CD38-NAD+ REGULATED METABOLIC AXIS IN ANTI-TUMOR IMMUNOTHERAPY
20240122986 · 2024-04-18
Assignee
Inventors
Cpc classification
A61K39/4632
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K39/464456
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C12N2501/599
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
A61K35/26
HUMAN NECESSITIES
A61K39/4611
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
C12N5/0638
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
International classification
A61K35/17
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61K35/26
HUMAN NECESSITIES
C07K16/24
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
The present disclosure provides methods of producing hybrid Th1/Th17 cells. Also provided herein are methods of treating cancer comprising targeting the CD38-mediated metabolic axis.
Claims
1. An ex vivo method for producing hybrid Th1/Th17 cells comprising: (a) obtaining a starting population of T cells; and (b) culturing the starting population of T cells in the presence of IL6, IL1?, IL23, TGF? and IL-12, thereby differentiating the starting population of T cells to hybrid Th1/Th17 cells.
2. The method of claim 1, wherein the TGF? is present at a concentration of 0.1 to 0.5 ng/mL.
3. The method of claim 1, wherein the IL1?, IL23, IL6, and/or IL12 are present at a concentration of about 5 to 30 ng/mL.
4. The method of claim 1, wherein the culture further comprises anti-CD3 and anti-CD28.
5. The method of claim 4, wherein the anti-CD3 and anti-CD28 are bound to a surface.
6. The method of claim 1, wherein the culture does not comprise IL-2.
7. The method of claim 1, wherein the culturing is for 2 to 5 days.
8. The method of claim 1, wherein the culture further comprises anti-IL4 and/or anti-IFN?.
9. The method of claim 1, wherein the starting population of T cells are CD.sup.4+ T cells.
10. The method of claim 9, wherein the CD.sup.4+ T cells are isolated from splenocytes.
11. The method of claim 1, wherein the starting population of T cells are human T cells.
12. The method of claim 1, wherein the isolation is performed by magnetic-bead sorting or fluorescence-activated cell sorting.
13. The method of claim 1, wherein the hybrid Th1/Th17 cells have an effector and stemness phenotype.
14. The method of claim 1, wherein the hybrid cells have increased NAD.sup.+ as compared to Th17 cells.
15. The method of claim 14, wherein the increase is at least 10-fold.
16. The method of claim 14, wherein the increase is at least 30-fold.
17. The method of claim 1, wherein the hybrid Th1/Th17 cells have reduced expression of CD38 as compared to Th1 or Th17 cells.
18. The method of claim 1, wherein the hybrid Th1/Th17 cells have at least two-fold higher Sirt1 activity as compared to Th17 cells.
19. The method of claim 1, wherein the hybrid Th1/Th17 cells co-express elevated levels of IFN? and IL17.
20. The method of claim 1, wherein the hybrid Th1/Th17 cells express transcription factors T-bet, ROR?, and/or IRF-4.
21. The method of claim 1, wherein the hybrid Th1/Th17 cells express chemokine receptors CXCR3 and/or CCR6.
22. The method of claim 1, wherein the hybrid Th1/Th17 cells express effector genes GzmB, Tbx21, and/or GM-CSF.
23. The method of claim 1, wherein the hybrid Th1/Th17 cells express stemness genes IL22, IL23R, TCF7, BCL6, and/or ?-catenin.
24. The method of claim 1, wherein the hybrid Th1/Th17 cells exhibit increased anti-tumor activity as compared to Th1 or Th17 cells.
25. A pharmaceutical composition comprising the hybrid Th1/Th17 cells with low or essentially no expression of CD38 and a pharmaceutical carrier.
26. The composition of claim 25, wherein the hybrid Th1/Th17 cells are produced according to any one of the claims 1-24.
27. A composition comprising an effective amount of hybrid Th1/Th17 cells with low or no expression of CD38 for the treatment of cancer in a subject.
28. The composition of claim 26, wherein the hybrid Th1/Th17 cells are produced according to any one of the claims 1-24.
29. A method of treating cancer comprising (a) administering an effective amount of T cells with low or no expression of CD38 and/or (b) administering anti-CD38 antibody in combination with an effective amount of T cells to the subject.
30. The method of claim 29, wherein the T cells with low or no expression of CD38 are the hybrid Th1/Th17 cells produced according to any one of claims 1-24.
31. The method of claim 29, wherein the T cells with low or no expression of CD38 are produced by activating T cells in the presence of an anti-CD38 antibody.
32. The method of claim 29, wherein the T cells with low or no expression of CD38 are produced by contacting the T cells with siRNA or shRNA.
33. The method of claim 29, wherein the T cells with low or no expression of CD38 are produced by genetically modifying the T cells.
34. The method of claim 33, wherein genetically modifying comprises the use of TALENs or the CRISPR/Cas9 system.
35. The method of claim 29, wherein the T cells of (b) are unprogrammed Th0 cells.
36. The method of claim 29, wherein the cancer is lung cancer, melanoma, or prostate cancer.
37. The method of claim 29, wherein the T cells are antigen-specific T cells.
38. The method of claim 37, wherein the T cells are genetically modified to express a recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region.
39. The method of claim 37 or 38, wherein the antigen is a tumor-associated antigen.
40. The method of claim 29, wherein the anti-CD38 antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a CDR- grafted antibody, a humanized antibody, a Fab, a Fab, a F(ab)2, a Fv, or a scFv.
41. The method of claim 29, further comprising administering an immune checkpoint inhibitor.
42. The method of claim 41, wherein the immune checkpoint inhibitor is an anti-PD1 antibody and/or an anti-CTLA antibody.
43. The method of claim 42, wherein the at least one checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.
44. The method of claim 42, wherein the PD-1 binding antagonist is nivolumab, pembrolizumab, pidillizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A or AMP-224.
45. The method of claim 42, wherein the anti-CTLA-4 antibody is tremelimumab or ipilimumab.
46. The method of claim 29, further comprising administering at least a second therapeutic therapy.
47. The method of claim 46, wherein the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy.
48. The method of claim 46, wherein the T cells and/or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
49. The method of claim 46, wherein the T cells are administered prior to or after the at least a second therapeutic agent.
50. The method of claim 46, wherein the T cells are administered simultaneously with the at least a second therapeutic agent.
51. The method of claim 29, wherein the subject is human.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0038] Adoptive T cell therapy (ACT) holds immense promise in eradicating large established tumors. Despite major advancements in understanding the functionality and the characteristics of various CD4 T cell subsets, the identification of T cell subsets with robust anti-tumor response is still obscure. Given that heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells respectively, are prerequisites of potent anti-tumor T cells, certain embodiments of the present disclosure provides optimized culture conditions to ex vivo program hybrid Th1/17 cells that exhibit characteristics of both Thl and Th17 cells.
[0039] Specifically, the method may comprise culturing a starting population of T cells in culture conditions to program the cells to a hybrid Th1/Th17 cell population. These hybrid Th1/Th17 cells can persist long-term in vivo while maintaining their effector function.
[0040] The present studies including transcriptional, metabolic, and proteomic profiling established that the enhanced anti-tumor property was attributed to increased NAD+mediated activity of histone deacetylase Sirtl in the hybrid Th1/17 cells. Inhibition of NAD+or Sirtl activity pharmacologically, or by using Sirt1-KO T cells exhibited a loss of stable anti-tumor control. Pharmacological or genetic inhibition of Sirtl activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD+, enhanced oxidative phosphorylation, higher glutaminolysis and altered mitochondrial dynamics, and vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using ThO anti-tumor T cells. Importantly, T cells with reduced surface expression of NADase CD38 and intrinsically higher NAD.sup.+ exhibited improved tumor control and enhanced oxidative phosphorylation that correlated with higher glutaminolysis and altered mitochondrial dynamics as discerned in hybrid Th1/17 cells. Lastly, replenishing NAD.sup.+ during the T cell expansion process or blocking the CD38 expression also resulted in improved tumor control even in the unpolarized anti-tumor T cells. Thus, strategies targeting the CD38-NAD.sup.+ axis may have translational potential to increase the efficacy of ACT therapy.
I. Definitions
[0041] As used herein, essentially free, in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0042] As used herein the specification, a or an may mean one or more. As used herein in the claim(s), when used in conjunction with the word comprising, the words a or an may mean one or more than one.
[0043] The use of the term or in the claims is used to mean and/or unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and and/or. As used herein another may mean at least a second or more.
[0044] Throughout this application, the term about is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0045] Treatment or treating includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease. For example, a treatment may include administration of a hybrid Th1/Th17 cell therapy.
[0046] As used herein, the term patient or subject refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.
[0047] The term effective, as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. Effective amount, Therapeutically effective amount or pharmaceutically effective amount when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.
[0048] An anti-cancer agent is capable of negatively affecting a cancer cell/tumor in a subject, for example, by promoting killing of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
[0049] The term antibody herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
[0050] The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier monoclonal indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
[0051] The phrases pharmaceutical or pharmacologically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
[0052] As used herein, pharmaceutically acceptable carrier includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.
[0053] The term unit dose or dosage refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 ?g/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 ?g/kg/body weight to about 100 mg/kg/body weight, about 5 ?g/kg/body weight to about 500 mg/kg/body weight, etc., can be administered. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0054] The term immune checkpoint refers to a molecule such as a protein in the immune system which provides signals to its components in order to balance immune reactions. Known immune checkpoint proteins comprise CTLA-4, PDI and its ligands PD-L1 and PD-L2 and in addition LAG-3, BTLA, B7H3, B7H4, TIM3, KIR. The pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012; Mellman et al., 2011).
[0055] An immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In particular the immune checkpoint protein is a human immune checkpoint protein. Thus the immune checkpoint protein inhibitor in particular is an inhibitor of a human immune checkpoint protein.
[0056] The term hybrid Th1/Th17 or Th1/17 cells refers to a population of T cells which co-express IFN? and IL17.
[0057] The term chimeric antigen receptors (CARs), as used herein, may refer to artificial T-cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. In some embodiments, CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3?, FcR, CD27, CD28, CD137, DAP10, and/or OX40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.
[0058] As used herein, the term antigen is a molecule capable of being bound by an antibody or T-cell receptor. An antigen may generally be used to induce a humoral immune response and/or a cellular immune response leading to the production of B and/or T lymphocytes.
