • a disclosed treated subject can have one or more types of cancers and/or tumors.
  • a method of expanding a desired T cell population 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.
  • 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.
  • Vd1 ⁇ T cells and Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells; and depleting residual ⁇ T cells from the expanded ⁇ T cells.
  • a method of generating CAR Vd1 ⁇ T cells and CAR Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells; depleting residual ⁇ T cells from the expanded ⁇ T cells; and transducing the expanded ⁇ T cells with a vector encoding a chimeric antigen receptor.
  • Vd1 ⁇ T cells and Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells, wherein purifying and expanding the isolated ⁇ T cells comprises preparing the autologous feeder cells and co-incubating the isolated ⁇ T cells with the autologous feeder cells; and depleting residual ⁇ T cells from the expanded ⁇ T cells.
  • a method of generating CAR Vd1 ⁇ T cells and CAR Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells; wherein purifying and expanding the isolated ⁇ T cells comprises preparing the autologous feeder cells and co- incubating the isolated ⁇ T cells with the autologous feeder cells; depleting residual ⁇ T cells from the expanded ⁇ T cells; and transducing the expanded ⁇ T cells with a vector encoding a chimeric antigen receptor.
  • Vd1 ⁇ T cells and Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells; depleting residual ⁇ T cells from the expanded ⁇ T cells; and characterizing the expanded ⁇ T cells.
  • a method of generating CAR Vd1 ⁇ T cells and CAR Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; [0175] isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells; depleting residual ⁇ T cells from the expanded ⁇ T cells; characterizing the expanded ⁇ T cells; and transducing the expanded ⁇ T cells with a vector encoding a chimeric antigen receptor.
  • a method of expanding Vd1 ⁇ T cells and Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells, wherein purifying and expanding the isolated ⁇ T cells comprises preparing the autologous feeder cells and co-incubating the isolated ⁇ T cells with the autologous feeder cells; depleting residual ⁇ T cells from the expanded ⁇ T cells; and characterizing the expanded ⁇ T cells.
  • a method of generating CAR Vd1 ⁇ T cells and CAR Vd3 ⁇ T cells comprising isolating total leukocytes from umbilical cord blood; isolating ⁇ T cells from the total leukocytes; purifying and expanding the isolated ⁇ T cells; wherein purifying and expanding the isolated ⁇ T cells comprises preparing the autologous feeder cells and co- incubating the isolated ⁇ T cells with the autologous feeder cells; depleting residual ⁇ T cells from the expanded ⁇ T cells; characterizing the expanded ⁇ T cells; and transducing the expanded ⁇ T cells with a vector encoding a chimeric antigen receptor.
  • a disclosed method of expanding a desired T cell population can be performed ex vivo.
  • a disclosed desired T cell population can be expanded ⁇ T-cells or certain types of expanded ⁇ T-cells.
  • a disclosed desired expanded T cell population can comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a disclosed desired expanded T cell population can comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a disclosed biological sample can be umbilical cord blood (UCB), PBMCs, induced pluripotent stem cells (iPSCs), or any combination thereof.
  • using UCB as a biological sample ensures that an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can comprise a broader repertoire of gamma and delta chains.
  • using PB as a biological sample yields a narrower repertoire of gamma and delta chains.
  • UCB can serve as a more tolerable source material than other sources such as, for example, PBMCs.
  • UCB can allow for less stringent HLA matching between a donor and a recipient.
  • UCB can serve as an enriched source of hematopoietic stem cells (HSCs).
  • HSCs hematopoietic stem cells
  • HSCs can self-renew and can be used to differentiate into T-cells.
  • T- cells isolated from UCB can have a unique antigen-naive status, which can be linked to the decreased alloreactivity observed.
  • 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.
  • NFAT impaired nuclear factor of activated T-cells
  • a disclosed biological sample can be obtained from PBMCs.
  • T- cells can be isolated from PBMCs.
  • stem cells and/or hematopoietic progenitor cells can be isolated from PBMCs, and can be activated and transduced into CAR T-cells.
  • a disclosed biological sample can be obtained from PBMCs.
  • T-cells can be derived from iPSCs.
  • iPSCs can have an unlimited capacity for self-renewal and can be banked.
  • T-cells derived from iPSCs can be transduced into CAR T-cells.
  • isolating a population of T cells from a biological sample can comprise isolating leukocytes from UCB, PBMCs, iPSCs, or any combination thereof.
  • isolating a population of T cells from a biological sample can comprise isolating leukocytes from UCB.
  • isolating a population of T cells from a biological sample such as cord blood can comprise any process known to the art.
  • 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.
  • 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.
  • a disclosed method can employ a commercially available product (e.g., EasySep or HetaSep by StemCell Technologies).
  • a disclosed cord blood sample can have high platelet counts, which can facilitate the formation of clots.
  • a disclosed method can further comprise using suitable anticoagulants (e.g., acid citrate dextrose (ACD) or EDTA).
  • suitable anticoagulants e.g., acid citrate dextrose (ACD) or EDTA.
  • use of Lymphoprep 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.
  • LymphoprepTM Using aseptic technique, reinsert the rubber septum; (4) add LymphoprepTM 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.
  • PBS + 2% FBS Fetal Bovine Serum
  • disclosed isolated leukocytes can be aliquoted.
  • an aliquot of disclosed isolated leukocytes can comprise about 5 million cells per aliquot to about 15 million cells per aliquot.
  • an aliquot of disclosed isolated leukocytes can comprise about 15 million cells per aliquot to about 25 million cells per aliquot.
  • 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.
  • disclosed isolated leukocytes can be cryopreserved.
  • disclosed isolated leukocytes can be aliquoted and can be cryopreserved.
  • a disclosed method can further comprise cryopreserving disclosed isolated leukocytes.
  • 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.
  • 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.
  • cryopreserving can comprise storing one or more disclosed aliquots of isolated leukocytes at about -20 °C or below -20 °C.
  • cryopreserving can comprise storing the disclosed aliquots of isolated leukocytes at about -80 °C or below -80 °C.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ T cells from a disclosed population of isolated leukocytes. In an aspect, a population of ⁇ T cells can be immunopurified from a disclosed aliquot of isolated leukocytes. In an aspect, a population of ⁇ T cells can be immunopurified from a disclosed aliquot of isolated leukocytes using immunomagnetic positive selection.
  • a population of ⁇ T cells can be immunopurified from a disclosed aliquot of isolated leukocytes using immunomagnetic negative selection.
  • immunopurification of ⁇ T cells can be achieved using a commercially available kit (e.g., EasySepTM Human Gamma/Delta T Cell Isolation Kit by StemCell Technologies).
  • 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.
  • unwanted cells can be labeled with antibodies and magnetic particles, and then separated using a magnet.
