A novel platform for the generation of "naive-like" cells of the t lineage and uses thereof

Abstract

The present disclosure provides methods for preparing improved populations of cells of the T lineage of, specifically, naive-like T cells that comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). The method is based on enrichment for cells expressing a marker of circulating innate lymphoid cell precursor cells. The present disclosure further provides populations of these naive-like T cells, compositions thereof, and uses thereof in treating immune-related disorders.

Description

[0001] A NOVEL PLATFORM FOR THE GENERATION OF "NAIVE-LIKE " CELLS OF THE T LINEAGE AND USES THEREOF

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to the field of immunotherapy. More specifically, the present disclosure relates to methods for preparing improved populations of cells of the T lineage.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. Priesner C, Aleksandrova K, Esser R, Mockel-Tenbrinck N, Leise J, Drechsel K, Marburger M, Quaiser A, Goudeva L, Arseniev L, Kaiser AD, Glienke W, Koehl U. Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L+ T Cells for Manufacturing of Gene Therapy Medicinal Products. Hum Gene Ther. 2016 Oct;27(10):860-869. doi: 10.1089 / hum.2016.091. PMID: 27562135; PMCID: PMC5035932.

[0007] 2. Wang D, Aguilar B, Starr R, Alizadeh D, Brito A, Sarkissian A, Ostberg JR, Forman SJ, Brown CE. Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI Insight. 2018 May 17;3(10):e99048. doi: 10.1172 / jci.insight.99048. PMID: 29769444; PMCID: PMC6012522.

[0008] 3. Joedicke JJ, GroBkinsky U, Gerlach K, Kiinkele A, Hdpken UE, Rehm A. Accelerating clinical-scale production of BCMA CAR T cells with defined maturation stages. Mol Ther Methods Clin Dev. 2021 Dec 25;24: 181-198. doi: 10.1016 / j.omtm.2021.12.005. PMID: 35118163; PMCID: PMC8791860.

[0009] 4. Du L, Nai Y, Shen M, Li T, Huang J, Han X, Wang W, Pang D, Jin A. IL-21 Optimizes the CAR-T Cell Preparation Through Improving Lentivirus Mediated Transfection Efficiency of T Cells and Enhancing CAR-T Cell Cytotoxic Activities. Front Mol Biosci. 2021 Jun 4;8:675179. doi: 10.3389 / fmolb.2021.675179. PMID: 34179083; PMCID: PMC8220804.

[0010] 5. Arcangeli S, Bove C, Mezzanotte C, Camisa B, Falcone L, Manfredi F, Bezzecchi E, El Khoury R, Norata R, Sanvito F, Ponzoni M, Greco B, Moresco MA, Carrabba MG, Ciceri F, Bonini C, Bondanza A, Casucci M. CAR T cell manufacturing from naive / stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. J Clin Invest. 2022 Jun 15;132(12):el50807. doi: 10.1172 / JCI150807. PMID: 35503659; PMCID: PMC9197529. 6. Dickinson MJ, Barba P, Jager U, Shah NN, Blaise D, Briones J, Shune L, Boissel N, Bondanza A, Mariconti L, Marchal AL, Quinn DS, Yang J, Price A, Sohoni A, Treanor LM, Orlando EJ, Mataraza J, Davis J, Lu D, Zhu X, Engels B, Moutouh-de Parseval L, Brogdon JL, Moschetta M, Flinn IW. A Novel Autologous CAR-T Therapy, YTB323, with Preserved T-cell Sternness Shows Enhanced CAR T-cell Efficacy in Preclinical and Early Clinical Development. Cancer Discov. 2023 Sep 6;13(9):1982-1997. doi: 10.1158 / 2159- 8290.CD-22-1276. PMID: 37249512; PMCID: PMC10481129.

[0011] 7. WO2021108661.

[0012] 8. WO2016044811.

[0013] 9. Meyran D. et al. , TSTEM-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models; Science Translational Medicine, 05 Apr 2023, 15(690).

[0014] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0015] BACKGROUND

[0016] CART cells are engineered T-cell expressing a chimeric antigen receptor targeted to a cell-surface tumor antigen. This modality of cancer immunotherapy has proven safe and efficacious for the treatment of hematologic malignancies (i.e., leukemia, lymphoma and multiple myeloma) for which the FDA and EMA agencies gave their approval for their use in human. Despite unprecedented outcomes, relapses following CART treatment remain frequent. Different reasons may explain resistance to CART therapy; some are related to the tumor (e.g., antigen escape, cytogenetic mutations, etc.) and some are related to the CART product (e.g., reduced proliferative capacity, exhaustion, etc.). While it is crucial to improve the clinical outcome of this “living-drug” to extend the remission periods, it is also evident that options impacting the tumor intrinsic features are very limited. On the other side, patients’ T-cells are often of reduced quality (e.g., aging, lympho-toxic regimens, tumor microenvironment, etc.), which subsequently results in CART products of reduced quality. Publications [1-9] disclose various methods for production of CAR T cells. GENERAL DESCRIPTION

[0017] The first aspect of the present disclosure relates to a method for preparing a population of naive- like T cells. More specifically, the naive-like T cells comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM)). The disclosed method comprises the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ)with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching cells expressing the at least one marker. Step (b), involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. A further aspect of the present disclosure relates to a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject, the method comprising the step of administering to the subject an effective amount of a cell population comprising cells of the T lineage, or a composition comprising the same. The cells of the T lineage comprise naive-like T cells. In some embodiments, the naive-like T cells comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). It should be further understood that the cell population is prepared by a method comprising the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days.

[0018] A further aspect of the present disclosure relates to an effective amount of a cell population comprising cells of the T lineage, or a composition comprising the same, for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject. The cells of the T lineage comprise naive-like T cells. The naive-like T cells comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). Still further, the naive-like T cell population used herein is prepared by a method comprising following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a), to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days.

[0019] Another aspect of the present disclosure relates to an isolated cell population comprising T cells. The T cells of the disclosed cell population comprise naive-like T cells. The naive-like T cells comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM)The T cells are characterized by a CD4+T cell to CD8+T cell ratio that is greater than one. A further aspect of the present disclosure relates to a composition comprising the isolated cell population prepared by the disclosed methods, and at least one of pharmaceutically acceptable carrier / s, diluent / s, excipient / s and additive / s.

[0020] Another aspect relates to a kit comprising: (a), a compound that specifically selects for cells expressing the CD62L marker; (b), T-cell activation mixture comprising: (i) a colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL-15) and Interleukin 21 (IL-21); (c), at least one exogeneous nucleic acid molecule encoding at least one CAR molecule and at least one transduction enhancer comprising Vectofusin-1; thereby obtaining a cell population comprising T cells harboring said CAR molecule; and (d), a low serum medium containing 1% or less human AB serum. The kit is adapted for operation in a single container or compartment for a period of up to 6 days.

[0021] A further aspect of the present disclosure relates to a method for preparing a population of naive- like T cells. The naive-like T cells comprise at least one of naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). The disclosed method comprising: (a), contacting a population of cells comprising cells of the T lineage subsets with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker; (b), subjecting the enriched cell population obtained in step (a) to T-cell activation; and (c), expanding the activated cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days.

