Cells for cell therapy and compositions and methods related thereto

Abstract

The present disclosure provides cells and cell populations which may be used for cell therapy and methods of producing such cells and cell populations. The present disclosure further provides pharmaceutical compositions and therapeutic methods and use relating to such cells and cell populations.

Description

[0001] 1143253.008213

[0002] CELLS FOR CELL THERAPY AND COMPOSITIONS AND METHODS RELATED THERETO

[0003] RELATED APPLICATIONS

[0004]

[0001] This application claims priority to U. S. Provisional Application No: 63 / 760,153, filed on February 19, 2025, the contents of which are incorporated by reference in their entirety.

[0005] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006]

[0002] This invention was made with government support under R01 CA257954, and R01 AI155015 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008]

[0003] Cell therapy has opened a new avenue for treating various diseases. While the classic adoptive T cell therapy isolates T cells from patients, activates and expands the T cells, and transfer them back to the patients, recent approaches administer cells engineered to express a chimeric or synthetic receptors which specifically binds to an antigen of interest to patients.

[0009]

[0004] For example, in chimeric antigen receptor (CAR)-based cell therapy, immune cells, such as T cells, NK cells, or macrophages, are genetically modified to express a receptor which has an antigen-binding domain specific for an antigen of interest and an intracellular signaling domain. Other synthetic receptors may also be used. In synthetic T cell receptor (TCR)-based cell therapy, T cells may be modified to express a TCR-like complex, in which (a) synthetic antigen-binding binding domain(s) is / are used in place of a TCR's antigen-binding site but the remainder of the complex is similar to or essentially that of an endogenous TCR complex containing a CDS signaling complex.

[0010]

[0005] Upon antigen recognition, these cells are activated via the signaling domains of the receptor, converting them into potent cell killers and / or immune stimulators. In 2017, an anti- CD19 CART cell product received FDA approval for B cell lymphoma, illustrating the potential of this therapeutic approach in cancer, e.g., hematological cancer (Leyfman Y, et la.. Cancer CeP Int. 2818 Nov 14:18:182..). While also showing great promise for the treatment of solid tumors (louis C. U. et al, Blood. 2011 Dec 1; 118(23): 6050-6056. Pre-published online 20111143253.008213

[0011] Ort?.), CAR-based cell therapy and other similar cell therapy have encountered additional challenges in these indications (Yong C.5. M. et ah, Immunol Cell Biol. 2017 Apr;95(4):356-363.) such as but not limited to: reduced and / or transient effects due to exhaustion of therapeutic cells; insufficient efficacy and / or the need for repeated dosing, due to the short in vivo persistence of the therapeutic cells.

[0012]

[0006] There exists a need for a technology to address one or more of such obstacles.

[0013] SUMMARY

[0014]

[0007] One aspect of the present disclosure provides a composition, which may for example be for treating cells. The composition may comprise: (I) a PI3K-AKT-mTOR pathway inhibitor; and (II) (ll-l) a Wnt / P-catenin pathway activator; and / or (11-2) an acetyl-CoA synthesis inhibitor. In some embodiments, the composition may be used for preparing lymphoid cells or cell populations comprising lymphoid cells according to the present disclosure.

[0015]

[0008] Another aspect of the present disclosure provides isolated or recombinant lymphoid cells treated with and / or comprising: (I) a PI3K-AKT-mTOR pathway inhibitor; and (II) (ll-l) a Wnt / p-catenin pathway activator; and / or (11-2) an acetyl-CoA synthesis inhibitor.

[0016]

[0009] In any of the embodiments disclosed herein, the PI3K-AKT-mTOR pathway inhibitor, the Wnt / P-catenin pathway activator, and / or the acetyl-CoA synthesis inhibitor may individually be of any modality.

[0017]

[0010] In some embodiments, the PI3K-AKT-mTOR pathway inhibitor may be or comprise a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen¬ binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide. In certain embodiments, the PI3K-AKT-mTOR pathway inhibitor is or comprises a small molecule. In particular embodiments, the PI3K-AKT-mTOR pathway inhibitor is or comprises a small molecule inhibitor.

[0018] [Oil] In some embodiments, the Wnt / P-catenin pathway activator may be or comprise a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide. In certain embodiments, the Wnt / P-catenin pathway activator is or comprises a small molecule. In particular embodiments, the Wnt / p-catenin pathway activator is or comprises a small molecule inhibitor.1143253.008213

[0019]

[0012] In some embodiments, the acetyl-CoA synthesis inhibitor may be or comprise a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide. In certain embodiments, the acetyl-CoA synthesis inhibitor is or comprises a small molecule. In particular embodiments, the acetyl-CoA synthesis inhibitor is or comprises a small molecule inhibitor.

[0020]

[0013] In some embodiments, the PI3K-AKT-mTOR pathway inhibitor may be or comprise: (a) a RISK inhibitor or a PI3K class I inhibitor; (b) an AKT inhibitor; and / or (c) a dual PI3K / mTOR inhibitor.

[0021]

[0014] In certain embodiments, the PI3K inhibitor or PI3K class I inhibitor may be (a-1) an isoform-selective PI3 K inhibitor or an inhibitor selective for one or more of PI3K 6 isoform, PI3K y isoform, PI3K a isoform, and / or PI3K 0 isoform; or (a-2) a pan-PI3K inhibitor, in some cases, an isoform-selective P 13 K inhibitor or an inhibitor selective for one or more of P 13 K 8 isoform, PI3 K y isoform, PI3 K a isoform, and / or PI3 K 0 isoform, in certain cases, the PI3K inhibitor may be selected from the group consisting of 5-fluoro-3-phenyl-2-[(lS)-l-(7H-purin-6-ylamino)propyl]quinazolin-4-one, idelalisib, ZYDELIG®, CAL-101, umbralisib, TGR1202, alpelisib, duvelisib, tenalisib, acalisib, dezapelisib, parsaclisib, linperlisib, zandelisib, BGB-10188, SHC014748M, amdizalisib, AZD8835, TQ-B3525, puqutinib, eganelisib, I PI-549, alpelisib, WX-037, inavolisib, serabelisib, CYH33, ASN003, RLY-2608, LOXO-783 / LOX-22783, STX-478, GSK2636771, AZD6482 / KIN193, AZD8186, KA2237, and taselisib. In some cases, a pan-PI3K inhibitor may be selected from the group consisting of wortmannin, PX866, BKM120, pictilisib, burparlisib, copanlisib, pilaralisib, izorlisib, ZSTK474, and / or fimepinostat.

[0022]

[0015] In certain embodiments, the AKT inhibitor may be selected from the group consisting of l,3-dihydro-l-[l-[[4-(6-phenyl-lH-imidazo[4,5-g]quinoxalin-7-yl)phenyl]methyl]-4-piperidinyl]-2H-benzimidazol-2-one, Akt Inhibitor VIII, MK2206, capivasertib, afuresertib, and / or ipatasertib. in certain embodiments, the AKT inhibitor may be a pan-AKT inhibitor. In certain embodiments, the AKT inhibitor may be an isoform-selective AKT inhibitor or an inhibitor selective for one or more of AKT1, AKT2, and AKT3. In certain embodiments, the AKT inhibitor may be an allosteric inhibitor. In some cases, the AKT inhibitor may be MK-2206, ARQ.092, ARQ751, and / or BAY1125976. In certain embodiments, the AKT inhibitor may be an ATP-competitive inhibitor. In some cases, the AKT inhibitor may be GSK2141795, GSK690693, LY2780301, GDC-0068, AZD5363, capivasertib, and / or ipatasertib.1143253.008213

[0023]

[0016] In certain embodiments, the dual PI3K / mTOR inhibitor may be LY294002, SF-1126, samotolisib, omipalisib, voxtalisib, apitolisib, gedatolisib, VS-5584 / SB2343, paxalisib, DS-7423, and / or PWT33597.

[0024]

[0017] In some embodiments, the Wnt / P-catenin pathway activator may comprise a GSK-3 inhibitor.

[0025]

[0018] In certain embodiments, the GSK-3 inhibitor may be capable of inhibiting GSK-3p, GSK-3a, or GSK-3P and GSK-3a. In certain embodiments, the GSK-3 inhibitor may be selected from the group consisting of 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol, TWS119, SB-415286, CHIR-99021, AR-A014418, lithium, LiCI, BIO, SB-216763, LY-2090314, PF-04802367, L807mts, tideglusib, elraglusib, AZD1080, BRD3731, rosmarinic acid, BRD0705, psoralidin, and / or an oxadiazole-based selective GSK3a inhibitor. In certain embodiments, the GSK-3 inhibitor may be: (1) a cation, optionally lithium or a metal ion, further optionally zinc or copper; (2) an ATP competitive inhibitor; (3) an allosteric non-ATP competitive inhibitor; (4) a substrate competitive inhibitor.

[0026]

[0019] In some embodiments, the acetyl-CoA synthesis inhibitor is or comprises an ATP-citrate lyase (ACLY) inhibitor.

[0027]

[0020] In certain embodiments, the ACLY inhibitor may be selected from the group consisting of 2-hydroxycitrate, a salt thereof (e.g., potassium hydroxycitrate or sodium hydroxycitrate), bempedoic acid, ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid, NEXLETOL®, ESP-55016, bempedoyl-CoA, BemA, hydroxycitric acid or a derivative thereof, SB204990, a 2-hydroxy-N-arylbenzenesulfonamide, B MS-303141, NDI-091143, SB-201076, citric acid tripotassium hydrate, citric acid, (-)- hydroxycitric acid lactone, MEDICA 16, 3-thiadicarboxylic acid, an emodin derivative, a furan carboxylate derivative, Hibiscus subdariffa extract, Garcinia cambogia extract, lithium citrate (Litarex) tetrahydrate, bempedoic acid-d4, 2-Furoic acid-d4, forrestiacids J, forrestiacids K, 2-Furoic acid, and / or bempedoic acid-d5.

[0028]

[0021] In particular embodiments, the PI3K-AKT-mTOR pathway inhibitor may be or comprise a PI3K 6 isoform inhibitor, optionally a small molecule inhibitor. In particular cases, the inhibitor may be or comprise 5-fluoro-3-phenyl-2-[(lS)-l-(7H-purin-6- ylamino)propyl]quinazolin-4-one, idelalisib, ZYDELIG®, CAL-101, umbralisib, and / or

[0029] TGR1202.1143253.008213

[0030]

[0022] In particular embodiments, the Wnt / -catenin pathway activator may be or comprise a GSK-3P inhibitor, optionally a small molecule inhibitor. In particular cases, the inhibitor may be or comprise3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol, TWS119, SB-415286, CHIR-99021, and / or AR-A014418.

[0031]

[0023] In particular embodiments, the acetyl-CoA synthesis inhibitor may be or comprise an ACLY inhibitor, optionally a small molecule inhibitor. In particular cases, the inhibitor may be or comprise 2-hydroxycitrate or a salt thereof (e.g., potassium hydroxycitrate or sodium hydroxycitrate).

[0032]

[0024] In further particular embodiments, (I) the PI3K-AKT-mTOR pathway inhibitor may be or comprise a PI3 K 6 isoform inhibitor and (II) ( 11-1) the Wnt / p-catenin pathway activator may be or comprise a GSK-3P inhibitor and / or (11-2) the acetyl-CoA synthesis inhibitor may be or comprise an ACLY inhibitor. In particular cases, (I) the PI3K-AKT-mTOR pathway inhibitor may be or comprise idelalisib and (II) ( 11-1) the Wnt / P-catenin pathway activator may be or comprise TWS119 and / or (11-2) the acetyl-CoA synthesis inhibitor may be or comprise 2-hydroxycitrate or a salt thereof.

[0033]

[0025] The isolated or recombinant lymphoid cell(s) according to the present disclosure may be of any appropriate cell type or subtype, may display any appropriate phenotype or express any appropriate markers, may be derived from any appropriate species, subject, tissue or organ source, or may be, comprise, or be derived from primary cells or cell-line cells.

[0034]

[0026] In some embodiments, the isolated or recombinant lymphoid cell(s) may be or comprise a cell of T cell lineage or NKT cell lineage.

[0035]

[0027] In certain embodiments, the isolated or recombinant lymphoid cell(s) may be or comprise a CD8+ cell, a CD4+ T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing.

[0036]

[0028] In particular embodiments, the isolated or recombinant lymphoid cell(s) may be or comprise at least CD8+ T cells, and in some cases both CD8+ T cells and CD4+ T cells.

[0037]

[0029] In some embodiments, the isolated or recombinant lymphoid cell(s) may be or comprise a memory or memory-like cell or memory T cell.

[0038]

[0030] In certain embodiments, the memory-like cell or memory T cell may be or comprise a stem cell-like memory T (Tscm) cell, a central memory T (Tcm) cell, an effector! cell memory T (Tern) cell, a resident memory! (Trm) cell or tissue-resident memory! cell, a memory1143253.008213

[0039] regulatory T (Tregm) cell, a peripheral memory T cell (Tpm), and / or a memory precursor effector T cell (Tmpec). In particular embodiments, the memory-like cell or memory T cell may be or comprise a stem cell-like memory T (Tscm) cell. Such cell subtype may be determined based on the expression of one or more markers associated with the cell type or subtype.

[0040]

[0031] In some embodiments, the isolated or recombinant lymphoid cell(s) may express a memory T cell marker. In certain embodiments, the isolated or recombinant lymphoid cells may express one or more of CCR7 (CD197), CD95, CD27, CD62L, CD122, IL-7Ra (CD127), CD103, CD69, CD44, CXCR3, TCF7, TCF1, LEF1, LFA-1, LFA-3, F0X01, BCL6, Sca-1, FOXP1, KLF2, CD45RA, and / or CD45RO. In particular embodiments, the isolated or recombinant lymphoid cells may express one or more Tscm markers. In some cases, the marker may be one or more of CCR7 (CD197), CD95, CD27, CD62L, CD122, IL-7 Ret (CD127), CD103, CD69, CD44, CXCR3, TCF7, TCF1, LEF1, LFA-1, LFA-3, FOXO1, BCL6, Sca-1, FOXP1, KLF2, CD45RA, and / or Ki67. In certain cases, the marker may be one or more of CD45RA, CCR7 (CD197), CD62L, CD28, CD27, CD12 CD95, CD27, IL-7Ra (CD127), CXCR3, CD95, CDlla, I L-2R (CD122), and / or CD58. In particular cases, the marker may be one or more of CD45RA, CD27, CD62L, and / or CD95. In further particular cases, the lymphoid cells may have the phenotype of: CD45RA+ CD45RO- CD27+ CD62L+ CD95+; CD45RO- CCR7+ CD28+ CD95+; CD45RO-CCR7+ CD62L+ CD95+ TCFlhiScalhi; CD25'° CD44'° CD62LhiCD122+ Scal+; and / or CCR7+ CD95+ CD45RO- CD27+ CD45RA+ CD62L+ CD122+.

[0041]

[0032] In some embodiments, the isolated or recombinant lymphoid cell(s) may, upon exposure to a cognate antigen (e.g., a cognate antigen of a CAR, a synthetic immune receptor, or an endogenous immune receptor expressed on the cell), express or express an increased level of a T cell activation marker. In certain embodiments, the activation marker may be or comprise one or more of CD25, CD26, CD27, CD28, CD30, CD38, CD43, CD44, CD69, CD71, CD107, I L-7Ra (CD127), 0X40 (CD134), CD40L (CD154), CXCR1, CX3CR1, H LA- DR, KLRG1, IFNγ, granzyme B, and / or perforin.

[0042]

[0033] In some embodiments, the isolated or recombinant lymphoid cell(s) may not express or may express at a lower level (e.g., compared to an activated or effector T cell) of a T cell exhaustion marker. In certain embodiments, the exhaustion marker may be or may comprise one or more of Tim-3 (CD366), Lag-3 (CD223), PD-1, CTLA-4, Slamf6 (CD352), CD39a, TIGIT, CD160, BTLA, and / or 2B4 (CD244).1143253.008213

[0043]

[0034] In some embodiments, the isolated or recombinant lymphoid cell(s) may be, comprise, or be derived from a splenocyte, a lymph node cell, a bone marrow cell, and / or a thymic cell.

[0044]

[0035] In some embodiments, the isolated or recombinant lymphoid cell(s) may be, comprise, or be derived from a stem cell, a multipotent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (i PS) cell.

[0045]

[0036] In some embodiments, the isolated or recombinant lymphoid cell(s) may be derived from a subject, optionally a mammal, further optionally a human, a non-human primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse.

[0046]

[0037] In some embodiments, the isolated or recombinant lymphoid cell(s) may be or comprise a mammalian cell, optionally a human cell, a non-human primate cell, a monkey cell, a rabbit cell, a rodent cell, a hamster cell, a rat cell, or a mouse cell.

[0047]

[0038] In some embodiments, the isolated or recombinant lymphoid cell(s) may be, comprise, or be derived from a cell line cell or a primary cell.

[0048]

[0039] In some embodiments, the isolated or recombinant lymphoid cell(s) may express or may be modified to express a polypeptide (or polypeptides) which specifically binds to an antigen of interest.

[0049]

[0040] In certain embodiments, the polypeptide(s) may be, comprise, or be comprised in a chimeric antigen receptor (CAR). In particular embodiments, the CAR may comprise: (1) an antigen-binding domain (e.g., comprising an immunoglobulin (Ig) heavy chain variable domain (VH) and / or light chain variable domain (V L)); (2) a transmembrane domain; and (3) at least one intracellular signaling domain (e.g., of or derived from CD3, CD3<(, CD27, CD28, CD44, DAP10, 4-1BB (CD137), OX-40, ICOS, NKG2D, a Fey receptor, FcRyl II (CD16), FcRyllA (CD32A), and / or 2B4 (CD244).

[0050]

[0041] In certain embodiments, the polypeptide(s) may be, comprise, or be comprised in a synthetic immune receptor. In particular embodiments, the synthetic immune receptor may comprise a synthetic T cell receptor (TCR) complex, optionally comprising: (1) an a chain comprising a TCR a constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain; (2) a p chain comprising a variable domain, a TCR constant domain and a transmembrane domain and optionally a TCR variable domain or1143253.008213

[0051] an Ig variable domain; (3) one or more CD3^ polypeptides; and (4) optionally one or more of CD3s, CD38, and / or CD3y polypeptides.

[0052]

[0042] In some embodiments, the isolated or recombinant lymphoid cell(s) may not express or may be modified to remove or suppress expression of an endogenous TCR and / or BCR. In certain embodiments, such modification may be achieved via gene editing or RNA interference (RNAi), e.g., via siRNA or shRNA.

[0053]

[0043] In some embodiments, the isolated or recombinant lymphoid cell(s) may not express or may be modified to remove or suppress expression of endogenous MHCI and / or MHCII. In certain embodiments, such modification may be achieved via gene editing or RNA interference (RNAi), e.g., via siRNA or shRNA.

[0054]

[0044] In some embodiments, the isolated or recombinant lymphoid cell(s) may be modified to express another molecule, optionally a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a hybrid or chimeric cytokine receptor, and / or a transcription factor.

[0055]

[0045] One aspect of the present disclosure provides cell populations. A cell population according to the present disclosure may comprise, essentially consist of, or consist of any one or more of isolated or recombinant lymphoid cells according to the present disclosure.

[0056]

[0046] Such a cell population may, for example, be used as part of a pharmaceutical composition and / or as cells for cell therapy according to the present disclosure.

[0057]

[0047] One aspect of the present disclosure provides pharmaceutical compositions.

[0058]

[0048] A pharmaceutical composition according to the present disclosure may comprise: (I) one or more isolated or recombinant lymphoid cells disclosed herein, or a cell population which comprises, essentially consists of, or consists of any one or more of isolated or recombinant lymphoid cells according to the present disclosure; and (II) a pharmaceutically acceptable carrier.

[0059]

[0049] In some embodiments, the pharmaceutical composition may further comprise an additional agent, optionally an adjuvant or another therapeutic agent.

[0060]

[0050] Another aspect of the disclosure provides methods of producing one or more isolated or recombinant lymphoid cells or a cell population comprising one or more isolated or recombinant lymphoid cells.

[0061]

[0051] In some embodiments, the one or more isolated or recombinant lymphoid cells may be any of those described herein.1143253.008213

[0062]

[0052] In some embodiments, the cell population may be any of the cell populations described herein.

[0063]

[0053] The method may comprise culturing one or more lymphoid cells or a population of cells comprising one or more lymphoid cells in the presence of: (I) a PI3K-AKT-mTOR pathway inhibitor; and (II) ( 11-1) a Wnt / p-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor.

[0064]

[0054] In some embodiments, any one or more of the foregoing inhibitors and / or activators are added to the cell culture at the same time.

[0065]

[0055] In some embodiments, any one or more of the foregoing inhibitors and / or activators are added to the cell culture separately or at different timepoints.

[0066]

[0056] In certain embodiments, any one or more of the foregoing inhibitors and / or activators may be added to the cell culture in any order.

[0067]

[0057] In certain embodiments, any one or more of the foregoing inhibitors and / or activators may be added to the cell culture in a pre-determined order. In some cases, a PIS K- AKT-mTOR pathway inhibitor is added and then a Wnt / p-catenin pathway activator is added. In some cases, a PI3K-AKT-mTOR pathway inhibitor is added and then an acetyl-CoA synthesis inhibitor is added. In some cases, a Wnt / p-catenin pathway activator is added and then a PI3K-AKT-mTOR pathway inhibitor is added. In some cases, an acetyl-CoA synthesis inhibitor is added and then a PI3K-AKT-mTOR pathway inhibitor is added. In particular cases, the differences in the times of adding may be about 5 minutes to 3 days, about 10 minutes to about 3 days, about 30 minutes to about 1 day, about 1 hour to about 12 hours, about 2 hours to about 6 hours, about 3 hours, about 4 hours, or about 5 hours.

[0068]

[0058] The PI3K-AKT-mTOR pathway inhibitor may be any appropriate agent which inhibits the PI3K-AKT-mTOR pathway. In some embodiments, the PI3K-AKT-mTOR pathway inhibitor may be any of those disclosed herein.

[0069]

[0059] The PI3K-AKT-mTOR pathway inhibitor may be present at any appropriate concentration. In some embodiments, the PI3K-AKT-mTOR pathway inhibitor may be present at about 10 nM to about 100 mM, about 100 nM to about 10 mM, about 1 pM to about 1 mM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM, optionally at the start of or at least at one point during the culturing.1143253.008213

[0070]

[0060] The Wnt / P-catenin pathway activator may be any appropriate agent which activates the Wnt / P-catenin pathway, in some embodiments, the Wnt / P-catenin pathway activator may be any of those disclosed herein.

[0071]

[0061] The Wnt / P-catenin pathway activator may be present at any appropriate concentration. In some embodiments, the Wnt / P-catenin pathway activator may be present at about 1 nM to about 10 mM, about 10 nM to about 1 mM, about 100 nM to about 100 pM, about 1 pM to about 10 pM, about 1 pM to about 5pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM, optionally at the start of or at least at one point during the culturing.

[0072]

[0062] The PI3K-AKT-mTOR pathway inhibitor and the Wnt / p-catenin pathway activator may be present at any appropriate molar ratio. In some embodiments, the PI3K-AKT-mTOR pathway inhibitor and the Wnt / P-catenin pathway activator may be present at a molar ratio of about 0.1:1 to about 100: 1, about 0.1:1, about 0.5:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, or about 100:1, at the start of or at least one point during the culturing.

[0073]

[0063] The acetyl-CoA synthesis inhibitor may be any appropriate agent which inhibits acetyl-CoA synthesis. In some embodiments, the acetyl-CoA synthesis inhibitor may be any of those disclosed herein.

[0074]

[0064] The acetyl-CoA synthesis inhibitor may be present at any appropriate concentration. In some embodiments, the acetyl-CoA synthesis inhibitor may be present at about 10 pM to about 500 mM, about 100 pM to about 50 mM, about 0.5 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, or about 5 mM, optionally at the start of or at least one point during the culturing.

[0075]

[0065] The PI3K-AKT-mTOR pathway inhibitor and the acetyl-CoA synthesis inhibitor may be present at any appropriate molar ratio. In some embodiments, the PI3K-AKT-mTOR pathway inhibitor and the acetyl-CoA synthesis inhibitor may be present at a molar ratio of about 10:1 to about 1: 10000, about 1:1 to about 1:10000, about 1:10 to about 5000, about 1:10 to about 5000, about 1:3000, about 1:2500, about 1:2000, about 1:1000, about 1:500, about 1:300, about 1:250, about 1:200, about 1:100, about 1:50, about 1:30, about 1:25, about 1:20, or about 1:10 at the start of or at least one point during the culturing.1143253.008213

[0076]

[0066] In some embodiments, the culturing may be performed in the presence of IL-2, optionally at about 0.1 U / mLto about 200 U / mL, about 0.5 U / mLto about 100 U / mL, about 1 U / mLto about 50 U / mL, about 5 U / mLto about 25 U / mL, about 25 U / mL, or about 10 U / mL.

[0077]

[0067] In some embodiments, the culturing may be performed in the presence of an CDS activating agent, optionally an anti-CDS antibody or antigen-binding fragment thereof, and / or an CD28 activating agent, optionally an anti-CD28 antibody or antigen-binding fragment thereof.

[0078]

[0068] In some embodiments, the culturing may be performed in the presence of an antigen of interest to which at least one of the one or more lymphoid cells specifically binds. In certain embodiments, the antigen is a cognate antigen of theTCR expressed on the cell. In certain embodiments, the antigen is a cognate antigen of the CAR expressed on the cell. In certain embodiments, the antigen is a cognate antigen of the synthetic immune receptor expressed on the cell.

[0079]

[0069] In some embodiments, the culturing in the presence of (I) the PIBK-AKT-mTOR pathway inhibitor and (II) ( 11-1) the Wnt / p-catenin pathway activator and / or (11-2) the acetyl- CoA synthesis inhibitor may be performed for about 0.5 to about 20 days, about 1 to about 15 days, about Iday, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days.

[0080]

[0070] In some embodiments, the one or more lymphoid cells (e.g., the cells to be treated in the method) may be or comprise a cell ofT cell lineage or NKT cell lineage, optionally a CD8+ T cell, a CD4+ T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing. In some embodiments, the one or more lymphoid cells (e.g., the cells to be treated in the method) may be, comprise, or be derived from one or more splenocytes, lymph node cells, bone marrow cells, and / or thymic cells. In some embodiments, the one or more lymphoid cells (e.g., the cells to be treated in the method) may be, comprise, or be derived from a stem cell, a multipotent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (iPS) cell. In some embodiments, the one or more lymphoid cells (e.g., the cells to be treated in the method) may be derived from a subject, optionally a mammal, further optionally a human, a non-human primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse. In some embodiments, the one or1143253.008213

[0081] more lymphoid cells (e.g., the cells to be treated in the method) may be or comprise a mammalian cell, optionally a human cell, a non-human primate cell, a monkey cell, a rabbit cell, a rodent cell, a hamster cell, a rat cell, or a mouse cell. In some embodiments, the one or more lymphoid cells (e.g., the cells to be treated in the method) may be, comprise, or be derived from a cell line cell or a primary cell.

[0082]

[0071] In some embodiments, the population of cells (e.g., the population of cells to be treated in the method) may be or comprise splenocytes, lymph node cells, bone marrow cells, and / or thymic cells. In some embodiments, the population of cells (e.g., the population of cells to be treated in the method) may be, comprise, or be derived from one or more stem cells, multipotent stem cells, pluripotent stem cells, hematopoietic stem cells, embryonic stem cells, and / or iPS cells. In some embodiments, the population of cells (e.g., the population of cells to be treated in the method) may be derived from a subject, optionally a mammal, further optionally a human, a non-human primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse. In some embodiments, the population of cells (e.g., the population of cells to be treated in the method) may be or comprise a population of mammalian cells, optionally human cells, non-human primate cells, monkey cells, rabbit cells, rodent cells, hamster cells, rat cells, or mouse cells. In some embodiments, the population of cells (e.g., the population of cells to be treated in the method) may be, comprise, or be derived from cell line cells or primary cells.