[0059] The terms tumor-associated antigen, tumor antigen and cancer cell antigen are used interchangeably herein. In each case, the terms refer to proteins, glycoproteins or carbohydrates that are specifically or preferentially expressed by cancer cells.
II. T Cells for Adoptive Cell Transfer (ACT)
[0060] Embodiments of the present disclosure concern obtaining and administering T cells with low or no expression of CD38 to a subject as an immunotherapy to target cancer cells. Several basic approaches for the derivation, activation and expansion of functional anti-tumor effector T cells have been described in the last two decades. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); T cells activated ex-vivo using autologous
[0061] DCs, lymphocytes, artificial antigen-presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or redirected to express tumor-reactive TCR or chimeric TCR molecules displaying antibody-like tumor recognition capacity known as T-bodies. These approaches have given rise to numerous protocols for T cell preparation and immunization which can be used in the methods of the present disclosure.
A. T Cell Preparation
[0062] In some embodiments, the T cells are derived from the blood, bone marrow, lymph, or lymphoid organs. In some aspects, the cells are human cells. In certain embodiments, T cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art. The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD.sup.4+ cells, CD.sup.8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
[0063] Among the sub-types and subpopulations of T cells (e.g., CD.sup.4+ and/or CD8+T cells) are naive T (T.sub.N) cells, effector T cells (T.sub.EFF), memory T cells and sub-types thereof, such as stem cell memory T (TSC.sub.M), central memory T (TCM.sub.), effector memory T (T.sub.EM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. In particular aspects, the T cells are helper T cells.
[0064] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).
[0065] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD.sup.4+ or CD.sup.8+ selection step is used to separate CD4.sup.+ helper and CD.sup.8+ cytotoxic T cells. Such CD.sup.4+ and CD.sup.8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. In particular aspects, the CD.sup.4+ T cells are sorted by methods known in the art, such as fluorescence assisted cell sorting (FACS).
[0066] In some embodiments, the T cells are autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS? Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2). The cells are cultured until confluence (e.g., about 2?10.sup.6 lymphocytes), e.g., from about 5 to about 21 days, preferably from about 10 to about 14 days.
[0067] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T-cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days, preferably about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days, preferably about 14 days.
[0068] Expansion can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The non-specific T-cell receptor stimulus can include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil?, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 IU/ml IL-2 or IL-15, with IL-2 being preferred. The in vitro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.
[0069] In particular aspects, the T cells are differentiated to hybrid Th1/Th17 cells by culturing the presence of IL6, ILI?, IL23, TGF? and IL12. The TGF? may be present at a concentration of about 0.1 to 0.5 ng/mL. The ILI?, IL23, IL6, and/or IL 12 may be present at a concentration of about 5 to 30 ng/mL. In particular aspects, the concentration of IL6 is about 25 ng/mL, ILI? is about 20 ng/mL, IL23 is about 20 ng/ml, TGF? is about 0.3 ng/ml and IL 12 is about 20 ng/mL. The differentiation may occur in the presence of anti-CD3 and/or anti-CD28 beads. The culture may further comprise anti-IL4 and/or anti-IFN?.
[0070] In other aspects, T cells with decreased or no expression of CD38 are produced by disruption of the expression of CD38. Disruption in some cases is transient or reversible and in other cases is permanent. Disruption in some cases is of a functional or full length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, gene activity or function, as opposed to expression, is disrupted. Gene disruption is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and/or by disruption of nucleic acid of or associated with the gene, such as at the DNA level. Exemplary methods for gene disruption include gene silencing, knockdown, knockout, and/or gene disruption techniques, such as gene editing. Examples include antisense technology, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, as well as gene editing techniques which result in targeted gene inactivation or disruption, e.g., by induction of breaks and/or homologous recombination. Examples include insertions, mutations, and deletions. In one method, the T cells are activated in the presence of anti-CD38 antibody. In another method, the T cells are contacted with siRNA or shRNA specific to CD38. Finally, the T cells may be genetically modified to disrupt CD38 using DNA-targeting molecule, such as a DNA-binding domain, e.g., a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA-binding domain, or a DNA-binding domain from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein.
B. Genetically Engineered Antigen Receptors
[0071] The T cells can be genetically engineered to express antigen receptors such as engineered TCRs and/or chimeric antigen receptors (CARs). For example, the autologous T-cells are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen. Suitable TCRs include, for example, those with antigenic specificity for a mesothelin antigen. Suitable methods of modification are known in the art. See, for instance, Sambrook and Ausubel, supra. For example, the T cells may be transduced to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen using transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.
[0072] In some embodiments, the T cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).
[0073] In some embodiments, the CAR contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.
[0074] Exemplary antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., Curr. Opin. Immunol., 2012; Wu et al., 2012. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Patent No .: 7,446,190, and those described in International Patent Application Publication No .: WO/2014055668 A1.
[0075] In some aspects, the tumor antigen is a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYPIB), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1). For example, the target antigen is hTERT or survivin. In some aspects, the target antigen is CD38. In other aspects, the target antigen is CD33 or TIM-3. In other aspects, it is CD26, CD30, CD53, CD92, CD148, CD150, CD200, CD261, CD262, or CD362. In some embodiments, the engineered immune cells can contain an antigen that targets one or more other antigens. In some embodiments, the one or more other antigens is a tumor antigen or cancer marker. Other antigens include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, mesothelin, MUCI, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gplOO, oncofetal antigen, RORI, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin Al (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
2. Chimeric Antigen Receptors
[0076] In certain embodiments, the T cells are genetically modified to express a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.
[0077] In some embodiments, the engineered antigen receptors include chimeric antigen receptors (CARs), including activating or stimulatory CARs, costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
[0078] In some embodiments, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). The antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment can also be any number of different antigen binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.
[0079] The arrangement could be multimeric, such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and/or dimerizes can serve this purpose. One could use just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin. One could also use the hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization. One could also use just the hinge portion of an immunoglobulin. One could also use portions of CD8alpha.
[0080] In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[0081] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
[0082] The CAR generally includes at least one intracellular signaling component or components. In some embodiments, the CAR includes an intracellular component of the TCR complex, such as a TCR CD3+chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the CAR further includes a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor ? and CD8, CD4, CD25 or CD16. In specific embodiments, intracellular receptor signaling domains in the CAR include those of the T-cell antigen receptor complex, such as the zeta chain of CD3, also Fc? RIII costimulatory signaling domains, CD28, CD27, DAP10, CD137, OX40, CD2, alone or in a series with CD3zeta, for example. In specific embodiments, the intracellular domain (which may be referred to as the cytoplasmic domain) comprises part or all of one or more of TCR zeta chain, CD28, CD27, OX40/CD134, 4-1BB/CD137, F?ERI?, ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, and CD40. In some embodiments, one employs any part of the endogenous T-cell receptor complex in the intracellular domain. One or multiple cytoplasmic domains may be employed, as so-called third generation CARs have at least two or three signaling domains fused together for additive or synergistic effect, for example.
[0083] It is contemplated that the chimeric construct can be introduced into T cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.
[0084] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into T cells. Suitable vectors for use in accordance with the method of the present invention are non-replicating in the T cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.
[0085] 3. T Cell Receptor (TCR)
[0086] In some embodiments, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells. A T cell receptor or TCR refers to a molecule that contains a variable a and B chains (also known as TCRa and TCRp, respectively) or a variable y and 8 chains (also known as TCRy and TCR5, respectively) and that is capable of specifically binding to an antigen peptide bound to a MHC receptor. In some embodiments, the TCR is in the a? form. Typically, TCRs that exist in a? and yo forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term TCR should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the ?? form or ?? form.
[0087] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC-peptide complex. An antigen-binding portion or antigen-binding fragment of a TCR, which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC-peptide complex) to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable ? chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions.
[0088] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; see also Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for recognizing processed antigen, although CDRI of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDRI of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the ?-chain can contain a further hypervariability (HV4) region.
[0089] In some embodiments, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain, ?-chain) can contain two immunoglobulin domains, a variable domain (e.g., V.sub.a or Vp; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., 1991) at the N-terminus, and one constant domain (e.g., a-chain constant domain or C.sub.a, typically amino acids 117 to 259 based on Kabat, ?-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. In some embodiments, a TCR may have an additional cysteine residue in each of the a and B chains such that the TCR contains two disulfide bonds in the constant domains.
[0090] In some embodiments, the TCR chains can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
[0091] Generally, CD3 is a multi-protein complex that can possess three distinct chains (?, ?, and ?) in mammals and the ?-chain. For example, in mammals the complex can contain a CD3? chain, a CD35 chain, two CD3? chains, and a homodimer of CD3? chains. The CD3?, CD35, and CD38 chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3?, CD35, and CD38 chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3?, CD35, and CD3? chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3? chain has three.
[0092] Generally, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The CD3- and ?-chains, together with the TCR, form what is known as the T cell receptor complex.
[0093] In some embodiments, the TCR may be a heterodimer of two chains ? and ? (or optionally ? and ?) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (? and ? chains or ? and ? chains) that are linked, such as by a disulfide bond or disulfide bonds.
[0094] In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into the cells. In some embodiments, nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T-cell hybridomas or other publicly available source. In some embodiments, the T cells can be obtained from in vivo isolated cells. In some embodiments, a high-affinity T cell clone can be isolated from a patient, and the TCR isolated. In some embodiments, the T cells can be a cultured T-cell hybridoma or clone. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005. In some embodiments, the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
[0095] 4. Antigen-Presenting Cells
[0096] Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of a particular MHC molecule. APCs internalize antigen and re-express a part of that antigen, together with the MHC molecule on their outer cell membrane. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, referred to as class I and class II MHCs. T helper lymphocytes generally recognize antigen associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans the MHC is referred to as the HLA complex and in mice the H-2 complex.
[0097] In some cases, aAPCs are useful in preparing therapeutic compositions and cell therapy products of the embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. WO2007/103009.
[0098] aAPC systems may comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (Fc?RI), 41BB ligand, and IL21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.