  • immunopurification and/or sorting of ⁇ T cells can be performed without a column. In an aspect, immunopurification and/or sorting of ⁇ T cells can be performed with a column. [0191] In an aspect, following immunopurification, a disclosed separated fraction can be a ⁇ T cell fraction. In an aspect, following immunopurification, a disclosed separated fraction can be a non- ⁇ 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.
  • a magnet can be used for as long 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.
  • using a commercial ⁇ T cells isolation kit and immunomagnetic negative selection can allow the desired cells (i.e., ⁇ T cells) to be poured into one or more new tubes.
  • the disclosed isolated ⁇ 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.
  • a population of disclosed immunopurified and/or sorted ⁇ T cells can be centrifuged and re-suspended in media.
  • the disclosed immunopurified and/or sorted ⁇ T cells can be re-suspended in a commercially available medium.
  • a disclosed medium can be AIM V (ThermoFisher, Cat. # 12055083).
  • AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 ⁇ g/mL), and gentamicin sulfate (e.g., 10 ⁇ g/mL).
  • a disclosed medium can further comprise one or more antibodies and/or one or more transcription factors.
  • a disclosed medium can comprise OKT3 (orthoclone-muromonab-CD3).
  • 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.
  • a disclosed medium can comprise IL-2.
  • AIM V medium can comprise about 6000 IU/mL of IL-2 and about 60 ng/mL OKT3.
  • immunopurified and/or sorted ⁇ 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.
  • immunopurified and/or sorted ⁇ 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 ⁇ T cells can be centrifuged at about 500 x g for about 5 minutes. In an aspect, centrifuging the disclosed immunopurified and/or sorted ⁇ T cells can be done at a temperature other than room temperature. In an aspect, centrifuging the disclosed immunopurified and/or sorted ⁇ T cells can be done at about 4 °C. In an aspect, immunopurified and/or sorted ⁇ T cells can be centrifuged at about 500 x g and at 4 °C for about 5 minutes.
  • a disclosed method can further comprise using a disclosed separated fraction comprising non- ⁇ T cells.
  • a disclosed separated fraction comprising non- ⁇ T cells that is, cells other than ⁇ T cells
  • disclosed feeder cells can be used for the immunopurified and/or sorted ⁇ T cells.
  • a disclosed method can further comprise transferring disclosed feeder cells (such as, for example, a disclosed separated fraction comprising non- ⁇ T cells) to a new medium.
  • a disclosed method can further comprise transferring a population of disclosed feeder cells (such as, for example, non- ⁇ T cells) to a new medium.
  • a disclosed medium can be a commercially available medium with or without modifications.
  • a population of disclosed feeder cells can be transferred and/or added to a 10% FBS RPMI medium.
  • a disclosed medium comprising disclosed feeder cells be centrifuged.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a disclosed method can further comprise centrifuging a disclosed medium comprising re-suspended feeder cells.
  • a disclosed medium having DNAse I and comprising re-suspended feeder cells can have been incubated.
  • a disclosed incubation can comprise any disclosed incubation.
  • 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.
  • 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.
  • 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.
  • 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.
  • a disclosed method can further comprise re-suspending disclosed feeder cells following a centrifugation step.
  • re-suspending disclosed feeder cells can be done in a commercially available medium with or without modifications.
  • AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 ⁇ g/mL), and gentamicin sulfate (e.g., 10 ⁇ g/mL).
  • a disclosed medium can further comprise one or more antibodies and/or one or more transcription factors.
  • a disclosed medium can comprise OKT3 (orthoclone-muromonab-CD3).
  • 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.
  • a disclosed medium can comprise IL-2.
  • AIM V medium can comprise about 6000 IU/mL of IL-2 and about 60 ng/mL OKT3.
  • 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.
  • a disclosed method can further comprise irradiating a population of disclosed feeder cells.
  • irradiating feeder cells can comprise any irradiation protocol known to the skilled person in the art.
  • irradiating the disclosed feeder cells can comprise using an X-ray irradiator.
  • use of an X-ray irradiator can comprise a protocol comprising 5000 rad
  • irradiating a disclosed population of feeder cells can induced expression of NKG2D ligands in autologous cells.
  • inducing expression of NKG2D ligands in autologous cells can cause preferential activation of ⁇ T cells.
  • a disclosed method can further comprise adding together a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells.
  • a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells can be added to a container for incubation (such as, e.g., a flask).
  • a disclosed flask can be an upright 25 cm 2 flask.
  • a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells can be added to a flask.
  • adding together a disclosed population of immunopurified and/or sorted ⁇ 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.
  • adding together a disclosed population of immunopurified and/or sorted ⁇ 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).
  • a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells.
  • changing the medium bathing a disclosed population of immunopurified and/or sorted ⁇ 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.
  • 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 ⁇ T cells and a disclosed population of irradiated feeder cells.
  • changing the medium can comprise removing spent medium from the flask containing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells, and adding fresh medium to the flask.
  • spent medium can be collected and subjected to immediate analysis.
  • spent medium can be collected and cryopreserved.
  • cryopreserved spent medium can be thawed and subjected to analysis.
  • changing the medium can be done without disrupting the cells.
  • fresh medium can be added slowly to the flask to avoid disruption of the cells.
  • 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.
  • fresh medium can comprise a pre-warmed medium.
  • fresh medium can comprise AIM V medium.
  • fresh AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 ⁇ g/mL), and gentamicin sulfate (e.g., 10 ⁇ g/mL).
  • a disclosed fresh medium can further comprise one or more antibodies and/or one or more transcription factors.
  • 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.
  • fresh AIM V medium can comprise about 3000 IU/mL of IL-2 and can be pre-warmed.
  • a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and/or sorted ⁇ 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.
  • 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 ⁇ T cells and a disclosed population of irradiated feeder cells, and adding fresh pre-warmed medium to the flask.
  • a disclosed flask containing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells can be placed upright.
  • a disclosed method can further comprise repeating the removing of spent medium and the adding of fresh medium.
  • a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells.
  • 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 ⁇ T cells and a disclosed population of irradiated feeder cells.
  • changing the medium can comprise removing spent medium from the flask containing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells, and adding fresh medium to the flask.
  • spent medium can be collected and subjected to immediate analysis.
  • spent medium can be collected and cryopreserved.
  • cryopreserved spent medium can be thawed and subjected to analysis.
  • changing the medium can be done without disrupting the cells.
  • fresh medium can be added slowly to the flask to avoid disruption of the cells.
  • 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.
  • fresh medium can comprise a pre-warmed medium.
  • fresh medium can comprise AIM V medium.
  • fresh AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 ⁇ g/mL), and gentamicin sulfate (e.g., 10 ⁇ g/mL).
  • a disclosed fresh medium can further comprise one or more antibodies and/or one or more transcription factors.
  • a disclosed fresh medium can comprise IL-2.