[0083]

[0072] In some embodiments, the method may be for producing one or more isolated or recombinant lymphoid cells or a cell population comprising one or more isolated or recombinant lymphoid cells for use in cell therapy, optionally a CAR cell therapy, further optionally a CAR T cell therapy, optionally for treating cancer.

[0084]

[0073] In some embodiments, the method further comprises modifying the one or more lymphoid cells or the population of cells to induce expression of a chimeric antigen receptor (CAR). In certain embodiments, the CAR may comprise: (1) an antigen-binding domain (e.g., comprising an immunoglobulin (Ig) heavy chain variable domain (VH) and / or light chain variable domain (VL)); (2) a transmembrane domain; and (3) at least one intracellular signaling domain (e.g., of or derived from CDS, CDS^, CD27, CD28, CD44, DAP10, 4-1BB (CD137), OX-40, ICOS, NKG2D, a Fey receptor, FcRyll I (CD16), FcRyllA (CD32A), and / or 2B4 (CD244).1143253.008213

[0085]

[0074] In some embodiments, the method further comprises modifying the one or more lymphoid cells or the population of cells to induce expression of a synthetic immune receptor, optionally a synthetic T cell receptor (TCR) complex. In certain embodiments, the synthetic TCR complex may comprise: (1) an a chain comprising a TCR a constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain; (2) a p chain comprising a variable domain, a TCR p constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain; (3) one or more CD3 polypeptides; and (4) optionally one or more of CD3e, CD36, and / or CD3y polypeptides.

[0086]

[0075] In some embodiments, the method further comprises modifying the one or more lymphoid cells or the population of cells to remove or suppress expression (e.g., functional expression) of or inhibit a function of an endogenous TCR and / or BCR. In certain embodiments, such modification may be achieved via gene editing or RNAi.

[0087]

[0076] In some embodiments, the method further comprises modifying the one or more lymphoid cells or the population of cells to not express or to remove or suppress expression of endogenous MHCI and / or MHCII. In certain embodiments, such modification may be achieved via gene editing or RNAi.

[0088]

[0077] In some embodiments, the method further comprises modifying the one or more lymphoid cells or the population of cells to express another molecule, optionally a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a hybrid cytokine receptor, and / or a transcription factor.

[0089]

[0078] Further encompassed by the present disclosure are one or more isolated or recombinant cells or cell populations comprising one or more isolated or recombinant lymphoid cells, which are produced using any of the methods of producing described herein.

[0090]

[0079] One aspect of the present disclosure provides methods of treating a disease, disorder, or condition in a subject. The method may comprise administering to the subject an effective amount of at least one of: (a) one or more isolated or recombinant cells according to the present disclosure; (b) a cell population according to the present disclosure; and / or (c) a pharmaceutical composition according to the present disclosure.

[0091]

[0080] In some embodiments, the subject may be a mammal, optionally a human, a nonhuman primate, a monkey, a horse, a cow, sheep, a goat, a pig, a dog, a cat, a rabbit, a1143253.008213

[0092] rodent, a hamster, a rat, or a mouse. In some embodiments, the subject may be a non¬ mammalian vertebrate, optionally a bird, fish, an amphibian, or a reptile.

[0093]

[0081] In some embodiments, the administering may be or comprise parenterally administering, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intradermal, intrathecal, intra-arterial, intraarticular, intraosseous, intraparenchymal, or intraperitoneal administration) or by inhalation. In some embodiments, the administering may be or comprise locally administering, optionally by direct administration into a diseased site (optionally intratumoral injection) or by inhalation or administering to the skin, liver, bone marrow, lymphoid tissue, the peritoneal cavity, gut, ovary, testis, eye, ear, nose (optionally intranasally), skin (optionally transdermally or epicutaneously), mucosa, or vagina. In some embodiments, the administering may be or comprise enterally administering, optionally orally, sublingually, buccally, or rectally.

[0094]

[0082] In some embodiments, the administering may be effected two or more times, optionally about 3-5 times, optionally about once a week, about every 2 weeks, or about every 3 weeks, about once a month, about every 3 months, about every 6 months, about every 9 months, or about once per year.

[0095]

[0083] In some embodiments, the method may further comprise administering to the subject an additional agent, optionally an adjuvant or another therapeutic agent. In certain embodiments, the additional agent may comprise a cytokine or a cytokine complex. In particular embodiments, the additional agent may comprise IL-2 or an IL-2 complex. In some cases, the IL-2 complex may be or comprise IL-2 complexed with an anti-IL-2 antibody. In certain cases, the antibody may be capable of blocking binding of IL-2 to an IL-2 receptor or one or more subunits thereof (e.g., one or more of a, P, or y subunits). In some cases, the IL-2 complex may be or comprise IL-2 complexed with an IL-2 receptor or one or more subunits thereof (e.g., one or more of a, P, or y subunits).

[0096]

[0084] In some embodiments, the disease, disorder, or condition (to be treated) comprises cancer, an infectious disease, an autoimmune disease, an inflammatory disease, a fibrotic disease, an allergic disease, a neural disease, a neurodegenerative disease, a metabolic disease, a cardiovascular disease, or another disease,

[0097]

[0085] In certain embodiments, the cancer may be a solid cancer, optionally chosen from: one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer,1143253.008213

[0098] pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharynx cancer, head and neck cancer, rectal cancer, esophagus cancer, or bladder cancer, or a metastasis thereof.

[0099]

[0086] In certain embodiments, the cancer may be a liquid cancer, optionally chosen from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma.

[0100]

[0087] In certain embodiments, the infectious disease may be a viral, bacterial, fungal, yeast, protozoan, prion or parasitic disease.

[0101]

[0088] In some cases, the viral disease may be human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C virus), human papillomavirus (HPV), herpes simplex virus (HSV) (optionally HSV-1 or HSV-2), enterovirus, human cytomegalovirus, adenovirus, rhinovirus, Pox virus, Influenza virus, coronavirus (optionally MERS-CoV, SARS- CoV, or SARS-CoV-2, or common human coronavirus), respiratory syncytial virus (RSV),1143253.008213

[0102] human metapneumovirus (HMPV), norovirus, West Nile Virus, Zika virus, poliovirus, Eboia virus, or dengue virus (DENV) infection.

[0103]

[0089] In some cases, the bacterial disease may be Salmonella, Escherichia coli, Mycobacterium tuberculosis, methicillin-resistant staphylococcus aureus (MRSA), Clostridium difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, Vibrio vulnificus.

[0104]

[0090] In some cases, the fungal disease is Aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucormycetes, Taloromyces, ringworm, Blastomyces, Coccidioides, Cryptococcus gattii, Histoplasma, Paracoccidioides, or Sporothrix infection.

[0105]

[0091] In certain embodiments, the autoimmune disease or the inflammatory disease may be psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjogren's syndrome, Addison's disease, Behcet's disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis, or fibrosis.

[0106]

[0092] In certain embodiments, the neurodegenerative disease may be Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, Creutzfeldt-Jakob disease.

[0107]

[0093] Another aspect of the present disclosure provides kits. A kit according to the present disclosure may comprise: (A) one or more lymphoid cells or a population of cells comprising one or more lymphoid cells; and (B) (I) a PI3K-AKT-mTOR pathway inhibitor; and (II) ( 11-1) a Wnt / P-catenin pathway activator; and / or (11-2) an acetyl-CoA synthesis inhibitor.

[0108]

[0094] The PI3K-AKT-mTOR pathway inhibitor may be any appropriate agent which inhibits the PI3K-AKT-mTOR pathway. In some embodiments, the PI3K-AKT-mTOR pathway inhibitor may be any of those disclosed herein.

[0109]

[0095] The Wnt / P-catenin pathway activator may be any appropriate agent which activates the Wnt / p-catenin pathway. In some embodiments, the Wnt / P-catenin pathway activator may be any of those disclosed herein.1143253.008213

[0110]

[0096] The acetyl-CoA synthesis inhibitor may be any appropriate agent which inhibits acetyl-CoA synthesis, in some embodiments, the acetyl-CoA synthesis inhibitor may be any of those disclosed herein.

[0111]

[0097] In some embodiments, a kit may further comprise one or more of a cytokine, a cytokine complex (e.g., a cytokine complexed with an antibody specific to the cytokine (e.g., an antibody which blocks binding of the cytokine to its receptor or one or more subunits thereof) or complexed with its receptor or one or more subunits thereof), IL-2, an IL-2 complex, an CDS activating agent, optionally an anti-CDS antibody or antigen-binding fragment thereof, an CD28 activating agent, optionally an anti-CD28 antibody or antigen¬ binding fragment thereof, an antigen of interest to which at least one of the one or more lymphoid cells specifically binds (e.g., a cognate antigen of a TCR, a CAR, and / or another synthetic immune receptor expressed on the cell).

[0112]

[0098] In certain embodiments, the IL-2 complex may be or comprise IL-2 complexed with an anti-IL-2 antibody. In some cases, the antibody may be capable of blocking binding of IL-2 to an IL-2 receptor or one or more subunits thereof (e.g., one or more of a, P, or y subunits). In certain embodiments, the IL-2 complex may be or comprise IL-2 complexed with an IL-2 receptor or one or more subunits thereof (e.g., one or more of a,, or y subunits).

[0113]

[0099] In some embodiments, the one or more lymphoid cells may be or comprise a cell of T cell lineage or NKT cell lineage, optionally a CD8+ T cell, a CD4+ T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing. In some embodiments, the one or more lymphoid cells may be, comprise, or be derived from a stem cell, a multipotent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (iPS) cell. In some embodiments, the one or more lymphoid cells may be derived from a subject, optionally a mammal, further optionally a human, a non-human primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse. In some embodiments, the one or more lymphoid cells may be or comprise a mammalian cell, optionally a human cell, a non- human primate cell, a monkey cell, a rabbit cell, a rodent cell, a hamster cell, a rat cell, or a mouse cell. In some embodiments, the one or more lymphoid cells may be, comprise, or be derived from a cell line cell or a primary cell.

[0114]

[0100] In some embodiments, the population of cells comprising one or more lymphoid cells may be or comprise splenocytes, lymph node cells, bone marrow cells, and / or thymic cells.1143253.008213

[0115]

[0101] In some embodiments, the kit may further comprise a label, optionally including an instruction for performing a method (e.g., a method of producing or a method of treating) according to the present disclosure. In certain embodiments, the kit may be for producing one or more isolated or recombinant lymphocytes or a cell population comprising such cells according to the present disclosure.

[0116]

[0102] Any of the isolated or recombinant lymphoid cells described herein, any of the cell populations containing such lymphoid cells described herein, and any of the pharmaceutical compositions described herein may be for use in medicine and / or for use in treating a disease, disorder, or a condition, which may optionally be any of the diseases, disorders, and conditions described herein.

[0117]

[0103] Further encompassed by the present disclosure herein include use of any of the isolated or recombinant lymphoid cells described herein, any of the cell populations containing such lymphoid cells disclosed herein, and / or any of the pharmaceutical compositions described herein, any of the kits described herein may be for the manufacture of a medicament, optionally for treatment of a disease, disorder, or a condition, which may optionally be any of the diseases, disorders, and conditions described herein.

[0118] BRIEF DESCRIPTION OF THE DRAWINGS

[0119]

[0104] FIGS. 1A-1D provide exemplary results from Example 1-1. FIG. 1A compares cell counts (number of cells contained per mL, when total cells were reconstituted in the same volume CD8+) observed on Days 3, 5, and 7 among different treatment groups. FIG. 1B compares cell viability (%) observed on Days 3, 5, and 7 among different treatment groups.

[0120] FIGS. 1C-1D compare expression (based on mean fluorescence intensity (MFI)) of different T cell markers on CD8+ T cells observed on Day 7.

[0121]

[0105] FIGS.2A-2D provide exemplary results from Example 1-2. FIG. 2A compares cell counts observed on Days 3, 5, and 6 among different treatment groups. FIG. 2B compares cell viability (%) observed on Days 3, 5, and 6 among different treatment groups. FIGS. 2C-2D compare expression (based on MFI) of different T cell markers on CD8+ T cells observed on Day 6.

[0122]

[0106] FIGS.3A-3B provide exemplary results from Example 1-3. FIG.3A compares cell viability (%) (left) and cell counts (right) observed on Days 3, 5, and 6 among different1143253.008213

[0123] treatment groups. FIG. 3B compares expression (based on MFI) of different T cell markers on CD8+ T cells observed on Day 6.

[0124]

[0107] FIGS. 4A-4E provide exemplary results from Example 1-4. FIG. 4A compares cell diameters (pm) observed on Day 5 (top), cell counts (number of cells contained per ml, when total cells were reconstituted in the same volume CD8+) observed on Days 3, 5, and 6 (middle left), cell viability (%) observed on Days 3, 5, and 6 (bottom left), CD8+ cell counts (number of cells contained per mL, when total CD8+ cells were reconstituted in the same volume across groups) observed on Day 6 (middle right), and CD8+ cell frequency (frequency of CD8+ cells among total live, single cell lymphocytes) observed on Day 6 (bottom right), among different treatment groups. FIGS. 4B-4E compare expression (based on MFI) of different T cell markers on CD8+ T cells observed on Day 6.

[0125]

[0108] FIGS. 5A-5E provide exemplary results from Example 1-5. FIG. 5A compares cell counts (left, number of cells contained per ml, when total cells were reconstituted in the same volume CD8+) and cell viability (%) (right) observed on Days 3 and 6, among different treatment groups. FIGS. 5B-5D compare expression (based on MFI) of different T cell markers on CD8+ T cells observed on Day 6.

[0126]

[0109] FIGS. 6A-6F provide exemplary results from Example 2. FIGS. 6A-6C compare expression (based on MFI) of different T cell markers on CD8+ T cells observed on Days 8 and 10. FIG. 6D-6F provide exemplary PCA results in which PCI scores are plotted against each other (based on parallel analyses) or PC2 scores are plotted against PCI scores (based on eigenvalue analyses), to show overall differences in T cell phenotypes observed on Days 8 and 10 depending on different treatment groups. For each analysis, a table listing markers in the order of the level of contribution to driving T cell phenotype differences (contribution is higher when the score values are larger).

[0127]

[0110] FIG. 7 provides exemplary results from Example 3-1. The top panel visualizes the experimental timeline from cancer inoculation to mouse treatment with OT-I cells. The bottom left graph provides comparison of changes in tumor sizes among different treatment groups, and bottom right table provides two-way ANOVA statistical analyses comparing tumor sizes among different treatment groups.

[0128]

[0111] FIG. 8 visualizes the experimental timeline from cancer inoculation to mouse treatment with OT-I cells and IL-2 complex in Example 3-2.1143253.008213

[0129]

[0112] FIG.9A & B contains the results of a killing assay wherein CD8 T cells and luminescent cancer cells (RMA-Rg-Luc cells incubated with SIINFEKL) were plated together and given IL-2 but no CAL or TW and killing was determined by luminescence on day 6, and 24 hours after co-culture. The results show that CAL + TW treated cells were the worst at killing, which is consistent with a TSCM phenotype. By contrast, the control cells (OT-1 Thyl.l Splenocytes stimulated with SIINFEKL) were the most effector-like and were the best at killing.

[0130]

[0113] FIG. 10A & B contain Western blot data showing the effects of control, CAL, TW, and CAL and TW on the levels of signaling-associated proteins (AKT, GSK3P, FOXO1, and P-catenin) in CD8 T cells. The results show the connections between the PI3K-AKT-mTOR pathway inhibitor and the Wnt / P-catenin pathway activator. The CAL and CAL + TW treated CD8T cells had less phosphorylated AKT, meaning less active AKT, resulting in less phosphorylation of its targets GS3KP and FOXO1. Phosphorylated GS3KP is inactivated decreasing its activity such as phosphorylating P-catenin, which targets it for degradation. Notably, TWS119 inhibition of GSK3P is independent of its phosphorylation. Phosphorylated FOXO1 is trapped in the cytoplasm. In summary, CAL + TW treated cells have inhibited GSK3p, less inhibited FOXO1, more p-catenin, and less p-catenin targeted for degradation. FIG. 10A contains the raw data.

[0131] FIG. 10B contains bar graphs quantifying the effects of control, CAL, TW and CAL and TW on the amounts of specific proteins (AKT, GSK3p, FOXO1, and p-catenin) based on the raw Western blot data.

[0132]

[0114] FIG. 11 compares MC38-OVA tumor protection data in mice comprising MC38-OVA tumors injected with the same number of control, CAL, TW or CAL + TW treated CD8 T cells. The results show that only the MC38ova tumor bearing mice treated with CAL / TW treated T cells had a statistically significant decrease in tumor volume, compared with control T cells..

[0133]

[0115] FIG. 12 contains cytokine staining data wherein the expression of specific effector cell-associated cytokines ( IFNy, TNFa and IL-2), was compared by stimulated (cocktail of phorbol 12-myristate 13-acetate (PMA) and ionomycin) and unstimulated control, CAL, TW and CAL + TW treated OT-1 T cells. The graphs show the percentage of alive CDS T cells that stain positive for each cytokine. The data shows that the CAL and CAL + TW treated OT-1 T cells express the least amount of the detected effector-associated cytokines.

[0134]

[0116] FIG. 13A and 13B contain single cell sequencing data comparing the number of tumor cells and other cell types (including specific immune cell types) in mice with B16-OVA1143253.008213

[0135] tumors which were treated with control, CAL, TW or CAL + TW treated CD8 T cells. The data in FIG 13A show the percentage of specific cell types respectively isolated from B16-ova tumor bearing mice treated with control, CAL, TW, and CAL + TW treated CD8 T cells. The data shows that the CAL-treated groups had many fewer melanoma cells and many more T cells proportionally in their sample compared to other cell types. The data in FIG 13A also shows the increase of CDS cells in mice injected with CAL, TW, and CAL + TW treated CDS T cells compared to control CD8 T cells. FIG. 13B shows the breakdown of the proportion of immune cell subsets only in cell samples isolated from B16-ova tumor bearing mice treated with control, CAL, TW, or CAL + TW treated CD8 T cells and the increase of CD8 cells in mice injected with CAL, TW, and CAL + TW treated T cells compared to mice injected with control T cells.

[0136] DETAILED DESCRIPTION

[0137]

[0117] The present disclosure generally relates to cells and cell therapy. Some aspects of the present disclosure relates to improved cells, such as lymphocytes or T cells, which may be used for and may improve cell therapy, methods of producing such improved cells, and methods of using such cells. Some aspects of the present disclosure relates to inducing a stem-like and / or memory T cell-like phenotype in cells of T cell lineage.

[0138] PI3K-AKT-mTOR pathway inhibitors

[0139]

[0118] The PI3K-AKT-mTOR pathway is an intracellular signaling pathway, which is downstream of various receptors and results in alteration of transcription. A number of signaling molecules are involved in a complex manner but, briefly, a receptor activation (e.g., upon binding to its ligand) results in PI3K activation, which then phosphorylates PIP2 to produce PIP3, which activates AKT (also called protein kinase B). AKT activation may occur directly or via activation of PDK1 or mTORC2. Activated Akt then inhibits migration of the transcription factor Foxo into the nucleus, resulting in suppression of Foxo-mediated transcription. Foxo is typically accompanied by p-catenin, a transcription co-activator.

[0140]

[0119] Therefore, an inhibitor of the PI3K-AKT-mTOR pathway would promote Foxo- mediated transcription.1143253.008213

[0141]

[0120] A PI3K-AKT-mTOR pathway inhibitor according to the present disclosure may be any appropriate agent which inhibits the PI3K-AKT-mTOR pathway, and may target any of the components of the PI3K-AKT-mTOR pathway. For example, any agents which inhibits PI3K or AKT may be used.

[0142]

[0121] In some embodiments, a PI3K inhibitor may be used. PI3K may be a class I PI3K. Largely there are four isoforms of PI3K, which are δ isoform, γ isoform, α isoform, and β isoform. While some PI3K inhibitors are pan-PI3K inhibitors capable of inhibiting all four isoforms (e.g., at the same or similar levels), some PI3 K inhibitors are selective for one or more of the four isoforms. Some may inhibit one isoform highly selectively, while some may have preference to certain isoform(s) over other isoforms. In some embodiments, a PI3K inhibitor which inhibits the 6 isoform may be used.

[0143]

[0122] In some embodiments, a PI3K inhibitor may be selected from the group consisting of 5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]quinazolin-4-one, idelalisib, ZYDELIG®, CAL-101, umbralisib, TGR1202, alpelisib, duvelisib, tenalisib, acalisib, dezapelisib, parsaclisib, linperlisib, zandelisib, BGB-10188, SHC014748M, amdizalisib, AZD8835, TQ-B3525, puqutinib, eganelisib, I PI-549, alpelisib, WX-037, inavolisib, serabelisib, CYH33, ASN003, RLY-2608, LOXO-783 / LOX-22783, STX-478, GSK2636771, AZD6482 / KIN193, AZD8186, KA2237, and taselisib.

[0144]

[0123] In certain embodiments, a small molecule which inhibits the 6 isoform may be used. In particular embodiments, the inhibitor may be 5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]quinazolin-4-one, idelalisib, ZYDELIG®, CAL-101, umbralisib, or TGR1202.

[0145]

[0124] In some embodiments, a AKT inhibitor may be used. Largely there are 3 isoforms of AKT, which are AKT1, AKT2, and AKT3. Many AKT inhibitors inhibit all three isoforms, although the degree of inhibition or preference to certain isoforms may vary. Some AKT inhibitors are selective for one or more of the three isoforms.

[0146]

[0125] In some embodiments, a AKT inhibitor may be selected from 1,3-dihydro-1-[1-[[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl]methyl]-4-piperidinyl]-2H-benzimidazol-2-one, Akt Inhibitor VIII, MK2206, capivasertib, afuresertib, and / or ipatasertib.

[0147]

[0126] A PI3K-AKT-mTOR pathway inhibitor according to the present disclosure may be of any modality. Exemplary modalities include but are not limited to a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide. For example, an mRNA or a DNA1143253.008213

[0148] encoding an siRNA, an shRNA, or antibody or antigen-binding fragment thereof may be used.

[0149]

[0127] In particular embodiments, a PI3K-AKT-mT0R pathway inhibitor according to the present disclosure may be a small molecule, such as a small molecule inhibitor.

[0150] Wnt / P-catenin pathway activators

[0151]

[0128] The Wnt / p-catenin pathway is an intracellular signaling pathway, which is initiated when a Wnt protein binds to the extracellular domain of a Frizzled (Fz) family receptor and results in alteration of transcription. A number of signaling molecules are involved in a complex manner but, briefly, in the canonical Wnt pathway, a Fz receptor activation signal (e.g., upon binding by Wnt) is transmitted to the phosphoprotein Dishevelled (Dvl). Activated Dvl triggers displacement of GSK-3P from the APC / Axin / GSK-30 complex ("a P-catenin destruction complex", which facilitates degradation of P-catenin in the absence of Wnt signaling), and consequently destabilizes free cytoplasmic P-catenin, which is no longer translocated to the nucleus. Thereby transcription via transcription factors such as TCF / LEF are suppressed.

[0152]

[0129] Therefore, an activator of the Wnt / p-catenin pathway would promote Wnt / p-catenin pathway-mediated transcription.

[0153]

[0130] A Wnt / p-catenin pathway activator according to the present disclosure may be any appropriate agent which activates the Wnt / p-catenin pathway, and may target any of the components of the Wnt / P-catenin pathway. For example, any agents which inhibit GSK-3P may be used (GSK-3P negatively regulates the Wnt / p-catenin pathway).

[0154]

[0131] Largely there are two isoforms of PI3K, which are P isoform and a isoform. While some GSK-3 inhibitors are capable of inhibiting both isoforms (e.g., at the same or similar levels), some GSK-3 inhibitors are selective for one of the two isoforms. In some embodiments, a GSK-3 inhibitor which inhibits the P isoform may be used.

[0155]

[0132] In some embodiments, a GSK-3 inhibitor may be 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol, TWS119, SB-415286, CHIR-99021, AR-A014418, lithium, LiCI, BIO, SB-216763, LY-2090314, PF-04802367, L807mts, tideglusib, elraglusib, AZD1080, BRD3731, rosmarinic acid, BRD0705, and / or psoralidin.1143253.008213

[0156]

[0133] In particular embodiments, a GSK-3 inhibitor may be 3-[[6-(3-aminophenyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol, TWS119, SB-415286, CHIR-99021, and / or AR-A014418.

[0157]

[0134] A Wnt / p-catenin pathway activator according to the present disclosure may be of any modality. Exemplary modalities include but are not limited to a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide. For example, an mRNA or a DNA encoding an siRNA, an shRNA, or antibody or antigen-binding fragment thereof may be used.

[0158]

[0135] In particular embodiments, a Wnt / p-catenin pathway activator according to the present disclosure may be a small molecule, such as a small molecule inhibitor. A small molecule may work in any appropriate biochemical mechanism of action. For example, the small molecule may be a cation, an ATP competitive inhibitor, an allosteric non-ATP competitive inhibitor, or a substrate competitive inhibitor.

[0159] Acetyl-CoA synthesis inhibitors

[0160]

[0136] Acetyl CoA can alter the conformation and thus activity of proteins (e.g., enzymes) it binds to, for example when acetyl CoA binds as an allosteric effector. For example, histone acetyltransferase activities in some cases are stabilized by acetyl-CoA. Therefore, acetyl CoA in the nucleus functions as a co-factor for various transcriptional activities.

[0161]

[0137] Therefore, an activator of acetyl-CoA synthesis would promote transcriptional activities.

[0162]

[0138] Acetyl CoA synthesis may be endogenously catalyzed by ATP-citrate lyase (ACLY), which converts citrate and Coenzyme A (CoA) to acetyl-CoA and oxaloacetate. This reaction is driven by hydrolysis of ATP, using ATP and CoA as co-factors.

[0163]

[0139] An acetyl-CoA synthesis inhibitor according to the present disclosure may be any appropriate agent which activates acetyl-CoA synthesis, and may target any of the components of the acetyl-CoA synthesis pathway. For example, any agents which inhibit ACLY may be used.

[0164]

[0140] In some embodiments, an ACLY inhibitor may be 2-hydroxycitrate, a salt thereof (e.g., potassium hydroxycitrate or sodium hydroxycitrate), bempedoic acid, ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid, NEXLETOL®, ESP-55016, bempedoyl-CoA,1143253.008213

[0165] BemA, hydroxycitric acid or a derivative thereof, SB204990, a 2-hydroxy-N-arylbenzenesulfonamide, BMS-303141, NDI-091143, SB-201076, citric acid tripotassium hydrate, citric acid, (-)-hydroxycitric acid lactone, MEDICA 16, 3-thiadicarboxylic acid, an emodin derivative, a furan carboxylate derivative, Hibiscus subdariffa extract, Garcinia cambogia extract, lithium citrate (Litarex) tetrahydrate, bempedoic acid-d4, 2-Furoic acid-d4, forrestiacids J, forrestiacids K, 2-Furoic acid, and / or bempedoic acid-d5.

[0166]

[0141] In particular embodiments, an ACLY inhibitor may be 2-hydroxycitrate or a salt thereof (e.g., potassium hydroxycitrate or sodium hydroxycitrate).

[0167]

[0142] A acetyl-CoA synthesis inhibitor according to the present disclosure may be of any modality. Exemplary modalities include but are not limited to a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide. For example, an mRNA or a DNA encoding an siRNA, an shRNA, or antibody or antigen-binding fragment thereof may be used.

[0168]

[0143] In particular embodiments, a acetyl-CoA synthesis inhibitor according to the present disclosure may be a small molecule, such as a small molecule inhibitor, e.g., an small molecule inhibitor of ACLY.

[0169] Methods of producing cells and cell populations

[0170]

[0144] The present disclosure provides methods of producing cells and cell populations.

[0171]

[0145] Applicant unexpectedly discovered that culturing splenocytes or T cells in the presence of (I) a PI3K-AKT-mT0R pathway inhibitor and (II) (11-1) a Wnt / p-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor results in T cells that are beneficial for cell therapy.

[0172]

[0146] As shown in Examples 1-2, culturing solenocytes or T cells in the presence of (I) a PI3K-AKT-mT0R pathway inhibitor and (II) (11-1) a Wnt / (3-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor skews T cells to present a phenotype of memory T cells, such as stem cell-like memory T (Tscm) cells.