[0022] These and other aspects of the present disclosure will become apparent by the hand of the present disclosure.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0025] Figure 1. T-cell subsets

[0026] Upon antigen stimulation, Naive / s tern-cell like memory T-cells (TNaive / SCM) differentiate into cytotoxic effector and memory T-cells (TEM), mediating tumor killing, before becoming functionally exhausted (TEMRA).

[0027] Figure 2A-2B. Impact of disease presentation on T-cell differentiation status

[0028] Fig. 2A. Peripheral blood mononuclear cells (PBMCs) derived from healthy donors (HDs, n=7), multiple myeloma (MM, n= 19) and light chain amyloidosis (AL, n=2) patients were characterized for T-cell subsets by flow cytometry. TNaive / SCMwere defined as: CD45RA+CCR7+; TCM, CD45RA-CCR7+; TEM, CD45RA-CCR7- and TEMRA, CD45RA+CCR7- (gated on CD3+). Each dot represents a healthy / patient apheresis donation.

[0029] Fig. 2B. T-cells from MM patients display increased proportions of senescent-like cells (defined as CD27-CD28-, gated on CD3+ cells).

[0030] Figure 3. CART-cell traditional manufacturing platform

[0031] Conventional CART cells are generated through a complex and time-consuming (8-14 days) process. Major steps related to this process include: 1) Leukapheresis collection (which might optionally be cryopreserved); 2) PBMCs purification (which might optionally be cryopreserved); 3) T-cell activation with anti-CD3 (0KT3) and IL-2; 4) T-cell retroviral transduction; 5) CART cell expansion (>5 days); 6-7) CART cell harvest and formulation. Though it has proven safe and efficient for the massive production of CART-cells, this process requires the use of several kinds of vessels (e.g., flasks, plates, G-Rex), and multiple centrifugation steps. More importantly, at the end of this process, CART cells display mainly a T-cell effector phenotype.

[0032] Figure 4. CART-cell “naive” manufacturing platform

[0033] “Naive-like” CART cells are generated through a streamline process involving one single-vessel within 6-days. Major steps related to this process include: 1) Leukapheresis collection (which might optionally be cryopreserved); 2) CD62L+ cells purification (CD62L microbeads, Miltenyi) (which might optionally be cryopreserved); 3) T-cell activation with Trans Act beads (Miltenyi) and IL-7 / 15 / 21 cytokines cocktail (Premium grade, Miltenyi); 4) T-cell retroviral transduction in the presence of Vectofusin-1 as enhancer; 5) CART cell short expansion in AIM-V 1% Human Serum and IL-7 / 15 / 21 cytokines cocktail (3-5 days); 6-8) CART cell harvest and formulation (CryoStor-5).

[0034] Figure 5A-5B. CART-cell “naive” vs. the traditional (TM) manufacturing platforms

[0035] The figure shows a simplified schematic of HBI0101 CART manufacturing process according to the conventional (Fig. 5A) or optimized (Fig. 5B) protocols. While the major steps required for the generation of CART cells are conserved for both platforms and include the T-cell activation, transduction, expansion and formulation of the CART product, the process in depicted in (Fig. 5B) is markedly more streamlined and shorter in comparison with the process depicted in (Fig. 5A).

[0036] Figure 6A-6B. Rationale for CD62L T-cell magnetic sorting

[0037] T-cell differentiation profile of leukapheresis product from a healthy donor (Fig. 6A) or multiple myeloma patient (Fig. 6B). T-cell sorting using CD45RA as selection marker (bold boxes) that results in enrichment of TNaive / SCMbut of TEMRA cells as well. T-cell sorting using CD62L as selection marker (bold box) that results in enrichment of TNaive / SCMand TCMcells as well.

[0038] Figure 7A-7C. Effect of CD62L+ selection on T-cell subsets in the starting material

[0039] Fig. 7A. CD62L+ magnetic sorting significantly increases CD4 / CD8 ratio in the starting material. CD4+ and CD8+ distribution before (circles) and after (squares) CD62L+ magnetic selection.

[0040] Fig. 7B. Increased proportions of naive / stem-cell like memory and central memory T-cell (TNaive / SCMand TCM) following CD62L+ magnetic sorting.

[0041] FIG.7C. Significant decrease in the proportion of senescent-like (CD27-CD28-) T-cells following CD62L+ enrichment. (n=5). The selection with CD62L is indicated in Figs. 7A, 7B, 7C, as "before" or "after" selection.

[0042] Figure 8A-8B. Traditional versus “naive” manufacturing platform

[0043] Fig. 8A. CD62L+ magnetic sorting significantly increases the proportion of “naive-like” T-cells in the starting material.

[0044] Fig. 8B. Significant increase in the proportion of “naive-like” CART-cells in the final product when using the “naive” platform in comparison with the traditional manufacture platform (n=5). The selection with CD62L is indicated in Figs. 8A, 8B, as "before" or "after" selection.

[0045] Figure 9A-9B. Gating strategy for the characterization of the starting material of the “naive- like” CART cells Differentiation status of CD3+ T cells at leukapheresis collection (MM patient) before (FIG. 9A) and after (FIG. 9B) CD62L magnetic sorting. CD62L enrichment increases the proportion of CD4+ cells in the starting material, the proportion of CD27+CD28+ and the proportion of TNaive / SCMcells (CD45RA+CCR7+ or alternatively CD45RA+CD62L+).

[0046] Figure 10A-10B. Gating strategy for the characterization of HBI0101 CART final product (FP) according to the manufacturing platform

[0047] Differentiation status of CART cells from MM patient at Day 6 (Fig. 10A: “naive” platform) or Day 8 (Fig. 10B: TM platform). Final product generated via the “naive” platform mostly consists of CD4+ cells, and display an early memory phenotype (TNaive / SCM: CD45RA+CCR7+ / CD62L+ and TCM: CD45RA-CCR7+ / CD62L+) with a high percentage of CD27+CD28+ CART cells, in contrast to the final product generated via the TM platform showing similar proportions of CD4+ and CD8+, and mainly consists of effector cells (TEM: CD45RA-CCR7- / CD62L-). Of note, similar percentages of transduced cells (CAR+) are observed in both platforms.

[0048] Figure 11. Cellular composition of the CART final product using “Traditional” versus “naive” manufacturing platform

[0049] Both manufacturing platforms generate a CAR product mostly constituted of CD3+ cells. The feature that significantly differs between the two processes is the CD4 / CD8 ratio which is reversed: increased proportion of CD4 in the “naive” process; increase CD8 in the “traditional” process (n=5).

[0050] Figure 12. Naive-like CART cells are highly functional in myeloma eradication even at low E:T ratios

[0051] CART cells were generated via the traditional (TM; Squares) or “naive” (circles) manufacturing platforms from mononuclear cells derived from a myeloma patient. The killing potency of these cells was assessed in co-culture experiments with a myeloma cell line (NCI-H929) expressing the BCMA molecule. Ratios represent the effector (CART) to target (Myeloma) ratios, 1:4, 1:8, 1:16, 1:32, or 1:64.