  • 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 ⁇ T cells and a disclosed population of irradiated feeder cells, and adding fresh pre-warmed medium to the flask.
  • a disclosed flask containing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells can be placed on its side or place on the flat surface.
  • changing the medium for a second time can comprise removing spent medium from the flask containing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells, and adding fresh medium to the flask.
  • spent medium can be collected for a second time and subjected to immediate analysis.
  • spent medium can be collected and cryopreserved.
  • cryopreserved spent medium can be thawed and subjected to analysis.
  • changing the medium for a second time can be done without disrupting the cells.
  • fresh medium can be added slowly to the flask to avoid disruption of the cells.
  • 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.
  • fresh medium can comprise a pre-warmed medium.
  • fresh medium can comprise AIM V medium.
  • fresh AIM V medium can be serum-free and can contain L-glutamine, streptomycin sulfate (e.g., 50 ⁇ g/mL), and gentamicin sulfate (e.g., 10 ⁇ g/mL).
  • a disclosed fresh medium can further comprise one or more antibodies and/or one or more transcription factors.
  • 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.
  • fresh AIM V medium can comprise about 3000 IU/mL of IL-2 and can be pre-warmed.
  • a disclosed method can further comprise changing the medium bathing a disclosed population of immunopurified and/or sorted ⁇ 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.
  • 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 ⁇ T cells and a disclosed population of irradiated feeder cells, and adding fresh pre-warmed medium to the flask.
  • a disclosed flask containing a disclosed population of immunopurified and/or sorted ⁇ T cells and a disclosed population of irradiated feeder cells can be placed upright.
  • a disclosed population of irradiated feeder cells can be dead.
  • a disclosed expanded population of ⁇ T cells can comprise at least 200 million ⁇ T cells, at least 250 million ⁇ T cells, at least 300 million ⁇ T cells, or more than at least 300 million ⁇ T cells.
  • a disclosed expanded population of ⁇ T cells can comprise predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells.
  • a disclosed expanded population of ⁇ T cells can comprise predominantly V ⁇ 1 ⁇ T cells.
  • a disclosed expanded population of ⁇ T cells can comprise predominantly V ⁇ 3 ⁇ T cells.
  • a disclosed expanded population can comprise clinically relevant amounts of V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells.
  • a disclosed method can further comprise transducing the expanded population of ⁇ T cells with one or more chimeric antigen receptors (CARs).
  • CARs are known to the art and discussed supra.
  • CARs include an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising one or more immunostimulatory domains.
  • a disclosed CAR can be a 1 st generation CAR, a 2 nd generation CAR, a 3 rd generation CAR, a 4 th generation CAR, or a 5 th generation CAR.
  • a disclosed method can further comprise transducing the expanded population of ⁇ T cells with one or more chimeric antigen receptors (CARs) using a transduction protocol known to the skilled person in the art.
  • a disclosed method can further comprise transducing the expanded population of ⁇ 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).
  • a lentiviral vector can be used to integrate the genetic material for one or more CARs into the host cell's genome.
  • 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.
  • a retroviral vector can be used to integrate the genetic material for one or more CARs into the host cell genome.
  • the advantage of retroviral vectors lies in their robustness and established methodologies.
  • a transposon system e.g., nonviral approaches like the Sleeping Beauty system
  • 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.
  • CAR-T therapy a transposon carrying the CAR gene is introduced into T-cells along with a transposase enzyme.
  • a disclosed expanded population of ⁇ T cells can be transduced using lentiviral or retroviral transduction, transfection, electroporation, or any combination thereof.
  • a disclosed expanded population of ⁇ T cells can be transduced using spin- infection. Spin-infection protocols are generally known in the art.
  • a disclosed expanded population of ⁇ T cells can be transduced with one or more desired CARs and then re- suspended in AIM media.
  • AIM V medium can be serum-free and can contain L- glutamine, streptomycin sulfate (e.g., 50 ⁇ g/mL), and gentamicin sulfate (e.g., 10 ⁇ g/mL).
  • a disclosed AIM V medium can further comprise one or more antibodies and/or one or more transcription factors.
  • a disclosed AIM V medium can comprise IL-2.
  • a disclosed transduced expanded population of ⁇ T cells can be incubated while laying flat in a flask or other container.
  • a disclosed antigen binding domain can comprise a binding domain for a known cancer-specific antigen or cancer-related antigen.
  • 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
  • a disclosed antigen binding domain can be a scFV and wherein the scFV can comprise a linker.
  • a disclosed linker can join the V H and V L regions of the ScFv.
  • a disclosed the extracellular domain can further comprise a signal peptide.
  • a disclosed signal peptide can comprise a CD8 signal peptide or a fragment thereof.
  • a disclosed chimeric antigen receptor can further comprise a spacer domain between the extracellular domain and the transmembrane domain.
  • 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.
  • a disclosed spacer domain can comprise a hinge region.
  • a disclosed hinge region can comprise a hinge region of CD8 ⁇ , CD28, IgG1, IgG2, IgG3, IgG4, IgA, IgD, or any combination thereof.
  • 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 (
  • a disclosed chimeric antigen receptor can further comprise a transmembrane domain.
  • a disclosed transmembrane can further comprise a transmembrane domain of CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD79B, CD80, CD86, CD95 (FAS), CD134 (OX40), CD137 (4-1BB), CD154, CD278(ICOS), TCR ⁇ , TCR ⁇ , NKG2D, 2B4, or any combination thereof.
  • 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,
  • a disclosed transmembrane domain can be from or can be derived from NKG2D, [0242]
  • a disclosed intracellular domain can comprise the intracellular domain of CD28.
  • a disclosed intracellular domain can comprise one or more immunoreceptor tyrosine-based activation domains (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation domains
  • a disclosed ITAM can comprise the signaling domain of DAP10, DAP12, TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), or any combination thereof.
  • a disclosed intracellular domain can further comprise one or more co- stimulatory domains.
  • 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.
  • 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, DNAM
  • a disclosed intracellular domain can comprise an ITAM and one or more co-stimulatory domains, wherein the ITAM can comprise the signaling domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , 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.
  • the ITAM can comprise the signaling domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD278 (ICOS), or any combination thereof
  • 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, NKG
  • 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 CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , 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.
  • ITAMs can comprise the signaling domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD278 (ICOS), or any combination thereof
  • 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, NKG
  • 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.
  • 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 IFN ⁇ , IL2 secretion and cytotoxicity do not increase, or (vii) any combination thereof.
  • a disclosed co-stimulatory domain can comprise 4-1BB.
  • 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.
  • a disclosed intracellular domain can further comprise a self-cleaving peptide.
  • a disclosed self-cleaving peptide can comprise a T2A, a P2A, a E2A, or F2A peptide.
  • a GSG linker can be added to the N-terminus of a disclosed 2A peptide.