[0173]

[0147] As shown in Example 3, administering T cells cultured in the presence of (I) a PI3K-AKT-mTOR pathway inhibitor and (II) a PI3K-AKT-mT0R pathway inhibitor slow cancer progression. Such a therapeutic benefit is not achieved by T cells from solenocytes cultured1143253.008213

[0174] in the presence of either (I) a PI3K-AKT-mT0R pathway inhibitor or (II) a Wnt / P-catenin pathway activator, i.e., single treatment is insufficient.

[0175]

[0148] Without wishing to be bound by theory, alteration in transcription induced by a PI3 K-AKT-mTOR pathway inhibitor and alteration in transcription induced by a Wnt / P-catenin pathway activator, even though such alteration individually are not sufficient to provide a desired effects on T cells, may work together to induce expression of one or more genes which promote development of memory T cell or sternness of T cells (e.g., Tscm phenotype) and / or which reverses or prevents T cell exhaustion.

[0176]

[0149] While CAL-101, a PI3K-AKT-mT0R pathway inhibitor, has been reported to lower nuclear levels of P-catenin, TWS119, a Wnt / P-catenin pathway activator, has been reported to increase nuclear levels of P-catenin (Le Xuan Truong Nguyen et al. Blood Cancer Journal, 2014, Lee et al., Clin Mol Hepatol. 2020).

[0177]

[0150] Therefore, methods of producing cells and cell populations according to the present disclosure comprises culturing one or more lymphoid cells or a population of cells comprising one or more lymphoid cells in the presence of: (I) a PI3K-AKT-mTOR pathway inhibitor; and (II) ( 11-1) a Wnt / P-catenin pathway activator; and / or (11-2) an acetyl-CoA synthesis inhibitor.

[0178] Cells

[0179]

[0151] Any cells which are or comprise lymphoid cells may be used.

[0180]

[0152] In some embodiments, the cells may comprise a cell of T cell lineage or NKT cell lineage, optionally a CD8+ T cell, a CD4+ T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing.

[0181]

[0153] In certain embodiments, cells may be or comprise leukocytes from a subject (e.g., containing or derived from splenocytes, lymph node cells, bone marrow cells, and / or thymic cells, or tissue-infiltrating leukocytes or lymphocytes), or lymphoid cells or cells of T cell lineage obtained from leukocytes.

[0182]

[0154] In certain embodiments, cells may be or comprise or may be derived from a stem cell, a multipotent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (iPS) cell. In some cases, such cells may have been developed into or may develop during culturing a T cell or a specific subset of T cell.

[0183] Culture conditions-- inhibitors and activators1143253.008213

[0184]

[0155] In some embodiments, such inhibitors and / or activators may be added to the cell culture at the same time, separately or together.

[0185]

[0156] In some embodiments, such inhibitors and / or activators may be added to the cell culture at different times and in any order.

[0186]

[0157] Any appropriate concentrations of a PI3K-AKT-mTOR pathway inhibitor, any appropriate concentrations of a Wnt / p-catenin pathway activator, and / or any appropriate concentrations of an acetyl-CoA synthesis inhibitor may be used in culture.

[0187]

[0158] In some embodiments, such concentrations may be determined based on the phenotype of marker expression of cells after treatment.

[0188]

[0159] In some embodiments, the concentration of the PI3K-AKT-mT0R pathway inhibitor may be about 10 nM to about 100 mM, about 100 nM to about 10 mM, about 1 pM to about 1 mM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM. Such a concentration may be achieved at the start a culture or at least one point during the culturing.

[0189]

[0160] In some embodiments, the concentration of the Wnt / P-catenin pathway activator may be about 1 nM to about 10 mM, about 10 nM to about 1 mM, about 100 nM to about 100 pM, about 1 pM to about 10 pM, about 1 pM to about 5pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. Such a concentration may be achieved at the start a culture or at least one point during the culturing.

[0190]

[0161] In certain embodiments, the PI3K-AKT-mTOR pathway inhibitor and the Wnt / p- catenin pathway activator may be present at a molar ratio of about 0.1:1 to about 100: 1, about 0.1:1, about 0.5:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, or about 100:1. Such a concentration ratio may be achieved at the start a culture or at least one point during the culturing.

[0191]

[0162] In some embodiments, the concentration of the acetyl-CoA synthesis inhibitor is present at about 10 pM to about 500 mM, about 100 pM to about 50 mM, about 0.5 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, or about 5 mM.1143253.008213

[0192] Such a concentration may be achieved at the start a culture or at least one point during the culturing.

[0193]

[0163] In certain embodiments, the PI3K-AKT-mTOR pathway inhibitor and the acetyl-CoA synthesis inhibitor may be present at a molar ratio of about 10:1 to about 1: 10000, about 1:1 to about 1:10000, about 1:10 to about 5000, about 1:10 to about 5000, about 1:3000, about 1:2500, about 1:2000, about 1:1000, about 1:500, about 1:300, about 1:250, about 1:200, about 1:100, about 1:50, about 1:30, about 1:25, about 1:20, or about 1:10. Such a concentration ratio may be achieved at the start a culture or at least one point during the culturing.

[0194] Culture conditions - additional stimulations

[0195]

[0164] The culture may further contain one or more factors, such as a factor which facilitates T cell activation, expansion, maintenance, or longevity.

[0196]

[0165] In some embodiments, cells may be cultured in the presence of IL-2. In certain embodiments, IL-2 may be present or exogenously added at about 0.1 U / mL to about 200 U / mL, about 0.5 U / mL to about 100 U / mL, about 1 U / mL to about 50 U / mL, about 5 U / mL to about 25 U / mL, about 25 U / mL, or about 10 U / mL. In certain embodiments, IL-2 may be present at about 10 U / mL.

[0197]

[0166] In some embodiments, cells may be cultured in the presence of IL-7. In certain embodiments, IL-7 may be present or exogenously added at about 0.01 ng / mLto about 100 ng / mL, about 0.05 ng / mLto about 50 ng / mL, about 0.1 ng / mLto about 10 ng / mL, about 0.5 ng / mLto about 5 ng / mL, or about 1 ng / mL.

[0198]

[0167] In some embodiments, cells may be cultured in the presence of an CD3 activating agent (e.g., anti-CD3 antibody or antigen-binding fragment thereof or beads comprising such an antibody), and / or an CD28 activating agent (e.g., an anti-CD28 antibody or antigen¬ binding fragment thereof or beads comprising such an antibody).

[0199]

[0168] In some embodiments, cells may be cultured in the presence of a cognate antigen or an antigenic peptide (e.g., an antigen or antigenic peptide which a TCR, BCR, CAR, or another synthetic immune receptor expressed on the cell is specific for). In certain embodiments, the antigen or antigenic peptide may be present or exogenously added at about 0.01 pg / mL to about 100 pg / mL, about 0.05 pg / mLto about 50 pg / mL, about 0.1 pg / mLto about 10 pg / mL, about 0.5 pg / mLto about 5 pg / mL, or about 0.5, 0.6. 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 pg / mL.1143253.008213

[0200] Culture conditions - duration

[0201]

[0169] The culture may be continued for any appropriate length. An appropriate length may for example determined based on cell number (e.g., cell count per a pre-determined volume, when the total cells are reconstituted to a specified volume), cell viability, phenotype, and / or marker expression.

[0202]

[0170] In some embodiments, cells may be cultured in the presence of (I) a PI3K-AKT-mTOR pathway inhibitor and (II) ( 11-1) a Wnt / p-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor for about 0.5 to about 20 days, about 1 to about 15 days, about lday, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days. In certain embodiments, the duration may be about 5 to about 10 days.

[0203]

[0171] In some cases, (I) a PI3K-AKT-mTOR pathway inhibitor and / or (II) (11-1) a Wnt / P-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor may be added at the start of cell culture (e.g., splenocyte culture or T cell culture or culture of cells of T cell lineage).

[0204]

[0172] In some cases, (I) a PI3K-AKT-mTOR pathway inhibitor and / or (II) (11-1) a Wnt / -catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor may be added after the cell culture (e.g., splenocyte culture or T cell culture or culture of cells of T cell lineage) is initiated, e.g., 0.5 day later, 1 day later, 2 days later, 3 days later, etc.

[0205]

[0173] In some cases, (I) a PI3K-AKT-mTOR pathway inhibitor and / or (II) (11-1) a Wnt / p- catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor may be replenished during the culture at least once, e.g., to maintain the approximate concentration of the inhibitor(s) / activator(s).

[0206]

[0174] In some cases, (I) a PI3K-AKT-mTOR pathway inhibitor and / or (II) (11-1) a Wnt / P-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor may be removed in the middle of cell culture (e.g., splenocyte culture or T cell culture or culture of cells of T cell lineage), e.g., 0.5 day, 1 day, 2 days, 3 days, 4 days, 5 days, etc after addition of the inhibitor(s) / activator(s).

[0207] Additional modifications1143253.008213

[0208]

[0175] The cells cultured or to be cultured in the presence of (I) a PI3K-AKT-mT0R pathway inhibitor and (II) (11-1) a Wnt / p-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor may be further modified.

[0209]

[0176] Exemplary modifications include induction of expression of an exogenous receptor, such as a CAR or another synthetic immune receptor, such as but not limited to those disclosed herein. Such expression may for example be achieved by using a nucleic acid (e.g., vector) encoding such a receptor, e.g., by transducing, transforming, or transfecting the cells.

[0210]

[0177] Exemplary modifications further include removal or suppression of expression (e.g., functional expression) of or inhibit a function of an endogenous TCR and / or BCR and / or endogenous MHCI and / or MHCII. Such removal or suppression may be achieved for example by using a nucleic acid (e.g., vector) encoding a dominant negative form of the molecule to be suppressed or to cause dominant negative suppression, e.g., by transducing, transforming, or transfecting the cells. Such removal or suppression may also be achieved via gene editing or RNAi, e.g., using siRNA, shRNA, or a DNA or mRNA encoding siRNA or shRNA.

[0211]

[0178] Further exemplary modifications include modification to express another molecule, optionally a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a hybrid cytokine receptor (also called flip receptor), and / or a transcription factor.

[0212]

[0179] The timing of any of such transduction, transformation, transfection, gene editing, RNAi, etc may be before, during, or after the culturing in the presence of (I) a PI3K-AKT-mTOR pathway inhibitor and (II) (ll-l) a Wnt / p-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor.

[0213] Acquisition of cells (cells used in the method of producing)

[0214]

[0180] For cells according to the present disclosure, prior to expansion and / or treatment, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and disease sites such as tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art, may be used. In some embodiments, cells can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In some embodiments, cells can be part of a mixed population of cells which present different phenotypic characteristics.1143253.008213

[0215]

[0181] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0216]

[0182] In some embodiments, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from a leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, tongue, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0217]

[0183] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some embodiments, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments contains cells other than red blood cells and platelets.

[0218]

[0184] Also provided herein are cell lines obtained from a transformed cell according to any of the above-described methods. Also provided herein are modified cells resistant to an immunosuppressive treatment. In some embodiments, the cells may be a cell line cell engineered to express a CSR according to the present disclosure. In some embodiments, such a cell may be used to further engineer to further express an immune receptor of a choice, specific for a target molecule of interest.

[0219] Cell purification

[0220]

[0185] In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove1143253.008213

[0221] unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0222]

[0186] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some embodiments, a washing step is accomplished a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some embodiments, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0223]

[0187] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid.

[0224] Exemplary markers that may be used include but not limited to CD45, CD3, CD8, and / or CD4. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some embodiments includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0225]

[0188] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a1143253.008213

[0226] heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0227]

[0189] In some embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. For example, CD3+ T cells can be positively selected using CD3 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0228]

[0190] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker high) on the positively or negatively selected cells, respectively.

[0229]

[0191] 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 embodiments, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ 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.

[0230]

[0192] In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.

[0231]

[0193] In some embodiments, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). The magnetically responsive material, e.g., particle, generally1143253.008213

[0232] is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0233]

[0194] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U. S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U. S. Pat. No. 4,795,698, and Liberti et al., U. S. Pat. No. 5,200,084 are other examples.

[0234]

[0195] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0235]

[0196] In some embodiments, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0236]

[0197] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0237]

[0198] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and / or engineered; in some1143253.008213

[0238] embodiments, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0239]

[0199] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the methods. In some embodiments, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and / or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009 / 072003, or US 20110003380 Al.

[0240]

[0200] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and / or in an automated or programmable fashion. In some embodiments, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various embodiments of the processing, isolation, engineering, and formulation steps.

[0241]

[0201] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[0242]

[0202] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some1143253.008213

[0243] examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0244]

[0203] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0245]

[0204] In any of the aforementioned separation steps, the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0246] Cell preparation and expansion

[0247]

[0205] In some embodiments, the provided methods include cultivation, incubation, culture, and / or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and / or engineering the depleted cell populations and cultureinitiating compositions.

[0248]

[0206] Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and / or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.

[0249]

[0207] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation.

[0250]

[0208] In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to1143253.008213

[0251] mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The cells of the disclosure can be activated and expanded, either prior to or after genetic modification of the cells, using methods as generally described, for example without limitation, in U. S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U. S. Patent Application Publication No. 20060121005. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0252]

[0209] T cells can be expanded in vitro or in vivo. Generally, the T cells of the disclosure can be expanded, for example, by contact with an agent that stimulates a CDS TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) or concanavalin A (ConA) can be used to create an activation signal for the T cell.

[0253]

[0210] In some embodiments, T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. In some embodiments, the T cell populations may be stimulated in vitro by contact with Muromonab-CD3 (OKT3). For co¬ stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640® or, X-vivo 5®, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-7, GM-CSF, IL-10, IL-15, IL-21, IL-4, TGF-P, and TNF, or any other additives for the growth of cells known to the skilled artisan. In a preferred embodiment, T cells are stimulated in vitro by exposure to OKT3 and IL-2. Other additives for the growth of cells include, but are not limited to, surfactant, Plasmanate, and1143253.008213

[0254] reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640®, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1®, and X-Vivo 20®, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°Celsius) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varied stimulation times may exhibit different characteristics.

[0255]

[0211] In some embodiments, the isolated cells of the disclosure can be expanded by coculturing with tissue or cells. The cells can also be expanded in vivo, for example in the subject's blood after administrating the cell into the subject.

[0256]

[0212] In some embodiments, when cells are expanded in vivo, at least one cell of the disclosure may be administered to a subject, and the administration may lead to an expansion of the cell in the subject, resulting in a population of cells. Alternatively, a polynucleotide or vector of the disclosure may be administered to a subject. Once the polynucleotide or vector is taken up by a cell within the subject and the cell proliferate or expand in the subject, this may result in a population of cells of the disclosure within the subject.

[0257]

[0213] In certain embodiments, the resulting population of cells persists in the subject for at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least two years, or at least three years after administration.

[0258]

[0214] In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the numbers of T cells). In some embodiments, the non-dividing feeder cells can comprise y-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with y rays in the range of about 3000 to 36001143253.008213

[0259] rads to prevent cell division. In some embodiments, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0260]

[0215] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and / or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some embodiments may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80° Celsius at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0261] Additional steps

[0262]

[0216] In some embodiments, once a method of producing according to the present disclosure is performed, the phenotype (e.g., expression of markers, such as memory T cell markers orTscm markers) and / or the function (e.g., cytotoxicity, cytokine release, gene expression changes, etc upon exposure to a cognate antigen) of the cells produced may be assessed. This may for example help confirm that a desired type of cells are produced and / or the percentage of cells displaying a desired phenotype and / or function.

[0263]

[0217] In certain embodiments, if the method comprises a step of modifying cells to express an exogenous receptor and / or an exogenous molecule and / or suppress expression of an endogenous molecule, expression of the exogenous receptor, exogenous molecule, and / or endogenous molecule may be analyzed.

[0264]

[0218] In some embodiments, the cells obtained after culturing in the presence of an aforementioned inhibitor(s) and / or activator(s) may be further subjected to selection. In certain embodiments, a subset of cells among the produced cells may be selected or sorted based on the phenotype (e.g., expression of markers, such as memory T cell markers orTscm markers) and / or the function (e.g., cytotoxicity, cytokine release, gene expression changes, etc upon exposure to a cognate antigen) of the cells may be assessed.

[0265]

[0219] For example, in some embodiments, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., TCF1, CD95, CCR7,1143253.008213

[0266] CD44, Sca-1, CD103, CD28, CD62L, CCR7, CD27, CD127, CD4, CD8, CD45RA, CD45RO, are isolated by positive or negative selection techniques.

[0267]

[0220] In some embodiments, enrichment for central memory T (Tcm) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some embodiments is particularly robust in such sub¬ populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining Tcm-enriched CD8+ T cells and CD4+ T cells further enhances efficacy. In embodiments, memory T cells are present in both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8 fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0268]

[0221] In some embodiments, the enrichment for central memory T (Tcm) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some embodiments, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (Tcm) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some embodiments are carried out simultaneously and in other embodiments are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0269]

[0222] In certain embodiments, if the method comprises a step of modifying the cells to express an exogenous receptor and / or an exogenous molecule and / or suppress expression of an endogenous molecule, cells may also be subjected to a selection based on the expression or expression level of the exogenous receptor (to be expressed), exogenous molecule (to be expressed), and / or endogenous molecule (to be suppressed).1143253.008213

[0270]

[0223] Any of these additional steps may for example enrich the desired cell subsets, which may further improve the efficacy when the cells are used in therapy. Such selection may be performed using any appropriate methods, including but not limited to flow cytometry¬ based sorting, beads (e.g., magnetic beads)-based selection, and / or antibody-based selection.

[0271] Cells and cell populations

[0272]

[0224] Also provided herein are cells comprising or treated with (I) a PI3K-AKT-mTOR pathway inhibitor and (II) ( 11-1) a PI3K-AKT-mTOR pathway inhibitor and / or (11-2) an acetyl-CoA synthesis inhibitor. In some embodiments, any of the methods of producing disclosed herein may be used to obtain such cells. The PI3K-AKT-mTOR pathway inhibitor, the PI3K- AKT-mTOR pathway inhibitor and / or the acetyl-CoA synthesis inhibitor. In some cases, such cells may present a phenotype of or express markers associated with memory T cells, such as stem cell-like memory T (Tscm) cells.

[0273] Cell types

[0274]

[0225] Cells or cell populations according to the present disclosure generally are or comprise eukaryotic cells, such as mammalian cells, and typically are human cells, more typically primary human cells, e.g., allogeneic or autologous donor cells. In some embodiments, the cells may be obtained from or derived from a subject. In certain embodiments, the subject from which the cell is isolated may be a healthy subject. In certain embodiments, the subject from which the cell is isolated may be one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments may be a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered, in some embodiments, the cells are derived from the spleen, blood, bone marrow, lymph node, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g.,, lymphoid cells, including lymphocytes (typically T cells, NK cells, B cells, NKT cells), myeloid cells, including monocytes, macrophages, dendritic cells, neutrophils, eosinophils, basophils, or mast cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including hematopoietic stem cells and induced pluripotent stem cells (iPSCs). Many of such cells typically are primary cells, such as those1143253.008213

[0275] isolated directly from a subject and / or isolated from a subject and frozen, or cells derived from primary cells.

[0276]

[0226] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD8+ cells, CD4+ 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.

[0277]

[0227] Alternatively, an immortalized cell or a cell line may be used to produce the cells of the present disclosure. Such examples include, but are not limited to, a T cell line, a CD8+ T cell line, a CD4+T cell line, a regulatory? cell line, an NK-T cell line, an NKcell line (e.g., NK-92), a monocyte line, a macrophage line, a dendritic cell line, and a mast cell line.

[0278] Furthermore, a desired cell type, for example T cells or NK cells, may be generated from a stem cell, such as an embryonic stem cell, iPSCs, or hematopoietic stem cell.

[0279]

[0228] The cells according to the present disclosure, when used in vivo, may be autologous. 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. Alternatively, cells may be allogeneic, and such cells may be for use in off-the-shelf methods. In some embodiments, 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).

[0280]

[0229] In some embodiments, the cells are T cells. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T cells, effector T cells (Teff), memory T cells, tumor-infiltrating lymphocytes (TIL), immature? cells, mature? cells, helper T cells, cytotoxic? cells, mucosa-associated invariant? (MAI?) cells, naturally occurring and adaptive regulatory? (Treg) cells, helper T cells, such asTHl cells, TH2 cells, THS cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, a / T cells, and 8 / y T cells.

[0281]

[0230] In particular embodiments, the cells according to the present disclosure are or comprise T cells presenting a phenotype of and / or expressing markers associated with memory? cells. There are several sub-types of memory? cells, such as stem cell memory? (Tscm), central memory? (Tcm), effector memory? (?em), or terminally differentiated effector memory? cells. In particular cases, the cells according to the present disclosure are1143253.008213

[0282] or comprise Tscm cells or cells presenting a phenotype of or expressing one or more markers associated with Tscm cells.

[0283]

[0231] In some cases, the markers may be or comprise one or more of CCR7 (CD197), CD95, CD27, CD62L, CD122, IL-7Ra (CD127), CD103, CD69, CD44, CXCR3, TCF7, TCF1, LEF1, LFA-1, LFA-3, FOXO1, BCL6, Sca-1, FOXP1, KLF2, CD45 A, and / or CD45RO.

[0284]

[0232] In some cases, the markers may be or comprise one or more Tscm markers. In certain cases, the marker may be or comprise one or more of CCR7 (CD197), CD95, CD27, CD62L, CD122, IL-7Ra (CD127), CD103, CD69, CD44, CXCR3, TCF7, TCF1, LEF1, LFA-1, LFA-3, FOXO1, BCL6, Sca-1, FOXP1, KLF2, CD45RA, and / or Ki67. In certain cases, the markers may be or comprise one or more of CD45RA, CCR7 (CD197), CD62L, CD28, CD27, CD12 CD95, CD27, IL- 7Ra (CD127), CXCR3, CD95, CDlla, I L-2R (CD122), and / or CD58. In particular cases, the markers may be or comprise one or more of CD45RA, CD27, CD62L, and / or CD95. In further particular cases, the cells may have the phenotype of: CD45RA+ CD45RO- CD27+ CD62L+ CD95+; CD45RO- CCR7+ CD28+ CD95+; CD45RO- CCR7+ CD62L+ CD95+ TCFlhiScalhi; CD25'° CD44'° CD62LhiCD122+ Scal+; and / or CCR7+ CD95+ CD45RO- CD27+ CD45RA+ CD62L+ CD122+.

[0285]

[0233] In particular embodiments, the cells according to the present disclosure are or comprise T cells which are less like exhausted T cells or which does not express or have reduced expression of one or more markers associated with T cell exhaustion.

[0286]

[0234] In some cases, the markers may be or comprise one or more of Tim-3 (CD366), Lag-3 (CD223), PD-1, CTLA-4, Slamf6 (CD352), CD39a, TIGIT, CD160, BTLA, and / or 2B4 (CD244).

[0287]

[0235] In particular embodiments, the cells according to the present disclosure are or comprise T cells which, upon exposure to a cognate antigen (e.g., a cognate antigen of TCR, BCR, CAR, or another synthetic immune receptor), expresses or increases expression of markers associated with activated T cells or effector T cells.

[0288]

[0236] In some cases, the markers may be CD25, CD26, CD27, CD28, CD30, CD38, CD43, CD44, CD69, CD71, CD107, IL-7Ra (CD127), 0X40 (CD134), CD40L (CD154), CXCR1, CX3CR1, HLA-DR, KLRG1, IFNy, granzyme B, and / or perforin.

[0289]

[0237] Marker expression may be determined or evaluated by any appropriate methods, including but not limited to flow cytometry.

[0290]

[0238] In some embodiments, the cells are natural killer (N K) cells, Natural Killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), lymphokine-1143253.008213

[0291] activated killer (LAK) cells, or the like. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. CAR-expressing phagocytic cells expressing may be able to bind to and phagocytose or nibble target cells (Morrissey M. A. et al., Elife. 2018 Jun 4, "7).

[0292]

[0239] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.

[0293] Further modifications - exogenous receptors, e.g., CARs and other immune receptors

[0240] In some embodiments, cells according to the present disclosure may be modified to express an exogenous receptor such as a chimeric antigen receptor (CAR) or another synthetic immune receptor specific to an antigen of interest.

[0294] Antigens of interest

[0295]

[0241] The antigen of interest may be any appropriate molecule, including but not limited to an antigen expressed on a cell and / or a secretory antigen.

[0296]

[0242] In some embodiments, the antigen of interest may be associated with a disease, disorder, or a condition.

[0297]

[0243] In certain embodiments, the antigen of interest may be a cancer antigen. In some cases, the cancer may be a solid cancer, optionally any of the solid cancers described herein. In some cases, the cancer may be a liquid cancer, optionally any of the liquid cancers described herein. In particular cases, the cancer may be pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer. In particular cases, the cancer may be bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, melanoma, oesophago-gastric adenocarcinoma, oligodendrioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, small cell lung cancer, stomach cancer, and thyroid cancer.

[0298]

[0244] In certain embodiments, the antigen of interest may be an antigen of an autoimmune or inflammatory disease, optionally selected from psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, chronic inflammatory demyelinating1143253.008213

[0299] polyneuropathy, pemphigus vulgaris, Sjogren's syndrome, Addison's disease, Behcet's disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis, or fibrosis.

[0300]

[0245] In certain embodiments, the antigen of interest may be an antigen of a neurodegenerative disease, optionally selected from Alzheimer’s disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, or Creutzfeldt-Jakob disease.

[0301]

[0246] In certain embodiments, the antigen of interest may be an antigen of an infectious disease, optionally a viral, bacterial, fungal, yeast, protozoan, prion or parasitic disease. In some cases, the viral disease may be any appropriate viral infection including but not limited to one of those described herein. In some cases, the bacterial disease may be any appropriate bacterial infection including but not limited to one of those described herein. In some cases, the fungal disease may be any appropriate fungal infection including but not limited to one of those described herein.

[0302] Receptor structure

[0303]

[0247] Non-limiting examples of immune receptors include chimeric antigen receptors (CARs), a synthetic immune receptor (SIR), a T cell receptor (TCR), or B cell receptor (BCR), comprising an antigen-binding region specific to a target molecule of interest.

[0304]

[0248] In some embodiments, immune receptor may be or may comprise a synthetic TCR complex comprising: a chimeric TCR comprising alpha and beta polypeptides, forming an antigen-binding region or site; and a CD3 complex, comprising one or more of CD3y, CD36, CD3e, and / or CD3^.

[0305]

[0249] In some embodiments, immune receptor may be or may comprise a CAR comprising: an antigen-binding region specific to a target molecule of interest; a transmembrane (TM) region; and a cytoplasmic (CY) region comprising one or more intercellular signaling (ICS) domains and / or one or more costimulatory (CS) domains.

[0306] Receptor structure - antigen-binding region

[0307]

[0250] The immune receptor used according to the present disclosure may comprise an antigen-binding region specific to a target molecule of interest.1143253.008213

[0308]

[0251] In some embodiments, the antigen-binding region of the immune receptor (e.g., CAR, synthetic TCR, SIR, BCR) may comprise an antibody or antigen-binding antibody fragment specific to a target molecule of interest, comprising either a combination of a VH and a VL or a VH if sufficient to provide binding specificity (e.g., in case of nanobody). In certain embodiments, the antigen-binding region may comprise an scFv. The scFv may comprise a VH and a VL and optionally a linker that connects the VH and VL, e.g., in a format of VH-linker-VL or VL-linker-VH.