[0052] Figure 13A-13C. Naive-like CART cells display enhanced proliferative capacity and improved long-term tumor control

[0053] CART cells were generated from four donors (two multiple myeloma (MM; BCMA#112, BCMA#120), one light chain amyloidosis (AL; BCMA#119) patients and one healthy donor (HD; PHE-011)) using the traditional (TM, squares) or Naive (circles) platform. The CART cells were cultivated at the indicated effector to target ratios (1:1; left panels; 1:4 right panels) with NCI- H929 myeloma cells for 3-4 days.

[0054] Fig. 13A-13B. Naive-like versus traditional CART cell proliferative capacity.

[0055] Fig. 13A. Cumulative CART cell count along rechallenge assay. Arrows indicate re-stimulation of left-over CART cells from the previous round with fresh tumor cells.

[0056] Fig. 13B. Proliferation index was calculated as the ratio of CART cell count at the end of each stimulation round by the CART cell count at the beginning of each co-culture initiation.

[0057] Fig. 13C. Tumor eradication is described as the percentage of tumor cells remaining at the end of each rechallenge round from the initially loaded cells.

[0058] Data are represented as the mean ± SEM. Statistical analysis by 2-way ANOVA. N=4.

[0059] Figure 14A-14C. Naive-like CART cells display enhanced proliferative capacity and improved long-term tumor control

[0060] CART cells were generated from four donors (two multiple myeloma (MM; BCMA#112, BCMA#120), one light chain amyloidosis (AL; BCMA#119) patients and one healthy donor (HD; PHE-011)) using the traditional (TM, squares) or Naive (circles) platform. CART cells were cultivated at the E:T ratio of 1:4 with MM1S myeloma cells for 3-4 days.

[0061] Fig. 14A-14B. Naive-like versus traditional CART cell proliferative capacity.

[0062] Fig. 14A. Cumulative CART cell count along rechallenge assay. Arrows indicate re-stimulation of left-over CART cells from the previous round with fresh tumor cells.

[0063] Fig. 14B. Proliferation index was calculated as the ratio of CART cell count at the end of each stimulation round by the CART cell count at the beginning of each co-culture initiation.

[0064] Fig. 14C. Tumor eradication is described as the percent of tumor cells remaining at the end of each rechallenge round from the initially loaded tumor cells.

[0065] Data are represented as the mean ± SEM. Statistical analysis by 2-way ANOVA. N=4. Figure 15A-15C. CART cells from MM and AL patients and generated via the NAIVE platform exhibit comparable proliferative capacity than naive-like CART cells derived from healthy donor

[0066] CART cells were generated from four donors (two multiple myeloma (MM; BCMA#112, BCMA#120), one light chain amyloidosis (AL; BCMA#119) patients and one healthy donor (HD; PHE-011))] using the traditional (TM) or Naive platform. CART cells were cultivated at effector to target ratios of 1:1 (Fig. 15A) or 1:4 (Fig. 15B-15C) with NCLH929 (Fig. 15A-15B) or MM1S (Fig. 15C) myeloma cells for 3-4 days.

[0067] Fig. 15A-15C. Naive-like versus traditional CART cell proliferative capacity. Cumulative CART cell count along rechallenge assay. Arrows indicate re-stimulation of left-over CART cells from the previous round with fresh tumor cells. Data are represented as the mean ± SEM. Statistical analysis by 2-way ANOVA. N=4.

[0068] For graph in Fig. 15C, 2-way ANOVA was performed until D18.

[0069] Figure 16A-16C. Analysis of the T-cell compartment along the rechallenge experiment with H929 myeloma cell line

[0070] The percent of CD3+ T-cells (Fig. 16A), the percent of CAR+ T-cells (gated on CD3+) (Fig. 16B), and the distribution of the CD4+ and CD8+ subsets (Fig. 16C) were determined by flow cytometry in co-culture of CART cells generated via the Naive or the traditional (TM) platforms. Left panels represent CART cell culture with H929 at the E:T of 1: 1, right panels represent CART cell culture with H929 at the E:T of 1:4. Data are represented as the mean ± SEM. Statistical analysis by 2- way ANOVA. N=4.

[0071] Figure 17A-17C. Analysis of the T-cell compartment along the rechallenge experiment with MM1S myeloma cell line

[0072] The percent of CD3+ T-cells in (Fig. 17A), the percent of CAR+ T-cells (gated on CD3+) (Fig. 17B), and the distribution of the CD4+ and CD8+ subsets (Fig. 17C) were determined by flow cytometry in co-culture of CART cells generated via the Naive or the traditional (TM) platforms. The CART cells were co-cultured with MM1S at E:T of 1:4. Data are represented as the mean ± SEM. Statistical analysis by 2-way ANOVA. N=4.

[0073] Figure 18A-18C. Correlation between the percent of CAR+CD4+ T-cell subset and myeloma cell eradication

[0074] The proportion of CD4+ T-cells within the CAR+ cell population was assessed by flow cytometry. Correlation curve of CD4+ CART cells and the tumor load in co-culture of traditionally manufactured (TM) CART cells (squares) and the tumor load (as the percent of initial load) at D+8, 15, 22 and 29 post co-culture initiation. The line represents the linear regression. N=4. Pearson’s test, r, Pearson correlation coefficient, R2, coefficient of determination. Dotted gray lines represent the 95% CI.

[0075] Fig. 18A. CART cell culture with H929 at the E:T of 1:1.

[0076] Fig. 18B. CART cell culture with H929 at the E:T of 1:4.

[0077] Fig. 18C. CART cell culture with MM1S at the E:T of 1:4.

[0078] Figure 19A-19B. CAR T-cell differentiation along recursive exposure to tumor cells

[0079] CART cells were generated from four leukapheresis source materials (two multiple myeloma (MM; BCMA#112, BCMA#120), one light chain amyloidosis (AL; BCMA#119) patients and one healthy donor (HD; PHE-011)) using the “NAIVE” (Fig. 19A) or traditional (Fig. 19B) platforms. Every three to four days, at the indicated timepoints, persistent CART cells from the precedent co- culture setting were rechallenged with fresh MM IS myeloma cells. Once weekly, at the indicated timepoints, the differentiation profile of CART cells was characterized by flow cytometry. Left panels indicate the percentages of the different CART-cell subsets (gated on CD3+CAR+ cells) and right panels indicate the absolute numbers. TNAIVE / SCM, described as CD45RA+CD62L+; TCM, described as CD45RA-CD62L+; TEM, described as CD45RA-CD62L-; TEMRA, described as CD45RA+CD62L-.

[0080] Figure 20A-20B. CAR T-cell exhaustion along recursive exposure to tumor cells

[0081] Fig. 20A. Expression of PD-1, TIM-3, LAG-3, TIGIT and CTLA-4 on Naive (circles) and TM (squares) CART cells was assessed by flow cytometry weekly along the rechallenge experiment with MM1S myeloma cells at a 1:4 E:T ratio.

[0082] Fig. 20B. Pie charts represent the percentage of Naive (right charts) and TM (left charts) CAR+ cells co-expressing 0 / 1 / 2 / 3 / 4 or 5 exhaustion markers.