  • a disclosed intracellular domain can further comprise a sequence encoding an EGFR domain or a truncated EGFR domain.
  • 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.
  • a disclosed method can further comprise removing and/or depleting residual ⁇ T cells from a disclosed expanded population of ⁇ T cells.
  • removing and/or depleting residual ⁇ T cells from a disclosed expanded population of ⁇ T cells can be performed using a commercially available kit (e.g., CliniMACS TCR ⁇ / ⁇ 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.
  • removing and/or depleting residual ⁇ T cells from a disclosed expanded population of ⁇ T cells can be performed at day 8, day 9, day 10, day 11, or day 12 of incubation.
  • removing and/or depleting residual ⁇ T cells from a disclosed expanded population of ⁇ T cells can be performed at day 10 of incubation.
  • removing and/or depleting ⁇ T cells from a disclosed expanded population of ⁇ T cells can be performed at day 12, day 13, day 14, day 15, or day 16 of incubation.
  • removing and/or depleting ⁇ T cells from a disclosed expanded population of ⁇ T cells can be performed at day 14 of incubation.
  • the remaining population of ⁇ T cells can comprise predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells.
  • the remaining population of CAR ⁇ T-cells can comprise predominantly V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • predominantly can mean that the number of V ⁇ 1 ⁇ T cells and/or V3 ⁇ T cells in a disclosed expanded population of ⁇ T cells significantly exceeds the number of V2 ⁇ T cells in the same expanded population. [0253] In an aspect, predominantly can mean that the number of V ⁇ 1 ⁇ T cells in a disclosed expanded population of ⁇ T cells significantly exceeds the number of V2 ⁇ T cells in the same expanded population. In an aspect, predominantly can mean that the number of V3 ⁇ T cells in a disclosed expanded population of ⁇ T cells significantly exceeds the number of V2 ⁇ T cells in the same expanded population.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells remains.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells remains.
  • a disclosed method can generate a disclosed expanded population of ⁇ T cells that comprises predominantly V ⁇ 1 ⁇ T cells and/or V3 ⁇ T cells (rather than V2 ⁇ T cells).
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells remains can be cryopreserved for future use and/or analysis.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T- cells can be cryopreserved for future use and/or analysis.
  • expanded ⁇ T cells and/or expanded CAR ⁇ 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.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells remains can be administered to one or more subjects having cancer.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered to one or more subjects having cancer.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered as an allogeneic cell therapy to a subject having cancer.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a subject having cancers.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancer.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a plurality of subjects having cancers.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered to a subject having an ⁇ T cell refractory cancer.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a subject having an ⁇ CAR T-cell refractory cancer.
  • a population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be administered to a plurality of subjects having an ⁇ T cell refractory cancer.
  • a population of predominantly purified V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells can be administered as an allogeneic cell therapy to a plurality subjects having an ⁇ CAR T-cell refractory cancer.
  • a disclosed method of expanding a desired T cell population can further comprise measuring the purity of a population of ⁇ T cells following expansion by a disclosed method and/or the purity of a population of CAR ⁇ T-cells following expansion by a disclosed method.
  • a disclosed method can increase of purity of a population of V ⁇ 1 T-cells.
  • a disclosed method can increase of purity of a population of V ⁇ 1- V ⁇ 2- T-cells.
  • a disclosed method can increase of purity of a population of V ⁇ 3 T-cells.
  • a disclosed method can increase of purity of a population of V ⁇ 1 T-cells and/or V ⁇ 1- V ⁇ 2- T-cells and/or V ⁇ 3 T-cells.
  • a disclosed method of expanding a desired T cell population can further comprise validating the efficacy of one or more of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method).
  • 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.
  • a disclosed method of expanding a desired T cell population can further comprise subjecting one or more disclosed population of expanded ⁇ T cells to a delta chain analysis using, for example, iRepertoire.
  • a disclosed method of expanding a desired T cell population can further comprise sorting one or more disclosed population of expanded ⁇ T cells using FACS or a similar type of flow cytometry.
  • a disclosed method of expanding a desired T cell population can further comprise evaluating one or more disclosed population of expanded ⁇ T cells in a cytotoxicity assay.
  • a disclosed flow panel can consider one or more of the following: (1) BD HorizonTM BV786 Mouse Anti-Human CD45. Clone: HI30; Cat No:563716; (2) BD HorizonTM BUV395 Mouse Anti-Human CD3. Clone: SK7. Cat No: 564001; (3) BD PharmingenTM Alexa Fluor® 700 Mouse Anti-Human CD14. Clone M5E2. Cat No:557923; (4) BD PharmingenTM PE Mouse Anti-Human TCR ⁇ . Clone: IP26 (also known as IP26A). Cat Number: 564728; (4) BD HorizonTM BV711 Mouse Anti-Human ⁇ TCR. Clone: 11F2.
  • a disclosed gating strategy can comprise viable CD45 + CD14 neg CD3 + TCR ⁇ neg TCR ⁇ + Vd1 + Vd2 + .
  • expression for specific markers IFN- ⁇ , Perforin, Granzyme B, CD27, TCF1/TCF7, Tim-3, NKG2D, LAG-3 and NKp30 can be evaluated separately for the V ⁇ 1 and V ⁇ 2 T cells subsets.
  • expecific markers IFN- ⁇ , Perforin, Granzyme B, and TCF1/TCF7 (an intracellular staining)
  • expression for expecific markers can be performed following the manufacturer's instructions (eBioscienceTM 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.
  • a disclosed method can further comprise cryopreserving a disclosed population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method).
  • a disclosed method can further comprise thawing a disclosed cryopreserved population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed cryopreserved population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell s(expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • a disclosed method can further comprise administering a subject having cancer a disclosed population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • a disclosed method can further comprise administering to a plurality of subjects having cancer an allogeneic cell therapy comprising a disclosed population of V ⁇ 1 ⁇ T- cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells (expanded by a disclosed method) of a pharmaceutical formulation thereof.
  • a disclosed population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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.
  • a method of expanding a desired T cell population comprising isolating leukocytes from a sample comprising cord blood, immunopurifying ⁇ T cells from the isolated leukocytes; sorting the isolated leukocytes into ⁇ + T cells and ⁇ ⁇ T cells; using the ⁇ ⁇ T cells as feeder cells; irradiating the feeder cells to create preferential activation of ⁇ + T cells; incubating the irradiated feeder cells and the activated ⁇ T cells at a ratio of about 1:200 ( ⁇ T cells: feeder cells) in an upright flask; on day 4, removing spent media from the top of 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 ⁇ T cells and removing and counting the ⁇ T cells.
  • the method can further comprise transducing the ⁇ T cells with a vector comprising a nucleic acid sequence encoding one or more chimeric antigen receptors on day 14.
  • a disclosed population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ 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.