[0309]

[0252] Various antigen-binding regions, combinations of VH and VL sequences, and VH sequences that provide specificity to a wide variety of antigens are known in the field, and any of such antigen-binding regions, combinations of VH and VL sequences, and VH sequences may be used in an immune receptor as long as they specifically bind to a target molecule of interest (e.g., those that bind to a target molecule of interest at a dissociation constant (KD) of 1000 nM or lower) or a cell expressing such a target molecule of interest (i.e., target cell). Whether a given antigen-binding region, a given combination of VH and VL sequences, or a given VH (e.g., nanobody) binds to the target molecule of interest may be assessed using any appropriate methods, techniques, and / or assays. Non-limiting examples of methods, techniques, and assays for evaluating binding to a target molecule may include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), BioLayer Interferometry (BLI) (e.g., using an OCTET® system), and surface plasmon resonance (SPR) (e.g., using a CARTERRA® system or a BIACORE® system). Non-limiting examples of methods, techniques, and assays for evaluating binding to a cell expressing a target molecule may include flow cytometry and cell chromatography. In some embodiments, antigen-binding regions, combinations of VH and VL sequences, or VH sequences may be considered to sufficiently bind to a target cell as long as the binding (e.g., in terms of histograms or mean, or median, fluorescence intensity when flow cytometry is used) is discernably higher than that with a control, non-specific antigen-binding region, combination of VH and VL sequences, or VH sequence, respectively.

[0310]

[0253] In some embodiments, the antigen-binding region of the immune receptor (e.g., TCR, CAR) may comprise a set of V domains (e.g., Va and Vp; or Vy and V8), optionally derived from a TCR specific to the target molecule of interest.

[0311]

[0254] Various antigen-binding regions of TCRs and combinations of V domains that provide specificity to a wide variety of antigens are known in the field, and any of such TCR antigen-1143253.008213

[0312] binding regions and combinations of V domains may be used in an immune receptor as long as they specifically bind to a target molecule of interest (e.g., those that bind to a target molecule of interest at a dissociation constant (KD) of 1000 nM or lower) or a cell expressing such a target molecule of interest. Whether a given TCR antigen-binding region or a combination of V domains binds to the target molecule of interest may be assessed using any appropriate methods, techniques, and / or assays, essentially in the same manner as assessing binding by an antigen-binding region of an antibody (e.g., using a TCR or an antigen-binding region thereof instead of an antibody or an antigen-binding fragment). Nonlimiting examples of methods, techniques, and assays for evaluating binding to a target molecule may include ELISA, RIA, BLI (e.g., using an OCTET® system), and SPR (e.g., using a CARTERRA® system or a BIACORE® system). Non-limiting examples of methods, techniques, and assays for evaluating binding to a cell expressing a target molecule may include flow cytometry and cell chromatography. In some embodiments, TCR antigen-binding regions or combinations of V domains may be considered to sufficiently bind to a target cell as long as the binding (e.g., in terms of histograms or mean, or median, fluorescence intensity when flow cytometry is used) is discernably higher than that with a control, non-specific TCR antigen-binding region or combination of V domains, respectively.

[0313]

[0255] In some embodiments, the antigen-binding region of the immune receptor (e.g., CAR) may comprise a sequence of or derived from a protein other than an antibody, TCR, or BCR. In certain embodiments, a target molecule may be a ligand and the protein may be a receptor having specificity to the ligand, and the antigen-binding region may comprise or may be derived from the ligand-binding region of the receptor. In certain embodiments, a target molecule may be a receptor and the protein may be a ligand specific to the receptor, and the antigen-binding region may comprise or may be derived from the receptor-binding region of the ligand. In certain embodiments, a target molecule may be a substrate and the protein may be an enzyme having specificity to the substrate, and the antigen-binding region may comprise or may be derived from the substrate-binding region of the enzyme. In certain embodiments, a target molecule may be an enzyme and the protein may be a substrate specific to the enzyme, and the antigen-binding region may comprise or may be derived from the enzyme-binding region of the substrate.

[0314]

[0256] Various combinations of a receptor and a ligand and of an enzyme and a substrate are known in the field, and any of such receptors, ligands, enzymes, and substrates may be1143253.008213

[0315] used in an immune receptor as long as they specifically bind to the respective binding partners which are target molecules of interest (e.g., with a dissociation constant (KD) of 1000 nM or lower) or a cell expressing such a target molecule of interest. Again, binding to the respective target molecules of interest may or cells expressing such molecules may be assessed using any appropriate methods, techniques, and / or assays. Non-limiting examples of methods, techniques, and assays for evaluating binding to a target molecule may include ELISA, RIA, BLI (e.g., using an OCTET® system), and SPR (e.g., using a CARTERRA® system or a BIACORE® system). Non-limiting examples of methods, techniques, and assays for evaluating binding to a cell expressing a target molecule may include flow cytometry and cell chromatography.

[0316] Receptor structure - hinge

[0317]

[0257] In some embodiments, for example when cells are modified to express a CAR or any other synthetic receptor, the receptor may comprise a hinge sequence between the antigen¬ binding region and the TM region. One of the ordinary skill in the art will appreciate that a hinge sequence is a short sequence of amino acids that facilitates flexibility (see e.g., Woof J. M. et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.

[0318]

[0258] In some embodiments, the length of the hinge sequence may be optimized based on the desired length of the extracellular portion of the receptor, which may be based on the location of the epitope within the target molecule. For example, if the epitope is in the membrane proximal region within the target molecule, longer hinges may be optimal.

[0319]

[0259] In some embodiments, the hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an lgG2 Fc region, an lgG3 Fc region, an lgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the hinge includes at least a portion of an IgGl, an lgG2, an lgG3, an lgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CHS domains. In some embodiments, the hinge may also include at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the hinge is derived from or includes at least a portion of a modified immunoglobulin Fc region, for example, a modified IgGl Fc region, a modified lgG2 Fc region, a modified lgG3 Fc region, a modified lgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations,1143253.008213

[0320] insertions, deletions, duplications) resulting in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the hinge to an Fc receptor (FcR). In some embodiments, the modified immunoglobulin Fc region may be designed with one or more mutations which result in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the hinge to one or more FcR including, but not limited to, FcyRI, FcyR2A, FcyR2Bl, Fcy2B2, Fey 3A, Fey 3B, FCERI, FCER2, FcaRI, Fca / pR, or FcRn.

[0321]

[0260] In some embodiments, a portion of the immunoglobulin constant region serves as a hinge between the antigen-binding region, for example scFv or nanobody, and the TM region. The hinge can be of a length that provides for increased responsiveness of the CAR (or another synthetic receptorj-expressing cell following antigen binding, as compared to in the absence of the hinge. In some examples, the hinge is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary hinges include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a hinge has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary hinges include a CD28 hinge, lgG4 hinge alone, lgG4 hinge linked to CH2 and CH3 domains, or lgG4 hinge linked to the CH3 domain.

[0322] Exemplary hinges include, but are not limited to, those described in Hudecek M. et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U. S. Pat. No. 8,822,647 or published App. No. US2014 / 0271635.

[0323]

[0261] In some embodiments, the hinge sequence is derived from CD8 a molecule or a CD28 molecule. In particular embodiment, the hinge sequence is derived from CD28.

[0324] Receptor structure - transmembrane domain

[0325]

[0262] In some embodiments, for example when cells are modified to express a CAR or any other synthetic receptor, the receptor can be designed to comprise a TM region that is fused to the antigen-binding region of the receptor. A hinge sequence may be inserted between the antigen-binding region and the TM region. In one embodiment, the TM region that naturally is associated with one of the regions and / or domains in the CAR is used. In some1143253.008213

[0326] instances, the TM region can be selected or modified by amino acid substitution to avoid binding of such regions to the transmembrane portions of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0327]

[0263] The TM region may be derived either from a natural or from a synthetic source. Where the source is natural, the region may be derived from any membrane-bound or transmembrane protein. Typically, the TM region denotes a single transmembrane a helix of a transmembrane protein, also known as an integral protein. TM regions of particular use in this disclosure may be derived from (i.e., comprise at least the transmembrane region(s) of) CD28, CDS e, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR a, TCR 0, or CDS zeta and / or TM regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof.

[0328]

[0264] Alternatively the TM region may be synthetic, in which case the TM region will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic TM region. A TM region of the disclosure is thermodynamically stable in a membrane. It may be a single a helix, a transmembrane 0 barrel, a 0-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length.

[0329]

[0265] In certain embodiments, the TM region in the CAR or the synthetic receptor of the disclosure is derived from the TM region of CD28.

[0330] Receptor structure - cytosolic region (e.g., ICS domain and CS domain)

[0331]

[0266] In some embodiments, for example when cells are modified to express a CAR or any other synthetic receptor, the receptor can be designed to comprise a cytosolic (CY) region which may comprise one or more intracellular signaling (ICS) domains and / or one or more costimulatory signaling (CS) domains.

[0332]

[0267] The ICS domain triggers or elicits activation of at least one of the normal effector functions of the cell (i.e., one or more specialized functions of a cell) in which the CAR has been placed. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. While usually the entire ICS domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may1143253.008213

[0333] be used in place of the intact chain as long as it transduces the effector function signal. In some cases, a truncated ICS domain may comprise an intact form of a particular motif.

[0334]

[0268] Preferred examples of ICS domains for use in the CAR of the disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0335]

[0269] Signals generated through one ICS domain alone may be insufficient for full activation of a cell, and a secondary or costimulatory signal may also be required. In such cases, or alternatively, one or more CS domains may be included in the CY region of a CAR. A CS domain is a domain that transduces such a secondary or costimulatory signal. Optionally, the CAR of the present disclosure may comprise two or more CS domains.

[0336]

[0270] When a CAR or another synthetic immune receptor comprises both an ICS domain and a CS domain(s), the CS domain(s) may be placed upstream of the ICS domain or downstream of the ICS domain.

[0337]

[0271] In some embodiments, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Such a cytoplasmic signaling sequence may be contained in the ICS and / or the CS domain of a CAR or another synthetic immune receptor of the present disclosure.

[0338]

[0272] Examples of ITAM-containing primary cytoplasmic signaling sequences that are of particular use in the disclosure include those derived from an ICS domain of a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3 FcRy, FcR, CD3y, CD38, CD3e, CD5, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), Fc e Rl, DAP10, and DAP12.1143253.008213

[0339]

[0273] It is particularly preferred that the ICS domain in a CAR or another synthetic immune receptor of the disclosure comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0340]

[0274] In a preferred embodiment, the cytoplasmic domain of the CAR or another synthetic immune receptor may be designed to comprise the CD3ζICS domain by itself. In another preferred embodiment, the CD3ζICS domain may be combined with one or more of any other desired cytoplasmic domain(s) useful in the context of the receptor of the disclosure. For example, the cytoplasmic domain of the receptor can comprise a CD3z ICS domain and a CS domain.

[0341]

[0275] Various CS domains have been reported to confer differing properties. For example, the 4-1BB CS domain showed enhanced persistence in in vivo xenograft models (Milone M. C. et al. Mol Ther 2009;17:1453-1464; Song D. G. et al. Cancer Res 2011;71:4617-4627). Additionally, these different CS domains produce different cytokine profiles, which in turn, may produce effects on target cell-mediated cytotoxicity and the disease microenvironment. Indeed, DAP10 signaling in NK cells has been associated with an increase in Thl and inhibition of Th2 type cytokine production in CD8+ T cells (Barber A. et al. Blood 2011;117:6571-6581).

[0342]

[0276] Examples of co-stimulatory molecules include an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7- H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8 a, CD8 p, CDlla, LFA-1 (CDlla / CD18), CDllb, CDllc, CDlld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, CRTAM, 0X40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2R P, IL2R y, IL7R a, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, a ligand that specifically binds with CD83, and the like. Thus, while the disclosure is exemplified primarily with regions of CD28, DAP10, and / or 4-1BB as the CS domain, other costimulatory elements are within the scope of the disclosure.1143253.008213

[0343]

[0277] The ICS domain and the CS domain(s) of the CAR of the disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0344]

[0278] In one embodiment, the CAR is designed to comprise a cytoplasmic signaling sequence of CD3ζ as the ICS domain and comprise a cytoplasmic signaling sequence of CD28 as the CS domain. In another embodiment, the CAR is designed to comprise a cytoplasmic signaling sequence of CD3ζ as the ICS domain and comprise a cytoplasmic signaling sequence of DAP10 as the CS domain. In yet another embodiment, the CAR is designed to comprise a cytoplasmic signaling sequence of CD3ζ as the ICS domain and comprise a cytoplasmic signaling sequence of 4-1BB as the CS domain.

[0345] Receptor introduction

[0346]

[0279] When cells according to the present disclosure are modified to express an exogenous receptor, the modification may be via any means. For example, cells may be transfected, transduced, and / or transformed, transiently or permanently, using a nucleic acid encoding the exogenous receptor. Such a nucleic acid may be a vector.

[0347]

[0280] The vector may be, for example, a DNA vector or a RNA vector. The vector may be, for example, but not limited to, a plasmid, a cosmid, a viral replicon, or a viral vector. The viral vector may be a vector of a DNA virus, which may be an adenovirus, or an RNA virus, which may be a retrovirus or lentivirus. Types of vectors suited for expressing exogenous receptors are well known in the art (for example, see Rita Costa A. et al., EurJ Pharm Biopharm. 2010 Feb;74(2):127-38.; Frenzel A. et al. Front Immunol. 2013; 4: 217.).

[0348]

[0281] For expressing vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. This would be particularly beneficial for expressing immune receptor (e.g., CAR) constructs.

[0349]

[0282] In general, the expression of an immune receptor (e.g., CAR) from one or more nucleic acids is typically achieved by operably linking a nucleic acid encoding the immune receptor (e.g., CAR) to a promoter, and incorporating the construct into an expression vector.1143253.008213

[0350] The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired polynucleotide.

[0351]

[0283] The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U. S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector.

[0352]

[0284] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, y-retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U. S. Pat. No. 6,326,193).

[0353]

[0285] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0354] Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0355]

[0286] Various promoter sequences may be used, including, but not limited to the immediate early cytomegalovirus (CMV) promoter, the CMV-actin-globin hybrid (CAG) promotor, Elongation Growth Factor-la (EF-la), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene1143253.008213

[0356] promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0357] Further modifications -- cytokines / chemokines, receptor thereof, hybrid receptor, transcription factors

[0358]

[0287] Cells according to the present disclosure may be further modified, engineered, optimized, or appended in order to express one or more of certain cytokines, chemokines, cytokine receptors, chemokine receptors, or a hybrid receptors.

[0359]

[0288] In some embodiments, cells may be modified to express an immunostimulatory cytokine or chemokine (e.g., IL-2, IL-7. IFNγ, TNFa, IL-12, IL-18, IL-6, IL-6, IL-8, IL-18, GM-CSF, G-CSF, IFN-P, TNFp, etc).

[0360]

[0289] In some embodiments, cells may be modified to express an immunostimulatory cytokine receptor and / or chemokine receptor (e.g., receptor for IL-2, IL-7. IFNy, TNFa, IL-12, IL-18, IL-6, IL-6, IL-8, IL-18, GM-CSF, G-CSF, IFN-p, TNFp, or CXCR3).

[0361]

[0290] An exemplary hybrid receptors include a receptor which has an extracellular domain of a first receptor and an intracellular domain of a second receptor. When an immunostimulatory signal is desired in an immunosuppressive environment, the first receptor may be a receptor for an immunosuppressive molecule (e.g., PD-1, PDL1, IL-10, TGF-P, IL-4, IL-13, CTLA-4, IL-22, etc) and the second receptor may be a receptor for an immunostimulatory molecule (e.g., IL-2, IL-7. IFNy, TNFa, IL-12, IL-18, IL-6, IL-6, IL-8, IL-18, GM-CSF, G-CSF, IFN-, TNFP, etc) or an immunostimulatory receptor (e.g., a TLR, such as TLR4, FcyR), or an ITAM motif-containing receptor.

[0362]

[0291] Cells according to the present disclosure may be further modified, engineered, optimized, or appended in order to express a transcription factor.

[0363]

[0292] In some embodiments, the transcription factor may be one which is associated with or promotes development of memory T cells, e.g., Tscm cells. In certain embodiments, the transcription factor may be or comprise Foxol, TCF1, Bcl-6, EOMES, and / or Thpok.1143253.008213

[0364] Further modifications - inhibition of endogenous molecules

[0365]

[0293] Cells according to the present disclosure may be further modified, engineered, optimized, or appended in order to provide or select for various features. These features may include, but are not limited to, efficacy, persistence, target specificity, reduced immunogenicity, multi-targeting, enhanced immune response, expansion, growth, reduced off-target effect, reduced subject toxicity, improved target cytotoxicity, improved attraction of disease alleviating immune cells, detection, selection, targeting, and the like. For example, the cells may be engineered to have a suicide mechanism.

[0366]

[0294] In some embodiments, the cell mays further comprise a disruption to one or more endogenous genes. In some embodiments, the endogenous gene encodes TCRa, TCRp, MHO, MHCII, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein such as, for example, PD1 or CTLA4.

[0367]

[0295] In some embodiments, the cells are further modified to evade or neutralize the activity of immunosuppressive mediators, including, but not limited to prostaglandin E2 (PGE2) and adenosine. In some embodiments, this evasion or neutralization is direct. In other embodiments, this evasion or neutralization is mediated via the inhibition of protein kinase A (PKA) with one or more binding partners, for example ezrin. In a specific embodiment, the cells further express the peptide "regulatory subunit I anchoring disruptor" (RIAD). RIAD is thought to inhibit the association of protein kinase A (PKA) with ezrin, which thus prevents PKA's inhibition of TCR activation (Newick K. et al. Cancer Immunol Res. 2016 Jun;4(6):541-51.).

[0368]

[0296] In some embodiments, the cells of the disclosure may induce a broad immune response, consistent with epitope spreading.

[0369]

[0297] In some embodiments, the cells of the disclosure further comprise a homing mechanism. For example, the cell may transgenically express one or more stimulatory chemokines or cytokines or receptors thereof, in particular embodiments, the cells are genetically modified to express one or more stimulatory cytokines. In certain embodiments, one or more homing mechanisms are used to assist the inventive cells to accumulate more effectively to the disease site. In some embodiments, the cells are further modified to release inducible cytokines upon activation, e.g., to attract or activate innate immune cells to a targeted cell (so-called fourth generation CARs or TRUCKS).

[0370] Effector function and evaluation thereof1143253.008213

[0371]

[0298] The ceils according to the present disclosure may be further modified to improve persistence, effector functions, efficacy, and / or specificity against cells expressing an antigen of interest (a target cell). The target cell may be cancer cells, vascular cells, or any other target disease-associated cells. In some embodiments, the improved efficacy may be measured by increased cytotoxicity against cells expressing the antigen of interest, for example cytotoxicity against cancer cells. In some embodiments, the improved efficacy may also be measured by increased production of cytotoxic mediators such as, but not limited to, IFNγ, perforin, granzyme B, TNFa, and GM-CSF, e.g., upon exposure to an antigen of interest. In some embodiments, the improved efficacy may be shown by reduction in the signature cytokines of the diseases, or alleviated symptoms of the disease when the cells are administered to a subject. Other cytokines that may be reduced include TGF-beta, I L-6, IL-4, IL-10, and / or IL-13. In case of cancer, improved efficacy may be shown by better tumor cytotoxicity, better infiltration into the tumor, reduction of immunosuppressive mediators, reduction in weight decrease, reduction in ascites, reduction in tumor burden, and / or increased lifespan. In some embodiments, gene expression profiles may be also investigated to evaluate the efficacy.

[0372] Pharmaceutical compositions

[0373]

[0299] In one aspect the present disclosure provides pharmaceutical compositions comprising: (A) one or more isolated or recombinant lymphoid cells of the present disclosure or a cell population of the disclosure; and (B) a pharmaceutically acceptable carrier and / or excipient. In some embodiments, a pharmaceutical composition according to the present disclosure may be used for any of the in vivo and / or treatment methods described herein.

[0374] Administration routes and dosage forms

[0375]

[0300] The compositions of the present disclosure may be suited for administration in a number of ways depending upon whether local or systemic treatment is desired.

[0376]

[0301] In general, a composition may be suited for topical, parenteral, and / or enteral administration. In some embodiments, compositions of the present disclosure may be suited for topical, parenteral, or enteral administration. Preferably, compositions are suitable for administration via parenteral administration for example, subcutaneous, intramuscular, intraperitoneal or intravenous injection.1143253.008213

[0377]

[0302] Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parenterally- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Such formulation may be, for example, made of a biodegradable, biocompatible polymer, such as, but not limited to, ethylene vinyl acetate, poly(alkyl cyanoacrylates), poly(anhydrides), poly(amides), poly(ester), poly(ester amides), poly(phosphoesters), polyglycolic acid (PGA), collagen, polyorthoester, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), or naturally occurring biodegradable polymers such as chitosan and hyaluronic acid-based polymers (Kamaly N. et al, Chem Rev. Author manuscript; available in PMC 2017 Jul 13).

[0378]

[0303] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids,1143253.008213

[0379] semi-solids, monophasic compositions, multiphasic compositions (e.g., oil-in-water, water-in- oil), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0380]

[0304] Compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0381]

[0305] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0382]

[0306] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0383]

[0307] The pharmaceutical compositions of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0384]

[0308] The compositions of the present disclosure may be formulated to provide appropriate in vivo distribution of the active ingredient. In many cases, concentrating the distribution of an anti-tumor drug in the tumor site is challenging, and it can be so even when a drug has a specificity to a molecule expressed by cancer cells. Various strategies have been developed to address the issue and any appropriate strategies may be applied for the current disclosure (for example, reviewed in Rosenblum D. et al., Nat Commun. 2018 Apr 12;9(l):1410). For delivering a drug to the brain, the drug needs to cross the blood-brain barrier (BBB). Any appropriate strategies to enable BBB crossing may be utilized to for the delivery of any of the cells and pharmaceutical compositions of the present disclosure (see for example, Dong X. et al., Theranostics. 2018; 8(6): 1481-1493, for exemplary strategies).1143253.008213

[0385]

[0309] The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0386]

[0310] In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (U. S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97 / 30731), also enhance the cellular uptake of oligonucleotides.

[0387]

[0311] The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0388] Carriers and excipients

[0389]

[0312] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as1143253.008213

[0390] 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 interstitial 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.

[0391]

[0313] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0392]

[0314] Compositions of the present disclosure may include pharmaceutically acceptable excipient(s). Excipients included in the formulations will have different purposes depending, for example, on the subpopulation of cells used, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water- for- infection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents. The formulations comprising populations of the CAR-expressing cells of the present disclosure will typically have been prepared and cultured in the absence of any nonhuman components, such as animal serum (e.g., bovine serum albumin).1143253.008213

[0393]

[0315] The pharmaceutical composition in some embodiments can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[0394] Additional agents

[0395]

[0316] In some embodiments, the pharmaceutical composition may be used alone without an additional agent.

[0396]

[0317] In some embodiments, the pharmaceutical composition may be used with an additional agent.

[0397]

[0318] In certain embodiments, the additional agent(s) may be or may comprise a chemotherapeutic agent, gene therapy agent, DNA therapy agent, viral therapy agent, RNA therapy agent, immunotherapy agent, nanotherapy agent, monoclonal antibody, or a combination of the foregoing. In certain embodiments, the additional therapeutic agent(s) may be or may comprise an adjuvant or neoadjuvant. In certain embodiments, the additional therapeutic agent(s) may be or may comprise a small molecule enzymatic inhibitor or anti-metastatic agent.

[0398]

[0319] In certain embodiments, the additional therapeutic agent(s) may be or may comprise a 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, including but not limited to a neurokinin-1 receptor antagonist (NK1 RA), serotonin receptor antagonist (5-HT3 RA), dexamethasone, olanzapine, and palonosetron, etc.).

[0399]

[0320] Non-limiting exemplary additional agents may include a chemotherapy agent, an antibody-drug conjugate (ADC), an immunotherapy agent, and / or a biological modifier.

[0400]

[0321] In certain embodiments, chemotherapy agents may be selected from alkylating agents, antimetabolites, plant alkaloids, and anti-cancer antibiotics, further optionally one or more selected from cyclophosphamide, cisplatin, carboplatin, oxaliplatin, etoposide, irinotecan, lurbinectedin, paclitaxel, docetaxel, cabazitaxel, altretamine, capecitabine, gemcitabine, ifosfamide, melphalan, pemetrexed, topotecan, vinorelbine, mitoxantrone, ixabepilone, eribulin, estramustine, vinblastine, vincristine, 5-fluorouracil (5-FU), doxorubicin, epirubicin, dactinomycin, or a derivative thereof. In certain embodiments, chemotherapy1143253.008213

[0401] agents may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP).

[0402]

[0322] In certain embodiments, ADC may be selected from an anti-CD79b antibody drug conjugate (such as anti-CD79b-MC-vc-PAB-MMAE or the anti-CD79b antibody drug conjugate described in any one of U. S. Pat. No. 8,088,378 and / or US 2014 / 0030280, or polatuzumab vedotin), an anti-CD19 antibody drug conjugate, an anti-CD22 antibody drug conjugate, an anti-CD45 antibody drug conjugate, and an anti-CD32 drug conjugate. A biological modifier may be selected from a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), a PI3K-delta inhibitor (such as idelalisib (ZYDELIG®)), a PD-1 axis binding antagonist, an agonist, e.g., agonist antibody, directed against an activating co-stimulatory molecule, e.g., CD40, CD226, CD28, 0X40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1 BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127, an antagonist, e.g., antagonist antibody, directed against an inhibitory co-stimulatory molecule, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g,, 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA / B, GITR (e.g., TRX518) or arginase, ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675,206, urelumab (also known as BMS-663513), MGA271, an antagonist directed against a TGF beta, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and an adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR), e.g., adoptive transfer of a T cell comprising a dominant-negative TGF beta receptor, e.g., a dominant-negative TGF beta type II receptor.

[0403]

[0323] In certain embodiments, immunotherapy agents and / or chemotherapy agents may be or may comprise an immune checkpoint inhibitor and / or a growth factor or growth factor receptor inhibitor, optionally an inhibitor of PD-L1, PD-1, CTLA-4, VISTA, EGF, EGFR, VEGF, and / or VEGFR, or an antibody or antigen-binding fragment against PD-L1, PD-1, CTLA-4, VISTA, EGF, EGFR, VEGF, and / or VEGFR, or an antibody or antigen-binding fragment against a cancer antigen.

[0404]

[0324] In certain embodiments, the additional agent(s) may be or may comprise a chemotherapeutic agent, cytotoxic agent, an anti-hormonal agent, growth inhibitory agent, cytotoxic agent, agent used in radiation therapy, anti-angiogenesis agent, apoptotic agent, anti-tubulin agent, or other agent, such as a epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (TARCEVA™)),1143253.008213

[0405] platelet derived growth factor inhibitor (e.g., GLEEVEC™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferon, cytokine, antibody other than the anti-CD3 antibody of the disclosure, such as an antibody that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA VEGF, or VEGF receptor(s), TRAIL / Apo2, PD-1, PD-L1, PD-L2, or another bioactive or organic chemical agent. In certain embodiments, the additional therapeutic agent(s) may be or may comprise a glucocorticoid, optionally dexamethasone.

[0406]

[0325] A pharmaceutical composition may further contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs. Such agents or drugs may be, but are not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an enzyme, a hormone, a toxin, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, or an imaging drug. Specific examples are, for instance, but are not limited to, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0407] Therapeutic application - methods and uses

[0408]

[0326] Lymphoid cells and cell populations of containing lymphoid cells described herein, cells obtained by the methods described herein, and / or pharmaceutical compositions described herein can be used for the treatment of a disease, disorder, or condition in a subject or for the manufacture of a medicament for the treatment of a disease, disorder, or condition in a subject.

[0409]

[0327] Therefore, another aspect of the present disclosure provides therapeutic methods, such as methods of treating a disease, disorder, or condition in a subject and uses therefor.1143253.008213

[0410] The methods or the uses may comprise administering one or more isolated or recombinant cells or a cell population according to the present disclosure or a pharmaceutical composition according to the present disclosure to a subject.

[0411] Target diseases and conditions

[0412]

[0328] The disease, disorder, or condition which may be any appropriate disease, disorder, or condition.

[0413]

[0329] In some embodiments, the disease, disorder, or condition may be cancer. In certain embodiments, the cancer may be a solid cancer, optionally any of the solid cancers described herein. In certain embodiments, the cancer may be a liquid cancer, optionally any of the liquid cancers described herein. In particular cases, the cancer may be melanoma, skin cancer, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer. In particular cases, the cancer may be bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, desmoid tumor, esophageal cancer, fibromatosis, glioblastoma, head and neck cancer, liver cancer, lung cancer, oesophago-gastric adenocarcinoma, oligodendrioma, oral cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, small cell lung cancer, stomach cancer, and thyroid cancer.