[0083] Figure 21A-21B. CAR T-cell activation following overnight co-culture

[0084] Fig. 21A. CART cell activation profile. Expression of CD25, CD69 and 4- IBB on Naive (circles) and TM (squares) CART cells was assessed by flow cytometry following an overnight co-culture with H929 myeloma cells at a E:T ratio of 1:1.

[0085] Fig. 21B. IFN-y secretion by Naive (circles) and TM (squares) CART cells was assessed by ELISA following an overnight co-culture with H929 myeloma cells at a E:T ratio of 1:1.

[0086] Figure 22A-22B. Intracellular cytokine and degranulation profiles

[0087] IFN-y, TNF-a and IL-2 (Fig. 22A) and granzyme B (GZM) intracellular staining and CD107a mobilization (Fig. 22B). Naive or TM CART cells from 4 independent donors were retrieved from rechallenge (RC) -4 (4 stimulations with tumor cells) at E:T ratio of 1:4, and cryopreserved. Upon thawing, RC-4 remaining CART cells were subjected to a 5-hrs incubation either alone (unstimulated), or with K562 (negative control), or PMA ionomycin (positive control) or with fresh MM1S myeloma cells (E:T ratio: 1:1). Staining of intracellular IFN-y, TNFa, IE-2, GZM and mobilized CD 107a was performed after cell fixation and permeabilization, and the percent of cytokine-expressing CAR+ cells was assessed by flow cytometry (gated on viable CD3+CAR+ cells). Error bars represent the mean ± SEM. N=4 individual CART products from two multiple myeloma (MM; BCMA#112, BCMA#120), one light chain amyloidosis (AL; BCMA#119) patients and one healthy donor (HD; PHE-011)). Statistical analysis by 2-way ANOVA.

[0088] Figure 23. CART exhaustion profile of CART cells upon rechallenges with fresh tumor cells Naive (circles) or TM (squares) CART cells from 4 independent donors were retrieved from rechallenge (RC) -4 (4 stimulations with tumor cells) at E:T ratio of 1:4, and cryopreserved. Upon thawing, RC-4 remaining CART cells were subjected to a 5-hrs incubation either alone (unstimulated), or with K562 (negative control), or PMA ionomycin (positive control) or with fresh MM1S myeloma cells (E:T ratio: 1:1). Cells were then analyzed for PD-1 and TIM-3 expression by flow cytometry (gated on viable CD3+CAR+ cells). Error bars represent the mean ± SEM. N=4 individual CART products from one healthy donor, two myeloma (MM) and one light chain amyloidosis (AL) patients. Statistical analysis by 2-way ANOVA.

[0089] Figure 24. Flow cytometry of CD4 and CD8 CART cell subsets following sequential stimulations with fresh myeloma cells

[0090] CD4 and CD 8 CART cell subsets following four sequential stimulations with fresh myeloma cells were assessed by flow cytometry. Naive (bottom panel) or TM (upper panel) CART cells from 4 independent donors were retrieved from RC-4 and cryopreserved. CART cell co-cultures with MM1S were analyzed for CD4 and CD8 expression by flow cytometry (gated on viable CD3+CAR+ cells). Flow cytometry dot plots are depicted.

[0091] Figure 25. Genes associated with naive and stem cell memory T cell phenotypes (scRNAseq) Dot plot showing signature genes for Stem cell like memory (TSCM) and Naive T cells in the CAR- positive Naive and TM samples. Dot size corresponds to the percentage of cells expressing each gene within each sample, and color indicates the average scaled (z-score) expression value.

[0092] Figure 26. Effector-associated genes (scRNAseq)

[0093] Dot plot showing signature genes for activated and exhausted T cells in the CAR-positive Naive and TM samples. Dot size corresponds to the percentage of cells expressing each gene within each sample, and color indicates the average scaled (z-score) expression value. Figure 27. Genes associated with naive and stem cell memory T cell phenotypes in CAR- positive CD8+ subset (scRNAseq)

[0094] Dot plot displaying selected signature genes for stem cell-like memory (TSCM) and Naive T cells within a subset of the CAR-positive Naive and TM samples, restricted to CD8 cells annotated based on transcriptomic markers (cells with CD8A / CD8B / CD8B2 transcript expression> 0). Dot size corresponds to the percentage of cells expressing each gene within each sample, and color indicates the average scaled (z-score) expression value.

[0095] Figure 28. Heatmap of selected signature genes for stem cell-like memory (TSCM) / Naive and effector T cells

[0096] Heatmap of selected signature genes for stem cell-like memory (TSCM) / Naive and effector T cells based on pseudo bulk RNA-seq of cell populations obtained from an integrated single-cell dataset comprising six samples (Naive and TM CART products derived from one healthy donor and two multiple myeloma patients). Rows represent genes, and columns represent samples. Expression values were scaled (z-score) across each gene (row), such that color intensity reflects expression relative to the average expression of that gene across all samples — red indicates higher-than- average expression, and blue indicates lower-than-average expression.

[0097] Figure 29A-29C. Characterization of MM patient’s CART final products

[0098] HBI0101 CART cells were generated using the “naive” (Fig. 29 A) or the traditional manufacture platforms (Fig. 29B). Cells were cryopreserved in CS-5 and kept frozen in liquid nitrogen. At the day of CART infusion, cells were thawed in warm medium and characterized by flow cytometry to assess the percent of transduced cells (Fig. 29C).

[0099] Figure 30. Efficacy of Naive CART cells in multiple myeloma control in comparison with TM CART cells: in situ experiment monitoring

[0100] Disseminated human xenograft myeloma was induced by injecting NSG mice with lx10˄6 NCI- MMls-luc cells i.v. in the vein tail. Two weeks following tumor inoculation, CART cells generated from a multiple myeloma (MM) patient via the “naive” or the traditional manufacture (TM) platforms were infused i.v. (vein tail) at escalating doses ranging from 0.1- to lx10˄6 CAR+ cells. Non-transduced (NonT) T control cells were adjusted to the highest dose of CART total cells as per each platform. Mice were monitored twice weekly by IVIS camera for the bioluminescence intensity. Ventral (V, top images) and dorsal (D, bottom images) imaging were performed for more accurate evaluation of the tumor. Mice were sacrificed if showing human end-points clinical symptoms as per ethical authorized form or if BEI intensity reached 0.5x10˄9 - 10˄10 p / sec. This experiment was terminated at D+92, as per ethical protocol. Figure 31A-31B. Multiple myeloma monitoring in NSG mice xenograft treated with naive or TM CART cells from a MM patient

[0101] Graphical representation of the experiment depicted above in Figure 30. This graph represents the bioluminescence (BLI) values in myeloma NSG xenograft treated with (Fig.31A) traditional (TM) or (Fig. 31B) Naive CART as the sum of the ventral and dorsal intensity values (photon / sec). Points represent the BLI mean of each group at a specific time point; bars represent SEM. Mice were sacrificed if showing human end-points clinical symptoms as per ethical protocol or if BLI intensity reached 0.5x10˄9 - 10˄10 p / sec. This experiment was terminated at D+92, as per ethical protocol.