[0099] In some embodiments, the presently disclosed process can be used to genetically modify CD26.sup.highT cells derived from peripheral blood and/or umbilical cord blood to express CAR(s) that can be numerically expanded in vitro using aAPC (Singh et al., 2008; Singh et al., 2011; Shah et al., 2013). The process has implications for cell and gene therapy, due to the relative ease of DNA plasmid production, electroporation, use of thawed ?-irradiated master-bank aAPC, and can be readily transferred to facilities operating in compliance with current good manufacturing practice (cGMP) for clinical trials.
[0100] In one embodiment, aAPCs are also subjected to a freeze-thaw cycle. In an exemplary freeze-thaw cycle, the aAPCs may be frozen by contacting a suitable receptacle containing the aAPCs with an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or similar low-temperature material, such that freezing occurs rapidly. The frozen aAPCs are then thawed, either by removal of the aAPCs from the low-temperature material and exposure to ambient room temperature conditions, or by a facilitated thawing process in which a lukewarm water bath or warm hand is employed to facilitate a shorter thawing time. Additionally, aAPCs may be frozen and stored for an extended period of time prior to thawing. Frozen aAPCs may also be thawed and then lyophilized before further use. Preferably, preservatives that might detrimentally impact the freeze-thaw procedures, such as dimethyl sulfoxide (DMSO), polyethylene glycols (PEGs), and other preservatives, are absent from media containing aAPCs that undergo the freeze-thaw cycle, or are essentially removed, such as by transfer of aAPCs to media that is essentially devoid of such preservatives.
III. Methods of Treatment
[0101] Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount T cell therapy, particularly T cells with decreased or no expression of CD38, such as hybrid Th1/Th17 of the present disclosure. Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.
[0102] In some embodiments, the individual has cancer that is resistant (has been demonstrated to be resistant) to one or more anti-cancer therapies. In some embodiments, resistance to anti-cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance to anti-cancer therapy includes progression of the cancer during treatment with the anti-cancer therapy. In some embodiments, the cancer is at early stage or at late stage.
[0103] In some embodiments, the T cells (e.g. unprogrammed Th0 cells) are administered in combination with at least one additional anti-cancer therapy, such as anti-CD38 antibody. The T cell therapy may be administered before, during, after, or in various combinations relative to an anti-cancer agent. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the T cell therapy is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the T therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.
[0104] In some embodiments, the subject can be administered nonmyeloablative lymphodepleting chemotherapy prior to the T cell therapy. The nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route. The nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which can be metastatic. An exemplary route of administering cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. In particular aspects, around 60 mg/kg of cyclophosphamide is administered for two days after which around 25 mg/m.sup.2 fludarabine is administered for five days.
[0105] In certain embodiments, a T cell growth factor that promotes the growth and activation of the autologous T cells is administered to the subject either concomitantly with the autologous T cells or subsequently to the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the growth and activation of the autologous T cells. Examples of suitable T-cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2. IL-12 is a preferred T cell growth factor.
[0106] The T cell therapy and anti-cancer agent may be administered by the same route of administration or by different routes of administration. In some embodiments, the T cell therapy and/or anti-cancer agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the T cell therapy and anti-cancer agent may be administered for prevention or treatment of disease. The appropriate dosage of the T cell therapy and anti-cancer agent be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
[0107] Intratumoral injection, or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate. For tumors of >4 cm, the volume to be administered will be about 4-10 ml (in particular 10 ml), while for tumors of <4 cm, a volume of about 1-3 ml will be used (in particular 3 ml). Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes.
B. Pharmaceutical Compositions
[0108] Also provided herein are pharmaceutical compositions and formulations comprising the activated T cell therapy, optionally an anti-cancer agent and a pharmaceutically acceptable carrier.
[0109] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX?, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
[0110] C. Anti-Cancer Therapy
[0111] In certain embodiments, the compositions and methods of the present embodiments involve a T cell therapy in combination with at least additional anti-cancer agent. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may another subset of T cells, such as CD8.sup.+ T cells. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.
[0112] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side- effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.
[0113] Various combinations may be employed. For the example below a T cell therapy is A and an anti-cancer therapy is B:
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0114] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.
[0115] 1. Chemotherapy
[0116] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term chemotherapy refers to the use of drugs to treat cancer. A chemotherapeutic agent is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
[0117] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegal1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2,2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.
[0118] 2. Radiotherapy
[0119] Other factors that cause DNA damage and have been used extensively include what are commonly known as ?-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
[0120] 3. Immunotherapy
[0121] The skilled artisan will understand that additional immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN?) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells
[0122] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Cancer is one of the leading causes of deaths in the world. Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in armed MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. The approval of two ADC drugs, ADCETRIS? (brentuximab vedotin) in 2011 and KADCYLA? (trastuzumab emtansine or T-DM1) in 2013 by FDA validated the approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment. As antibody engineering and linker-payload optimization are becoming more and more mature, the discovery and development of new ADCs are increasingly dependent on the identification and validation of new targets that are suitable to this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated/high levels of expression in tumor cells and robust internalization.
[0123] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
[0124] Examples of immunotherapies include, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169); cytokine therapy, e.g., interferons ?, ?, and ?, IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53 (U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
[0125] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints are molecules in the immune system that either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory checkpoint molecules that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.
[0126] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll 2012; both incorporated herein by reference). Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present invention. For example it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
[0127] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDLI binding antagonist is a molecule that inhibits the binding of PDLI to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art such as described in U.S. Patent Application No. 20140294898, 2014022021, and 20110008369, all incorporated herein by reference.
[0128] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDLI or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO?, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA?, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
[0129] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an off switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
[0130] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0131] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; Camacho et al., 2004; Mokyr et al., 1998 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
[0132] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy?) or antigen binding fragments and variants thereof (see, e.g., WOO 1/14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
[0133] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Pat. Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesions such as described in U.S. Patent No. US8329867, incorporated herein by reference.
[0134] 4. Surgery
[0135] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery.
[0136] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
[0137] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
[0138] 5. Other Agents
[0139] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
IV. Articles of Manufacture or Kits
[0140] An article of manufacture or a kit is provided comprising the hybrid Th1/Th17 T cells is also provided herein. The article of manufacture or kit can further comprise a package insert comprising instructions for using the hybrid Th1/Th17 T cells to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the hybrid Th1/Th17 T cells described herein may be included in the article of manufacture or kits. In some embodiments, the hybrid Th1/Th17 T cells are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