[0414]

[0330] In some embodiments, the disease, disorder, or condition may be an autoimmune or inflammatory disease. In certain embodiments, the autoimmune disease may be any of those described herein.

[0415]

[0331] In some embodiments, the disease, disorder, or condition may be a neurodegenerative disease. In certain embodiments, the neurodegenerative disease may be any of those described herein.

[0416]

[0332] In some embodiments, the disease, disorder, or condition may be an infectious disease, such as viral, bacterial, fungal, yeast, protozoan, prion or parasitic infection. In certain embodiments, the infectious disease may be any of those described herein.

[0417] Subject

[0418]

[0419]

[0333] The subject referred to herein may be any living subject. In a preferred embodiment, the subject is a mammal. The mammal referred to herein can be any mammal as described herein. In some embodiments, the mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In particular embodiments, the mammals may be humans.1143253.008213

[0420]

[0334] The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects, in some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing toxic outcomes such as cytokine release syndrome (CRS).

[0421]

[0335] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another immunotherapy and / or other therapy. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.

[0422]

[0336] In some embodiments, the methods include administration of a cell, a cell population, or composition according to the present disclosure to a subject, tissue, or cell, such as one having, at risk for, or suspected of having a disease, condition or disorder associated with a target molecule of interest, such as cancer. In some embodiments, the cells and / or compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells and / or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some embodiments, the methods thereby treat, e.g., ameliorate one or more symptoms of the disease or condition, for example, by reducing, inhibiting, or inactivating target cells expressing an antigen of interest.

[0423] Cell origin

[0424]

[0337] For purposes of the methods of cell therapy, wherein host cells or populations of cells are administered, the cells can be cells that are xenogeneic, allogeneic or autologous to the subject.

[0425]

[0338] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some embodiments, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0426]

[0339] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise1143253.008213

[0427] prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0428]

[0340] In certain embodiments, where cells are T cells and are not autologous to the subject, the expression of the cell's endogenous T cell receptors (TCRs) may be suppressed or disrupted. The endogenous TCR expression may be suppressed via any appropriate technique, for example, by silencing any compartment of the endogenous TCRs using tools such as, but not limited to, an siRNA, shRNA, micro RNA, or artificial microRNA. Alternatively, TCR gene may be disrupted or deleted via any appropriate technique, for example using the CRISPR / Cas system, transcription activator-like effector nucleases (e.g., TALEN®), or Zinc finger nucleases (ZFNs). The suppression or disruption of TCR may allow for reduction or prevention of undesired effects in which the TCRs recognize antigens in the subject as foreign and cause immune responses against the subject, an immune attack often called as graft-versus-host disease (GVHD).

[0429]

[0341] In certain embodiments, where cells (donor cells) are not autologous to the subject, the expression of endogenous MHC or HLA gene(s) may be suppressed or disrupted, which may be achieved via any appropriate technique, such as but not limited to, an siRNA, shRNA, micro RNA, artificial microRNA, or gene editing using the CRISPR / Cas system, transcription activator-like effector nucleases (e.g., TALEN®), or Zinc finger nucleases (ZFNs). The suppression or disruption of MHC or HLA gene(s) may allow for reduction or prevention of undesired effects in which the subject's endogenous T cells recognize the donor cell's antigens presented on donor cell's MHC molecules as foreign and cause immune responses against the donor cells and increase the persistence of the administered cells within the subject.

[0430] Functional activity

[0431]

[0342] In one embodiment, the present disclosure includes a type of cellular therapy where isolated cells are treated with (I) a PI3K-AKT-mTOR pathway inhibitor and (II) ( 11-1) a Wnt / p-catenin pathway activator and / or (11-2) an acetyl-CoA synthesis inhibitor, and the cell is infused into a subject in need thereof. Such administration can promote activation of the cells (e.g., T cell activation), optionally in a manner specific to an antigen of interest (e.g., when the cells1143253.008213

[0432] express a receptor specific to the antigen of interest) such that the cells of the disease or disorder are targeted for destruction, in the case where the cells are or comprise T cells, the T cells may replicate in vivo resulting in long-term persistence that may lead to sustained control of diseases, disorders, or conditions associated with the antigen of interest, such as cancer.

[0433]

[0343] In one embodiment, the isolated cells of the disclosure can undergo in vivo expansion and / or are long-living and can persist for an extended amount of time. In another embodiment, where the isolated ceils are or comprise a T cell, the T cells may be or may evolve into specific memory T ceils that can be reactivated to inhibit growth of any additional cells expressing the antigen of interest. T cells may present or further differentiate in vivo into a central memory-like state upon encounter and subsequent elimination of target cells expressing the antigen. Similarly, in certain embodiments, where the isolated cells are B cells, the isolated B cells may be or further evolve into memory B cells that can be reactivated to inhibit the growth of any cells expressing the antigen of interest.

[0434]

[0344] Once the cells are administered to a subject (e.g., a human), the biological activity of the engineered cell populations is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and / or secretion of certain mediators, such as GM-CSF, IL-6, RANTES (CCL5), TNF-a, IL-4, IL-10, IL-13, IFN-y, granzyme B, perforin, CD 107a, or IL-2.

[0435]

[0345] In some embodiments the biological activity elicited by a cell or a composition described herein is measured by assessing clinical outcome, such as the reduction in disease symptoms. In case of cancer, improved efficacy may be shown by better infiltration of diseaseresolving immune cells into the tumor, reduced tumor sizes, or reduced ascites. In some embodiments, gene expression profiles may be also investigated to evaluate the activity. Target ceils1143253.008213

[0436]

[0346] Cells that may be targeted by any cells, cell populations, and / or pharmaceutical compositions according to the present disclosure include any cells expressing an antigen of interest. The target cell may be present in any part of the body of a subject, including blood or lymphatic circulation, and disease-affected tissues. For example, when the target disease is solid cancer, the disease-affected tissues include, but are not limited to, the skin, breast, brain, bone, pancreas, testis, cervix, endometrium, ovary, stomach, colon, rectum, lung, liver, mesothelium, or tongue, or another oral tissue. Alternatively, target cells may blood cells or hematopoietic cells.

[0437]

[0347] Preferably, the cells of the disclosure are used to treat cancer, wherein the target antigen of interest is upregulated. In particular, the cells of the disclosure may be used to treat pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.

[0438]

[0348] In general, cells that are positive for a target antigen of interest may be identified via known methods, for example, immunofluorescence or flow cytometry using specific antibodies, or alternatively, through cytotoxicity against target cells. Methods of testing a cell or an immune receptor expressed thereon for the ability to recognize target cells and for antigen specificity are known in the art. For instance, Clay et al., J. Immunol., 163: 507-513 (1999), teaches methods of measuring the release of cytokines (e.g., interferon-y, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL-2)). In addition, CAR function can be evaluated by measurement of cellular cytotoxicity, as described in Zhao et al., J. Immunol., 174: 4415-4423 (2005).

[0439]

[0349] A biopsy is the removal of tissue and / or cells from an individual. Such removal may be to collect tissue and / or cells from the individual in order to perform experimentation on the removed tissue and / or cells. This experimentation may include experiments to determine if the individual has and / or is suffering from a certain condition or disease-state. The condition or disease may be, e.g., cancer. With respect to detecting the presence of cells of the present disclosure in a host, the sample comprising cells of the host can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. If the sample comprises whole cells, the cells can be any cells of the host, e.g., the cells of any organ or tissue, including blood cells or endothelial cells.

[0440] Method and / or mode of administration1143253.008213

[0441]

[0350] The administration route used in the method of the present disclosure may be any appropriate route, which depends upon whether local or systemic treatment is desired.

[0442]

[0351] In general, administration may be topical, parenteral, or enteral.

[0443]

[0352] Preferably, the administration may be parenteral, for example, via subcutaneous, intramuscular, intraperitoneal or intravenous injection.

[0444]

[0353] In some cases, the administration may be direct injection into a diseased site, such as but not limited to intratumoral injection.

[0445]

[0354] In the case of adoptive cell therapy, methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; U. S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0446]

[0355] In some embodiments, the composition of the present disclosure may be administered using any appropriate medical devices (for example, reviewed in Richter B. B., J. BioDrugs (2018) 32: 425).

[0447] Dosing

[0448]

[0356] For administration of any of the cells, nucleic acids, vectors, and compositions of the present disclosure, the dosage will vary and depend on, for example, the target disease, the severity of the disease, the route of administration, and pharmacokinetic factors. Dosing may be modified based on the response observed in the subject.

[0449]

[0357] Dosing frequency may be, for example, three times per day, twice per day, once per day, every other day, once per week, every other week, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per seven weeks, once per eight weeks, once per nine weeks, once per ten weeks, once per three months, once per four months, once per six months, once per year, or even less frequent.

[0450]

[0358] The pharmaceutical composition in some embodiments contains cells of the present disclosure in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the1143253.008213

[0451] treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0452]

[0359] In certain embodiments, a subject may be administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and / or such a number of cells per kilogram of body weight of the subject. For example, in some embodiments the administration of the cells or population of cells can comprise administration of about 100 to about 200 cells per kg body weight including all integer values of cell numbers within those ranges.

[0453]

[0360] The cells or population of cells can be administrated in one or more doses. In some embodiments, said effective amount of cells can be administrated as a single dose. In some embodiments, said effective amount of cells can be administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In some embodiments, an effective amount of cells or composition comprising those cells are administrated parenterally.1143253.008213

[0454] In some embodiments, administration can be an intravenous administration. In some embodiments, administration can be directly done by injection into the disease site.

[0455]

[0361] For purposes of the disclosure, the amount or dose of the inventive cells and / or compositions administered should be sufficient to effect a therapeutic or prophylactic response in the subject or animal over a reasonable timeframe. For example, the dose of the inventive cells and / or compositions should be sufficient to bind to antigen, or detect, treat or prevent disease in a period of from about 2 hours or longer, e.g., about 12 to about 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive anti-ALPPL2 material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0456]

[0362] For purposes of the disclosure, an assay, which comprises, for example, comparing the extent to which target cells are lysed or, I FN-y is secreted by T cells expressing the inventive CSR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal, among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or I FN-y is secreted upon administration of a certain dose can be assayed by methods known in the art.

[0457] Combination therapy and additional steps

[0458]

[0363] In some embodiments, the cells and / or compositions of the present disclosure are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells and / or compositions are co-administered with another therapy sufficiently close in time such that the cells and / or compositions enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells and / or compositions are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells and / or compositions are administered after the one or more additional therapeutic agents. Furthermore, the compositions of the present disclosure may1143253.008213

[0459] be given to a subject along with one or more of other therapies, which may be surgery, or a radiotherapy.

[0460]

[0364] In some embodiments, the subject is further administered with an additional agent, which may be administered together with or separately from (e.g., at the same time or at separate times) one or more cells according to the present disclosure. In certain embodiments, the additional agent may comprise a cytokine such as IL-2, IL-7, IL-12, etc, or a cytokine complex, such as IL-2 complexed with anti-IL-2 antibody or with an IL-2 receptor (or a subunit thereof), IL-7 complexed with anti-IL-7 antibody or with an IL-7 receptor (or a subunit thereof), IL-12 complexed with anti-IL-12 antibody or with an IL-12 receptor (or a subunit thereof), etc.

[0461]

[0365] In particular embodiments, the additional agent may comprise IL-2 or an IL-2 complex. In some cases, an IL-2 complex may be or comprise IL-2 complexed with an anti-IL-2 antibody. For example, binding of the antibody to the IL-2 may block binding between IL-2 and an IL-2 receptor or one or more subunits thereof (e.g., one or more of a, P, or y subunits). In some cases, an IL-2 complex may be or comprise IL-2 complexed with an IL-2 receptor or one or more subunits thereof (e.g., one or more of a, P, or y subunits). For example, binding of IL-2 receptor or one or more subunit thereof may block binding between IL-2 and an IL-2 receptor or between IL-2 and a particular IL-2 receptor subunit of interest, in which case binding of the IL-2 to an IL-2 receptor may be skewed towards a specific IL-2 receptor subtype. High affinity IL-2 receptor, often expressed on Treg cells and recently activated T cells, is comprised of a, p, and y subunits (i.e., CD25, CD122, and CD132, respectively), while IL-2 receptor of intermediate affinity, often expressed on CD8+ memory T cells, resting T cells, and cytotoxic CD8+ T cells, is comprised of p and y subunits (i.e., CD122 and CD132, respectively), and low affinity IL-2 receptor, which may be found on activated T cells, is comprised of a subunit and is capable of mediating trans-presentation of IL-2 to IL-2 receptor comprised of P and y subunits (i.e., CD25). The y subunit (i.e., CD132) is shared among several cytokine receptors such as IL-2 receptor, IL-7 receptor, IL-4 receptor, IL-9 receptor, IL-15, and IL-21 receptor. The P subunit (i.e., CD122) is shared between IL-2 receptor and IL-15 receptor.

[0462]

[0366] In some embodiments, in cell therapy, a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of cells. In an example, the lymphodepleting chemotherapy is administered to the subject prior to administration of the cells. For example, the lymphodepleting chemotherapy ends 1-1143253.008213

[0463] 4 days (e.g., 1, 2, 3, or 4 days) prior to cel I infusion. In embodiments, multiple doses of cells are administered, e.g., as described herein. In embodiments, a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of a cell described herein. Examples of lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc. Examples of lymphodepleting agents include, but are not limited to, anti-thymocyte globulin, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD52 antibodies, anti-CD2 antibodies, TCRaP blockers, anti-CD20 antibodies, anti-CD19 antibodies, Bortezomib, rituximab, anti-CD154 antibodies, rapamycin, CD3 immunotoxin, fludarabine, cyclophosphamide, busulfan, melphalan, Mabthera, Tacrolimus, alefacept, alemtuzumab, OKT3, OKT4, OKT8, OKT11, fingolimod, anti-CD40 antibodies, anti-BR3 antibodies, Campath- 1H, anti-CD25 antibodies, calcineurin inhibitors, mycophenolate, and steroids, which may be used alone or in combination.

[0464] Kits

[0465]

[0367] Also provided herein are kits.

[0466]

[0368] A kit according to the present disclosure may comprise (A) one or more lymphoid cells or a population of cells comprising one or more lymphoid cells; and (B) (I) a PI3K-AKT-mTOR pathway inhibitor; and (II) (ll-l) a Wnt / -catenin pathway activator; and / or (11-2) an acetyl-CoA synthesis inhibitor.

[0467]

[0369] The PI3K-AKT-mTOR pathway inhibitor may be any agent which inhibits the PI3K-AKT-mTOR pathway, optionally any of those disclosed herein. The Wnt / P-catenin pathway activator may be any agent which activates the Wnt / -catenin pathway activator, optionally any of those disclosed herein. The acetyl-CoA synthesis inhibitor may be any agent which inhibits acetyl-CoA synthesis, optionally any of those disclosed herein.

[0468]

[0370] The one or more lymphoid cells or a population of cells comprised in the kit may be any appropriate cells or a population of any appropriate cells, such as but not limited to those disclosed herein, e.g., cell type, subtype, origin, species, etc disclosed herein.

[0469]

[0371] The kit may be, for example a kit for producing cells or cell populations according to the present disclosure, of for use in any of the methods of treating according to the present1143253.008213

[0470] disclosure. Therefore, in some embodiments, the kit may further comprise a label, which optionally includes an instruction for performing such a method.

[0471]

[0372] When a kit is for use in a method of producing according to the present disclosure, the kit may be for producing cells for cell therapy. The resultant cells or a population of cells may be or may comprise T cells presenting a phenotype of and / or expressing one or more markers associated with memory T cells, such as Tscm cells.

[0472] Definitions

[0473]

[0373] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0474]

[0374] Although various embodiments and examples of the present invention have been described referring to certain molecules, compositions, methods, or protocols, it is to be understood that the present invention is not limited to the particular molecules, compositions, methods, or protocols described herein, as theses may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0475]

[0375] It should be understood that, unless clearly indicated otherwise, in any methods disclosed or claimed herein that comprise more than one step, the order of the steps to be performed is not restricted by the order of the steps cited.

[0476]

[0376] Throughout this disclosure, numerical features are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and1143253.008213

[0477] are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0478]

[0377] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0479]

[0378] It must also be noted that, unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" as used herein and in the appended claims include plural refence. Thus, the reference to "a cell" refers to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the reference to "a nucleic acid" refers to one or more nucleic acid molecules and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by a person of skilled in the art.

[0480]

[0379] As used herein, the term "about" or "approximately" when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 10%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values in between (e.g., 91, 92, 93, 99, 99.1, 99.2, 99.3, 99.4, 100, 100.8, 100.9, 101, 106, 107, 108, 109, etc.).

[0481]

[0380] It is understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments. Transitional phrases such as "comprising," "including," "having," "containing," "involving," "composed of," and the like are to be understood to be open-ended, namely, to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0482]

[0381] It will be further understood that all transitional terms such as "comprises," "comprising," "including," "having," "containing," "involving," "composed of," and the like, when used in this specification, are to be understood to be open-ended, namely, to specify the presence of stated features, integers, steps, operations, elements, and / or components, but not to preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Only the transitional phrases1143253.008213

[0483] "consists of" or "consisting of" shall be closed transitional phrases. The semi-closed transitional phrase "consists essentially of" or "consisting essentially of" shall be understood to specify the presence of stated features, integers, steps, operations, elements, and / or components and to allow the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof as long as they do not materially affect the basic characteristics of stated features, integers, steps, operations, elements, and / or components. For example, in some embodiments, "cells consisting essentially of T cells" may encompass a population of cells about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of which are T cells.

[0484]

[0382] The term "allogeneic" as used herein refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0485]

[0383] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0486]

[0384] A standard form of "antibody" or "immunoglobulin (lg)" molecules may comprise two heavy chains and two immunoglobulin light chains or multiple units each comprising two pairs of heavy and light chains interconnected by disulfide bonds. Antibodies may be of one of the five major classes, IgA, IgD, IgE, IgG, and IgM and may be further classified based on the subclasses (isotypes), e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, s, y, and [i, respectively. Each heavy chain is comprised of: a heavy chain variable domain (VH); and a heavy chain constant region (CH), which is typically comprised of a CHI domain, a hinge, a CH2 domain and a CH3 domain.

[0487]

[0385] The term "apheresis" as used herein refers to the art-recognized extracorporeal process by which the blood of a donor or patient is removed from the donor or patient and passed through an apparatus that separates out selected particular constituent(s) and returns the remainder to the circulation of the donor or patient, e.g., by re-transfusion. Thus, in the context of "an apheresis sample" refers to a sample obtained using apheresis. In some embodiments, PBMCs may be obtained from an apheresis sample, T cells (or a specific subset1143253.008213

[0488] thereof) may be purified from the PBMCS, and cells according to the present disclosure may be manufactured according to a method described herein.

[0489]

[0386] ATP-citrate lyase (ACLY) is an enzyme which is responsible for endogenous acetyl CoA synthesis. ACLY catalyzes conversion of citrate and Coenzyme A (CoA) into acetyl-CoA and oxaloacetate via ATP hydrolysis. Acetyl CoA also has a role in transcription regulation as a transcription co-factor. For example, Acetyl CoA can alter the conformation of and thereby stabilize histone acetyltransferase, which results in increased histone acetylation and thus increased transcription. Human ACLY is encoded by the ACLY gene on chromosome 17 with the gene location 17q21.2 in humans. Human ACLY may for example have the amino acid sequences provided as NCBI Reference Sequence: NP_001087.2.

[0490]

[0387] The term "autologous" or "donor-derived" as used herein refers to any material derived from the same individual to whom it is later to be re-introduced.

[0491]

[0388] The term " B cell receptor" or " BCR" as used herein includes native BCRs as well as functional BCR variants. In its native form, the BCR are typically expressed on B cells as a membrane-bound immunoglobulin (mlg), which comprises two immunoglobulin heavy chains (each comprising a VH and a heavy chain constant region) and two immunoglobulin light chains (each comprising a LH and a CL) as described above, wherein each heavy chain further comprises a transmembrane domain and a short cytoplasmic domain. Typically, BCRs non-covalently associate with a heterodimer of Iga and Igp to form a BCR complex. The Iga / lgp heterodimer is important in conveying BCR signaling, e.g., via the ITAM motif(s). In some embodiments, BCRs which may be used as an immune receptor herein may comprise a combination of VH and VL or an antigen binding site advantageously capable of specifically recognizing a target antigen of interest. In certain embodiments, the combination of VH and VL or the antigen binding site of a BCR used herein may be replaced with a combination of V domains of a TCR specific to a target antigen of interest. In certain embodiments, one or more of the VH and / or VL or the antigen binding site of a BCR used herein may be replaced with or an antibody fragment such as but not limited to a scFv or a Fab. In some cases, such a BCR may comprise two or four scFvs. In certain embodiments, the VH and / or VL or the antigen binding site of a BCR may be replaced with a ligand specific for a receptor that is a target molecule of interest. In certain embodiments, the VH and / or VL or the antigen binding site of a BCR may be replaced with a receptor (or a fragment thereof) specific to a ligand that is a target molecule of interest. In certain embodiments, the VH and / or VL or the antigen binding1143253.008213

[0492] site of a BCR may be replaced with a substrate specific to an enzyme that is a target molecule of interest. In certain embodiments, the VH and / or VL or the antigen binding site of a BCR may be replaced with an enzyme (or a fragment thereof) specific to a substrate that is a target molecule of interest.

[0493]

[0389] " Cancer" refers to a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies; with more particular examples including squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases. In certain embodiments, cancers that are amenable to treatment by cytokine hybrid proteins of the disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer1143253.008213

[0494] (CRC), and hepatocellular carcinoma. Yet, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers. In other embodiments, the cancer is selected from a class of mature B-Cell cancers excluding Hodgkin's Lymphoma but including germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cell leukemia, Splenic lymphoma / leukemia, unclassifiable, Splenic diffuse red pulp small B-cell lymphoma, Hairy cell leukemia variant, Waldenstrom macroglobulinemia, Heavy chain diseases, a Heavy chain disease, y Heavy chain disease, p Heavy chain disease, Plasma cell myeloma, Solitary plasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicular lymphoma, Primary cutaneous follicle centre lymphoma, T-cell / histiocyte rich large B-cell lymphoma, Primary DLBCL of the CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, Lymphomatoid granulomatosis, Primary mediastinal (thymic) large B-cell lymphoma, Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Primary effusion lymphoma: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0495]

[0390] The term " CD28" refers to the protein Cluster of Differentiation 28, one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. Human CD28 protein may have at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI Reference No: NP_006130 or a fragment thereof that has stimulatory activity. The term " CD28 transmembrane domain," also referred to as " CD28 TM domain" or " CD28TM" refers to the amino acid residues derived from the transmembrane domain of CD28. The term " CD28 costimulatory domain," also referred to as " CD28CS domain" or " CD28CS," refers to the amino acid residues derived from the cytoplasmic domain of CD28.1143253.008213

[0496]

[0391] The term " CDS zeta," or alternatively, "zeta," "zeta chain," " CDS-zeta," " CD3z," or " CD247," is a protein encoded by the CD247 gene on chromosome 1, with gene location lq24.2, in humans. CD3 zeta, together with T cell receptor (TCR) and CDS (a protein complex composed of a CDS gamma, a CD3 delta, and two CDS epsilon), forms the TCR complex. Human CDS zeta may have an amino acid sequence provided as NP_000725 or NP_932170, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. The term " CDS zeta intracellular signaling domain," or alternatively " CDS zeta ICS domain" or a " CDSzICS," is defined as the amino acid residues from the cytoplasmic domain of the CDS zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation.

[0497]

[0392] The term " Chimeric Antigen Receptor" or alternatively a " CAR" refers to a polypeptide which when present on a cell, typically an immune effector cell, provides the cell with specificity for a target molecule of interest or a cell expressing such a molecule ("target cell"), and with intracellular signal generation upon binding to the target molecule of interest. In some embodiments, a CAR comprises at least an extracellular antigen recognition domain, a transmembrane domain (TM domain) and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain (ICS domain)") comprising a functional signaling domain of or derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some embodiments, the stimulatory molecule may be CD3, FcRy, FcR, CD3y, CD36, CDSs, CD5, CD22, CD79a, CD79b or CD66d. In particular embodiments, the ICS domain may be derived from CD3. In some embodiments, the cytoplasmic portion of a CAR further comprises one, two, or more costimulatory domains (CS domains) each comprising one or more functional signaling domains (same or different) derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is individually chosen from the costimulatory molecules described herein, e.g., CD28, 4-1BB (i.e., CD137), DAP10, CD8, CD27, ICOS, or 0X40 (CD134). In some embodiments, the ICS domain and / or the CS domain may comprise an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the antigen recognition domain may comprise an antibody fragment (e.g., scFv, Fab, F(ab'), F(ab')2, or Fv, etc.) specific to a target molecule of interest. In some embodiments, the antigen recognition domain may comprise a combination of V domains of or an antigen-binding site of a TCR specific to a target molecule of interest. In some embodiments, the antigen recognition domain may comprise a ligand specific to a receptor that is a target1143253.008213

[0498] molecule of interest. In some embodiments, the antigen recognition domain may comprise a receptor (or a fragment thereof) specific to a ligand that is a target molecule of interest. In some embodiments, the antigen recognition domain may comprise a substrate specific to an enzyme that is a target molecule of interest. In some embodiments, the antigen recognition domain may comprise an enzyme (or a fragment thereof) specific to a substrate that is a target molecule of interest. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR may comprise a hinge between the antigen recognition domain and the TM domain. In certain embodiments, the hinge domain may comprise an immunoglobulin (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, lgA2, IgM, IgD or IgE), CH2CH3 region of immunoglobulin and optionally portions of CDS, and CD8a. In some embodiments, the CAR further comprises a leader sequence atthe N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain during cellular processing and localization of the CAR to the cellular membrane.

[0499]

[0393] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to a protein selected from the group consisting of an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CD5, CD7, CD8alpha, CD8beta, CDlla, LFA-1 (CDlla / CD18), CDllb, CDllc, CDlld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLA1, VLA-6, and a ligand that specifically binds with CD83. In embodiments wherein a CAR comprises one or more CS domain, each CS domain comprises1143253.008213

[0500] a functional signaling domain derived from a costimulatory molecule. In some embodiments, the CS domain comprises 4-1BB, CD28, or DAP10.

[0501]

[0394] The term "cytokines" as used herein refers to a broad category of small proteins that are involved in cell signaling. Generally, their release has some effect on the behavior of cells around them. Cytokines may be involved in autocrine signaling, paracrine signaling, and / or endocrine signaling as immunomodulating agents. Cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a broad range of cells, including but not limited to immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, epithelial cells, and various stromal cells. " Chemokines" are a family of cytokines generally involved in mediating chemotaxis.

[0502]

[0395] The term "cytotoxicity" generally refers to any cytocidal activity resulting from the exposure of a cell according to the present disclosure to cells expressing a target molecule of interest ("target cell"). This activity may be measured by any appropriate cytotoxicity assays, including measurement of release of a cytocidal molecule IFN-y, perforin, or granzyme B or measurement of luminescence released by live cells. When the target cell is a cancer or tumor cell, the term "anti-cancer cytotoxicity" or "anti-tumor cytotoxicity" may be used.

[0503]

[0396] The term " DAP10" refers to a protein, which in humans is encoded by the HSCT gene. It may also be referred to as HSCT, KAP10, PIK3AP, or hematopoietic cell signal transducer. In some embodiments, DAP10 may have the sequence provided in Genbank Accession No.: Q9UBK5.1. The term " DAP10 costimulatory domain," also referred to as " DAP10 CS domain" or " DAP10CS," refers to the amino acid residues derived from the cytoplasmic domain of DAP10.