[0102] Figure 32A-32B. Survival analysis

[0103] Fig. 32A. Averaged bioluminescence as per treatment group.

[0104] Fig. 32B. Kaplan-Meier survival curves.

[0105] Figure 33A-33B. Naive versus TM CART cell pharmacokinetic

[0106] CAR+ cells were enumerated in the peripheral blood of NSG mice xenograft at the indicated time points.

[0107] Fig. 33A. CAR+ cells were stained for the G4S linker and processed for flow cytometry, using Flow count fluorospheres (Beckman Coulter) for cell count.

[0108] Fig. 33B. CAR+ cells were detected by qRT-PCR of the vector transgene. Vector copies were then adjusted to the copy number per transduced cell at the time of CART infusion and normalized to the amount of genomic DNA for each sample.

[0109] Figure 34A-34C. Naive versus TM CART cell pharmacokinetic

[0110] Fig. 34A. CAR+ cell maximal concentration in the peripheral blood of NSG xenograft treated with Naive or TM CART cells.

[0111] Fig. 34B. The area under the curve (AUC) which represents the total CART cell expansion, was calculated for each treatment group for the period from CART infusion (DO) and until D+28 post CART infusion. After D+28, mice from the TM group were sacrificed.

[0112] Fig. 34C. AUC for the entire period the experiment lasted (90 days).

[0113] Values represent the mean for each treatment group + / - SEM. Statistical analysis by multiple unpaired t-test. Figure 35. Characterization of CAR+ cells in the blood of NSG myeloma xenograft treated with Naive or TM CART cells

[0114] The percentage of CD3+, of CD3+CAR+ and of the CART-cell subsets (CD4+ and CD8+) were determined by flow cytometry. Bars represent the mean for each treatment group + / - SEM. Statistical analysis 2-way ANOVA.

[0115] Figure 36. CART-cell differentiation profile at peak in the mice peripheral blood

[0116] Proportion of the T-cell subset in the blood of mice treated with Naive or TM CART cells determined by flow cytometry. TNaive / SCM: CD45RA+ CD62+; TCM, CD45RA- CD62+, TEM, CD45RA- CD62-; TEMRA, CD45RA+ CD62-. Bars represent the mean for each treatment group + / - SEM. Statistical analysis 2-way ANOVA.

[0117] Figure 37. Proportion of senescent-like CART cells in the mouse peripheral blood

[0118] Percentages of senescent-like (described as CD27-CD28-) and functional (described as CD27+CD28+) CART cells (gated on CD3+CAR+ cells) in the were determined in the peripheral blood of Naive and TM mice at CART peak. Bars represent the mean for each treatment group + / - SEM. Statistical analysis 2-way ANOVA.

[0119] Figure 38A-38D. CD62L expression recovery post thaw, upon PBMCs cultivation overnight and post CD62L enrichment

[0120] Maturation profile of MM patient’s T-cells upon thawing (Fig. 38A) of the leukapheresis bag and the day afterwards following recovery overnight in AIMV+1% HS+IL7 / 15 / 21 at 37°C (Fig. 38B), and post CD62L enrichment (Fig. 38C) was assessed by flow cytometry. (Fig. 38D) CD62L expression on T-cells in the CD62L-negative fraction as well. Mean fluorescence intensity (MFI) of the CD62L marker is described to evaluate the effect of overnight incubation at 37°C in Naive- T-cell media supplemented with IL-7 / 15 / 21 on CD62L marker recovery.

[0121] Figure 39A-39C. Multiple myeloma monitoring in NSG mice xenograft treated with naive or TM CART cells from a MM patient

[0122] Fig. 39A. Graphical representation of the BLI values as the sum of the ventral and dorsal intensity values (photon / sec) as per treatment group. Each curve represents a single mouse subject. Mice were sacrificed if showing human end-points clinical symptoms as per ethical protocol or if BLI intensity reached 10˄9 - 10˄10 p / sec.

[0123] Fig. 39B. Kaplan-Meir survival curve.

[0124] Fig. 39C. Body weight of the mice involved in was recorded over time. Each line represents one mouse. Figure 40. Naive versus TM CART cell pharmacokinetic

[0125] CAR+ cells were enumerated in the peripheral blood of NSG mice xenograft infused with Naive (red lines; red triangles: Ix106CAR+; light red circles: O. lx106CAR+) and TM (blue lines; blue triangles: Ix106CAR+; light blue circles: O.lx106CAR+) CAR T products at the indicated time points. CAR+ cells were stained for the G4S linker and processed for flow cytometry, using Flow count fluorospheres (Beckman Coulter) for cell count.

[0126] DETAILED DESCRIPTION OF EMBODIMENTS

[0127] CAR-T cells are a “living-drug”, which manufacture, and in vivo performance are tightly related. Moreover, the manufacture process has a direct impact on T-cell function and durability in vivo. The traditional manufacturing process of CART cells is a long-lasting process (generally 9-14 days) and involves generally the use of IL-2 as growth factor. While IL-2 promotes T-cell survival and proliferation, it also drives T-cell differentiation to more terminal stages. More specifically, IL-2 also promotes regulatory T cells (Treg) cells formation, which presence in the CART final product, was found to negatively correlate with CART therapy outcome. While this way to expand T-cell ex vivo has proven efficient to yield hundreds of millions CART cells, these cells turn to become exhausted and senescent shortly following their injection into patients. Technologies aiming at improving CART cell fitness and persistence are therefore crucial to improve their function and persistence in vivo, and thus to improve patients’ clinical outcome.

[0128] Upon antigen stimulation {ex vivo or in vivo'), naive T-cells differentiate into cytotoxic effector and memory T-cells, before becoming functionally exhausted (as illustrated by Figure 1). As explained above, traditional manufacture of CART cells depletes essential T-cell subsets from the CART final product, namely the naive and stem-cell like T-cells (TNaive / SCM), while enriching effector T-cells (TEMand TEMRA). This results in early relapses and thus reduced clinical outcome.

[0129] This is even more critical when it comes to patients, which T-cell are often of poorer quality, due to aging, but also to the lympho-toxic regimen patients have endured. TNaive / SCMhave the capacity to self-renew, they exhibit a high proliferative capacity, while they can also differentiate into T-cells with potent effector functions upon antigen stimulation. Preserving a pool of TNaive / SCMin the CART product, is essential to provide sufficient amount and highly performant CART-cells with long-lasting durability in vivo, even upon recursive stimulation with tumor cells. Such cells may even play a role in the case of relapse prevention.

[0130] The development of novel manufacturing platforms for the generation of CART cells with stem-cell like features is crucial to improve cancer immunotherapies outcome. In the past decade, naive / stem-cell like memory and central memory T-cells (TNaive / SCMand TCM) have been placed under the spotlights for their capacity to self-renew, highly proliferate, and differentiate into effector T-cells mediating tumor killing. However, traditional manufacture process for CART cell generation is a long-lasting process (generally 9-14 days) which involves generally the use of IL-2 as growth factor. While IL-2 promotes T-cell survival and proliferation, it also drives T-cell differentiation, to more terminal stages. It also promotes regulatory T cells (Treg) cells formation, which presence in the CART final product, were found to negatively correlate with CART therapy outcome. While this way to expand T-cell ex vivo have proven efficient to yield hundreds of millions CART cells, these cells turn to become exhausted and senescent shortly following their injection into patients. This is even more critical when it comes to patients, which T-cell are often of poorer quality, due to aging, but also to the lympho-toxic regimens patients have endured.