  • stressed cells e.g., tumor or virally infected cells
  • IgG targeting molecules on the tumor cell surface
  • Methods of Treating Cancer 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 ⁇ T cells expanded by a disclosed method.
  • 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 ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 CAR ⁇ T- cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • 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 ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer the method comprising administering to a subject having cancer a therapeutically effective amount of a population of CAR ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T- cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a population of CAR ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T-cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a method of treating cancer comprising administering to the subject a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a population of ⁇ 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 ⁇ T-cells expanded by a disclosed method.
  • 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 ⁇ T cells expanded by a disclosed method.
  • 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 ⁇ T-cells expanded by a disclosed method.
  • a method of treating cancer comprising treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising a population of ⁇ T cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer 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 ⁇ T-cells expanded by a disclosed method, wherein the expanded population of ⁇ T cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a method of treating cancer comprising administering to a subject having cancer a therapeutically effective amount of a pharmaceutical formulation comprising a population of ⁇ 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 ⁇ T-cells expanded by a disclosed method.
  • 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 ⁇ T-cells expanded by a disclosed method, wherein the expanded population of ⁇ T-cells predominantly comprise V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells.
  • a method of treating cancer 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 ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T-cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • a pharmaceutical formulation comprising a population of CAR ⁇ T-cells expanded by a disclosed method, wherein the expanded population of CAR ⁇ T-cells predominantly comprise V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells.
  • Use of a population of V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T-cells expanded by a disclosed method to treat a subject having cancer Use of a population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ T-cells expanded by a disclosed method to treat a subject having cancer.
  • Use of a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells to treat a subject having cancer.
  • Use of a population ⁇ 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 ⁇ T-cells expanded by a disclosed method in the manufacture of a medicament to treat a subject having cancer.
  • a disclosed ⁇ CAR T-cell can target one or more antigens on the surface of a cancer cell.
  • a disclosed ⁇ CAR T-cell can target a cancer cell in a solid tumor.
  • a disclosed ⁇ 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, Flt
  • a disclosed population of expanded ⁇ T-cells or a disclosed population of expanded CAR ⁇ T-cells can target an antigen on a ⁇ T-cell refractory tumor or an ⁇ CAR T-cell refractory cancer cell or tumor cell.
  • a disclosed method of treating cancer can comprise stimulating an effector cell mediated immune modulator response to a cancer cell targeted by a disclosed ⁇ T-cell or a disclosed ⁇ CAR T-cell .
  • a disclosed method of treating cancer can induce a tumor reducing immune response.
  • a disclosed method of treating cancer can induce phagocytosis of cancer cells in the subject.
  • a disclosed method of treating cancer can cross-prime an anti-tumor T cell response.
  • a disclosed method of treating cancer can induce a tumor eliminating immune response.
  • a disclosed method of treating cancer can effect tumor cell death.
  • a disclosed ⁇ T cell and/or a disclosed ⁇ CAR T-cell can be allogeneic to a subject having cancer and/or a subject in need thereof.
  • the disclosed V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells (expanded by a disclosed method) and/or the disclosed V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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,
  • a disclosed subject can have an ⁇ T-cell refractory tumor or an ⁇ CAR T-cell refractory cancer.
  • a disclosed subject can one or more ⁇ T-cell refractory tumors or one or more ⁇ CAR T-cell refractory tumors.
  • 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. For example, in an aspect, a blood sample and/or a biological sample can be collected from a subject at a pre-determined interval.
  • 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.
  • 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.
  • 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.
  • 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.
  • a therapeutically effective amount of a disclosed expanded population of ⁇ T cells predominantly comprising V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell and/or a disclosed expanded population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell or a pharmaceutical formulation can be about 1 x 10 3 cells to about 1 x 10 9 cells per subject.
  • a therapeutically effective amount of a disclosed expanded population of ⁇ T cells predominantly comprising V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell and/or a disclosed expanded population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell or a pharmaceutical formulation can be about 1 x 10 4 cells/kg per subject, about 1 x 10 5 cells/kg per subject, about 1 x 10 6 cells/kg per subject, about 1 x 10 7 cells/kg per subject, about 1 x 10 8 cells/kg per subject, or about 1 x 10 9 cells/kg per subject.
  • a disclosed expanded population of ⁇ T cells predominantly comprising V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell can be delivered a dosing regimen.
  • a first dose of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered to the subject.
  • a third dose V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered to the subject.
  • a disclosed expanded population of ⁇ T cells predominantly comprising V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell can be delivered a dosing regimen.
  • a first dose of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered.
  • a second dose of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR 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 V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) can be administered to the subject.
  • a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of the disclosed V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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.
  • administering can comprise systemic or direct administration.
  • administering can comprise oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof.
  • V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered by any method of administration disclosed herein.
  • V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation can be administered via multiple routes either concurrently or sequentially.
  • V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof can be first administered intratumorally and then be administered intravenously.
  • V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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.
  • local administration of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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.
  • 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.
  • a disclosed method of treating cancer can further comprise repeating the administering of the V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) a pharmaceutical formulation thereof.
  • a disclosed method of treating cancer can comprise protecting the subject from metastasis.
  • a disclosed method of treating cancer can comprise reducing the risk of developing metastasis.
  • a disclosed method of treating cancer can comprise preventing or inhibiting metastasis.
  • a disclosed method can comprise monitoring the subject for adverse effects.
  • a disclosed method can comprise continuing to treat the subject.
  • continuing to treat the subject can comprise continuing to administer V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • a disclosed method in the presence of adverse effects, can comprise modifying one or more steps of the method.
  • modifying one or more steps of a disclosed method can comprise modifying the administering step.
  • modifying the administering step can comprise changing the amount of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof administered to the subject, changing the frequency of administration of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, changing the duration of administration of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method)
  • 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.
  • a disclosed anti-cancer therapy can comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof.
  • a disclosed anti-cancer therapy can comprise one or more chemotherapeutic agents.
  • 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.
  • GITR TNFR glucocorticoid induced TNFR related protein
  • 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 (C
  • a disclosed method of treating cancer can comprise administering to the subject an anti-chemokine therapy.
  • 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.
  • a disclosed method of treating cancer can comprise administering to the subject one or more immune modulators.
  • 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-
  • a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV).
  • a disclosed immune modulator can be bortezomib or SVP-Rapamycin.
  • an immune modulator can be administered by any suitable route of administration.
  • 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.
  • a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi.
  • a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable.
  • a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease.
  • a disclosed endonuclease can be Cas9.
  • CRISPR/Cas9 systems and methods are known to the art.
  • a disclosed method of treating cancer can further comprise preventing or inhibiting metastasis of cancer cells.
  • 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., V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T- cell (expanded by a disclosed method) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T- cell (expanded by
  • 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., V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof).