[0504]

[0397] An "effective amount" of a cell disclosed herein or a composition (e.g., pharmaceutical composition) described herein, is at least the minimum amount required to achieve the desired therapeutic or prophylactic result, e.g., a measurable improvement (e.g., in a symptom, severity, grade, or progression) of or prevention of a particular disease, disorder, or condition, e.g., a cell proliferative disorder, e.g., cancer, preferably with minimal or no toxic or detrimental effects. An effective amount may vary according to inter alia disease state, age, sex, and weight of the patient, and the ability of the active ingredient (e.g., cell) to elicit a desired response in the individual and, in some instances, by co-administering one or more additional therapeutic agents._For therapeutic use, beneficial or desired results include clinical1143253.008213

[0505] results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and / or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. An "effective amount" may be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0506]

[0398] The terms "enteral,” "enterally," "oral," "orally," "non-parenteral," "non- parenterally," and the like, refer to administration of a compound or composition to an individual by a route or mode along the alimentary canal. Examples of "oral" routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.

[0507]

[0399] The term "exogenous" or "exogenously added" as used herein in reference to a PI3K-AKT-mTOR pathway inhibitor, a Wnt / -catenin pathway activator, and / or an acetyl-CoA synthesis inhibitor, a culture of one or more cells (e.g., cells to be engineered, being engineered, or have been engineered to express an immune receptor specific to an antigen of interest) indicates that the inhibitor and / or activator is / are not released into the culture by said one or more cells but rather are from another source.1143253.008213

[0508]

[0400] Glycogen synthase kinase-3 (GSK-3) a serine / threonine protein kinase and has a role in various biological activities such as glycogen metabolism, cell signaling, and cell transport. The GSK-3 family has two isozymes: GSK-3a and GSK-3P, which are encoded by two homolog genes. Human GSK-3a and GSK-3p are encoded by the GSK3A gene on chromosome 19 with the gene location 19ql3.2 and the GSK3B gene on chromosome 3 with the gene location 3ql3.33, respectively, in humans. Human GSK-3aand GSK-3p may for exampie have the amino acid sequences provided as NCBI Reference Sequences: NP_063937.2 and NP_002084.2, respectively. GSK-3 piays negatively regulates the Wnt / P-catenin pathway. In the canonical Wnt pathway, Wnt activates its cognate receptor Fz, which activates the phosphoprotein Dishevelled (Dvl). Activated Dvl releases GSK-3 from the APC / Axin / GSK-3 complex, and GSK-3 phosphorylates and thereby destabilizes P-catenin stabilizes. Cytoplasmic P-catenin can no longer be translocated to the nucleus, and thus downstream transcription activities (e.g., mediated by TCF / LEF transcription factor) are suppressed.

[0509]

[0401] The term "immune cell" refers to a cell of hematopoietic origin functionally involved in the initiation and / or execution of innate and / or adaptive immune response.

[0510]

[0402] The term "immune receptor" specific to a target molecule of interest as used herein is a polypeptide or a combination of polypeptides which (A) comprises (a) an extracellular domain which is capable of binding to an antigen of interest and (b) a transmembrane domain and (B) (i) further comprises an intracellular signaling domain capable of transducing a signal in a cell and / or (ii) is capable of associating with another polypeptide (e.g., CDS, CD3<(, Iga, Igp, etc) or a combination of polypeptide (e.g., a CD3 complex, an Igct / lgP complex) which comprises an intracellular signaling domain capable of transducing a signal in an immune cell. In some embodiments, the signal may activate the cell and / or elicit certain function of the cell (e.g., cytokine production, cytotoxicity, etc). In some embodiments, an immune receptor may comprise at least one domain of or derived from antibodies, TCRs (or the associated CD3 complex), BCRs (or the associated Igct / lgP complex), and / or costimulatory molecules (e.g., CD28, etc). For example, such domain may be a VH, a VL, a CHI, a CH2, a CH3, a CL, a V domain of a TCR, C domain of a TCR, and / or a cytoplasmic portion (or a fragment thereof) of a costimulatory molecule.

[0511]

[0403] The term "intracellular signaling domain" or " ICS domain" as used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the cell transduced with a polynucleotide1143253.008213

[0512] comprising a CAR, e.g., a CAR T cell. Examples of immune effector function, e.g., in a CAR T cell, include cytolytic activity and helper activity, including the secretion of cytokines. In some embodiments, ICS domains may comprise or may be an ICS domain of a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3 zeta, FcR gamma, FcR beta, CDS gamma, CD3 delta, CDS epsilon, CD5, CD22, CD28, CD79a, CD79b, CD66d, CD278(ICOS), Fc epsilon Rl, 4-1BB, DAP10, or DAP12.

[0513]

[0404] An "isolated" biological component (such as an isolated protein, nucleic acid, vector, or cell) refers to a component that has been substantially separated or purified away from its environment or other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins may be "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis. An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as, for example, in a host cell.

[0514]

[0405] The term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice, rats, and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).

[0515]

[0406] The term "nucleic acid" or "polynucleotide" refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps,1143253.008213

[0516] substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis, recombinantly, or derived from a microorganism.

[0517]

[0407] The term "parenteral" or "parenterally" as used herein includes any route of administration of a compound or composition, characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In a preferred embodiment, parenteral administration of the compositions of the present invention comprises subcutaneous or intraperitoneal administration.

[0518]

[0408] A "pharmaceutical composition" refers to a preparation in such form as to permit the biological activity of an active ingredient contained therein, such as a cell described herein, to be effective and which preferably contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.

[0519]

[0409] A "pharmaceutical carrier," as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, and absorption delaying agents that are physiologically compatible. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In one embodiment, the carrier is suitable for parenteral, intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of1143253.008213

[0520] the present disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the carrier may be a liquid, in which an active therapeutic agent is formulated. The excipient generally does not provide any pharmacological activity to the formulation, though it may provide chemical and / or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, Gennaro, A. editor, 19th edition, Philadelphia, PA: Williams and Wilkins (1995), which is incorporated by reference.

[0521]

[0410] Phosphoinositide 3-kinase ( PI3 K), also referred to as phosphatidylinositol-3 kinase or PI3 kinase, is an enzyme that transmits signals in cells and have a role in controlling cell growth. The PI3K family is classified into Classes I through IV. Class I PI3Ks are a heterodimer of a regulatory subunit (often p85) and a catalytic subunit (pllO). There are four variants of the pllO catalytic subunit: pllOa, pliop, pllOy, and pll08, which are encoded by separate Pi3kc genes. PI3K comprising pllOa, pliop, pllOy, and pll08 are referred to herein as PI3K a isoform, P isoform, y isoform, and 8 isoform, respectively. The 8 isoform is expressed primarily in leukocytes. Human PI3K catalytic subunit pll06 is encoded by the PIK3CD gene on chromosome 1, with the gene location lp36.22 in humans. Human PI3K catalytic subunit pll06 may for example have the amino acid sequence provided as NCBI Reference Sequence: NP_005017.3. PI3K plays a pivotal role in the PI3K-AKT-mTOR pathway, and activation of PI3K results in activation of downstream AKT.

[0522]

[0411] As used herein, the term "polypeptide" refers to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0523]

[0412] A "promoter" as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species or cell-type specific, tissue-specific, or species specific. Promoters may be "constitutive," meaning continually active, or "inducible," meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the present disclosure are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent1143253.008213

[0524] gene expression and may be located in the 5' or 3' regions of the native gene. " Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0525]

[0413] As used herein, the term "protein" refers to a compound comprised of one or more polypeptides. When a protein comprises two or more polypeptides, the polypeptides may covalently be joined (e.g., disulfide bond) to or noncovalently paired (e.g., hydrogen bond) with each other to form a complex.

[0526] The term "recombinant" generally refers to any protein, polypeptide, polynucleotide, or cell encoding and / or expressing a gene of interest that is produced by genetic engineering methods. Therefore, the term "recombinant" as used with respect to a protein or polypeptide, means a protein or polypeptide produced by expression of a recombinant polynucleotide. " Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. The term "host cell" refers to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. In some embodiments, a cell according to the present disclosure may be host cell. The term "recombinant cell" or "host cell" refers to cells into which an exogenous nucleic acid sequence has been introduced, including the progeny of such cells. Such cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.

[0527]

[0414] Protein kinase B, " PKB," or " AKT" as used herein is a serine / threonine-specific protein kinas and has a role in various cellular pathways such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. The AKT family has three isoforms: AKT1, ALK2, and AKT3, which are encoded by separate Akt genes. Human AKT1, AKT2, and AKT3 are encoded by the AKT1 gene on chromosome 14 with the gene location 14q32.33, the AKT2 gene on chromosome 19 with the gene location 19ql3.2, and the AKT3 gene on chromosome 1 with the gene location Iq43-q44, respectively, in humans. Human AKT1, AKT2, and AKT31143253.008213

[0528] may for example have the amino acid sequences provided as NCBI Reference Sequences: NP_001014431.1, NP_001617.1, and NP_005456.1, respectively. AKT plays a pivotal role in the PI3K-AKT-mTOR pathway. Activated PI3K phosphorylates phosphatidylinositol bisphosphate (PIP2) to form phosphatidylinositol trisphosphate (PIP3), which recruits to the membrane AKT (via AKT's pleckstrin homology (PH) domain) and phosphoinositide-dependent kinase 1 (PDK1). PDK1 activates AKT via phosphorylation, and activated AKT inhibits transcription various factors including Foxo.

[0529]

[0415] The term "scFv," “single-chain Fv," or “single-chain variable fragment" refers to a fusion protein comprising at least one antibody fragment comprising a VH and at least one antibody fragment comprising a VL, wherein the VH and VL are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. The linker may comprise portions of the framework sequences. In scFvs, the VH may be placed upstream of the VL, and the two domains may optionally be linked via a linker (for example, the G4S X3 linker). Alternatively, the VH may be placed downstream of the VL, and the two domains may optionally be linked via a linker (for example, the G4S X3 linker).

[0530]

[0416] The term "signaling domain" refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0531]

[0417] The term "subject" as used herein may be any living organisms, preferably a mammal. In some embodiments, the subject is a primate such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the patient or subject is a validated animal model for disease and / or for assessing toxic outcomes. The subject may also be referred to as “patient" in the art. The subject may have a disease or may be healthy.

[0532]

[0418] The term “synthetic immune receptor" or “SIR" as used herein refers to a non-natu rally occurring set of polypeptides, typically two in the some embodiments, which when expressed1143253.008213

[0533] in an effector cell (e.g., an immune cell, such as but not limited to a T cell, a NK cell, a B cell, a NK T cell, a macrophage, a dendritic cell, etc) provides the cell with specificity for a target molecule of interest and / or a target cell expressing such target molecule of interest (e.g., a cancer cell) and with intracellular signal generation. In some embodiments, a SIR comprises one or more antigen recognition domains (e.g., antibody or antibody fragment, a ligand or a receptor, etc) that bind to a target molecule of interest, and are joined to one or more T cell receptor constant chains or regions via an optional linker. In certain embodiments, the set of polypeptides are contiguous with each other. In certain embodiments, a SIR comprises two or more sets of two or more polypeptides. The polypeptides of each set of SIR are contiguous with each other (functional polypeptide unit 1) but are not contiguous with the polypeptides of the other set (functional polypeptide unit 2). In some aspects, the T cell receptor constant chains (or regions) of the SIR is chosen from the constant chain of human T cell receptor-alpha (TCR-alpha or TCRa or TCRa or hTCR-alpha or hTCRa or hTCRa or Ca), human T cell receptor-betal (TCR-betal or TCRpi or TCRbl or hTCR-betal or hTCRpi or hTCRbl or cpi), human T cell receptor-beta 2 (TCR-beta2 or TCRp2 or TCRb2 or hTCR-beta2 or hTCRP2 or hTCRb2 or CP2 also designated TCR-beta, TCRp orTCRb or Cp), human Pre-T cell receptor alpha (preTCR-alpha or preTCRa or preTCRa or preCa), human T cell receptor-gamma (TCR-gamma orTCRy orTCRg or hTCR-gamma or hTCRy or hTCRg or hTCRyl or hTCRgammal, or Cy), or human T cell receptor-delta (TCR-delta or TCRd or TCR6 or hTCR-delta or hTCRd or hTCRS or C6). In some embodiments, the TCR constant chains of SIR are encoded by their wild-type nucleotide sequences while in other embodiments the TCR constant chains of SIR are encoded by the nucleotide sequences that are not wild-type. The TCR constant chain / domain of a SIR can be derived from the same species in which the SIR will ultimately be used. For example, for use in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or comprised of human TCR constant chains. However, in some instances, it is beneficial for the TCR constant chain to be derived from the same species in which the SIR will ultimately be used in, but modified to carry one or more amino acid substitutions that enhance the expression of the TCR constant chains. For example, for use in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or comprised of human TCR constant chains but in which certain amino acids are replaced by the corresponding amino acids from the murine TCR constant chains. Such murinized TCR constant chains may provide increased expression of the SIR. The SIR orfunctional portion thereof, can include additional amino acids1143253.008213

[0534] at the amino or carboxy terminus, or at both termini, which additional amino acids are not found in the amino acid sequence of the TCR or antigen binding domain which make up the SIR. Desirably, the additional amino acids do not interfere with the biological function of the SIR or functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent SIR. Exemplary SIRs include but not limited to those described in US20220204582A1 and US20210137977A1.

[0535]

[0419] The term "target molecule" or "target molecule of interest," also referred to as "antigen of interest" or "cognate antigen," as used herein refers to a molecule or an antigen that is targeted by an immune receptor that a cell according to the present disclosure is engineered to express. In some embodiments, the target molecule may be a molecule associated with a disease, disorder, or condition such as cancer.

[0536]

[0420] The term " T cell receptor" or " TCR" as used herein includes native TCRs as well as functional TCR variants. In its native form, the TCR are typically expressed on T cells as a heterodimer of alpha (TCRa) and beta (TCRp) chains (" TCRaP") or as a heterodimer of gamma (TCRy) and delta (TCRS) chains (" TCRyS"). Each chain comprises one N-terminal immunoglobulin-like (Ig)-variable (V) domain (referred to as " Va", " VP", " Vy", and " VS" for TCRa, TCRP, TCRy, and TCRS chains, respectively), one Ig-constant-like (C) domain (referred to as " Ca", " CP", " Cy", and " C8" for TCRa, TCRp, TCRy, and TCRS chains, respectively), a connecting domain, a transmembrane domain anchoring the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminal end. Naive TCRs comprise a single antigen¬ binding site, formed by Va and VP domains in case of TCRap or by Vy and VS domains in case of TCRyS, and recognize antigenic peptides bound to ("presented / displayed on") major histocompatibility complex (MHC) molecules (pMHCs).

[0537]

[0421] Each V domain of a TCR comprises three CDRs (CDR1, CDR2, and CDR3) and a hypervariable region (HV4), which have increased amino acid variability and may participate in recognition of a pMHC. In some cases, CDR1, CDR2, and HV4 interact with the MHC molecule itself, while CDR3 preferentially contacts with the antigenic peptide bound to the MCH molecule, thereby providing specificity (i.e., the ability to recognize and interact with a specific antigen). In some cases, CDR1 and / or CDR2 interact with part of the presented peptide, while CDRS contacts with MCH, and HV4 may not have a major contribution to pMHC binding.1143253.008213

[0538]

[0422] Typically, TCRs non-covalently associate with a CDS complex to form a TCR complex. The CDS complex is important in (i) TCR signaling, e.g., via the ITAM motif(s) and (ii) TCR surface expression. The CD3 complex typically contains three dimers made up of variable combinations of CD3y, CD36, CD3e, CD3, and CD3q polypeptides. The y chain of a Fc receptor can occasionally participate in the CD3 complex due to its structural similarity to CD3(J. In some cases, a CDS complex may be composed of one heterodimer of CDSe and CD36, one heterodimer of CDSe and CD3y, and one homodimer of CD3. In some cases, a CD3 complex may be composed of one heterodimer of CD3e and CD38, one heterodimer of CDSe and CDSy, and one heterodimer of CD3(J and CD3q. In some cases, a CDS complex may be composed of one heterodimer of CD3e and CD38, one heterodimer of CDSe and CDSy, and one heterodimer of CD3 and FceRly.

[0539]

[0423] In some embodiments, TCRs which may be used as an immune receptor herein may comprise V domains or an antigen binding site advantageously capable of specifically recognizing a target antigen of interest, in particular a target antigen of interest in its MHC bound form. In certain embodiments, the one or more V domains of a TCR used herein may be replaced with an antibody fragment specific to a target antigen of interest, such as but not limited to a scFv or a Fab. In some cases, such a TCR may comprise two scFvs. In certain embodiments, the two V domains of a TCR may be replaced with a combination of VH and VL of an antibody specific to a target antigen of interest. In some cases, such a TCR (optionally in combination with a CDS complex) may be a SIR. In certain embodiments, the V domain(s) of a TCR may be replaced with a ligand specific for a receptor that is a target molecule of interest. In certain embodiments, the V domain(s) of a TCR may be replaced with a receptor (or a fragment thereof) specific to a ligand that is a target molecule of interest. In certain embodiments, the V domain(s) of a TCR may be replaced with a substrate specific to an enzyme that is a target molecule of interest. In certain embodiments, the V domain(s) of a TCR may be replaced with an enzyme (or a fragment thereof) specific to a substrate that is a target molecule of interest.

[0540]

[0424] The term "transfected," "transformed," or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.1143253.008213

[0541]

[0425] By the term "transmembrane domain" or " TM domain / ' what is implied is any three- dimensional protein structure which is thermodynamically stable in a membrane. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an integral protein.

[0542]

[0426] As used herein, the term "treat," "treatment," or "treating" generally refers to the clinical procedure for reducing or ameliorating the progression, severity, and / or duration of a disease or of a condition, or for ameliorating one or more conditions or symptoms (preferably, one or more discernible ones) of a disease. The type of disease or condition to be treated may be, for example, but are not limited to, cancer and cancer-associated diseases and conditions. In specific embodiments, the effect of the "treatment" may be evaluated by the amelioration of at least one measurable physical parameter of a disease, resulting from the administration of one or more cells and / or composition according to the present disclosure. The parameter may be, for example, gene expression profiles, the mass of disease-affected tissues, inflammation-associated markers, cancer-associated markers, the number or frequency of disease-associated cells, tumor / cancer burden, the presence or absence of certain cytokines or chemokines or other disease-associated molecules, the presence or absence of a certain cell type e.g., a certain immune cells such as T cells or a specific subset thereof, and may not necessarily discernible by the patient. In other embodiments "treat", "treatment," or "treating" may result in the inhibition of the progression of a disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms "treat", "treatment" and "treating" refer to the reduction or stabilization of cancerous tissue or cells. Additionally, the terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete cure or prevention. Rather, there are varying degrees of treatment effects or prevention effects of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention effects of a disease in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented.1143253.008213

[0543] Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0544]

[0427] The term "tumor" as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.

[0545]

[0428] A "vector" is a compound or a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, viruses, and virus¬ like particles (VLPs). Thus, the term "vector" includes an autonomously replicating plasmid, a self-replicating RNA, or a viral particle. The term should also be construed to include non¬ plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. An "expression vector" is a vector, e.g., plasmid, minicircle, viral vector, liposome, and the like as discussed herein or as known in the art, comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell. An expression vector also comprises control elements, e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc., operatively linked to the encoding region to facilitate expression of the gene product in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0546]

[0429] The term "xenogeneic" or "xeno-" refers to a graft derived from an animal of a different species.

[0547]

[0430]

[0548]

[0431] Examples are provided below to illustrate embodiments of the present disclosure. These examples are not meant to constrain the present invention to any particular application or theory of operation.1143253.008213

[0549] EXAMPLES

[0550] Example 1: Analyses on OT-I T cells treated with a PI3K-AKT-mTOR pathway inhibitor, a Wnt / P-catenin pathway activator, and / or an acetyl-CoA synthesis inhibitor.

[0551] Example 1-1: Cell count, viability, and T cell phenotype upon treatment with 30 pM CAL, 5 iM TW, and / or 5 mM 2HC, with 25 U / mL IL-2 and 1 pg / mL SIINFEKL.

[0552] Methods

[0553]

[0432] On Day 0, splenocytes were harvested from OT-I mice and a cell culture was initiated in the presence of 25 U / mL IL-2 and 1 pg / mL SIINFEKL peptide, with or without (i) 5 pM TWS119 (also referred to as TW) ("5TW"), a Wnt / p-catenin pathway activator, and / or (ii) 5 mM 2-hydroxycitrate (also referred to as 2HC), an acetyl-CoA synthesis inhibitor. 3 hours later, (iii) 30 pM idelalisib (also referred to as CAL-101 or CAL) ("30CAL"), a PI3K-AKT-mTOR pathway inhibitor, was added to the culture of appropriate treatment groups. None of CAL, TW, and 2HC were added to cells of Control group (Ctrl). On Days 3 and 5, cell number and viability were evaluated, SIINFEKL peptide was removed, cells were split for further culture, and IL-2 and CAL / TW / 2HC treatments were simultaneously replenished at the same concentrations as those used on Day 0.

[0554]

[0433] On Day 7, cells were harvested, and cell number and viability were evaluated. Cells were then stained with antibodies against CD8, CD62L, CD44, CD127, CD95, PD-1, Sca-1, Lag-3, Slamf6, CCR7, TCF1, Foxol, CD27, Tim-3, and CX3CR1, conjugated with different fluorescent tags. Cell staining was measured by flow cytometry, and expression of CD62L, CD44, CD127, CD95, PD-1, Sca-1, Lag-3, Slamf6, CCR7, TCF1, Foxol, CD27, CD8, Tim-3, and CX3CR1 on CD8+ T cells were analyzed using FLOWJO®.

[0555] Results

[0556]

[0434] Cell counts on Days 3, 5, and 7 are shown in FIG. 1A, comparing all treatment groups (top) or comparing Ctrl, 30CAL, 5TW, and 5TW+30CAL groups (botom).

[0557]

[0435] Cell viability values (%) on Days 3, 5, and 7 for individual treatment groups are shown in FIG. IB, comparing all treatment groups (top) or comparing Ctrl, 30CAL, 5TW, and 5TW+30CAL groups (bottom).1143253.008213

[0558]

[0436] MFI values for CD62L, CD44, CD127, CD95, PD-1, Sca-1, Lag-3, Slamf6, CCR7, Foxol, CD27, CD8, Tim-3, and CX3CR1 staining on CD8+ T cells on Day 7 are shown in FIG. 1C, comparing Ctrl, 30CAL, 5TW, and 5TW+30CAL groups.

[0559]

[0437] MFI values for Foxol, TCF1, PD-1, CDS, Lag-3, CD27, CD127, Sca-1, CD62L, Tim-3, Slamf6, CD44, CX3CR1, CCR7, and CD95 staining on CD8+ T ceils on Day 7 are shown in FIG.

[0560] ID, comparing Ctrl, 30CAL, 2HC, and 2HC+30CAL groups.

[0561] Example 1-2: Cell count, viability, and T cell phenotype upon treatment with 10 or 30 pM CAL, 5 pM TW, and / or 5 mM 2HC, with 10 U / mL IL-2 and 1 pg / mL SIINFEKL.

[0562]

[0438] In this Example, a reduced IL-2 concentration of 10 U / mL was used, and a reduced CAL concentration of 10 pM was included.

[0563] Methods

[0564]

[0439] On Day 0, splenocytes were harvested from OT-I mice and CD8+ cells were isolated. A CD8+ cell culture was initiated in the presence of 10 U / mL IL-2 and 1 pg / mL SIINFEKL peptide. To that (i) 5 pM TW ("5TW") and / or (ii) 5 mM 2HC immediately, and / or (iii) 3 hours later, 10 or 30 pM CAL ("10CAL" or "30CAL", respectively) were added to the culture of appropriate treatment groups. None of CAL, TW, and 2HC were added to cells of Control group (Ctrl). On Days 3 and 5, cell number and viability were evaluated, SIINFEKL peptide was removed, cells were split for further culture, and IL-2 and CAL / TW / 2HC treatments were simultaneously replenished at the same concentrations as those used on Day 0.

[0565]

[0440] On Day 6, cells were harvested, and cell viability was evaluated. Cells were then stained with antibodies against CD3, CD4, CD8, CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF1, Foxol, Tim-3, Lag-3, and PD-1, conjugated with different fluorescent tags and L / D NIR. Cell staining was measured by flow cytometry, and expression of CD62L, CD44, CX3CR1, CD27, CD8, CD127, Sca-1, CD95, CCR7, Slamf6, TCFl, Foxol, Tim-3, Lag-3, and PD-1 on CD3+CD8+ T cells were analyzed using FLOWJO®.

[0566] Results

[0567]

[0441] Cell counts on Days 3, 5, and 6 are shown in FIG. 2A, comparing all treatment groups (top) or comparing Ctrl, 30CAL, 5TW, 5TW+10CAL, and 5TW+30CAL groups (bottom).

[0568]

[0442] Cell viability values (%) on Days 3, 5, and 6 for individual treatment groups are shown in FIG. 2B, comparing all treatment groups (top) or comparing Ctrl, 30CAL, 5TW, 5TW+10CAL, and 5TW+30CAL groups (bottom).1143253.008213

[0569]

[0443] MFI values for CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, CD8, Slamf6, Foxol, Tim-3, Lag-3, and PD-1 staining on CD3+CD8+ T cells on Day 6 are shown in FIG. 2C, comparing Ctrl, 30CAL, 5TW, 5TW+10CAL, and 5TW+30CAL groups.

[0570]

[0444] MFI values for Foxol, TCF1, PD-1, CDS, Lag-3, CD27, CD127, Sca-1, CD62L, Tim-3, Slamf6, CD44, CX3CR1, CCR7, and CD95 staining on CD8+ T cells on Day 6 are shown in FIG.

[0571] 2D, comparing Ctrl, 30CAL, 2HC, and 2HC+30CAL groups.

[0572] Example 1-3: Cell viability and T cell phenotype upon treatment with 10 pM CAL and 1, 2, 3, or 5 pM TW, with 10 U / mL IL-2 and 1 pg / mL SIINFEKL.

[0573]

[0445] In this Example, effects of different TW concentrations and different TW addition timings on cell viability were compared.

[0574] Methods

[0575]

[0446] On Day 0, splenocytes were harvested from OT-I mice. A splenocyte culture was initiated at 106cells / mL on a 24-well plate in the presence of 10 U / mL IL-2 and 1 pg / mL SIINFEKL peptide. To compare effects of different TW concentrations, some wells further received or did not receive (i) 1, 2, 3, or 5 pM TW (1TW, 2TW, 3TW, or 5TW) immediately at culture initiation and / or (ii) 10 pM CAL (10CAL) 3 hours after culture initiation. To allow for comparison of effects of different TW addition timings, 3 hours after culture initiation, other wells received (i) 3 pM TW and 10 pM CAL ("3hr 3TW+10CAL") or (ii) 3 pM TW ("3hr 3TW"). Neither CAL nor TW was added to cells of Control group (Ctrl). On Days 3 and 5, cell number and viability were evaluated, SIINFEKL peptide was removed, cells were split for further culture, and IL-2 and CAL / TW treatments were simultaneously replenished at the same concentrations as those used on Day 0.

[0576]

[0447] On Day 6, cells were harvested, and cell viability were evaluated. Cells were then stained with antibodies against CD8, CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF1, Tim-3, Lag-3, PD-1, conjugated with different fluorescent tags, and L / D ghost UV450. Cell staining was measured by flow cytometry, and expression of CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF7, Tim-3, Lag-3, and PD-1 on CD8+ T cells were analyzed using FLOWJO®.

[0577] Results

[0578]

[0448] Cell viability values (%) and cell counts on Days 3, 5, and 6 for individual treatment groups are shown in FIG. 3A (left and right, respectively), comparing Ctrl, 10CAL, 1TW, 2TW,1143253.008213

[0579] 3TW, 5TW, 1TW+10CAL, 2TW+10CAL, 3TW+10CAL, 5TW+10CAL, 3hr 3TW, and 3h 3TW+10CAL groups.