V. Examples
[0141] The following examples are included to demonstrate preferred embodiments
[0142] of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1HYBRID Th1/Th17 CELLS
[0143] Combined culture conditions of Th1 and Th17 cells generate hybrid Th1/17 cells: To merge the best functional traits of Th1 to Th17 cells, the culture conditions of Th17.sup.ILI?+TGF? cells (generated in presence of IL1? plus low dose of TGF?) were modified since they exhibited superior anti-tumor response as compared to conventional Th17 cells (generated in the presence of TGFB) due to reduced ectonucleotidase expression (Chatterjee et al., 2014). It was observed that using the culture conditions of IL6, ILI?, IL23, TGF62 .sup.lo with IL12 resulted in hybrid Th1/17 cells, which not only co-express elevated levels of IFN? and IL17 (
[0144] Next, a comparative Illumina microarray analysis showed that 589 genes were exclusively expressed in hybrid Th1/17 cells (
[0145] Hybrid Th1/17 cells exhibit enhanced anti-tumor potential: A comparative determination of the anti-tumor property between Th1, Th17 or Th1/17 cells differentiated ex vivo using melanoma epitope tyrosinase reactive CD.sup.4+ T cells obtained from h3T TCR transgenic mice (Mehrotra et al., 2012), showed that adoptive transfer of hybrid Th1/17 cells exhibit superior tumor control as compared to Th1, Th17 (
[0146] Given long-term tumor control observed with murine hybrid Th1/17 cells, similar strategies to generate hybrid Tc1/17 using class I restricted epitope reactive T cells also exhibited vastly improved tumor control as compared to Tc1 and Tc17 cells alone in both murine melanoma (using gp 100 reactive T cells), and a spontaneous prostate tumor model (using Sv40 reactive T cells) (FIGA. 10A-H). Next, it was determined if the ex vivo programming strategy has the potential to generate tumor-reactive human T cells for ACT. Using purified CD.sup.4+ T cells from normal healthy donors human Th1/17 hybrid cells were able to be generated, as indicated by the IFN? and IL17 cytokine profile (
[0147] Metabolically unique Th1/17 cells depend upon glutaminolysis: Metabolic commitment plays an important role in function and survival of T cells in a tumor microenvironment (Scharping et al., 2016; Sukumar et al., 2013). It was observed that uptake of 2NBDG, a fluorescent glucose analogue which indicates glycolytic commitment, was highest in Th1 cells followed by intermediate level in Th1/17 and lowest in Th17 cells (Thl>Th1/17>Th17) (
[0148] Since fatty acid oxidation (FAO) has been shown to modulate OXPHOS by providing acetyl-CoA, a substrate for TCA cycle, the influence of FAO was evaluated in affecting OXPHOS of different T cell subsets. Consistent with lower OXPHOS, mRNA level of CPTla, a rate-limiting enzyme that regulates the entry of fatty acid from cytosol to mitochondria (Jogl et al., 2004), was significantly reduced in Th1 and Th1/17 cells as compared to Th17 cells (
[0149] Because activation of T cells is also accompanied by the up-regulation of glutamine catabolismthat provides intermediates such as ?-ketoglutarate which can be metabolised through TCA cycle to fuel OXPHOS (Newsholme et al., 1985), it was next sought to determine the impact of glutaminolysis in the metabolic commitment of different T cell subsets. The genes associated with glutamine uptake (e.g., glutamine transporters Slcla5, Slc3a2 and Slc7a5), and its catabolismwere considerably higher in hybrid Th1/17 cells as compared to Th1 and Th17 cells (
[0150] The functional consequence of differential gluatamine metabolism in Th subsets was supported by the observation that Th17 cells with low glutaminolysis express a sizable population positive for FoxP3, whereas its expression is significantly reduced in Th1/17 with high glutaminolysis (
[0151] To comprehensively determine the dependence of Th1/17 cells on glutaminolysis for its effector function and antitumor potential, glutaminolysis was inhibited in Th1/17 cells using 6-Diazo-5-oxo-L-norleucine (DON), a glutaminase inhibitor. Similar to 2DG which has been shown to affect IFN? production by T cells (Chang et al., 2013), the data suggest that blocking of glutaminolysis also reduces the differentiation of Th1/17 cells as evident by reduction in IFN?+IL-17.sup.+ secreting fraction (
[0152] Increased levels of metabolite nicotinamide adenine dinucleotide (NAD.sup.+) in Th1/17 cell: A comprehensive evaluation of principle metabolites (using services from Metabolon, Inc.) shows that hybrid Th1/17 cells possess a metabolite signature that is also intermediate between the Th1 and Th17 cells (
[0153] Interestingly, it was found that there was a striking 34-fold increase in NAD.sup.+levels in Th1/17 cells as compared to Th17 cells (
[0154] NAD.sup.+-Sirt1 axis is required to exert anti-tumor response by Th1/17 cells: Sirt1, an NAD.sup.+ dependent protein deacetylase, acts as an epigenetic modulator of key transcription factors regulating immune cell function (Zhang and Kraus, 2010). It was observed that Sirtl deacetylase activity was two-fold higher in Th1/17 cells (
[0155] Expression of the stemness-associated genes was also markedly reduced in Th1/17 cells when using Sirtl pharmacological inhibitor (
[0156] NAD.sup.+ mediated Sirt1 deacetylase regulates Foxo1 activity, a key determinant of anti-tumor response of Th1/17 cells: Because the deacetylase activity of Sirt1 modulates the acetylation/deacetylation status of various transcription factors involved in the regulation of key cellular responses (Zhang and Kraus, 2010), the degree of acetylation was compared in hybrid Th1/17 cells. Using the global acetylation antibody that tracks the acetylation sites on the lysine residues, it was observed that Th1/17 cells indeed have reduced acetylation, which was reversed to a certain extent when NAD.sup.+levels were lowered due to Nampt inhibitor FK866 treatment (
[0157] Given that Foxo1 has been implicated in the generation of T cell memory response, migration (Rao et al., 2012) and is regulated both by phosphorylation and acetylation levels (Daitoku et al., 2011), the next studies focused on evaluating this protein between the Th subsets. The Th1/17 cells exhibited intermediate level of Foxo1 phosphorylation (
[0158] In further studies, pharmacological inhibition of NAD.sup.+ and Sirt1 in Th1/17 cells not only resulted in increased acetylation in Th1/17 cells (
[0159] Next, it was sought to determine whether maintenance of transcriptionally active nuclear Foxo1 is a key regulator of anti-tumor property in general, and could ameliorate the anti-tumor response of Th1 cells (that exhibit high NAD.sup.+ and Sirt1 but low nuclear Foxol). Phosphorylation of AKT (S473) inversely correlates with the nuclear Foxo1 level, as its kinase activity phosphorylates Foxo1 and facilitates nuclear export (Rao et al., 2012). To interrogate whether reduced nuclear Foxo1 in Th1 cells is due to increased activity of AKT, both pAKT (S473) level and their kinase activity was determined in Th subsets. It was observed that pAKT (S473) level and their kinase activity are markedly elevated in Th1 cells as compared to Th17 and Th1/17 cells (
TABLE-US-00002 TABLE 1 p value for glycolytic genes expression in Th1, Th17, and Th1/Th17 cells (see FIG. 3) Th1 vs. Th1 vs. Th17 vs. Gene name Th17 Th1/17 Th1/17 Glucose transporter 1 (Glut1) 0.04013 0.00181 0.96403 Hexokinase 1 (HK1) 0.15463 0.01092 0.80529 Hexokinase 2 (HK2) 0.05468 0.00251 0.94528 Glucose-6-phosphate isomerase (Gpi) 0.08248 0.07438 0.91092 Phosphofructokinase b 1 (Pfkb1) 0.01116 0.01188 0.12497 Phosphofructokinase b 2 (Pfkb2) 0.22222 0.22473 0.88340 Phosphofructokinase b 3 (Pfkb3) 0.84016 0.78659 0.99723 Phosphofructokinase 1 (Pfk1) 0.00816 0.00261 0.17861 Triose Phosphate isomerase (Tpi) 0.01430 0.02120 0.10837 Aldolase A 0.04038 0.05181 0.80448 Phosphoglycerate mutase 1 (Pgam1) 0.04068 0.48843 0.00561 Phosphoglucomutase 2 (Pgm2) 0.09448 0.12308 0.05527 Enolase (Enol) 0.00278 0.00909 0.12473 Pyruvate kinase 2 (Pkm2) 0.03252 0.03600 0.01143 Lactate dehrodenase A (LdhA) 0.01342 0.00862 0.04871 Monocarboxylate transporter 4 (MCT4) 0.01342 0.00862 0.04871
TABLE-US-00003 TABLE 2 p value for glutaminolysis genes expression in Th1, Th17, and Th1/17 cells (see FIG. 3). Th1 vs. Th1 vs. Th17 vs. Gene name Th17 Th1/17 Th1/17 Solute carrier family 1 member 5 0.10571 0.01140 0.02497 (Slc1a5) Solute carrier family 3 member 2 0.04713 0.03538 0.01239 (Slc3a2) Solute carrier family 7 member 5 0.05303 0.01560 0.00765 (Slc7a5) Glutamine-fructose-6-phosphate 0.00856 0.02245 0.00669 transaminase 1 (Gfpt1) Glutamine-fructose-6-phosphate 0.01416 0.15260 0.12616 transaminase 2 (Gfpt2) Phosphoribosyl pyrophosphate 0.01199 0.19293 0.01605 Aminotransferase (Ppat) Glutaminase 1 (Gls1) 0.35285 0.14691 0.09248 Glutaminase 2 (Gls2) 0.05839 0.09548 0.02848 Glutamate dehydrogenase 1 (Glud1) 0.90391 0.01053 0.01413 Glutamate oxaloacetate 0.04930 0.24485 0.02564 transaminase 1 (Got1) Glutamate oxaloacetate 0.17816 0.15652 0.00212 transaminase 2 (Got2) Ornithine aminotransferase (Oat) 0.04263 0.11451 0.02350
TABLE-US-00004 TABLE 3 p value for glutaminolysis genes expression in WT and CD38KO CD4.sup.+ T cells (FIG. 7). WT vs. CD38KO Gene name CD4.sup.+ T cells Solute carrier family 1 member 5 (Slc1a5) 0.113438 Solute carrier family 3 member 2 (Slc3a2) 0.045317 Solute carrier family 7 member 5 (Slc7a5) 0.021036 Glutamine-fructose-6-phosphate transaminase 1 0.049051 (Gfpt1) Glutamine-fructose-6-phosphate transaminase 2 0.057432 (Gfpt2) Phosphoribosyl pyrophosphate 0.194268 Aminotransferase (Ppat) Glutaminase 1 (Gls1) 0.039745 Glutaminase 2 (Gls2) 0.043545 Glutamate dehydrogenase 1 (Glud1) 0.137267 Glutamate oxaloacetate transaminase 1 (Got1) 0.050179 Glutamate oxaloacetate transaminase 2 (Got2) 0.019091 Ornithine aminotransferase (Oat) 0.034764