[0131] This is why the development of novel protocols preserving T-cell “sternness” are highly required. Herein, the inventors describe a novel streamlined, GMP-compliant, 6-day manufacturing platform for the generation of CART cells with preserved sternness in a single culture vessel (namely, G-Rex). The inventors believe that by integration of concepts already described in the literature (namely, the modification of the autologous CAR-T traditional manufacturing process with the aim to retain these less-differentiated T-cell populations, enhance the potency of the product, and increase the scalability of the manufacturing process), they have generated a novel manufacturing process by which a unique CART cell population with improved proliferative and cytotoxic capacity, and long-term persistence can be generated. As a “proof-of-concept” for the feasibility of such new technology, the inventors made use of HBI0101, an anti-BCMA CAR construct, originally developed for the treatment of multiple myeloma and light chain amyloidosis. Importantly, this novel platform was tested on peripheral blood apheresis products from healthy donors and myeloma / amyloidosis patients as well.

[0132] Thus, in a first aspect, the present disclosure relates to a method for preparing a population of naive-like T cells. More specifically, the naive-like T cells prepared by the disclosed method, comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). The disclosed methods comprise the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage, specifically, T cells, with at least one compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), the activated cells obtained in step (b), are modified, e.g., genetically modified with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. Step (d) involves expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the disclosed method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days.

[0133] In some alternative embodiments, the present disclosure provides methods for preparing a population of naive-like T cells. More specifically, the naive-like T cells prepared herein comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM)). The disclosed method comprises the following steps. Step (a) involves contacting a population of cells comprising cells of the T lineage, specifically, T cells, with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to a T-cell activation. Next, in step (c), expanding the activated genetically modified cell population, thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. More specifically, in some embodiments, the disclosed methods, either in the version that includes genetically modifying the cells (step c, above), or a version that does not comprise genetic modification of the cells, (expansion of the cells immediately after activation), may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. However, it should be understood that if the number of cells obtained by the disclosed process is insufficient for the intended use, the cells may undergo additional expansion. Accordingly, the present disclosure further contemplates that the method may comprise expanding the cells obtained in step (d) for up to two additional days. In particular, the cells may be expanded for one or two extra days, such that the total duration of the process is extended to seven or up to eight days, provided that such additional expansion does not alter the defining properties of the naive-like cell population generated by the process, e.g., the CD4+ / CD8+ratio and / or the genetic signature, as further discussed herein.

[0134] In some optional embodiments, the disclosed methods may further comprise modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. The present disclosure provides methods for preparing population of lymphocyte, specifically, any lymphocyte of the T lineage. "Lymphocytes" are mononuclear nonphagocytic leukocytes found in the blood, lymph, and lymphoid tissues. They are divided on the basis of ontogeny and function into two classes, B and T lymphocytes, responsible for humoral and cellular immunity, respectively. Most are small lymphocytes 7-10 pm in diameter with a round or slightly indented heterochromatic nucleus that almost fills the entire cell and a thin rim of basophilic cytoplasm that contains few granules. When "activated" by contact with antigen, small lymphocytes begin macromolecular synthesis, the cytoplasm enlarges until the cells are 10-30 pm in diameter, and the nucleus becomes less completely heterochromatic; they are then referred to as large lymphocytes or lymphoblasts. These cells then proliferate and differentiate into B and T memory cells and into the various effector cell types: B cells into plasma cells and T cells into helper, cytotoxic, and suppressor cells.

[0135] As indicated by the present disclosure, the cells prepared by the methods of the present disclosure, are cells of the T lineage, Specifically, the present disclosure relates to naive-like T cells, which represent a distinct subset of T cells, as will be further discussed herein after. The cells are prepared by enriching a population of cells (referred to herein as a "source material," "starting material," or "starting population") comprising cells of the T lineage, using a specific compound that selects for a marker characteristic of circulating innate lymphoid cell precursor cells. In accordance with the present disclosure, a suitable source material is any population of cells that includes at least one cell of the T lineage. More specifically, at least about 1% of the cells in the starting or source population are T lineage cells. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% of the cells in the starting population are T lineage cells.

[0136] A "cell of the T lineage", ”T cell” or ”T lymphocyte” as used herein is characterized by the presence of a T-cell receptor (TCR) on the cell surface. More specifically, The T cell receptor (TCR) is a heterodimeric membrane-bound receptor expressed on the surface of T lymphocytes that is responsible for recognizing antigenic peptides presented by major histocompatibility complex (MHC) molecules on antigen-presenting cells. Because the TCR itself lacks intrinsic signaling capacity, it is noncovalently associated with the CD3 complex, which is composed of invariant signaling chains (CD3γ, CD3δ, CD3ε. and CD3ξ) containing immunoreceptor tyrosine- based activation motifs (ITAMs). Together, the TCR and CD3 form the TCR-CD3 complex, which ensures both specific recognition of antigen-MHC complexes through the TCR and the initiation of intracellular signaling cascades via the CD3 subunits, ultimately leading to T cell activation, proliferation, and effector function. It should be noted that T-cells in general, include helper T cells ("effector T cells" or "Th cells"), cytotoxic T cells ("Tc," "CTL" or "killer T cell"), memory T cells, and regulatory T cells as well as Natural killer T cells, Mucosal associated invariants and Gamma delta T cells. More specifically, Thymocytes are hematopoietic progenitor cells present in the thymus. Thymopoiesis is the process in the thymus by which thymocytes differentiate into mature T lymphocytes. The thymus provides an inductive environment, which allows for the development and selection of physiologically useful T cells. The processes of beta-selection, positive selection, and negative selection shape the population of thymocytes into a peripheral pool of T cells that are able to respond to foreign pathogens and are immunologically tolerant towards self- antigens.

[0137] Thymocytes are classified into a number of distinct maturational stages based on the expression of cell surface markers. The earliest thymocyte stage is the double negative (DN) stage (negative for both CD4 and CD8), which more recently has been better described as Lineage-negative, and which can be divided into four sub-stages. The next major stage is the double positive (DP) stage (positive for both CD4 and CD8). The final stage in maturation is the single positive (SP) stage (positive for either CD4 or CD8).

[0138] More specifically, the maturational stages of thymocytes may include the following substages: Double negative 1 (DN1) or ETP (Early T lineage Progenitor) is characterized by CD44+CD25- CD117+ defining surface markers, thymocytes are located in the cortex and proliferation, loss of B and myeloid potentials are observed; Double negative 2 (DN2) is characterized by CD44+CD25+CD117+ defining surface markers and thymocytes are located in the cortex; Double negative 3 (DN3) is characterized by CD44-CD25+ defining surface markers, thymocytes are located in the cortex and TCR-beta rearrangement and beta selection are observed; Double negative 4 (DN4) is characterized by CD44-CD25- defining surface markers and thymocytes are located in the cortex; Double positive is characterized by CD4+CD8+ defining surface markers, thymocytes are located in the cortex and TCR-alpha rearrangement, positive selection, negative selection are observed; Single positive is characterized by CD4+CD8- or CD4-CD8+ defining surface markers, thymocytes are located in the medulla and Negative selection is observed.