[0580]

[0449] MFI values for CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF1, Tim-3, Lag-3, and PD-1 staining on CD8+ T cells on Day 6 are shown in FIG. 3B, comparing Ctrl, 10CAL, 1TW, 2TW, 3TW, 5TW, 1TW+10CAL, 2TW+10CAL, 3TW+10CAL, 5TW+10CAL, 3hr 3TW, and 3hr 3TW+10CAL groups.

[0581] Example 1-4: Cell number, viability, and size and T cell phenotype upon treatment with 10 or 30 pM CAL, 3 pM TW, and / or 5 mM 2HC, with 10 U / mL IL-2 and 1 pg / mL SIINFEKL.

[0582]

[0450] This Example is similar to Example 1-3 but 30 pM CAL and 5 mM 2HC were also included.

[0583] Methods

[0584]

[0451] On Day 0, splenocytes were harvested from OT-I mice. A splenocyte culture was initiated at 106cells / mL on a 24-well plate in the presence of 10 U / mL IL-2 and 1 pg / mL SIINFEKL peptide, and some wells additionally got (i) 3 pM TW (3TW) or (ii) 5 mM 2HC. 3 hours later, (iii) 10 or 30 pM CAL (10CAL or 30CAL, respectively) was added to the culture of appropriate treatment groups. None of CAL, TW, and 2HC were added to cells of Control group (Ctrl). On Day 3, SIINFEKL peptide was removed. On Days 3 and 5, cell number, viability, and sizes were evaluated using ViCellBLU cell counter, cells were split for further culture, and IL-2 and CAL / TW / 2HC treatments were simultaneously replenished at the same concentrations as those used on Day 0.

[0585]

[0452] On Day 6, cells were harvested, and cell number, viability, and sizes were evaluated. Cells were then stained with (i) antibodies against CD8, CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF7, Tim-3, Lag-3, and PD-1, conjugated with different fluorescent tags, and L / D NIR or (ii) antibodies against CD8, CD69, CD103, CD122, EOMES, CD25, Bcl2, LEF1, CXCR6, Tbet, CXCR3, Granzyme B, and pAKT (ser473), conjugated with different fluorescent tags, and L / D NIR. Cell staining was measured by flow cytometry, and expression of CD8, CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF1, Tim-3, Lag-3, PD-1, CD69, CD103, CD122, EOMES, CD25, Bcl2, LEF1, CXCR6, Tbet, CXCR3, Granzyme B, and pAKT (ser473) on CD8+ T cells were analyzed using FLOWJO®, and principal component analyses (PCA) on different treatment group and group combinations were performed using eigenvalues based on Iog2 transformed MFI values using GraphPad Prism™. Uniform Manifold1143253.008213

[0586] Approximation and Projection (UMAP) analyses were also performed using FLOWJO®. Replicates were concatenated and an equal number of cells from each group was used to make the UMAPs. see also e.g., https: / / umap- learn. readthedocs.io / sn / latest / parameters.html.

[0587] Results

[0588]

[0453] Cell diameters on Day 5, and cell count (xlO6 / mL) and viability values (%) on Days 3, 5, and 6 are shown in FIG. 4A (top, middle left, and bottom left, respectively), and CD8+ cell count and frequency data on Day 6 are shown in FIG.4A (middle and bottom right), comparing Ctrl, 10CAL, 30CAL, 2HC, 3TW, 2HC+30CAL, 3TW+10CAL, and 3TW+30CAL groups.

[0589]

[0454] MFI values for CD62L, CD44, CX3CR1, CD27, CD127, Sca-1, CD95, CCR7, Slamf6, TCF1, Tim-3, Lag-3, and PD-1, CD69, CD103, CD122, EOMES, and CD25 staining on CD8+ T cells on Day 6 are shown in FIG. 4B, comparing Ctrl, 10CAL, 3TW, and 3TW+10CAL groups.

[0590]

[0455] MFI values for CD8, PD-1, CD127, CX3CR1, CD69, CD103, CCR7, CD95, TCF1, CD44, CD27, CD62L, Slamf6, Sca-1, Tim-3, Lag-3, EOMES, CD122, and CD25 staining on CD8+ T cells on Day 6 are shown in FIGS. 4C-4D, comparing Ctrl, 30CAL, 2HC, and 2HC+30CAL groups.

[0591]

[0456] MFI values for Bcl2, LEF1, CXCR6, Tbet, CXCR3, Granzyme B, and pAKT (ser473) staining on CD8+ T cells on Day 6 are shown in FIG. 4E, comparing Ctrl, 10CAL, 30CAL, 2HC, 3TW, 2HC+30CAL, 3TW+10CAL, and 3TW+30CAL groups.

[0592] Example 1-5: T cell phenotype upon treatment with 10 pM CAL, 1 pM AKTi, 5 pM AKTi, and 3 pM TW, with 10 U / mL IL-2 and 1 pg / mL SIINFEKL.

[0593] Methods

[0594]

[0457] On Day 0, splenocytes were harvested from OT-I mice and a cell culture was initiated in the presence of 10 U / mL IL-2 and 1 pg / mL SIINFEKL peptide, with or without (I) 3 pM TW ("3TW"). 3 hours later, (ii) 0.75 pM, 3.75 pM, or 7.5 pM AKT inhibitor VIII (also referred to as AKTi) ("0.75AKTi," "3.75AKTi," or "7.5AKTi," respectively) or (iii) 7.5 pM CAL ("7.5CAL") was added to the culture of appropriate treatment groups. None of CAL, TW, and AKTi were added to cells of Control group (Ctrl). On Days 3 and 5, cell number and viability were evaluated, SIINFEKL peptide was removed, cells were split for further culture, and IL-2 and CAL / TW / AKTi treatments were simultaneously replenished at the same concentrations as those used on Day 0, except that 1 pM AKTi was used instead of 0.75 pM AKTi, 5 pM AKTi was used instead of1143253.008213

[0595] 3.75 pM AKTi, 10 pM AKTi was used instead of 7.5 pM AKTi, and 10 pM CAL was used instead of 7.5 ptM CAL.

[0596]

[0458] On Day 6, cells were harvested, and cell number and viability were evaluated. Cells were then stained with antibodies against CD8, CD62L, CD44, CD127, CD95, CD69, PD-1, Sca-1, Lag-3, Slamf6, CCR7, TCF1, CD27, CD103, Tim-3, CXCR3, GranzymeB, Foxol, LEF1, Blimpl, Tigit, and Tox, conjugated with different fluorescent tags, and L / D NIR. Cell staining was measured by flow cytometry, and expression of CD62L, CD44, CD127, CD95, CD69, PD-1, Sca-1, Lag-3, Slamf6, CCR7, TCF1, CD27, CD103, Tim-3, CXCR3, GranzymeB, Foxol, LEF1, Blimpl, Tigit, and Tox, on CD8+ T cells were analyzed using FLOWJO®.

[0597] Results

[0598]

[0459] Cell count (xlO6 / mL) and viability values (%) on Days 3 and 6 are shown in FIG. 5A, comparing Ctrl, 0.75AKTI, 3.75AKTi, 7.5AKTi, 7.5CAL, 3TW, 3TW+7.5CAL, 3TW+0.75AKTi, 3TW+3.75AKTi, and 3TW+7.5AKTi groups.

[0599]

[0460] MFI values for CD62L, CD44, CD127, CD95, CD69, PD-1, Sca-1, Lag-3, Slamf6, CCR7, TCF1, CD27, CD103, Tim-3, CXCR3, GranzymeB, Foxol, LEF1, Blimpl, Tigit, and Tox, staining on CD8+ T cells on Day 6 are shown in FIG. 5B-5D, comparing Ctrl, 0.75AKTi, 3.75AKTi, 7.5CAL, 3TW, 3TW+7.5CAL, 3TW+0.75AKTi, and 3TW+3.75AKTI groups.

[0600] Example 2: Effects of chronic antigen exposure on the phenotype of OT-I T cells treated with a PI3K-AKT-mTOR pathway inhibitor, a Wnt / 3-catenin pathway activator, and / or an acetyl-CoA synthesis inhibitor.

[0601]

[0461] In this Example, effects of repeated exposure to a cognate antigen on the phenotype of OT-1 T cells treated with CAL, TW, and / or 2HC were tested.

[0602] Methods

[0603]

[0462] On Day 0, splenocytes were harvested from OT-I mice (Thyl.l+). An OT-I splenocyte culture was initiated in the presence of 10 U / mL IL-2 and 1 pg / mL SIINFEKL peptide, and some wells additionally got (i) 3 pM TW (3TW) or (ii) 5 mM 2HC. 3 hours later, (iii) 10 or 30 pM CAL (10CAL or 30CAL, respectively) was added to the culture of appropriate treatment groups. None of CAL, TW, and 2HC were added to cells of Control group (Ctrl). On Day 3, SIINFEKL peptide was removed, and on Days 3, 5, 7, and 9 cells were split for further culture. On Days 3, 5, 7, and 9, IL-2 and CAL / TW / 2HC treatments were simultaneously replenished at the same concentrations as those used on Day 0. On Day 7, splenic antigen-presenting cells (APCs) from1143253.008213

[0604] a non-OT-l mouse (Thyl.2+) were treated with SI INFEKL for 2 hours and added to the OT-1 splenocytes in culture at a ratio of 1:1 ratio to stimulate the OT-I splenocytes. On Days 8 and 9, 1 pg / ml SI I NFEKL peptide was added to the culture to restimulate the OT-I splenocytes.

[0605]

[0463] On Days 8 (before Sil N FEKL restimulation) and 10, cells (part of the cultured cells in case of Day 8) were harvested and stained with antibodies against CDS, CD127, CD95, CX3CR1, CCR7, TCF1, CD44, CD62L, CD27, Slamf6, PD-1, Tim-3, Lag-3, Thyl.l (marker for OT-I strain), Sca-1, CD45.1, and Live / Dead marker, conjugated with different fluorescent tags. Cell staining was measured by flow cytometry, and expression of CD127, CD95, CX3CR1, CCR7, TCF1, CD44, CD62L, CD27, Slamf6, PD-1, Tim-3, Lag-3, Thyl.l, Sca-1, and CD45.1 on Thyl.l+CD8+ T cells were analyzed using FLOWJO®, and principal component analyses (PCA) on different treatment group and group combinations were performed using parallel analyses or eigenvalues based on Iog2 transformed MFI values using GraphPad Prism™.

[0606] Results

[0607]

[0464] MFI values for TCF1, CD95, CCR7, CD62L, CD44, Sca-1, CD127, CD27, CX3CR1, PD-1, Tim-3, Lag-3, and Slamf6 staining on Thyl.l+CD8+T cells on Days 8 and 10 are shown in FIGS.

[0608] 6A and 6B, comparing Ctrl, 10CAL, 3TW, and 3TW+10CAL groups.

[0609]

[0465] MFI values for TCF1, Slamf6, CD127, CCR7, CD62L, PD-1, Tim-3, Lag-3, Sca-1, and CD44 staining on Thyl.l+CD8+T cells on Days 8 and 10 are shown in FIG. 6C, comparing Ctrl, 30CAL, 2HC, and 2HC+30CAL groups.

[0610]

[0466] PCA results comparing Ctrl, 10CAL, 30CAL, 3TW, 2HC, 3TW+10CAL, and 2HC+30CAL groups are shown in FIG.6D. PCA results comparing Ctrl, 10CAL, 3TW, and 3TW+10CAL groups are shown in FIG. 6E. PCA results comparing Ctrl, 30CAL, 2HC, and 2HC+30CAL groups are shown in FIG. 6F. The PC scores by parallel analyses indicated that: major markers driving T cell phenotype differences between 3TW+10CAL vs single treatment and control groups were CD95, TCF1, CD44, CD62L, and CCR7 on Day 8 and CX3CR1, Slamf6, CD44, and Tim-3 on Day 10; and major markers driving T cell phenotype differences between 2HC+30CAL vs single treatment and control groups were CD62L and CCR7 on Day 8 and CD62L, TCF1, and Slamf6 on Day 10.

[0611] Example 3: Therapeutic effects in a cancer mouse model by OT-I T cells treated with a PI3K-AKT-mTOR pathway inhibitor and / or a Wnt / p-catenin pathway activator.1143253.008213

[0612] Example 3-1: Tumor model diagram, tumor burden over time, and statistical differences between groups.

[0613] Methods

[0614]

[0467] B16-OVA cancer cell preparation and cancer model

[0615]

[0468] On Day -5, B16-OVA mouse melanoma cells were thawed and seeded at 106cells cells / flask. On Day -1, cell culture medium was changed for further culturing. On Day 0, B16-OVA cells were harvested, and 250,000 cells were intradermally administered to each of 20 mice.

[0616]

[0469] OT-1 cell preparation

[0617]

[0470] On Day 1, splenocytes were harvested from OT-I mice (Thyl.l+). An OT-I splenocyte culture was initiated in 24 well plates in the presence of 10 U / mL IL-2 and 1 pg / mL SI IN FEKL peptide, and some wells had (i) 3 pM TW (3TW) added to the culture. 3 hours later, (ii) 10 pM CAL (10CAL) was added to the culture of appropriate treatment groups. Neither CAL nor TW were added to cells of Control group (Ctrl). On Day 4, SI I N FEKL peptide was removed. On Days 4 and 6, cells were split for further culture, and IL-2 and CAL / TW treatments were replenished simultaneously at the same concentrations as those used on Day 1.

[0618]

[0471] Mouse treatment and monitoring

[0619]

[0472] On Day 6, mice were irradiated at 500 cGy. On Day 7, 1x10sOT-I cells treated or not treated with CAL and / or TW were administered to mice harboring B16-OA cancer cells in the dermis via retro-orbital injection. Dermal tumor sizes were measured every other day. Statistical differences in the tumor sizes on Days 23, 26, 28, and 30 were evaluated by two- way ANOVA analyses using GraphPad Prism™.

[0620]

[0473] The experimental scheme is visually explained in FIG. 7 (top).

[0621] Results

[0622]

[0474] Changes in the tumor size are shown in FIG. 7 (bottom left), comparing Ctrl, 10CAL, 3TW, and 3TW+10CAL groups. Results of the statistical analyses are summarized in FIG. 7 (bottom right). While tumors in single treatment groups (10CAL or 3TW) were statistically not different from those in the control group (Ctrl), tumors in 3TW+10CAL group were statistically smaller than those of 10CAL single treatment group on Days 28 and 30 than those of the 3TW single treatment group and the control groups on Days 23, 26, 28, and 30.1143253.008213

[0623] Example 3-2: Tumor model diagram, tumor burden over time, and statistical differences between groups with different amount of T cells transferred, different concentrations of CAL tested, and with the addition of IL-2 complex

[0624]

[0475] In this example, the effects of different CAL concentrations were compared, and the number of T cells transferred was 2 million per mouse. In addition, an IL-2 complex was added on Day 9 and Day 11.

[0625] Methods

[0626]

[0476] B16-OVA cancer cell preparation and cancer model

[0627]

[0477] On Day -5, B16-OVA mouse melanoma cells were thawed and seeded at 106cells cells / flask. On Day -1, cell culture medium was changed for further culturing. On Day 0, B16-OVA cells were harvested, and 250,000 cells were intradermally administered to each of 50 mice.

[0628]

[0478] OT-1 cell preparation

[0629]

[0479] On Day 1, splenocytes were harvested from OT-I mice (Thyl.l+). An OT-I splenocyte culture was initiated in 24 well plates the presence of 10 U / mL IL-2 and 1 pg / mL SIINFEKL peptide, some wells had (i) 3 pM TW (3TW) added to the culture. 3 hours later, (ii) 10 pM CAL (10CAL) or 30 pM CAL (30CAL) was added to the culture. Neither CAL norTW were added to cells of Control group (Ctrl). On Day 4, SIINFEKL peptide was removed. On Days 4 and 6, cells were split for further culture, and IL-2 and CAL / TW treatments were simultaneously replenished at the same concentrations as those used on Day 1.

[0630]

[0480] Mouse treatment and monitoring

[0631]

[0481] On Day 6, mice were irradiated at 500 cGy. On Day 7, 2xl06OT-I cells treated or not treated with CAL and / or TW were administered to mice harboring B16-OA cancer cells in the dermis via retro-orbital injection. On Day 9 and Day 11, IL-2 complex (IL-2c) injections were given to mice via intra-peritoneal (IP) injections. Specifically, IL-2c is 1.5 pg / mouse IL-2 complexed with 7.5 pg / mouse in PBS at 37°C for 15-30 minutes before intraperitoneal administration in 100 pL PBS. Dermal tumor sizes were measured every other day. Statistical differences in the tumor sizes were evaluated by two-way ANOVA analyses using GraphPad Prism™.

[0632]

[0482] The experimental results are shown visually in FIG. 8.1143253.008213

[0633] Example 4: Luminescence Killing Assays Which Compare the Effects of Control, CAL, TW and CAL + TW treated OT-1 T cells on Tumor Cell Killing

[0634]

[0483] This example relates to experiments wherein tumor cell killing by control OT-1 Thyl.l splenocytes and CAL, TW and CAL + TW treated OT-1 Thyl.l splenocytes was evaluated using a luminescence assay. The results of these experiments are contained in FIG. 9A & B.

[0635] Methods

[0636]

[0484] In these experiments OT-1 Thyl.l splenocytes were stimulated with Sil N FEKL peptide (antigen specific to OTls) on Day 0 and antigen was removed on Day 3. 10 uM CAL was then added 3 hours after antigen stimulation on Day 0. The resultant cell culture was then split on Day 3 and Day 5.

[0637]

[0485] Drugs and IL-2 were added to the split cell cultures on Day 0 and Day 3. The concentrations used in these experiments are below:

[0638]

[0486] Drug, Antigen and IL-2 Concentrations:

[0639]

[0487] IL-2: 10 U / ml

[0640]

[0488] SIINFEKL: 1 ug / ml

[0641]

[0489] CAL: 10 uM

[0642]

[0490] TW: 3uM

[0643]

[0491] Co-Culture Conditions

[0644]

[0492] On day 5, cancer cells and OT-1 T cells were plated together and incubated with I L-2 (10 U / ml) but no CALorTW. The cancer cells (RMA-Rg-Luc cells) were incubated with SIINFEKL peptide at 1 ug / ml for 2 hours at 37 °C to permit take-up of the antigen by the OT1 T cells in the co-culture.

[0645]

[0493] Luminescence Cell Killing Assay

[0646]

[0494] The degree of tumor ceil killing was then determined by luminescence on day 6, and 24 hours in the co-culture. A luciferase-based killing assay was used because the cancer cells used in the assay (RMA-Rg-Luc cells) luminesce when exposed to luciferin.

[0647] Results

[0648]

[0495] The results of these luminescence assays are contained in FIG. 9A & B. In the experiments, each well comprised 50k RMA cells, and triplicate wells were run. The error bars in the figures are SEM. In the figure, a higher luminescence value means that there are more cancer cells left after treatment.1143253.008213

[0649]

[0496] The results in FIG, 9A & B clearly show that the CAL + TW treated cells were the worst at killing, which is in line with a TSCM phenotype (these cells are believed to be the least differentiated). The control cells were the most effector-like and, accordingly were the best at tumor cell killing.

[0650] Example 5: Western Blot Experiments Further Evidencing the Combined Effects of TW and CAL on T cells

[0651]

[0497] The Materials and Methods of the Western Blot protocol used to obtain the Western Blot images in Figure 10 followed standard protocols. It should be understood that the specifics of this protocol are exemplary and that similar Western Blots could be obtained and analyzed using other known protocols and materials.

[0652] Methods

[0653]

[0498] CDS T cells were isolated from splenocytes with a StemCell kit. These cells stimulated with IL-2, anti-CD3 and anti-CD28. Cells were then harvested after 24 hours and washed 3 times with PBS.

[0654]

[0499] 2-2.5 million of the resultant stimulated cells were then frozen in liquid nitrogen and stored at -80°C until ready to run in a Western blot (using the Western Blot protocol described above).

[0655]

[0500] Prior to conducting the Western Blot the frozen cells were resuspended at a concentration of 2 x 107cells / mL in RIPA buffer (Radioimmunoprecipitation assay buffer) to which was added protease and phosphatase inhibitors.

[0656]

[0501] The concentrations of IL-2, anti-CD3, anti-CD28, and protease and phosphatase inhibitors were as follows:

[0657] IL-2: 10 U / ml

[0658] CAL: 10 uML

[0659] TW: 3uM

[0660] anti-CD3: 10 pg / mL

[0661] anti-CD28: 5 pg / mL1143253.008213

[0662]

[0502] Using this protocol Western blot images were obtained for T cells treated with IL-2, CAL, TW or a combination of TW and CAL. The resultant Western blots were imaged using specific antibodies in order to compare the effects of IL-2, CAL, TW or a combination of TW + CAL on AKT, GSK3p, FOXO1 and p-catenin transcription. The resultant Western blot images are contained in Figure 10A. FIG 10B contains bar graphs based on the raw data, wherein expression of specific genes was normalized as denoted in FIG 10B.

[0663] Results

[0664]

[0503] As can be seen from the Western blot images in Figure 10A and the bar graphs based on the raw Western blot data contained in FIG 10B, wherein expression of the referenced genes was normalized as denoted in FIG 10B treatment of CD8T cells with CAL + TW compared to treatment with IL-2, CAL or TW had disparate effects on the expression of the selected genes. Particularly, it can be seen that combined treatment with the PI3K-AKT-mTOR pathway inhibitor (CAL-101) and the Wnt / P-catenin pathway activator (TWS-119), had joint effects on the expression of genes which promote development of memory T cells or the sternness of T cells (e.g., Tscm phenotype) and / or genes which reverse or prevent T cell exhaustion.

[0665]

[0504] As is shown in Figure 10A and 10B the effects of CAL + TW on phosphorylation result in:

[0666] AKT inactivation which is shown through reduced AKT Phosphorylation;

[0667] GSK3p not inactivated by AKT but is inactivated by TWS119 not detectable by blot which is shown by reduced levels of inhibitory GSK3p phosphorylation from AKT in CAL-treated conditions. The inhibition of GSK3 by TW does not directly target phosphorylation;

[0668] FOXO1 not inhibited as shown by reduced levels of inhibitory FOXO1 phosphorylation from AKT; and

[0669] P-catenin much less targeted for degradation as shown by reduced levels of inhibitory P-catenin phosphorylation from GSK3p.

[0670]

[0505] As noted previously, while CAL-101, a PI3K-AKT-mTOR pathway inhibitor, has been reported to lower nuclear levels of P-catenin, TWS119, a Wnt / P-catenin pathway activator, has been reported to increase nuclear levels of p-catenin (Le Xuan Truong Nguyen et al. Blood Cancer Journal, 2014, Lee et al., Clin Mol Hepatol. 2020). Therefore, the combined effect between the PI3K-AKT-mTOR pathway inhibitor and the Wnt / p-catenin pathway activator as evidenced by its suppressive effects on p-catenin expression is unexpected.1143253.008213

[0671]

[0506] More specifically, P-catenin is targeted for degradation when phosphorylated by active GSK3p. TWS119 inhibits GSK3p pharmacologically and does not directly target a phosphorylation site, and although GSK3P phosphorylation effects have been reported in the literature and are observed in some of our data, the primary outcome is increased total p-catenin with reduced phosphorylation. With CAL-101, reduced AKT signaling is consistent with reduced inhibitory phosphorylation of GSK3p. Consistent with this mechanism, the CAL + TWS119 treated cells show the lowest levels of phosphorylated p-catenin, which is targeted for degradation.

[0672]

[0507] The results of the present Western blot assays therefore further suggest that administering T cells, e.g., CAR-T cells, treated with (I) a PI3K-AKT-mTOR pathway inhibitor and (II) a PI3K-AKT-mTOR pathway inhibitor to cancer patients should better slow cancer progression compared to T cells not treated with CAS + TW.

[0673] Example 6: Combination of CAL + TW Reduce Tumor Burden in MC38-OVA Tumor Mode!

[0674]

[0508] This example relates to experiments which compared the effects of CAL, TW and a combination of CAL + TW on tumor protection in the MC38-OVA tumor model.

[0675] Methods

[0676]

[0509] The experimental methods were as follows:

[0677]

[0510] OT-1 Thyl.l splenocytes were obtained and stimulated with SIINFEKL on Day -2 and then the SIINFEKL was removed on Day 1.

[0678]

[0511] 10 uM CAL was added to the stimulated T cell culture 3 hours after stimulation on Day -2.

[0679]

[0512] The stimulated cell cultures were split on Day 1 and Day 3.

[0680]

[0513] Drugs and IL-2 were added to the stimulated cell cultures on Day -2 and Day 1.

[0681]

[0514] On day 0, 1 million MC38-OVA cancer cells were injected subcutaneously into mice.

[0682]

[0515] On day 3, 1 million T cells were injected retro-orbitally into mice.

[0683]

[0516] The concentrations used were as follows:

[0684] • 10 U / ml IL-2

[0685] • SIINFEKL 1 ug / ml

[0686] • CAL lO uM

[0687] • TW 3uM1143253.008213

[0688]

[0517] In these experiments mice with MC38-OVA tumors were treated with either control CD8 T cells, or CAL, TW or CAL + TW treated CD8 T cells and tumor burden was assessed at times ranging from Day 3 to Day 20 by measuring tumor area in mm2in the treated MC38- OVA tumor bearing mice. Statistical differences in the tumor sizes were evaluated by two-way ANOVA analyses using GraphPad Prism™.

[0689] Results

[0690]

[0518] The results of these experiments are contained in Figure 11. These results further demonstrate the combined effect of the combination of CAL + TW treated T cells on antitumor immunity (based on the significant reduction in tumor volumes in the MC38-OVA tumor bearing mice treated with TW + CAL-treated T cells in comparison to the MC38-OVA tumor bearing mice treated with control T cells, or T cells treated with TW or CAL alone). In the figure * significance (p<0.05) is demonstrated upon comparing the effects of the controls versus CAL + TW treated T cells on tumor volumes, wherein the error bars are SEM.

[0691]

[0519] The results further show the joint impact elicited CAL+TW treatment since only the MC38-OVA tumor bearing mice administered CAL + TW treated T cells were observed to have a statistically significant reduction in tumor burden.

[0692] Example 7: Effects of CAL, TW and CAL + TW on Cytokine Expression By Stimulated and Non-Stimulated CD8 T Cells

[0693]

[0520] This example relates to experiments which compared the effects of CAL, TW and a combination of CAL and TW on cytokine expression by stimulated or non-stimulated CD8 T cells and control CD8 T cells.

[0694] Methods

[0695]

[0521] The experimental methods were as follows:

[0696]

[0522] OT-1 Thyl.l Splenocytes were obtained and stimulated with SIINFEKL on Day 0 and then removed on Day 3.

[0697]

[0523] 10 uM CAL was added to the resultant OT-1 Thyl.l splenocytes 3 hours after antigen stimulation on Day 0.

[0698]

[0524] The OT-1 Thyl.l splenocyte cell cultures were then split cells on Day 3, Day 5, and Day 6.1143253.008213

[0699]

[0525] Drugs and IL-2 were added to the OT-1 Thyl.l splenocyte cell cultures on Day 0, Day 3, and Day 5 at the following concentrations:

[0700] • 10 U / ml IL-2

[0701] • SIINFEKL 1 ug / ml

[0702] • CAL lO uM

[0703] • TW 3uM

[0704]

[0526] On day 6 of culture, some cells were stimulated for 5 hrs with Cell Stimulation Cocktail (Invitrogen 00-4970-93) and others were not as controls, and all cells during this 5 hr incubation also were in Brefeldin A (BFA) (10 ug / ml), and IL-2 (10 U / ml IL-2) in cell culture media at 37 °C, prior to flow cytometry. In these experiments BFA was kept in the buffer at all times to keep cytokines in the cells.

[0705] Results

[0706]

[0527] The results of these experiments are shown in Figure 12. These results further corroborate the validity of the luminescence assay results in Example 4 (showing CD8 T cell effector functions are reduced upon CAL + TW treatment). Particularly the experiments in Figure 12 compare the expression of specific proinflammatory (effector-associated) cytokines (IL-2, I FN-y, and TNFa) by stimulated ( Cell Stim cocktail of phorbol 12-myristate 13-acetate (PIVIA) and ionomycin) or non-stimulated OT-1 Thyl.l splenocytes, wherein such cells were untreated or were treated with TW, CAL or a combination of TW and CAL.