TABLE-US-00005 TABLE4 STARmethods. Gene Forward Reverse Source MouseqPCRprimers:5-3 Glut1 CAGTTCGGCTATAACACTGGTG GCCCCCGACAGAGAAGATG IDT,Coralville HKI CGGAATGGGGAGCCTTTGG GCCTTCCTTATCCGTTTCAATGG IDT,Coralville HKII GGAACCGCCTAGAAATCTCC GGAGCTCAACCAAAACCAAG IDT,Coralville Gpi TCAAGCTGCGCGAACTTTTTG GGTTCTTGGAGTAGTCCACCAG IDT,Coralville Pfkb1 ATGAGCTGCCCTATCTCAAGT GTCCCGGTGTGTGTTCACAG IDT,Coralville Pfkb2 GACAAGCCAACTCACAACTTCC ACACTGTAATTTCTTGGACGCC IDT,Coralville Pfkb3 CCCAGAGCCGGGTACAGAA GAGCCCCACCATCACAATCAC IDT,Coralville Pfk AGGAGGGCAAAGGAGTGTTT TTGGCAGAAATCTTGGTTCC IDT,Coralville TpI CTTACATCGACTTTGCCAGACA CTAGGGCGTGGCTCACTTT IDT,Coralville AldoA TCAGTGCTGGGTATGGGTG GCTCCTTAGTCCTTTCGCCT IDT,Coralville Pgam1 TCTGTGCAGAAGAGAGCAATCC CTGTCAGACCGCCATAGTGT IDT,Coralville Pgm2 AGTGAAGACGCAGGCATATCC GGCTCCACGGTAGAGACGA IDT,Coralville Enol AAAGATCTCTCTGGCGTGGA CTTAACGCTCTCCTCGGTGT IDT,Coralville Pkm2 GTCTGAATGAAGGCAGTCCC GTCCGCTCTAGGTATCGCAG IDT,Coralville LdhA TGTCTCCAGCAAAGACTACTGT GACTGTACTTGACAATGTTGGGA IDT,Coralville MCT4 TCACGGGTTTCTCCTACGC GCCAAAGCGGTTCACACAC IDT,Coralville Sic1a5 GGACGTCTTCTATCTCCACAA ACTCCTTCAATGATGCCACC IDT,Coralville Sic3a2 TGCAACCAAGAACTCAGAGC TCATTTTGGACCTCACTCCC IDT,Coralville Sic7a5 ACAGCTGTGAGGAGCAGCAC TCTTCGCCACCTACTTGCTC IDT,Coralville Gfpt1 GCCAACGCCTGCAAAATCC GCCCAACGGGTATGAGCTAT IDT,Coralville Gfpt2 TGATGGGAATAACCACGAAGTCA CGAAGTGTGTCTCAAACTCCAC IDT,Coralville Ppaf GGGAGTGCAGTGCCTAAATTC GTACCTCGTATGTCCGATTCCA IDT,Coralville Gls GCTGTGCTCTATTGAAGTGACA TTGGGCAGAAACCACCATTAG IDT,Coralville Gls2 TGCATATAGTGGAGATGTCTCG GCTCCATATCCATGGCCGACAA IDT,Coralville Glud1 CCCAACTTCTTCAAGATGGTGG AGAGGCTCAACACATGGTTGC IDT,Coralville Got1 ACCGCACAGATGAATCTCAGC ATGGGCAGGTACTCGTGGT IDT,Coralville Got2 TGGGCGAGAACAATGAAGTGT CCCAGGATGGTTTGGGCAG IDT,Coralville Oaf TGCCACCCAAAGATCATAGATGC TGTACTCCTCGTATTCACCAAGG IDT,Coralville Kif2 CTCAGCGAGCCTATCTTGCC CACGTTGTTTAGGTCCTCATCC IDT,Coralville CCR7 GTCTCTCTCCAGCTAGCCCA CAAACAGGAGCTGATGTCCA IDT,Coralville Cpt1a TCTATGAGGGCTCGCG CGTCAGGGTTGTAGCA IDT,Coralville PGC1? TGAGGACCGCTAGCAAGTTT TGTAGCGACCAATCGGAAAT IDT,Coralville Csf2 GGCCTTGGAAGCATGTAGAGG GGAGAACTCGTTAGAGACGACTT IDT,Coralville IL23r TTCAGATGGGCATGAATGTTTCT CCAAATCCGAGCTGTTGTTCTAT IDT,Coralville IL22 ATGAGTTTTTCCCTTATGGGGAC GCTGGAAGTTGGACACCTCAA IDT,Coralville GzmB GCCCACAACATCAAAGAACAG AACCAGCCACATAGCACACAT IDT,Coralville Tbx21 AGCAAGGACGGCGAATGTT GGGTGGACATATAAGCGGTTC IDT,Coralville Tcf7 GTGGACTGCTGAAATGTTCG AGCATCCGCAGCCTCAAC IDT,Coralville Bcl6 GATACAGCTGTCAGCCGGG AGTTTCTAGGAAAGGCCGGA IDT,Coralville ?-catenin ATGGAGCCGGACAGAAAAGC CTTGCCACTCAGGGAAGGA IDT,Coralville ?-actin ACGTAGCCATCCAGGCTGGTG TGGCGTGAGGGAGAGCAT IDT,Coralville HumanqPCRprimer:5-3 Tcf7 AGAGAGAGAGTTGGGGGACA TCTGCTCATGCATTACCCAC IDT,Coralville Bcl6 CTGGCTTTTGTGACGGAAAT GTTTCCGGCACCTTCAGACT IDT,Coralville Lef1 CACTGTAAGTGATGAGGGGG TGGATCTCTTTCTCCACCCA IDT,Coralville CD62L CTTTCACCAAGGGCGATTTA GGCATTTATCATTTGGCTGG IDT,Coralville Hprt1 TTTGCTTTCCTTGGTCAGGC GCTTGCGACCTTGACCATCT IDT,Coralville
[0160] Inverse correlation between CD38 and NAD.sup.+ controls long-term anti-tumor response by T cells. Recent studies have implicated that in addition to being a part of non-canonical adenosinergic pathway (Morandi et al., 2015), the cell surface molecule CD38 is also a NADase and its expression inversely correlates with NAD.sup.+levels (Chini, 2009). While the expression of CD38 was found to be upregulated on Th17 cells, hybrid Th1/17 cells exhibited reduced expression of CD38 and other ectonucleotidases (CD39, CD73) (
[0161] A comparative analysis of key metabolic regulators identified reduced glucose uptake (measured using 2-NBDG) (
[0162] It was also observed that there was almost a 50% reduction in the cytokine secretion from WT T cells infiltrating tumors as compared to the CD38KO T cells (
[0163] Given a recent report that blockade of PD1 is not sufficient to reverse mitochondrial insufficiency observed in tumor infiltrating lymphocytes (TILs) (Scharping et al., 2016), it was postulated that it is the expression of CD38 on the PD1 expressing T cells which regulates its metabolism. This idea was confirmed when the CD38-KO T cells did not lose functionality and maintained higher levels of PGC1? as compared to WT T cells after overnight exposure to EL4 tumor ascitis (
[0164] Since adoptively transferred T cells could acquire TGF? mediated CD38 expression in a tumor microenvironment (
[0165] The data showed that a hybrid Th1/17 subset has a significantly improved anti-tumor activity compared to Th1 or Th17 cells. Importantly, hybrid Tc1/17 cells also exhibited potent anti-tumor phenotype The long persisting hybrid T cells exclusively secreted IFN?, and showed loss of anti-tumor phenotype upon blocking IFNy. Thus, the strategy to generate both Th1/17 and Tc1/17 cells using similar programming conditions is of translational value, since it will overcome the current limitation where Th17 cells are better at anti-tumor activity than Th1 cells (Muranski et al., 2008), and Tel are better than Tc17 cells (Yu et al., 2013).
EXAMPLE 2MATERIALS AND METHODS
[0166] Mice: C57BL/6, B6-HLA-A2.sup.+, B6-Rag.sup.?/?, CD38KO, Sirt1.sup.fl/fl, CD4.sup.cre, B6 (CD45.1), IFN?.sup.?/?, TRP-1, Pmel, TRAMP mice were obtained from Jackson Laboratory (Bar Harbor, ME). IFN?.sup.Thy1.1 knock-in, Foxo1.sup.fl/flLck.sup.Cre and T Ag TCR-I mice were kind gift from Casey T. Weaver, UAB, Melanie Gubbels Bupp, Randolph-Macon College, VA and Jennifer Wu, MUSC respectively. CD38KO-IFN?.sup.Thy1.1 knock-in mice, HLA-A2.sup.+-Rag.sup.?/? mice and Pmel-IFN?.sup.?/? mice were developed in the lab. Melanoma epitope tyrosinase reactive HLA-A2 restricted TCR transgenic mouse (referred as h3T in the text) was developed in the lab and reported earlier (Mehrotra et al., 2012). Animals were maintained in pathogen-free facilities and experimental procedures were approved by Institutional Animal Care and Use Committees of Medical University of South Carolina, Charleston. Mice were bred and housed under standard housing conditions (group housing up to 5 mice per cage) except B6-Rag.sup.?/? and HLA-A2.sup.+-Rag.sup.?/? mice which require high barrier facility (also group housing up to 5 mice per cage). For tumor experiments, equal number of age and gender matched (both male and female) mice were randomly assigned for the experiments when they were between 8-10 weeks old. No influence of sex on the result of the studies was observed.
[0167] Cell Lines: B16-F10 (RRID: CVCL_0159) was obtained from American Type Culture Collection (ATCC), suggested to be of male origin.
[0168] T cell differentiation: Na?ve CD.sup.4+ T cells were purified from the total splenocytes of 6-9 weeks old WT (C57BL/6) and KO (Foxo1.sup.fl/flLck.sup.Cre, Sirt1.sup.fl/flCD4.sup.cre, CD38KO) mice using CD.sup.4+ T cell isolation kit (Miltenyi Biotec) according to manufacturer's protocol. Total splenocytes from 6-9 weeks old TCR transgenic mouse TRP-1 (bears class-II restricted CD.sup.4+ T cells) or Pmel (bears class-I restricted CD.sup.8+ T cells) or TCR-I (bears class-I restricted CD.sup.8+ T cells) were also used. Within experiments mice were age and sex matched. Purified na?ve CD.sup.4+ T cells or total splenocytes were differentiated to Th1 or Tel (10 ng/ml IL12, 50 U/ml IL2 and 10 ?g/ml anti-IL4), Th17 or Tc17 (3 ng/ml TGFB, 25 ng/ml IL6, 10 ?g/ml anti-IL4 and 10 ?g/ml anti-IFN?), Th1/17 or Tc1/17 (20 ng/ml IL1?, 25 ng/ml IL6, 10 ng/ml IL12, 20 ng/ml IL23, 400 pg/ml TGFB, 10 ?g/ml anti-IL4, 5 ?g/ml anti-IFN?), iTreg (5 ng/ml TGFB, 100 IU/ml IL2, 10 ?g/ml anti-IL4 and 10 ?g/ml anti-IFN? or Th0 (50 IU/ml IL2) in presence of plate bound anti-CD3 (5 ?g/ml) and anti-CD28 (5 ?g/ml). For TRP-1, gp100 or Sv40 specific generation of different Th or Tc subsets, total splenocytes from either TRP-1, Pmel or TCR-I TCR transgenic mice were stimulated with 1 ?g/ml respective peptides in presence of above-mentioned polarizing conditions. T cells were differentiated for three days in IMDM media supplemented with 10% FCS, 4mM L-glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, 55 ?M beta-mercaptoethanol under 7% CO.sub.2, atmospheric oxygen at 37? C. in a humidified incubator. For evaluation of intracellular cytokines by flow cytometry, T cells were re-stimulated with PMA (500 ng/ml) and Ionomycin (500 ng/ml) for 4 hr in presence of Glogi inhibitors. In some experiments, in vitro differentiated T cells were either treated with the vehicle control or inhibitor of AKT, AKTi (1?M; added into the differentiation media from day 0) or activator of AKT, SC-79 (0.5 ?g/ml; added into the differentiation media for last 24 hr) or FK866 (10 nM; added into the differentiation media from day 0) or Ex527 (10?M; added into the differentiation media from day 0) or 2DG (1 mM; added into the differentiation media from day 0) or DON (3?M; added into the differentiation media from day 0).
[0169] Retroviral Transduction: One day before transfection, 5?10.sup.6 Platinum-E ecotropic packaging cells (Cell Biolabs) were seeded in 10 ml antibiotic-free medium in 100 mm dishes (Corning). Packaging cells were transfected with 18 ?g retroviral plasmid DNA encoding either the TIL 13831 TCR or the MSGV-1 TRP-1 TCR and the helper plasmid pCL-Eco using 36 ?l Lipofectamine 2000 in OptiMEM (Invitrogen). After 24 hr, medium was replaced and the cells were incubated for additional 24 hr, after which the retrovirus-containing supernatant was collected and filtered. The viral supernatant was spun at 2,000g for 2 hr at 32? C. onto non-tissue-culture-treated 24-well plates (USA Scientific) coated overnight with Retronectin (Takara Bio). Freshly isolated mouse CD.sup.4+ T cells were activated with CD3/CD28-coated beads (Dynabeads, Life Technologies) at a 1:1 bead:cell ratio along with either T cell differentiation media (as described above) or IL2 containing media (100 U/ml) the same day as packaging cell transfection. Beads were removed 48 hr post-activation, just prior to transduction, and re-suspended to a concentration of 2?10.sup.6 cells ml.sup.?1 in fresh medium. After removing the virus from the Retronectin-coated plate following the first spin, 1 ml of the activated T cells were then plated in the same wells and 1 ml of fresh virus was added on top of the cells. The plate was spun for an additional 2 hr at 1100 g, 32? C. Post-spin, 1 ml of media was removed and replaced with fresh media containing 200 IU IL-2 ml.sup.?1 before the cells were incubated overnight. The following day the cells were collected, washed and plated at 0.8-1?10.sup.6 cells ml.sup.?1.