[0139] In human, circulating CD34+ hematopoietic stem cells (HSC) reside in bone marrow. They produce precursors of T lymphocytes, which seed the thymus (thus becoming thymocytes) and differentiate under influence of the Notch and its ligands. Early, double negative thymocytes express (and can be identified by) CD2, CD5 and CD7. Still during the double negative stage, CD34 expression stops and CD1 is expressed. Expression of both CD4 and CD8 makes them double positive and matures into either CD4+ or CD8+ cells. It should be appreciated that a cell of the T lineage as disclosed herein may be any of the thymocytes disclosed herein at any stage / substage and / or expressing any of the disclosed markers.

[0140] As discussed herein after, the disclosed methods provide populations of cells, specifically, T cells that display increased viability and effectiveness (as being composed of an increased ratio of naive and naive-like stem cells), reduced exhaustion, increases CD4+ / CD8+ ratio and potentially increases the trafficking potency. More specifically, as mentioned above, the T cell population prepared by the disclosed methods are enriched and mainly composed of naive-like cells. T cell compartment includes T cell subsets that are at different stages of differentiation. These subsets arise from differentiation of Naive T cells (TN), which are CD45RA+, CD62L+, CCR7+, CD27+, CD28+, and CD95-Upon encounter with antigen, TN cells upregulate the CD95 marker to become stem cell-like memory T cells (TSCM), designated as CD45RA+, CD62L+, CCR7+, CD27+, CD28+, and CD95+. These cells can further differentiate into Central Memory Cells (TCM), which are CD45RO+, CD62L+, CD28+, and CD95+ or into Effector Memory Cells (TEM), which are CD45RO+, CD62L-, CCR7-, CD28+ / -, and CD95+. Fully differentiated TEM, upregulate back their CD45RA, to become effector T cells, known as TE or TEMRA, and are described as CD45RO+, CD62L-, CCR7-, CD27-, CD28-, and CD95+.

[0141] Stem-cell like memory T Cells (TSCM) are present at a low level in the T cell compartment but appear to have significant self-renewal and proliferative potential. While they resemble naive T cells (TN) in that they express CD45RA+ and CD62L+, they can be distinguished from TN by their expression of CD95. TSCMcan be artificially generated from TN by stimulation with CD3 / CD28 beads in the presence of IL-7 and IL- 15. They also can be expanded in the presence of Wnt / p- catenin pathway activation.

[0142] Central Memory T Cells (TCM), which are more abundant in PBMCs, are a well-defined memory T cell subset with high self-renewal and proliferative potential. TCMpersists following adoptive transfer better than Effector T cells (TE). TNaive / SCMand TCMcan be enriched from PBMC for T cell therapy manufacturing based on their CD62L+ phenotype.

[0143] As demonstrated in Figures 6, 8, 9 and 10, the population of T cells prepared by the disclosed method compromise naive-like T cells, as specified above, specifically, cells composed of the TN, TSCM, and TCMsubsets. In more specific embodiments, the resulting population of T cells is enriched in TN, TSCM, and TCMsubsets. In some embodiments, the population of T cells prepared according to the methods described herein comprises enriched proportions of TN, TSCM, and TCMsubsets. As used herein, the term “enriched” when referring to a T cell population prepared by the disclosed methods with specific naive subsets of cells, denotes that the proportion or frequency of the naive T cell subset(s), specifically, the at least one of the TN, TSCM, and TCMsubsets, within the population is increased relative to a reference or control population, for example, any T cell population not prepared by the disclosed methods, and / or T cell populations in untreated and / or natural peripheral cell sample, and / or T cell populations prepared by methods other than the methods of the present disclosure, and / or to T cell populations prepared by a traditional method, the TM method as disclosed herein. In some embodiments of the present disclosure, the enrichment may result from the isolation, selection, sorting, of the cells as specified in step (a), but however does not require the naive subset to constitute the majority of the resulting population. Enrichment encompasses both relative and absolute increases in the number of naive T cells, and may be expressed, for example, as a fold increase, a percentage increase, or another quantitative measure compared to the reference. For example, enrichment may also encompass an increase of 5% to 100% or more, in the naive T cells, as compared to the source population, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. Still further, in other embodiments, "enriched" encompasses that the relative proportion of the naive T cells is increased in the resulting population. In more specific and non-limiting embodiments, the combined proportion of these subsets is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total T cell population. In more specific embodiments, the population of T cells prepared according to the method described herein comprises enriched proportions of TN, TSCM, and TCMsubsets, wherein the combined proportion of these subsets is at least 50% of the entire population.

[0144] If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can be isolated from tumor sites (i.e., tumor-infiltrating lymphocytes or TILs; marrow-infiltrating lymphocytes or MILs; etc.). T cells can also be enriched for or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. Thus, the present disclosure further envisaged a source cell population, specifically, the population of cells in step (a), that comprises at least one of: peripheral blood cells, a leukapheresis cell product, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ. In more specific embodiments, cell population for use in step (a) of the disclosed methods may be derived from infiltrating tissue microenvironment. The term “infiltrating tissue microenvironment” refers to the local milieu of a tissue into which cells, most commonly immune cells such as lymphocytes, macrophages, or other leukocytes, have migrated from the circulation. This microenvironment is composed of a complex network of resident cells, extracellular matrix components, signaling molecules such as cytokines and chemokines, metabolic factors, and vascular or stromal elements that collectively influence the survival, phenotype, and function of the infiltrating cells. The dynamic interactions between the infiltrating cells and the tissue microenvironment determine the outcome of immune responses. Examples of infiltrating tissue microenvironments include the tumor microenvironment (TME), in which T cells, macrophages, dendritic cells, and other immune cells infiltrate solid tumors and interact with cancer cells, stromal fibroblasts, and immunosuppressive mediators; sites of chronic inflammation, such as synovial tissue in rheumatoid arthritis, where activated T cells, B cells, and macrophages infiltrate and engage with fibroblast-like synoviocytes and pro-inflammatory cytokines; infected tissues, such as the lung during viral pneumonia, where lymphocytes and neutrophils migrate into the alveolar spaces and respond to viral antigens and inflammatory signals; and ischemic or injured tissues, such as the myocardium following myocardial infarction, where infiltrating monocytes and neutrophils participate in tissue remodeling and repair in concert with resident stromal and endothelial cells. Still further, in some embodiments, the cell population is derived from at least one of inflamed tissues and tumor tissue. It is further envisaged that the starting cell population in step (a) may be of a primary or secondary lymphoid organ, for example, bone marrow or lymph nodes. It should be understood that T cells at the infiltrated sites display a broad repertoire of endogenous TCRs capable of recognizing their cognate antigen. They are also properly equipped to traffic to the site. Though infiltrating lymphocytes may become dysfunctional under chronic antigen exposure and under a suppressive environment. Thus, enriching for the early-memory potentially still present even though at low frequency, may restore T-cell functionality while preserving their poly clonally and trafficking abilities.