  • a control subject such as, for example, a subject that has not received a disclosed treatment (e.g., V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇
  • a disclosed method of treating cancer can comprise surgically 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.
  • 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.
  • 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.
  • a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.
  • 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.
  • 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.
  • metabolic dysregulation can be associated with cancer or cancerous cells.
  • 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.
  • 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.
  • restoration can be a partial or incomplete restoration.
  • 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.
  • restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise preventing or inhibiting metastasis of cancer cells in the subject.
  • 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
  • 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.
  • 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.
  • a disclosed method of treating cancer can comprise titrating the dose of a disclosed treatment regimen in a specific or disclosed subject.
  • 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.
  • administering to a subject V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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.
  • the order of administering the one or more disclosed populations of expanded ⁇ T cells, one or more disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method), or the one or more disclosed populations of V ⁇ 1 CAR ⁇ T- cells and/or V ⁇ 3 CAR ⁇ 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.
  • 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.
  • 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.
  • 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.
  • a disclosed cfDNA analysis can be repeated one or more times.
  • a disclosed obtaining step can be repeated one or more times.
  • a disclosed cfDNA analysis can comprise next generation sequencing.
  • next generation sequencing (NGS) can comprise using one or more commercially available platforms.
  • next generation sequencing can comprise sequencing one or more cancer related genes.
  • sequencing one or more cancer related genes can comprise identifying one or more genomic aberrations.
  • one or more genomic aberrations can comprise somatic genomic aberrations.
  • the disclosed one or more somatic genomic aberrations can comprise mutations, insertions, deletions, chromosomal rearrangements, copy number aberrations, or any combination thereof.
  • one or more genomic aberrations in the biological sample can be detected.
  • a disclosed method can further comprise detecting one or more genomic aberrations in the biological sample.
  • a disclosed method can further comprise identifying one or more genomic aberrations in a panel of genes.
  • disclosed genomic aberrations can be identified in a panel of genes.
  • 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.
  • 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.
  • a disclosed panel of genes can comprise any combination of disclosed genes or disclosed cancer-related genes.
  • a disclosed method can comprise detecting the expression of one or more disclosed genes and/or one or more disclosed gene aberrations.
  • a disclosed method can comprise generating a control sample and/or a pooled control sample.
  • a disclosed method can comprise generating a reference sample and/or a pooled reference sample.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • a disclosed method of treating cancer can further comprise measuring the subject's tumor response to the administering of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • a subject's tumor response can comprise a partial response or a complete response.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof.
  • a disclosed partial response can comprise a decrease in the size of 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.
  • a complete response can be defined as complete disappearance of all tumors with no recurrence of tumors for at least four weeks.
  • a partial response can be defined as a 50% reduction in total tumor size with such reduction lasting at least four weeks.
  • 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.
  • a disclosed method of treating cancer can further comprise measuring the subject's molecular response to a disclosed treatment regimen.
  • a disclosed molecular response can comprise a decrease in the number of somatic genomic aberrations in a disclosed biological sample obtained from the subject.
  • a subject can be a human patient.
  • 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.
  • a subject can have a terminal cancer.
  • a disclosed subject can an ⁇ T-cell refractory tumor or an ⁇ CAR T-cell refractory cancer.
  • a disclosed subject can one or more ⁇ T-cell refractory tumors or one or more ⁇ CAR T-cell refractory tumors.
  • a disclosed targeted therapy can comprise one or more monoclonal antibodies.
  • 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.
  • a disclosed targeted therapy can comprise one or more small molecule inhibitors.
  • 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.
  • a tyrosine kinase inhibitor e.g., erlotinib, sunitinib, imatinib, dasatinib, etc.
  • a mTOR inhibitor e.g., everolimus
  • PARP inhibitor e.g., olaparib
  • CDK inhibitor e.g., palbociclib, ribociclib, abermaciclib, etc.
  • 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, bavit
  • 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, elot
  • 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, osimertini
  • 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,
  • a disclosed targeted therapy for subcutaneous administration can comprise denosumab, ofatumumab, or any combination thereof.
  • 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).
  • a targeted immunotherapy agent e.g., fam- trastuzumab
  • a disclosed method can be modified or one or more steps of a disclosed method can be modified.
  • 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.
  • a method can be altered by changing the amount of a disclosed treatment regimen, the disclosed V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the disclosed V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cell (expanded by a disclosed method) and/or the disclosed V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 ⁇ CAR T-cell (expanded by a disclosed method) or a pharmaceutical formulation thereof, a disclosed targeted therapy, a disclosed pharmaceutical
  • a disclosed population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ 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.
  • stressed cells e.g., tumor or virally infected cells
  • IgG targeting molecules on the tumor cell surface, thereby addressing the problem of antigen heterogeneity.
  • a disclosed expanded population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be used to avoid T cell exhaustion.
  • using UCB as a biological sample for a disclosed method ensures that an expanded population of predominantly purified V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can comprise a broader repertoire of gamma and delta chains.
  • using PB as a biological sample yields a narrower repertoire of gamma and delta chains.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or disclosed populations of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • kits comprising one or more disclosed populations of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or disclosed populations of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • a kit comprising a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells made according to the rapid expansion protocol set forth in Example 1, infra.
  • kits comprising a population of expanded ⁇ T cells comprising predominantly V ⁇ 1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells made according to the rapid expansion protocol set forth in Example 2, infra.
  • 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.
  • kits for use in a method of expanding a desired T cell population 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.
  • a disclosed kit can comprise one or more additional active agents and/or therapeutic agents (such as, for example, a disclosed anti-cancer agent).
  • the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject.
  • 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.
  • 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.
  • 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.
  • a kit for use in a disclosed method can comprise one or more containers holding a disclosed population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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 or package insert with instructions for use.
  • 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed populations of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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 V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • a disclosed kit can be used in a disclosed method of expanding a diesred T cell population, such as for example, ⁇ T cells or CAR ⁇ T-cells.
  • a disclosed kit can be used to treat cancer.
  • a disclosed kit can be used to preventing or inhibiting metastasis of cancer cells.
  • a disclosed kit can be used to risk of developing metastases.
  • a disclosed kit can be used to validate the efficacy and/or toxicity of a disclosed population of V ⁇ 1 ⁇ T-cells and/or V ⁇ 3 ⁇ T-cells (expanded by a disclosed method) and/or a disclosed population of V ⁇ 1 CAR ⁇ T-cells and/or V ⁇ 3 CAR ⁇ 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.
  • a disclosed kit comprising an expanded population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be used to generate an allogeneic CAR T-cell therapy.
  • a disclosed kit comprising an expanded population of predominantly purified Vd1 ⁇ T cells and/or V ⁇ 3 ⁇ T cells can be used to avoid T cell exhaustion.