[0170] Adoptive T cell protocol: B16-F10 or B16-F10-A2.sup.+ mouse melanoma tumor cells (2.5?10.sup.6) were injected subcutaneously (s.c.) into left flank of 8-10 weeks old C57BL/6 or HLA-A2.sup.+ (or HLA-A2.sup.+-RAG.sup.?/?) mice respectively. After 9 days of tumor establishment, recipient mice were injected (i.p) with cyclophosphamide (4 mg/mice) before adoptively transferring (i.v) either h3T (CD4.sup.+V?12.sup.+) or TRP-1 (CD4.sup.+V?14.sup.+) Th1, Th17, Th1/17 cells on day 10. Similarly, Pmel (CD8.sup.+V?13.sup.+) and TCR-I (CD.sup.8+Sv40 antigen specific) Tc1, Tc17 and Tc1/17 cells were adoptively transferred (i.v) into either 9 days established B16-F10 tumor bearing mice or TRAMP (spontaneously develop prostate tumor) respectively. For in vivo neutralization of IFN? and IL17, neutralizing antibody to IFN? and IL17 or matched isotype antibody was injected (100 ?g/ mouse; i.p.) in tumor bearing mice following adoptive transfer of T cells. In some experiments, tumor bearing mice received anti-CD38 antibody (50 ?g/ mouse; i.p.) after adoptive TRP-1 Th0 cells transfer. Recipient mice were given IL2 (50,000 U/mouse; i.p) for 3 consecutive days after ACT.
[0171] Flow cytometry: Staining for cell surface markers was performed by incubating cells with antibody at 1:200 dilutions in FACS buffer (0.1% BSA in PBS) for 30 min at 4? C. For intracellular cytokine staining of IFN? and IL17a, surface markers were stained before fixation/permeabilization (BD Cytofix/Cytoperm Kit, BD Biosciences, San Jose, CA).
[0172] For staining of transcription factors (T-bet, ROR?, IRF-4) and PGC1? cells were stained with surface markers and fixed/permeabilized with FoxP3 staining buffer set (eBioscience, San Diego, CA). For pFoxo1 staining, cells were fixed/permeabilized using BD perm III buffer set (BD Bioscience, San Jose, CA) before staining with cell surface markers and pFoxo1 primary antibody (Cell Signaling Technologies, Danvers, MA) followed by fluorochrome conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Samples were acquired on LSRFortessa and analysed with FlowJo software (Tree Star, OR).
[0173] Real-time quantitative-PCR: Total RNA was extracted from pellets of the indicated T cell subsets (2?10.sup.6 cells) using Trizol reagent (Life technologies, Grand Island, NY). cDNA was generated from 1 ?g total RNA using iScript cDNA Synthesis Kit (BioRad, Hercules, CA). SYBR Green incorporation quantitative real-time PCR was performed using a SYBR Green mix (Biorad, Hercules, CA) in the CFX96 Detection System (BioRad, Hercules, CA). Expression of different genes was quantified relative to Actb.
[0174] Transmission Electron Microscopy (TEM): The cells were pelletized and fixed in 2% Phosphate Buffered Glutaraldehyde for 1 hr. The pellets were rinsed in 0.1M Phosphate Buffered Rinse and then post fixed in 2% Aqueous Osmium Tetroxide for 1 hr. After rinsing in distilled water the pellets were dehydrated through a series of graded Ethyl Alcohol; 50% ETOH for 15 min, 70% ETOH for 15 min, 95% ETOH for 15 min and finally twice with 100% ETOH for 15 min each. The dehydrant was removed using the intermediate fluid, Propylene Oxide, 1 changes of 10 min each. The pellets were infiltrated with a 1: 1 solution of propylene oxide and Embed 812 (Electron Microscopy Sciences, Ft. Washington, PA) for 1 hr. The infiltration was continued using a 1:2 solution of propylene oxide and Embed 812, overnight. The pellets were embedded in Embed812 the following day and polymerized in a 60? C. oven for 48 hr. Preliminary 1/2 micron sections were cut and stained with Toluidine Blue and examined using a light microscope. Then with the cell types identified the 70nm thin sections are cut and stained with uranyl acetate and led citrate, allowed to dry. The sections are view on the JEOL 1010 and images are taken with a Hamamatsu electron microscope camera.
[0175] Immunoblotting and Immunoprecipitation of Foxo1: For evaluation of global protein acetylation, cells were lysed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA) and nuclear fraction was isolated according to the manufacturer's protocol. Equal amounts of nuclear protein (20 ?g) were separated by 4%-12% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were probed with anti-Acetyl-Lysine antibody (Cell Signaling Technologies, Danvers, MA) followed by incubation with secondary antibody (HRP-conjugated goat anti-rabbit IgG at 1:3000 dilutions) for overnight at 4? C. Pre-stained molecular weight and magic markers were run in parallel to identify the molecular weight of proteins. For chemiluminescent detection, the membranes were treated with enhanced chemiluminescent reagent, and the signal was monitored using a Biorad Versadoc imaging system (Biorad, Hercules, CA). Same membrane was stripped with stripping buffer (Bioland Scientific, Paramount, CA) according to manufacturer's protocol and re-probed with antibody specific to histone H3 (Cell Signaling Technologies, Danvers, MA) as reference control.
[0176] For evaluation of the protein level of glutamine transporters and glutaminolytic enzymes, cell pellets were washed in PBS and lysed in 80 ul RIPA buffer containing protease inhibitor cocktail. After 30 min incubation on ice cell lysates were centrifuged at 12000g for 15 min at 4? C. Protein concentration in lysates was determined by Bradford method. For immunoblot analyses, 40 ?g of protein lysates per sample were denatured in 2 X SDS-PAGE sample buffer and subjected to SDS-PAGE on 10% Tris-glycine gel. The separated proteins were transferred onto PVDF membrane followed by blocking with 5% BSA (w/v) in TBS (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature. Membrane was probed with either anti-Slcla5 antibody (Thermo Fisher Scientific, Waltham, MA) or x-CT (Abcam, Cambridge, MA) or Gludl (Cell Signaling Technology, Danvers, MA) or Gls (Novus Biologicals, Littleton, CO) overnight at 4? C. followed by 1 hr incubation with HRP-conjugated secondary antibody and using a chemiluminescence kit (SuperSignal West Dura Extended Duration Substrate, Thermo Fisher Scientific, Waltham, MA).
[0177] For immunoprecipitation of Foxol, T cells were first washed with phosphate buffer saline (PBS) and lysed in 500 ?L of RIPA buffer containing a protease inhibitor cocktail for 30 min on ice. Cell lysates were centrifuged at 6000g for 10 min. Foxol was isolated by immunoprecipitation using the immunoprecipitation kit (Cat # K286, BioVision, Milpitas, CA). Briefly, 500 ?L of supernatant (200 ?g protein) was incubated with anti-Foxo1 monoclonal antibodies (1:100 dilution) for 16 hr at 4? C. followed by incubation with Protein A/G-Sepharose for 1 hr at 4? C. The immunoprecipitate was washed multiple times with wash buffer and extracted with Tris-Glycine HCI SDS (2?) buffer and heated at 95? C. as per the manufacturer's direction. The proteins were resolved on 10% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were probed with anti-Acetyl-Lysine antibodies followed by incubation with secondary antibodies (HRP-conjugated goat anti-rabbit IgG at 1:3000 dilution) for 1 hr at 20? C. Pre-stained molecular weight and magic markers were run in parallel to identify the molecular weight of proteins of interest. For chemiluminescent detection, the membranes were treated with enhanced chemiluminescent reagent, and the signal was monitored using a Biorad Versadoc imaging system (Biorad, Hercules, CA).
[0178] Metabolism Assays: Glucose uptake by ex vivo differentiated T cells were determined by incubating cells with 100?M 2NBDG (Cayman chemical, Ann Arbor, MI) for 30 min before measuring fluorescence by flow cytometry.
[0179] Oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR) was determined using Seahorse Xfe96 analyser (Agilent Technologies, Santa Clara, CA). Briefly, T cells (0.5?10.sup.6/well) were plated on Cell-Tak coated Seahorse culture plate for 30 min. OCR, a measure of OXPHOS, was analysed under basal condition or in response to 200 ?M Etomoxir (Sigma-Aldrich, St. Louis, MO) followed by 1.0 ?M oligomycin (Sigma-Aldrich, St. Louis, MO), 1.0 ?M fluoro-carbonyl cyanide phenylhydrazone (FCCP) (Sigma-Aldrich, St. Louis, MO) and 2 ?M rotenone (Sigma-Aldrich, St. Louis, MO) plus 100 nM antimycin A (Sigma-Aldrich, St. Louis, MO). ECAR, a measure of glycolysis was measured under basal conditions and in response to glucose (5.5 mM), Oligomycin (1.0 ?M), 2-deoxyglucose (2-DG) (100 mM) (Sigma-Aldrich, St. Louis, MO).
[0180] Metabolomics: Intracellular level of different metabolites was determined by performing metabolomics analysis using UPHPLC/MS/MS and GC/MS platform (Metabolon Inc. Durham, NC). Data were then grouped by unsupervised clustering using MetaboAnalyst software. Samples were loaded in an equivalent manner across the platform and normalized to Bradford values prior to statistical analysis.
[0181] Glutamine uptake assay: Ex vivo programmed Th cell subsets (1?10.sup.6 cells) were starved in glutamine free RPMI-1640 media (Thermo Fisher Scientific, Cat# 21870-076) for 15 min before incubated with L-2,3,4-[.sup.3H] glutamine (0.5 mCi, PerkinElmer) for 10 min at room temperature. Cells were lysed in 500 ?l of lysis buffer (Sigma-Aldrich, St. Louis, MO) and radioactivity was measured by liquid scintillation.