[0707]

[0528] As was predicted based on our other data, the cytokine staining data in Figure 12 show a smaller proportion of CAL + TW treated CD8 T cells that produce effector cytokines (IL-2 I FN-y and TNFa) compared with control CDS T cells.

[0708] Example 8: Single Cell Sequencing Data Showing the Effects of CAL + TW on Specific Cell Types in B16-OVA Tumor Bearing Mice

[0709]

[0529] This example relates to experiments which compared the effects of control, CAL, TW and a combination of CAL and TW treated OT-1 T cells on the relative numbers of specific cell types in tumor samples obtained from B16-OVA Tumor bearing mice (e.g., the proportions of tumor cells and immune cells including CD8 T cells cells).

[0710] Methods

[0711]

[0530] The experimental methods were as follows:1143253.008213

[0712]

[0531] Single-cell Assays

[0713]

[0532] The experimental details are the same as the experiment described in Example 3-2, the results of which are contained in Figure 8, except for the fact that 4 million OT-1 T cells (control, CAL, TW or CAL + TW treated) were injected on day 7 instead of 2 million OT-1 T cells in B16-Ova Tumor bearing mice. Mice were sacrificed on day 14. Three tumors were pooled separately from each of the control, CAL, TW and CAL + TW OT-1 T cell treated B16- Ova Tumor mice groups.

[0714]

[0533] Using cell samples from these pooled tumor samples on-chip multiplexing (using GEM-X Universal 3’v4 and 5'v3 Gene Expression 4-plex assays according to 10X Genomics® protocol (see What is Sample

[0715]

[0716]

[0717] :< ■' a p. a: ■■ r-ip) and single-cell RNA-seq from 10X Genomics® was run.

[0718] Results

[0719]

[0534] The data generated from these assays are contained in Figure 13A and Figure 13B. The single cell sequencing data in Figure 13A shows the percentage of different cell types obtained from tumor samples respectively in tumor cell samples obtained from the control, CAL, TW and CAL + TW OT-1 T cell treated B16-OVA Tumor mice groups.

[0720]

[0535] It can be seen from the data in Figure 13A that the CAL-treated B16-OVA Tumor mice groups had a smaller proportion of melanoma cells and a larger proportion of CD8 T cells in their tumor cell samples. The single cell sequencing data in Figure 13A also shows that the proportion of CD8 T cells is increased in the tumor cell samples isolated from B16-OVA Tumor mice which were injected with CAL treated OT-1 T cells relative to tumor cell samples isolated from the control and other OT-1 T cell treated groups.

[0721]

[0536] The single cell sequencing data in Figure 13B shows the breakdown of the different immune cell subsets in tumor samples, respectively derived from the control, CAL, TW and CAL + TW OT-1 T cell treated B16-OVA Tumor mice groups. As with Figure 13A, data in Figure 13B shows that the proportion of CD8 T cells is higher in the B16-OVA Tumor bearing mice which were injected with CAL-treated OT-1 T cells relative to the control and other mice groups.

Claims

1143253.008213CLAIMSWhat is claimed is:

1. A composition comprising:(I) a PI3K-AKT-mTOR pathway inhibitor; and(II) ( 11-1) a Wnt / p-catenin pathway activator; and / or(11-2) an acetyl-CoA synthesis inhibitor.

2. An isolated or recombinant lymphoid cell treated with and / or comprising:(I) a PI3K-AKT-mTOR pathway inhibitor; and(II) ( 11-1) a Wnt / p-catenin pathway activator; and / or(11-2) an acetyl-CoA synthesis inhibitor.

3. The composition of claim 1 or the isolated or recombinant lymphoid cell of claim 2, wherein:(I) the PI3K-AKT-mT0R pathway inhibitor is or comprises a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide; and / or(II) (ll-l) the Wnt / 0-catenin pathway activator is or comprises a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, an aptamer, and / or a peptide; and / or(ll-l) the acetyl-CoA synthesis inhibitor is or comprises a small molecule, a small molecule inhibitor, an siRNA, an shRNA, an antibody or antigen-binding fragment thereof, a miRNA, a mRNA, a DNA, aptamer, and / or a peptide.

4. The composition of claim 1 or 3 or the isolated or recombinant lymphoid cell of claim 2 or 3, wherein:(I) the PI3K-AKT-mTOR pathway inhibitor is or comprises:(a) a PI3K inhibitor or a PI3K class I inhibitor, optionally(a-1 ) an isoform-selective PI3K inhibitor or an inhibitor selective for one or more of PI3K 6 isoform, PI3K y isoform, PI3K a isoform, and / or PI3K p isoform, optionally selected from the group consisting of 5-fluoro-3-phenyl-2-[(lS)-l-(7H-purin-6- ylamino)propyl]quinazolin-4-one, idela lisib, ZYDELIG®, CAL-101, umbralisib, TGR1202, alpelisib, duvelisib, tenalisib, acalisib, dezapelisib, pa rsac lisib, linperiisib,1143253,008213zandelisib, BGB-10188, SHC014748M, amdizalisib, AZD8835, TQ-B3525, puqutinib, eganelisib, I PI-549, alpelisib, WX-037, inavolisib, serabelisib, CYH33, ASN003, RLY- 2608, LOXO-783 / LOX-22783, STX-478, GSK2636771, AZD6482 / KIN193, AZD8186, KA2237, and tase lisib; and / or(a-2) a pan-PI3K inhibitor, optionally selected from the group consisting of wortmannin, PX866, BKM120, pictilisib, burparlisib, copanlisib, pilaralisib, izorlisib, ZSTK474, and / or fimepinostat;(b) an AKT inhibitor, optionally:( b-1) selected from the group consisting of l,3-dihydro-l-[l-[[4-(6-phenyl-lH- imidazo[4,5-g]quinoxalin-7-yl)phenyl]methyl]-4-piperidinyl]-2H-benzimidazol-2- one, Akt Inhibitor VIII, MK2206, capivasertib, afuresertib, and / or ipatasertib;(b-2) a pan-AKT inhibitor;(b-3) an isoform-selective AKT inhibitor or an inhibitor selective for one or more of AKT1, AKT2, and AKT3;(b-4) an allosteric inhibitor, optionally MK-2206, ARQ.092, ARQ751, BAY1125976; and / or(b-5) an ATP-competitive inhibitor, optionally GSK2141795, GSK690693, LY2780301, G DC-0068, AZD5363, capivasertib, and / or ipatasertib; and / or(c) a dual PI3K / mTOR inhibitor, optionally LY294002, SF-1126, samotolisib, omipalisib, voxtalisib, apitolisib, gedatolisib, VS-5584 / SB2343, paxalisib, DS-7423, and / or PWT33597; and / or(II) ( 11-1) the Wnt / P-catenin pathway activator is or comprises a GSK-3 inhibitor, optionally:(a) capable of inhibiting GSK-3P, GSK-3a, or GSK-3P and GSK-3a;(b) selected from the group consisting of 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl]oxy]phenol, TWS119, SB-415286, CHIR-99021, AR-A014418, lithium, LiCI, BIO, SB-216763, LY-2090314, PF-04802367, L807mts, tideglusib, elraglusib, AZD1080, BRD3731, rosmarinic acid, BRD0705, psoralidin, and / or an oxadiazole-based selective GSK3a inhibitor; and / or(c) (c-1) a cation, optionally lithium or a metal ion, further optionally zinc or copper,(c-2) an ATP competitive inhibitor,(c-3) an allosteric non-ATP competitive inhibitor, and / or(c-4) a substrate competitive inhibitor; and / or1143253.008213(11-2) the acetyl-CoA synthesis inhibitor is or comprises an ATP-citrate lyase (ACLY) inhibitor, optionally selected from the group consisting of 2-hydroxycitrate, a salt thereof (e.g., potassium hydroxycitrate or sodium hydroxycitrate), bempedoic acid, ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid, NEXLETOL®, ESP- 55016, bempedoyl-CoA, BemA, hydroxycitric acid or a derivative thereof, SB204990, a 2-hydroxy-N-arylbenzenesulfonamide, BMS-303141, NDI-091143, SB-201076, citric acid tripotassium hydrate, citric acid, (-)-hydroxycitric acid lactone, MEDICA 16, 3- thiadicarboxylic acid, an emodin derivative, a furan carboxylate derivative, Hibiscus subdariffa extract, Garcinia cambogia extract, lithium citrate (Lita rex) tetrahydrate, bempedoic acid-d, 2-Furoic acid-d4, forrestiacids J, forrestiacids K, 2-Furoic acid, and / or bempedoic acid-d5.

5. The composition of claim 1, 3, or 4 or the isolated or recombinant lymphoid cell of any one of claims 2-4, wherein:(I) the PI3K-AKT-mT0R pathway inhibitor is or comprises a PI3K 6 isoform inhibitor, optionally a small molecule inhibitor, further optionally 5-fluoro-3-phenyl-2-[(lS)-l-(7H- purin-6-ylamino)propyl]quinazolin-4-one, idela lisib, ZYDELIG®, CAL-101, umbralisib, and / or TGR1202; and(II) ( 11-1) the Wnt / P-catenin pathway activator is or comprises a GSK-3 inhibitor, optionally a small molecule inhibitor, further optionally 3-[[6-(3-aminophenyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol, TWS119, SB-415286, CHIR-99021, and / or AR-A014418; and / or(11-2) the acetyl-CoA synthesis inhibitor is or comprises an ACLY inhibitor, optionally a small molecule inhibitor, further optionally 2-hydroxycitrate or a salt thereof (e.g., potassium hydroxycitrate or sodium hydroxycitrate).

6. The isolated or recombinant lymphoid cell of any one of claims 2-5, which:(A) is or comprises a cell of T cell lineage or NKT cell lineage, optionally a CD8+ T cell, a CD4+ T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing;(B) is or comprises a memory or memory-like cell or memory T cell, optionally a stem cell-like memory T (Tscm) cell, a central memory T (Tern) cell, an effector T cell memory T (Tern) cell, a resident memory T (Trm) cell or tissue-resident memory T cell, a memory regulatory T (Tregm) cell, a peripheral memory T cell (Tpm), and / or a memory precursor effector T cell (Tmpec):1143253.008213(C) expresses a memory T cell marker and / or one or more of CCR7 (CD197), CD95, CD27, CD62L, CD122, IL-7Ra (CD127), CD103, CD69, CD44, CXCR3, TCF7, TCF1, LEF1, LFA-1, LFA- 3, FOXO1, BCL6, Sca-1, FOXP1, KLF2, CD45RA, and / or CD45RO;(D) is or comprises a T cell which:(i) upon exposure to a cognate antigen (e.g., a cognate antigen of a CAR, a synthetic immune receptor, or an endogenous immune receptor expressed on the cell), expresses or expresses an increased level of a T cell activation marker or one or more of CD25, CD26, CD27, CD28, CD30, CD38, CD43, CD44, CD69, CD71, CD107, IL-7Rcc (CD127), 0X40 (CD134), CD40L (CD154), CXCR1, CX3CR1, H LA-DR, KLRG1, IFNy, granzyme B, perforin; and / or(ii) does not express or express at a lower level (e.g., compared to an activated or effector T cell) of a T cell exhaustion marker or one or more of Tim-3 (CD366), Lag-3 (CD223), PD-1, CTLA-4, Slamf6 (CD352), CD39a, TIG IT, CD160, BTLA, and / or 2B4 (CD244);(E) is, comprises, or is derived from a splenocyte, a lymph node cell, a bone marrow cell, and / or a thymic cell;(F) is, comprises, or is derived from a stem cell, a multi otent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (IPS) cell;(G) is derived from a subject, optionally a mammal, further optionally a human, a non¬ human primate, a monkey, a rabbit, a rodent, a hamster, a rat, ora mouse;(H) is or comprises a mammalian cell, optionally a human cell, a non-human primate cell, a monkey cell, a rabbit cell, a rodent cell, a hamster cell, a rat cell, or a mouse cell; and / or(I) is, comprises, or is derived from a cell line cell or a primary cell.

7. The isolated or recombinant lymphoid cell of any one of claims 2-6, which:(A) expresses or is modified to express a polypeptide which specifically binds to an antigen of interest, optionally wherein the polypeptide is, comprises, or comprised in:(i) a chimeric antigen receptor (CAR), optionally comprising:(i-1) an antigen-binding domain (e.g., comprising an immunoglobulin (Ig) heavy chain variable domain (VH) and / or light chain variable domain (VL));(i-2) a transmembrane domain; and1143253.008213( i-3) at least one intracellular signaling domain (e.g., of or derived from CD3, CD3^ CD27, CD28, CD44, DAP10, 4-1BB (CD137), OX-40, ICOS, NKG2D, a Fey receptor, FcRylll (CD16), FcRyllA (CD32A), and / or 2B4 (CD244); and / or (ii) a synthetic immune receptor, optionally a synthetic T cell receptor (TCR) complex, optionally comprising:( ii-1) an a chain comprising a TCR a constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain; ( ii-2) a p chain comprising a variable domain, a TCR constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain;( ii-3) one or more CD3^ polypeptides; and( ii-4) optionally one or more of CD3e, CD36, and / or CD3y polypeptides; (B) does not express or is modified to remove or suppress expression of an endogenous TCR and / or BCR, optionally via gene editing or RNAi;(C) does not express or is modified to remove or suppress expression of endogenous MHCI and / or MHCII, optionally via gene editing or RNAi; and / or(D) is modified to express another molecule, optionally a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a hybrid or chimeric cytokine receptor, and / or a transcription factor.

8. A cell population, which comprises, essentially consists of, or consists of one or more isolated or recombinant lymphoid cells of any one of claims 2-7.

9. A pharmaceutical composition comprising:(A) one or more isolated or recombinant lymphoid cells of any one of claims 2-7 and / or; and / or a cell population of claim 8; and(B) a pharmaceutically acceptable carrier and / or excipient,optionally wherein the pharmaceutical composition further comprises an additional agent, optionally an adjuvant or another therapeutic agent.

10. A method of producing one or more isolated or recombinant lymphoid cells, optionally of any one of claims 2-7, or a cell population comprising one or more isolated or1143253.008213recombinant lymphoid cells, optionally of claim 8, the method comprising culturing one or more lymphoid cells or a population of cells comprising one or more lymphoid cells in the presence of:(I) a PI3K-AKT-mTOR pathway inhibitor; and(II) ( 11-1) a Wnt / p-catenin pathway activator; and / or(11-2) an acetyl-CoA synthesis inhibitor,optionally wherein any of the foregoing inhibitors and / or activators are added to the cell culture at the same or different times (e.g., about 1-12 hours, about 1-6 hours, or about 1, 2, 3, 4, 5, or 6 hours apart) and added to the cell culture in any order, and preferably are added to the cell culture at the same time:further optionally wherein:(i) the PI3K-AKT-mTOR pathway inhibitor is a PI3K-AKT-mTOR pathway inhibitor of any one of claims 3-5;(ii) the PI3K-AKT-mT0R pathway inhibitor is present at about 10 nM to about 100 mM, about 100 nM to about 10 mM, about 1 pM to about 1 mM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM, optionally at the start of or at least at one point during the culturing;(iii) the Wnt / P-catenin pathway activator is the Wnt / P-catenin pathway activator of any one of ciaims 3-5;(iv) the Wnt / p-catenin pathway activator is present at about 1 nM to about 10 M, about 10 nM to about 1 mM, about 100 nM to about 100 pM, about 1 pM to about 10 pM, about 1 pM to about 5pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM, optionally at the start of or at least at one point during the culturing;(v) the PI3K-AKT-mTOR pathway inhibitor and the Wnt / p-catenin pathway activator are present at a molar ratio of about 0.1:1 to about 100: 1, about 0.1:1, about 0.5:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, or about 100:1, at the start of or at least one point during the culturing;(vi) the acetyl-CoA synthesis inhibitor is the acetyl-CoA synthesis inhibitor of any one of claims 3-5;1143253.008213(vii) the acetyl-CoA synthesis inhibitor is present at about 10 pM to about 500 mM, about 100 pM to about 50 mM, about 0.5 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, or about 5 mM, optionally at the start of or at least one point during the culturing;(viii) the PI3K-AKT-mTOR pathway inhibitor and the acetyl-CoA synthesis inhibitor are present at a molar ratio of about 10:1 to about 1: 10000, about 1:1 to about 1:10000, about 1:10 to about 5000, about 1:10 to about 5000, about 1:3000, about 1:2500, about 1:2000, about 1:1000, about 1:500, about 1:300, about 1:250, about 1:200, about 1:100, about 1:50, about 1:30, about 1:25, about 1:20, or about 1:10 at the start of or at least one point during the culturing.

11. The method of claim 10, wherein:(i) the culturing is performed in the presence of:( i-1) IL-2, optionally at about 0.1 U / mL to about 200 U / mL, about 0.5 U / mL to about 100 U / mL, about 1 U / mL to about 50 U / mL, about 5 U / mL to about 25 U / mL, about 25 U / mL, or about 10 U / mL;( i-2) an CDS activating agent, optionally an anti-CD3 antibody or antigen-binding fragment thereof, and / or an CD28 activating agent, optionally an anti-CD28 antibody or antigen-binding fragment thereof;(i-3) an antigen of interest to which at least one of the one or more lymphoid cells specifically binds;(ii) the culturing in the presence of (I) the PI3K-AKT-mTOR pathway inhibitor and (II) (11-1) the Wnt / p-catenin pathway activator and / or (11-2) the acetyl-CoA synthesis inhibitor is performed for about 0.5 to about 20 days, about 1 to about 15 days, about lday, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days;(iii) the one or more lymphoid cells:are or comprise a cell of T cell lineage or NKT cell lineage, optionally a CD8+ T cell, a CD4+T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing;are, comprise, or are derived from one or more splenocytes, lymph node ceils, bone marrow cells, and / or thymic cells;are, comprise, or are derived from a stem cell, a multipotent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (iPS) cell;1143253.008213are derived from a subject, optionally a mammal, further optionally a human, a non¬ human primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse;are or comprise a mammalian cell, optionally a human cell, a non-human primate cell, a monkey cell, a rabbit cell, a rodent cell, a hamster cell, a rat cell, or a mouse cell; and / orare, comprise, or are derived from a cell line cell or a primary cell; and / or(iv) the population of cells:is or comprises splenocytes, lymph node cells, bone marrow cells, and / or thymic cells;is, comprises or is derived from one or more stem cells, multi potent stem cells, pluripotent stem cells, hematopoietic stem cells, embryonic stem cells, and / or iPS cells;is derived from a subject, optionally a mammal, further optionally a human, a non- human primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse;is or comprise a population of mammalian cells, optionally human cells, non-human primate cells, monkey cells, rabbit cells, rodent cells, hamster cells, rat cells, or mouse cells; and / oris, comprises or is derived from cell line cells or primary cells; and / or( v) the method is for producing one or more isolated or recombinant lymphoid cells or a cell population comprising one or more isolated or recombinant lymphoid cells for use in cell therapy, optionally a CAR cell therapy, further optionally a CAR T cell therapy.

12. The method of claim 10 or 11, further comprising modifying the one or more lymphoid cells or the population of cells:(A) to induce expression of:(i) a chimeric antigen receptor (CAR), optionally comprising:(i-1) an antigen-binding domain (e.g., comprising an immunoglobulin (Ig) heavy chain variable domain (VH) and / or light chain variable domain (VL));(i-2) a transmembrane domain; and( i-3) at least one intracellular signaling domain (e.g., of or derived from CDS, CD3^, CD27, CD28, CD44, DAP10, 4-1BB (CD137), OX-40, ICOS, NKG2D, a Fey receptor, FcRylll (CD16), FcRyllA (CD32A), and / or 2B4 (CD244); and / or1143253.008213(ii) a synthetic immune receptor, optionally a synthetic T cell receptor (TCR) complex, optionally comprising:( ii-1) an a chain comprising a TCR a constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain; (ii-2) a P chain comprising a variable domain, a TCR (3 constant domain and a transmembrane domain and optionally a TCR variable domain or an Ig variable domain;( ii-3) one or more CD3^ polypeptides; and( ii-4) optionally one or more of CD3e, CD36, and / or CD3y polypeptides; and / or (B) to remove or suppress expression (e.g., functional expression) of or inhibit a function of an endogenous TCR and / or BCR, optionally via gene editing or RNAi;(C) to not express or to remove or suppress expression of endogenous MHCI and / or MHCII, optionally via gene editing or RNAi; and / or(D) to express another molecule, optionally a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a hybrid cytokine receptor, and / or a transcription factor.

13. One or more isolated or recombinant cells or a cell population comprising one or more isolated or recombinant lymphoid cells, which is / are produced using the method of any one of claims 10-12.

14. A method of treating a disease, disorder, or condition in a subject, the method comprising administering to the subject an effective amount of at least one of:(a) one or more isolated or recombinant cells of any one of claims 2-7 and 13;(b) a cell population of claim 8 or 13; and / or(c) a pharmaceutical composition of claim 9,optionally wherein:(i) the subject is:( i-1) a mammal, optionally a human, a non-human primate, a monkey, a horse, a cow, sheep, a goat, a pig, a dog, a cat, a rabbit, a rodent, a hamster, a rat, or a mouse; or (i-2) a non-mammalian vertebrate, optionally a bird, fish, an amphibian, ora reptile; (ii) the administering is or comprises:1143253.008213(ii-1) parenterally administering, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intradermal, intrathecal, intra-arterial, intraarticular, intraosseous, intra parenchymal, or intraperitoneal administration) or by inhalation; (ii-2) locally administering, optionally by direct administration into a diseased site (optionally intratu moral injection) or by inhalation or administering to the skin, liver, bone marrow, lymphoid tissue, the peritoneal cavity, gut, ovary, testis, eye, ear, nose (optionally intranasally), skin (optionally transdermally or epicutaneously), mucosa, or vagina; or( ii-3) enterally administering, optionally orally, sublingually, buccally, or rectally; (iii) the administering is effected two or more times, optionally about 3-5 times, optionally about once a week, about every 2 weeks, or about every 3 weeks, about once a month, about every 3 months, about every 6 months, about every 9 months, or about once per year; and / or(iv) the method further comprises administering to the subject an additional agent, optionally an adjuvant or another therapeutic agent, optionally a cytokine or a cytokine complex, further optionally IL-2 or an IL-2 complex (e.g., IL-2 complexed with an anti-IL-2 antibody (e.g., an antibody which blocks binding of IL-2 to an IL-2 receptor or one or more subunits thereof) or with an IL-2 receptor or one or more subunits thereof).

15. The method of claim 14, wherein the disease, disorder, or condition comprises cancer, an infectious disease, an autoimmune disease, an inflammatory disease, a fibrotic disease, an allergic disease, a neural disease, a neurodegenerative disease, a metabolic disease, a cardiovascular disease, or another disease, optionally wherein:(i) the cancer is:( I 1) a solid cancer, optionally chosen from: one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharynx cancer, head and neck cancer, rectal cancer, esophagus cancer, or bladder cancer, or a metastasis thereof; and / or( i-2) a liquid cancer, optionally chosen from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic1143253,008213myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma;(ii) the infectious disease is a viral, bacterial, fungal, yeast, protozoan, prion or parasitic disease, optionally wherein( ii-1) the viral disease is human immunodeficiency virus (HIV), hepatitis virus (optionally hepatitis A, B, or C virus), human papillomavirus (HPV), herpes simplex virus (HSV) (optionally HSV-1 or HSV-2), enterovirus, human cytomegalovirus, adenovirus, rhinovirus, Pox virus, Influenza virus, coronavirus (optionally MERS-CoV, SARS-CoV, or SARS-CoV-2, or common human coronavirus), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), norovirus, West Nile Virus, Zika virus, poliovirus, Ebola virus, or dengue virus (DENV) infection,( ii-2) the bacterial disease is Salmonella, Escherichia coil, Mycobacterium tuberculosis, methicillin-resistant staphylococcus aureus (MRSA), Clostridium difficile, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Helicobacter pylori, Neisseria gonorrhoeae, Vibrio vulnificus, and / or(ii-3) the fungal disease is Aspergillosis, Candida, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Mucormycetes, Taloromyces, ringworm, Blastomyces, Coccidioides, Cryptococcus gatii, Histoplasma, Paracoccidioides, or Sporothrix infection;(iii) the autoimmune disease or the inflammatory disease is psoriasis, rheumatoid arthritis, autoimmune arthritis, type I diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, scleroderma, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, pemphigus vulgaris, Sjogren's syndrome, Addison's disease, Behcet's disease, Schmidt syndrome, celiac disease, dermatomyositis, autoimmune vitiligo, Graves' disease, Hashimoto thyroiditis, Kawasaki disease, pernicious anemia, autoimmune vasculitis, or fibrosis; and / .or1143253.008213(iv) the neurodegenerative disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Baten disease, Creutzfeldt-Jakob disease.

16. One or more of the isolated or recombinant cells of any one of claims 2-7 and 13, the cell population of claim 8 or 13, and / or the pharmaceutical composition of claim 9,(i) for use in medicine; and / or(ii) for use in treating or preventing a disease, disorder, or condition, optionally wherein the disease, disorder, or condition comprises any one or more of those according to claim 15.

17. Use of the composition of any one of claims 1 and 3-5, one or more of the isolated or recombinant cells of any one of claims 2-7 and 13, and / or the cell population of claim 8 and 13, and / or the pharmaceutical composition of claim 9, for the manufacture of a medicament for treating or preventing a disease, disorder, or condition, optionally wherein the disease, disorder, or condition comprises any one or more of those according to claim 15.

18. A kit comprising:(A) one or more lymphoid cells or a population of cells comprising one or more lymphoid cells; and(B) (I) a PI3K-AKT-mTOR pathway inhibitor; and(II) (11-1) a Wnt / P-catenin pathway activator; and / or(11-2) an acetyl-CoA synthesis inhibitor,optionally wherein:(i) the PI3K-AKT-mTOR pathway inhibitor is according to the PI3K-AKT-mTOR pathway inhibitor of any one of claims 3-5;(ii) the Wnt / P-catenin pathway activator is according to the Wnt / P-catenin pathway activator of any one of claims 3-5,(iii) the acetyl-CoA synthesis inhibitor is according to the acetyl-CoA synthesis inhibitor of any one of claims 3-5;(iv) the kit further comprises one or more of a cytokine, a cytokine complex (e.g., a cytokine complexed with an antibody specific to the cytokine (e.g., an antibody which blocks binding of the cytokine to its receptor or one or more subunits thereof) or1143253.008213complexed with its receptor or one or more subunits thereof), IL-2, an IL-2 complex (e.g., IL-2 complexed with an anti-IL-2 antibody (e.g., an antibody which blocks binding of IL-2 to an IL-2 receptor or one or more subunits thereof) or with an IL-2 receptor or one or more subunits thereof), an CD3 activating agent, optionally an anti-CD3 antibody or antigen-binding fragment thereof, an CD28 activating agent, optionally an anti-CD28 antibody or antigen-binding fragment thereof, an antigen of interest to which at least one of the one or more lymphoid cells specifically binds;(v) the one or more lymphoid cells:are or comprises a cell of T cell lineage or NKT cell lineage, optionally a CD8+ T cell, a CD4+T cell, a CD4+ / CD8+ cell, and / or a mixture of any of the foregoing;are, comprise, or are derived from a stem cell, a multipotent stem cell, a pluripotent stem cell, a hematopoietic stem cell, an embryonic stem cell, and / or an induced pluripotent stem (iPS) cell;are derived from a subject, optionally a mammal, further optionally a human, a nonhuman primate, a monkey, a rabbit, a rodent, a hamster, a rat, or a mouse;are or comprise a mammalian cell, optionally a human cell, a non-human primate cell, a monkey cell, a rabbit cell, a rodent cell, a hamster cell, a rat cell, or a mouse cell;are, comprise, or are derived from a cell line cell or a primary cell; and / or(vi) the population of cell is or comprises splenocytes, lymph node cells, bone marrow cells, and / or thymic cells; and / or(vii) the kit further comprises a label, optionally including an instruction for performing the method of any one of claims 10-12 and 14-15 or the use of claim 17.

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