  • H. Miscellaneous Disclosed herein is a method for the isolation and expansion of polyclonal V ⁇ 1 and V ⁇ 1- V ⁇ 2- ⁇ T cells from a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) isolating leukocytes from the sample; (c) immunopurifying ⁇ T cells from the isolated leukocytes; (d) sorting the isolated leukocytes into ⁇ + T cells and ⁇ ⁇ T cells; (e) using the ⁇ ⁇ T cells as feeder cells; (f) irradiating the feeder cells to created preferential activation of ⁇ T cells; (g) incubating the feeder cells and activated ⁇ T cells at a ratio of about 1:200 ( ⁇ T cells: Feeder cells) in an upright flask for at least
  • a therapeutic composition comprising isolated V ⁇ 1 and V ⁇ 1-V ⁇ 2- ⁇ T cells and/or ⁇ CAR-T cells and a pharmaceutically acceptable carrier, diluent and/or excipient.
  • a method of preventing and/or treating a subject suffering from a disease comprising administering to the subject a therapeutically effective amount of an isolated V ⁇ 1 and V ⁇ 1-V ⁇ 2- ⁇ T cells and/or ⁇ 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.
  • a disease can comprise cancer.
  • a method can further comprise administering one or more additional therapeutic agents.
  • one or more additional therapeutic agents can be administered prior to the V ⁇ 1 and V ⁇ 1-V ⁇ 2- ⁇ T cells and/or ⁇ CAR-T cells and/or therapeutic composition thereof. In an aspect, one or more additional therapeutic agents can be administered concurrently with the V ⁇ 1 and V ⁇ 1- V ⁇ 2- ⁇ T cells and/or ⁇ CAR-T cells and/or therapeutic composition thereof. In an aspect, the one or more additional therapeutic agents can be administered after the V ⁇ 1 and V ⁇ 1-V ⁇ 2- ⁇ T cells and/or ⁇ CAR-T cells and/or therapeutic composition thereof. VIII. EXAMPLES [0345] Little is known about the role of heterogeneous ⁇ T cells infiltrating human tumors.
  • Vd2 ⁇ T cells are found in tiny numbers in solid tumors, indicating that Vd2 likely circulate in blood. And while, Vd2 ⁇ T cells can be important to treat hematological tumors or bone marrow metastases, Vd2 ⁇ T cells do not appear to significant treat a solid tumor. Therefore, those ⁇ T cells that found in the tumor microenvironment are Vd1 and Vd3 ⁇ T cells.
  • TILs tumor-infiltrating lymphocytes
  • TILs were ⁇ T cells and that ⁇ 1 T cells outnumbered ⁇ 2 lymphocytes ⁇ 4:1.
  • encapsulation and running with 10X (single-cell) analyses, with sorted, bar-coded, ⁇ 1 plus ⁇ 2 TILs were performed.
  • ⁇ 1 TIL subsets showed distinctive clusters of (TCF7 + CD27 + IL7R dim ) stem-like cells ( ⁇ 1 stem ); and effector T cells expressing high levels of granzymes, IFNG and perforin, plus NK receptors ( ⁇ 1Eff).
  • V ⁇ 1 and V ⁇ 1-V ⁇ 2- T cells could be effective as an allogeneic CAR T-cell platform against ⁇ CAR T-cell-refractory tumors.
  • effective and clinically relevant methods for selective expansion of V ⁇ 2- ⁇ T cells from human umbilical cord blood were optimized.
  • the majority of ⁇ T cells in umbilical cord blood are naive V ⁇ 2- T cells expressing all V ⁇ chains, including V ⁇ 2/V ⁇ 5.
  • ⁇ T cells From 40 mL of cord blood, > 250 million viable cells were purified. Using 18 million cells from 2 different donors for optimization, bead-purified ⁇ T cells (initially > 8 x 10 4 ) were incubated with ⁇ -irradiated autologous feeder PBMCs ( ⁇ 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").
  • PBMCs ⁇ T cell-negative fraction expressing NKG2D ligands; 1:200, T cell:irradiated feeder cells
  • 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.
  • Step 2. 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.
  • Step 3 Second, by following the manufacturer's instructions (Cat #85450), ⁇ 250 million of total leukocytes were isolated using SepMateTM- 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 EasySepTM Human Gamma/Delta T Cell Isolation Kit and ⁇ 5 minutes of magnetic separation, ⁇ 80,000 ⁇ - ⁇ T cells were immunopurified from each 18 million cell aliquot, , . [0355] Step 4.
  • 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 ⁇ g/mL, and gentamicin sulfate at 10 ⁇ g/mL.
  • the ⁇ 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
  • Step 7 The ⁇ T cells and feeder cells were added to upright 25 cm 2 flasks at a 1:200 ratio (T cell:feeder cell).
  • 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.
  • Step 10 The 25 cm 2 flask were kept upright in the incubator. [0362] Step 11. At day 7, Steps 8-10 were repeated, but the 25 cm 2 flask were now placed lying flat. [0363] Step 12. At day 10, feeder cells were dead and T cells could be counted. Approximately 256 million ⁇ T cells were expanded at this point, with ⁇ 63% of them representing V ⁇ 1 T cells and ⁇ 34% representing V ⁇ 3 ⁇ 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 cm 2 flasks, which are deposited laying flat into the incubator.
  • Step 14 Following the manufacturer's instructions, residual human ⁇ T cells are depleted by using the CliniMACS TCR ⁇ / ⁇ product line (Miltenyi Biotec; Cat#.200-070-409). This step is performed at day 10, or if chimeric antigen receptors are transduced, then it is performed at day 14.
  • Step 15 Following the manufacturer's instructions, residual human ⁇ T cells are depleted by using the CliniMACS TCR ⁇ / ⁇ product line (Miltenyi Biotec; Cat#.200-070-409). This step is performed at day 10, or if chimeric antigen receptors are transduced, then it is performed at day 14.
  • V ⁇ 1 ⁇ T cells and V ⁇ 3 ⁇ T cells and/or the expanded V ⁇ 1 ⁇ CAR T cells and the V ⁇ 3 ⁇ CAR T cells are used for allogeneic administration into cancer patients.
  • Example 2 Optimization of an Isolation and Expansion Protocol for V ⁇ 1 and V ⁇ 3 T Cells [0367] Building on the protocol set forth in Example 1, a protocol for expanding human V ⁇ 1 and V ⁇ 3 ⁇ T cells from umbilical cord blood (UCB) was further optimized. Notably, V ⁇ 3 + T cells are almost absent from peripheral blood. This expansion protocol recapitulated proportions of ⁇ 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 lymphoprep TM (StemCEll Technologies; Cat. #07801) in according to the manufacturer's instructions. SepMateTM-50 tubes were used for the cell's separation in according to the manufacturer's instructions (Cat. #85450). Isolation of gamma delta human ( ⁇ ) T cells from total leukocytes. Human ⁇ 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.