[0182] NAD.sup.+, Sirt1 activity and Foxo1 activity assay: Intracellular NAD.sup.+ level in T cells were measured spectrophotometrically following the manufacturer's protocol (Cayman chemical, Ann Arbor, MI). Using equal amounts of nuclear protein extracted by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA), Sirt1 activity (Universal Sirt1 Activity Assay Kit, Abcam, Cambridge, MA) and Foxo1 activity (FKHR transcription Factor Assay Kit, Active Motif, Carlsbad, CA) were determined as per manufacturer's protocol.
[0183] Isolation of tumor infiltrating T cells: To obtain tumor infiltrating T cells (TILs) from subcutaneously established solid B16-F10 melanoma bearing mice, tumors were excised, chopped finely using tweezers and scissors and then digested with 2 mg/ml collagenase type IV (Stemcell technologies, Vancouver, BC) for 45 min. Following digestion, tumors were filtered through 70 ?M cells strainers (BD Biosciences, San Jose, CA) and layered over ficoll-paque (GE Healthcare). After centrifugation at 1200 rpm for 30 min, mononuclear cell layer containing TILs was isolated. For isolation of T cells from EL-4 tumor site, tumor was established as ascites in the peritoneal cavity for 12 days in mice and fluid was withdrawn to obtain TILs. To isolate TILs from prostate tumor sites, the resected prostate tumors were quickly minced with scalpels into small fragments (1-2 mm.sup.2) in cold RPMI media with 10% FBS, after trimming away the seminal vesicles, fat and connective tissue. The fragments were then placed on a 70 ?m strainer and homogenized using plunger end of a syringe (one fragment at a time to ensure complete homogenization). This process rapidly produces a single cell suspension without the requirement of enzymatic digestion. The cell suspension was washed in culture medium twice by centrifugation at 1500 rpm for 10 min at 4? C. After the second wash, the cells were re-suspended in 6 ml PBS and layered carefully over 3ml Ficoll-paque (GE healthcare) followed by centrifugation at 1500 rpm for 30 min at room temperature. The enriched TILs obtained at the interface as a thin buffy layer, were washed with PBS twice and finally re-suspended in FACS staining buffer for further staining procedures.
[0184] Activation induced T cell death: Differentiated Th1, Th17 and Th1/17 cells were re-stimulated overnight with plate bound anti-CD3 (2 ?g/ml) plus anti-CD28 (1 ?g/ml). Apoptosis was measured by Annexin V (BD Biosciences, San Jose, CA) vs. 7AAD staining according to the manufacturer's protocol, followed by analysis by flow cytometry. Data were analysed with FlowJo software (Tree Star, OR).
[0185] DNA Microarray Experiments: Biotinylated cRNA was prepared using the Illumina RNA Amplification Kit, Catalog #1L1791 (Ambion, Inc., Austin, TX) according to the manufacturer's specifications starting with 250 ng total RNA. For microarray analysis, the MouseWG-6 v2.0 Expression BeadChip Kit was used (Illumina, San Diego). Hybridization of 67abelled cRNA to the BeadChip, washing and scanning were performed according to the Illumina BeadStation 500x manual. Essentially the amplified, biotin-labeled mouse cRNA samples were re-suspended in a solution of Hyb El buffer (Illumina) and 25% (v/v) formamide at a final concentration of 25ng/?L. 1.5 ?g of each cRNA were hybridized. Hybridization was allowed to proceed at 55? C., for 18 hours after which the bead array matrix was washed for 10 min with 1? High temperature buffer (Illumina), followed by a subsequent 10 min wash in Wash ElBC buffer. The arrays were then washed with 100% ethanol for 10 min to strip off any remaining adhesive on the chip. A 2 minute E1BC wash was performed to remove residual ethanol. The arrays were blocked for 5 min with 1% (w/v) casein-PBS (Pierce). The array signal was developed via 10 minute incubation with Streptavidin-Cy3 at a final concentration of lug/mL solution of (GE Healthcare) in 1% casein-PBS blocking solution. The Expression BeadChip was washed a final time in Wash E1BC buffer for 5 min and subsequently dried via centrifugation for 4 min at a setting of 275 rcf.
[0186] The arrays were scanned on the Illumina BeadArray Reader, a confocal-type imaging system with 532 (cye3) nm laser illuminations. Image analysis and data extraction was carried out as in accordance with Illumina specifications. Preliminary data analysis and QC was carried out using the GenomeStudio software (Illumina). All array data has been deposited in the EBI ArrayExpress Database. The ArrayExpress accession E-MTAB-5237.
[0187] Analysis of microarray data: Normalization of microarray data: Expression level data from the Illumina Bead Studio software were normalized using quantile normalization as implemented in the PIMENTo package (Sasik et al., 2004). Probes whose expression level exceeds a threshold value in at least one sample are called detected. The threshold value is found by inspection from the distribution plots of (log) expression levels.
[0188] Sorting the probes according to significance: Detected probes are sorted according to their q-value, which is the smallest false discovery rate (FDR) at which the gene is called significant. FDR is the expected fraction of false positive tests among significant tests (Benjamini and Hochberg, 1995). We evaluate FDR using Significance Analysis of Microarrays (SAM) and its implementation in the official statistical package samr (Tusher et al., 2001).
[0189] Statistical analysis of pathways and gene ontology terms: Each gene ontology term or a pathway is treated simply as a set of genes. The probe list, sorted by q-value in ascending order, is translated into Entrez gene ID's and parsed so that whenever several different probes represent the same gene, only the highest-ranking probe is kept for further analysis.
[0190] The sorted list of genes is subjected to analysis using the ToppGene Suite (http://toppgene.cchmc.org), a one-stop portal for (i) gene list functional enrichment, (ii) candidate gene prioritization using either functional annotations or network analysis and (iii) identification and prioritization of novel disease candidate genes in the interactome. Functional annotation-based disease candidate gene prioritization uses a fuzzy-based similarity measure to compute the similarity between any two genes based on semantic annotations. The similarity scores from individual features are combined into an overall score using statistical meta-analysis. A P-value of each annotation of a test gene is derived by random sampling of the whole genome (Chen et al., 2009). Area-proportional Venn diagrams were created using Venny.
[0191] Heatmaps of expression levels were created using the heatmap.2 hierarchical clustering software. The colors qualitatively correspond to fold changes with respect to a reference which is calculated as the mid-point between compared groups.
[0192] Statistical analysis: All data reported are the arithmetic mean from 3 or independent experiments performed in triplicate ?SD unless stated otherwise. The unpaired Student t test was used to evaluate the significance of differences observed between groups, accepting P<0.05 as a threshold of significance. Data analyses were performed using the Prism software (GraphPad, San Diego, CA). Tumor size data was modeled using longitudinal linear regression with exchangeable correlation. Comparisons across groups at individual time points were made across groups using Wald tests based on linear combinations of coefficients.
[0193] Data And Software Availability: All array data has been deposited in the EBI ArrayExpress Database. The ArrayExpress accession number is E-MTAB-5237.