  • 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 ⁇ g/mL Lonza), sodium pyruvate (1 mM) (ThermoScientific) and 10% Fetal Bovine Serum (FBS) (Sigma). Feeder cells were obtained from the same donor.
  • L-glutamine 2 mM, Gen Clone
  • penicillin 100 U/mL, Lonza
  • streptomycin 100 ⁇ g/mL Lonza
  • sodium pyruvate (1 mM) (ThermoScientific)
  • FBS Fetal Bovine Serum
  • 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.
  • 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.
  • 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 were counted.
  • 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.
  • X-RAD 160 X-ray irradiator
  • 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.
  • the ⁇ T cells were co-incubated with the autologous feeder cells. After the isolation of gamma-delta T cells by the EasySep TM kit, the cells were counted.
  • 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 ReadyTM Anti-Human CD3, clone UCHT1 (Tonbo TM ), and 1 ⁇ g/mL of In Vivo ReadyTM Anti-Human CD28 (clone CD28.2) (Tonbo TM ).
  • the T cells were added to a 25 cm 2 flask 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 ⁇ g/mL).
  • the flask was kept upright and stored in an incubator at 37 °C and 5% CO 2 .
  • AIM V medium was used.
  • 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.
  • 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 cm 2 flask. 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 cm 2 flask and transferred to the 75 cm 2 flask.
  • the bottom of the original 25 cm 2 flask was rinsed a few times (3x-5x) with the new medium, then the cells were transferred to the 75 cm 2 flask.
  • the new 75 cm 2 flask 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 cm 2 flask.
  • 40 mL of AIM-V medium containing 3000 IU/mL of hIL-2 was prepared.
  • the contents of the 75 cm 2 flask were transferred to a 150 cm 2 flask.
  • the 75 cm 2 flask was carefully rinsed with some of the new medium, then the cells were transferred to the 175 cm 2 flask.
  • 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 cm 2 flask 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.
  • IFN- ⁇ IFN- ⁇ , Perforin, Granzyme B, CD27, TCF1/TCF7, Tim-3, NKG2D, LAG-3 and NKp30 was evaluated separately for the V ⁇ 1 and V ⁇ 2 T cells subsets.
  • IFN- ⁇ , Perforin, Granzyme B, and TCF1/TCF7 an intracellular staining
  • was performed following the manufacturer's instructions eBioscienceTM Foxp3 / Transcription Factor Staining Buffer Set Cat. #00-5523-00; ThermoFisher).
  • Fluorescence-activated cell sorting 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 KRAS G12D transduced OVCAR3 cells were placed in 6-well plates.
  • the wells were washed, fresh medium was added, and the cells were treated with anti-human KRAS G12D -IgA1 or KRAS G12D -IgG4 or non-specific human IgA (Abcam, ab91025) at 0.5 ⁇ g/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.
  • 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).
  • 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
  • the ⁇ CAR T cells which were expanded using a disclosed rapid expansion protocol, were tested in a reduction to practice against cancer cells (FIG.11).
  • 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.
  • red blood cells lysis on healthy human apheresis samples was performed.
  • T cells were activated in AIM-V medium with 5% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 300 U/mL IL-2, and 0.05 ⁇ g/mL anti-CD3 at a concentration of 10 6 cells/mL.
  • Umbilical cord blood samples were obtained. ⁇ 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 10 6 cells/mL.
  • Transduction efficiencies and T cell populations were measured with a BD LSRII flow cytometer.
  • cytotoxicity assays about 10 5 target cells per well were plated in a flat bottom 96 well plate with 100 ⁇ L 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.
  • TAS2R13-CD28 CAR TAS2R13-CD28 CAR
  • mock transduced T cells TAS2R13-CD28 CAR
  • 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.
  • flank tumors were initiated by injecting 1 ⁇ 10 7 KRAS G12D A427 NCSCL cells (ATCC) in mice. Tumor volume was calculated as: 0.5 ⁇ (L ⁇ W 2 ), where L is length and W is width. Intraperitoneal injections of CAR T-cells were done at day 7.
  • Described herein is a protocol for expanding human V ⁇ 1/3 ⁇ 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.
  • the data provided herein show (i) that ⁇ CAR-T outperformed ⁇ CAR-T cells, demonstrating enhanced cytotoxic activity, (ii) that ⁇ CAR-T cells exhibited heightened responsiveness against solid tumors, and that the integration of NKG2D domains enhanced the functionality of ⁇ CAR-T.
  • Vd1 ⁇ T cells from aphaeresis is technically feasible, it is ineffective.
  • Vd3 ⁇ T cells are not found in sufficient amounts in peripheral blood to provide a meaningful means for expansion.
  • UB umbilical cord blood
  • the disclosed expansion protocol yielded high numbers of expanded ⁇ T cells.
  • the proportion of Vd1 and Vd3 ⁇ T cells in that expanded population mimicked that seen in the microenvironment of different human solid cancers.
  • 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.
  • Another significant advantage provided by the disclosed compositions and methods concerns the ability of the disclosed compositions and methods to generate expanded populations of ⁇ T cells, especially Vd1 ⁇ T cells and Vd3 ⁇ T cells, from UCB, which is an easily accessible and nearly inexhaustible source of material.
  • Still another significant advantage provided by the disclosed compositions and methods concerns cost.
  • CAR ⁇ 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.

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Abstract

L'invention concerne des procédés de fabrication et d'utilisation d'une population étendue de lymphocytes T γδ. L'invention concerne une thérapie par lymphocytes T CAR γδ allogéniques.
PCT/US2024/034197 2023-06-14 2024-06-14 COMPOSITIONS ET PROCÉDÉS D'EXPANSION DE LYMPHOCYTES T γδ Pending WO2024259371A2 (fr)

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WO2025235413A1 (fr) * 2024-05-05 2025-11-13 Duke University Compositions ciblant des antigènes cancéreux et leurs procédés d'utilisation

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KR20180029201A (ko) * 2015-05-18 2018-03-20 티씨알2 테라퓨틱스 인크. 융합 단백질을 이용하는 tcr 리프로그래밍을 위한 조성물 및 방법
US20220290101A1 (en) * 2019-08-16 2022-09-15 GammaDelta Therapeutics Limited Ex vivo gamma delta t cell populations
WO2022216574A1 (fr) * 2021-04-05 2022-10-13 Janssen Biotech, Inc. Récepteurs des lymphocytes t calr et jak2

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025235413A1 (fr) * 2024-05-05 2025-11-13 Duke University Compositions ciblant des antigènes cancéreux et leurs procédés d'utilisation
WO2025235408A1 (fr) * 2024-05-05 2025-11-13 Duke University Compositions comprenant des séquences scfv ciblant des antigènes cancéreux et leurs méthodes d'utilisation

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