TABLE-US-00006 TABLE 5 Materials REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse CD3 BioXCell Clone: 145-2C11; Cat# BE0001-1; RRID: AB_1107634 Anti-mouse CD28 BioXCell Clone: 37.51; Cat# BE0015-1; RRID: AB_1107624 Anti-mouse IL4 BioXCell Clone: 11B11; Cat# BE0045; RRID: AB_1107707 Anti-mouse IFN? BioXCell Clone: XMG1.2; Cat# BE0055; RRID: AB_1107694 CD4-PE eBioscience Clone: GK5.1; Cat# 12-0041-83; RRID: AB_465506 T-bet-Percp/Cy5.5 eBioscience Clone: 4B10; Cat# 45-5825-80; RRID: AB_953657 ROR?-APC eBioscience Clone: AFKJS-9; Cat# 17-6988-80; RRID: AB_10609207 IRF-4-eFluor 660 eBioscience Clone: 3E4; Cat# 50-9858-80; RRID: AB_2574393 CXCR3-PE eBioscience Clone: CXCR3-173; Cat# 12-1831- 80; RRID: AB_1210734 CD45.2-PE eBioscience Clone: 104; Cat# 12-0454-81; RRID: AB_465678 CD39-PE/Cy7 eBioscience Clone: eBioA1 (A1); Cat# 25-0391- 80; RRID: AB_1582280 CD90.1-APC eBioscience Clone: HIS51; Cat# 17-0900-82; RRID: AB_1272252 CD90.1-Pacific Blue eBioscience Clone: HIS51; Cat# 48-0900-80; RRID: AB_1272254 EOMES-PE eBioscience Clone: Dan11mag; Cat# 12-4875-80; RRID: AB_1603275 CD4-PE/Cy7 Biolegend Clone: GK5.1; Cat# 100422; RRID: AB_312707 CD4-APC Biolegend Clone: GK5.1; Cat# 100412; RRID: AB_312697 CD4-APC/Cy7 Biolegend Clone: GK5.1; Cat# 100414; RRID: AB_312699 CD8-PE/Cy7 Biolegend Clone: 53-6.7; Cat# 100722; RRID: AB_312761 CD8-APC Biolegend Clone: 53-6.7; Cat# 100712; RRID: AB_312751 CD8-FITC Biolegend Clone: 53-6.7; Cat# 100706; RRID: AB_312745 IL17-PE Biolegend Clone: TC11-18H10.1; Cat# 506904; RRID: AB_315464 IL17-Pacific Blue Biolegend Clone: TC11-18H10.1; Cat# 506918; RRID: AB_893544 IFN?-PE Biolegend Clone: XMG1.2; Cat# 505808; RRID: AB_315402 IFN?-Alexa647 Biolegend Clone: XMG1.2; Cat# 505814; RRID: AB_493314 T-bet-PE Biolegend Clone: 4B10; Cat# 644809; RRID: AB_2028583 CCR5-PE Biolegend Clone: HM-CCR5; Cat# 107005; RRID: AB_313300 CCR6-PE Biolegend Clone: 29-2L17; Cat# 129803; RRID: AB_1279139 CCR7-PE Biolegend Clone: 4B12; Cat# 120105; RRID: AB_389357 CD73-Pacific Blue Biolegend Clone: Ty/11.8; Cat# 127212; RRID: AB_11219190 CD38-PE Biolegend Clone: 90; Cat# 102707; RRID: AB_312928 CD38-APC Biolegend Clone: 90; Cat# 102711; RRID: AB_312932 CD71-PE/Cy7 Biolegend Clone: RI7217; Cat# 113811; RRID: AB_2203383 PD1-PE Biolegend Clone: RMP1-30; Cat# 109103; RRID: AB_313420 PD1-PE/Cy7 Biolegend Clone: RMP1-30; Cat# 109109; RRID: AB_572016 CD98-PE Biolegend Clone: RL388; Cat# 128207; RRID: AB_1186107 Annexin V FITC BD Biosciences Clone: N/A; Cat# 51-65874X; RRID: N/A CD45.1-APC BD Biosciences Clone: A20; Cat# 558701; RRID: AB_1645214 V?13-FITC BD Biosciences Clone: MR1 2-3; Cat# 553204; RRID: AB_394706 V?14-FITC BD Biosciences Clone: 14-2; Cat# 553258; RRID: AB_394738 V?12-FITC Thermo Fisher Clone: S511; Cat# TCR2654; RRID: Scientific AB_417092 Slc1a5 Thermo Fisher Clone: N/A; Cat# PA5-50527; RRID: Scientific AB_2635980 Anti-Goat Alexa647 Thermo Fisher Clone: N/A; Cat# A21447; RRID: Scientific AB_141844 pFoxo1 (S256) Cell Signaling Clone: N/A; Cat# 9461; RRID: Technology AB_329831 Phospho-S6 Ribosomal Protein Cell Signaling Clone: D57.2.2E; Cat# 4851; RRID: (Ser235/236)- Alexa647 Technology AB_916160 Hexokinase I (HK I) Cell Signaling Clone: C35C4; Cat# 2024S; RRID: Technology AB_2116996 Hexokinase II (HK II) Cell Signaling Clone: C64G5; Cat# 2867S; RRID: Technology AB_2232946 Platelet-type phosphofructokinase (PFKP) Cell Signaling Clone: D4B2; Cat# 8164S; RRID: Technology N/A Glyceraldehyde-3-phosphate dehydrogenase Cell Signaling Clone: D16H11; Cat# 5174S; RRID: (GAPDH) Technology AB_10622025 Lactate dehydrogenase (LDH) Cell Signaling Clone: C4B5; Cat# 3582S; RRID: Technology AB_2066887 Pyruvate kinase M 1/2 (PKM1/2) Cell Signaling Clone: C103A3; Cat# 3190S; RRID: Technology AB_2163695 Pyruvate Dehydrogenase Cell Signaling Clone: C54G1; Cat# 3205S; RRID: Technology AB_2162926 Glutamate Dehydrogenase 1/2 (Glud1/12) Cell Signaling Clone: D9F7P; Cat# 12793S; RRID: Technology N/A Foxo1 Cell Signaling Clone: C29H4; Cat# 2880S; RRID: Technology AB_2106495 Acetylated-Lysine Cell Signaling Clone: N/A; Cat# 9441S; RRID: N/A Technology Anti-Rabbit HRP Cell Signaling Clone: N/A; Cat# 7074S; RRID: N/A Technology PGC1?-Alexa647 Novus Biologicals Clone: N/A; Cat# NBP1- 04676AF647; RRID: N/A Glutaminase (Gls) Novus Biologicals Clone: N/A; Cat# NBP2-29940; RRID: N/A Anti-Rabbit PE Jackson Clone: N/A; Cat# 111-116-144; ImmunoResearch RRID: AB_2337985 Laboratories Anti-Rabbit Alexa647 Jackson Clone: N/A; Cat# 111-607-003; ImmunoResearch RRID: AB_2338084 Laboratories xCT Santa Cruz Clone: Q-18; Cat# Sc79360; RRID: Biotechnology AB_2190856 InVivoMab anti-mouse IL-17A BioXCell Clone: 17F3; Cat# BE0173; RRID: AB_10950102 InVivoMAb anti-mouse IFN? BioXCell Clone: XMG1.2; Cat# BE0055; RRID: AB_1107694 Anti CD38 VHH Single Domain Antibody Creative Biolabs Clone: N/A; Cat# NABL-079; RRID: N/A Chemicals, Peptides, and Recombinant Proteins FK866 Cayman Cat# 13287 Chemical Ex527 Cayman Cat# 10009798 Chemical Etomoxir Cayman Cat# 11969 Chemical AKTi Cayman Cat# 14870 Chemical SC-79 Sigma Aldrich Cat# SML0749 6-Diazo-5-oxo-L-norleucine (DON) Sigma Aldrich Cat# D2141 2-Deoxy-D-glucose (2DG) Sigma Aldrich Cat# D6134 Antimycin A Sigma Aldrich Cat# A8674 Rotenone Sigma Aldrich Cat# R8875 Oligomycin Sigma Aldrich Cat# O4876 FCCP Sigma Aldrich Cat# C2920 IMDM GE Healthcare, Cat# SH30228.01 HyClone RPMI-1640 (Glucose free) Thermo Fisher Cat# 11879-020 Scientific RPMI-1640 (glutamine free) Thermo Fisher Cat# 21870-076 Scientific Penicillin-Streptomycin Corning Cat# 30-001-CI Fetal Bovine Serum (FBS) Atlanta Cat# S11150 Biologicals rIL12 Biolegend Cat# 577004 rIL6 Biolegend Cat# 575704 rIL1? Biolegend Cat# 575104 rIL23 Biolegend Cat# 589002 rTGF? Biolegend Cat# 580702 rhIL2 NCI, Biological https://ncifrederick.cancer.gov/ Resources research/brb/productDataSheets/ Branch cytokineHumanInterleukins/IL-2Bulk.aspx Foxp3/Transcription Factor Staining Buffer Thermo Fisher Cat# 00-5523 Set Scientific Fixation/Permeabilization Solution Kit BD Biosciences Cat# 554714 Mouse CD4.sup.+ T cell isolation kit Miltenyi Biotec Cat# 130-104-454 hgp100.sub.25-33 peptide (KVPRNQDW) Genscript Cat# RP20344 TRP-1.sub.106-130 peptide NeoMPS Custom synthesized (SGHNCGTCRPGWRGAACNQKILTVR) TCR-I peptide (SAINNYAQKL) Sydlabs Cat# 075472 RIPA Lysis Buffer Thermo Fisher Cat# 89900 Scientific NE-PER? Nuclear and Cytoplasmic Thermo Fisher Cat# 78833 Extraction Reagents Scientific Critical Commercial Assays CellTrace? CFSE Cell Proliferation Kit Thermo Fisher Cat# C34554 Scientific CellTrace? Violet Cell Proliferation Kit Thermo Fisher Cat# C34557 Scientific NAD/NADH Cell-Based Assay Kit Cayman Cat# 600480 Chemical Universal SIRT Activity Assay Kit abcam Cat# ab156915 TransAM? FKHR (FOXO1) Transcription Active Motif Cat# 46396 Factor ELISA Kits Akt kinase activity kit Enzo Life Cat# ADI-EKS-400A Sciences Immunoprecipitation (IP) Kit Biovision Cat# K286-25 iScript? cDNA Synthesis Kit Biorad Cat# 1708891 SsoAdvanced? Universal SYBR? Green Biorad Cat# 1725274 Supermix Deposited Data Gene microarray dataset Present study ArrayExpress accession # E-MTAB- 5237 Experimental Models: Cell Lines B16-F10 ATCC CRL-6475 B16-F10-A2.sup.+ Rolf Kiessling, N/A Karolinska Institute, Stockholm, Sweden. Mycoplasma testing was done in Mehrotra lab. Experimental Models: Organisms/Strains C57BL/6 Jackson Stock# 000664 Laboratory C57BL/6-Tg(HLA-A2.1)1Enge/J Jackson Stock# 003475 Laboratory B6.129S7-Rag1.sup.tm1Mom/J Jackson Stock# 002216 Laboratory B6.129P2-Cd38.sup.tm1Lnd/J Jackson Stock# 003727 Laboratory B6;129-Sirt1.sup.tm1Ygu/J Jackson Stock# 008041 Laboratory Tg(Cd4-cre)1Cwi/BfluJ Jackson Stock# 017336 Laboratory B6.SJL-Ptprc.sup.a Pepc.sup.b/BoyJ Jackson Stock# 002014 Laboratory B6.129S7-Ifng.sup.tm1Ts/J Jackson Stock# 002287 Laboratory B6.Cg-Rag1.sup.tm1Mom Tyrp1.sup.B-w Jackson Stock# 008684 Tg(Tcra, Tcrb)9Rest/J Laboratory B6.Cg-Thy1.sup.a/Cy Tg (TcraTcrb)8Rest/J Jackson Stock# 005023 Laboratory C57BL/6-Tg(TRAMP)8247Ng/J Jackson Stock# 003135 Laboratory IFN?.sup.Thy1.1 knock-in Casey T. (Harrington et al., 2008) Weaver, University of Alabama at Birmingham (UAB) Foxo1.sup.fl/flLck.sup.cre Melanie Gubbels (Gubbels Bupp et al., 2009) Bupp, Randolph- Macon College, VA TCR-I Jennifer Wu, (Staveley-O'Carroll et al., 2003) MUSC Oligonucleotides Primers for qPCR, see Table S4 Recombinant DNA MSGV1-TRP-1 vector (Kerkar et al., N/A 2011) LV-TCR/sr39TK-GFP vector (Koya et al., N/A 2010) Software and Algorithms FlowJo 10.2 TreeStar, OR https://www.flowjo.com/solutions/ flowjo/downloads/ Prism 5 GraphPad https://www.graphpad.com/scientific- software/prism/ Agilent Seahorse Wave 2.4 Agilent http://www.agilent.com/en- us/products/cell-analysis- (seahorse)/seahorse-wave-software CFX Manager 3.1 Biorad http://www.bio-rad.com/en- us/sku/soft-cfx-31-patch-cfx- manager-software-v3-1-upgrade GenomeStudio software Illumina https://www.illumina.com/techniques/ microarrays/array-data-analysis- experimental-design/genomestudio.html PIMENTo (Sasik et al., github.com/MUSC-CGM/PIMENTo 2004) ToppGene Suite (Chen et al., toppgene.cchmc.org 2009) Venny Present study bioinfogp.cnb.csic.es/tools/venny/ index.html
[0194] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents 10 described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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