[0145] The disclosed methods involve the step of using a population of cells originated from peripheral blood. As used herein, the term “cell population of peripheral blood” refers to a population of cells derived from the peripheral blood of a mammalian subject, including but not limited to humans. Such a population may comprise one or more lymphocyte subsets, monocytes, granulocytes, dendritic cells, or other circulating immune cells, either in unmodified form or following manipulation. The cell population may be heterogeneous, encompassing a variety of cell types present in the circulation, or may be enriched for one or more specific subsets, such as T cells, B cells, natural killer (NK) cells, monocytes, or progenitor cells, depending on the intended use. The cell population of peripheral blood may be obtained by any method suitable for isolating cells from circulating blood. Such methods include, for example, the collection of whole blood by venipuncture or apheresis, followed by separation of peripheral blood mononuclear cells through density gradient centrifugation, leukapheresis and the like. The term also encompasses cell populations that have been cryopreserved, thawed, expanded ex vivo, or otherwise manipulated, provided that they are originally derived from peripheral blood.

[0146] It should be however noted that several well-characterized blood-derived preparations contain blood cells, including cells of the T lineage and may be used herein as a starting population. For example, Leukapheresis cell products are obtained by apheresis procedures that selectively collect leukocytes from peripheral blood while returning red blood cells and plasma to the donor; these products provide a large number of white blood cells and are frequently used as starting material for adoptive cell therapies or ex vivo manipulation. Whole blood-derived buffy coats are prepared by centrifugation of whole blood donations and contain a concentrated fraction of leukocytes and platelets. Bone marrow aspirates are another source of heterogeneous blood and progenitor cells, including hematopoietic stem cells. Umbilical cord blood preparations contain a mixture of hematopoietic stem / progenitor cells, lymphocytes, and other leukocytes and are widely used in transplantation and regenerative medicine. Mobilized peripheral blood stem cell (PBSC) products, obtained after administration of cytokines such as G-CSF, are enriched in hematopoietic stem and progenitor cells collected via apheresis. Collectively, these preparations represent key sources of cells for the method for preparing the naive T cell population of the present disclosure.

[0147] As shown by the following examples, e.g., Example 12, the cell population may be obtained from cryopreserved leukapheresis cell product and / or any cryopreserved blood product. Thus, in some embodiments, step (a) of the disclosed methods may comprise the step of using cells of cryopreserved peripheral blood for the disclosed methods. Cryopreserved peripheral blood as used herein may be prepared by any of the methods discussed herein after in connection with other embodiments of the disclosure that encompass the term cryopreservation. The definition of the term therein is applicable for the same term in the current embodiment.

[0148] In some embodiments, all the steps of the disclosed methods are performed in the same cell culture vessel, specifically, in a single container or chamber. More specifically, a cell culture vessel as used herein relates to any apparatus or device designed to support the growth and maintenance of cells in a controlled environment. These structures are typically made from materials that are biocompatible and may be transparent to allow for the observation of cells. They can also be treated or coated to promote cell adhesion, depending on the type of cell culture. The vessel, container, or chamber may include additional features such as ports for gas exchange, access for adding or removing culture media, and surfaces designed for specific cell growth characteristics. The volume capacities of cell culture vessels, containers, or chambers vary widely depending on the type and application. The present disclosure encompasses the use of any size of culture vessel, from petri dishes or multi-well plates to large bioreactors. More specifically, petri dishes that typically range from 5 mL to 60 mL, T-Flasks cover a similar volume range but are defined by surface area, from 25 cm2to 225 cm2. Multi-well plates, depending on configuration, offer volumes from 0.1 mL per well to 5 mL per well. Spinner flasks provide larger volume options, ranging from 50 mL to 3,000 mL. Bioreactors, which can be scaled for industrial applications, range from 500 mL to several hundred liters, accommodating large-scale culture needs.

[0149] In yet some further embodiments, the disclosed methods further comprise the step of (e), harvesting the expanded cell population obtained in step (d). It should be understood that in methods that do not include the genetic modification step (c), step (d) is considered as step (c), step (e) is considered herein as step (d) and involves harvesting the expanded cell population obtained in step (c).

[0150] More specifically, harvesting the expanded cell population refers to the process of collecting or retrieving cells that have been cultured and proliferated to achieve a desired quantity or density. This process typically follows the in vitro expansion of cells, where the initial cell population is subjected to growth conditions that promote cell division and increase cell numbers. Harvesting involves the careful separation of the expanded cells from the culture medium, substrates, or growth surfaces, ensuring the cells are viable and suitable for subsequent applications. The harvested cells may be used herein for therapeutic purposes, such as infusion into a patient, or for further storage, or processing. The disclosed methods encompass various methods of cell retrieval, including enzymatic detachment, centrifugation, filtration, or other techniques designed to preserve the integrity and functionality of the expanded cell population.

[0151] In yet some further embodiments, the disclosed methods further comprise the step of (f) (this step may be optionally referred to as step (e)), that involves formulating the expanded cell population. In some specific embodiments, formulating the expanded cell population means suspending the cells in an infusion solution, thereby providing an infusion composition. Infusion composition as used herein, refers to a composition comprising a sterile liquid medium that contains a suspension of viable cells (e.g., the T cells prepared by the disclosed methods), intended for administration into a subject, typically via intravenous or other parenteral routes. In some embodiments, the solution not only serves as a vehicle for delivering the cells but may also contain additional components, such as electrolytes, nutrients, cryoprotective agents, or stabilizers, which support cell viability and functionality during storage, transportation, and infusion. An infusion composition as used herein encompass various formulations, methods of preparation, and conditions for maintaining cell stability and efficacy within the infusion solution, and depends on whether the infusion is delivered as fresh or cryopreserved / thawed suspension. An infusion solution useful in the present disclosure may be any buffer approved by the Ministry of Health for clinical use. More specifically, in some non-limiting embodiment, the infusion solution could comprise saline+2.5% human serum albumin, or alternatively, CryoStor™ (BioLife).

[0152] Thus, as indicated above, the population of cells used in step (a) of the disclosed methods comprises at least one of: peripheral blood cells, a leukapheresis cell product, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ. In some embodiments, such population of cells from peripheral blood comprises peripheral blood mononuclear cell(s) (PBMC(s)), the infiltrating tissue microenvironment is derived from at least one of inflamed tissues and tumor tissue, and the primary or secondary lymphoid organ is bone marrow or lymph nodes.

[0153] Alternatively, or additionally, the expanded cell population may be suspended in cryopreservation solution, thereby providing a composition suitable for cryopreservation. Cryopreservation, as used herein, refers to the process of preserving cells, tissues, organs, or other biological materials at ultra-low temperatures, typically in liquid nitrogen at temperatures of around -196°C. In some embodiments, the method may thus further involve the use of cryoprotective agents to prevent ice crystal formation, which can damage cellular structures. This procedure is commonly employed to maintain the viability and functionality of biological samples for long-term storage, enabling their retrieval and use at a later time. In the context of the present disclosure, this step enables storage of the cells for therapeutic use during different stages of the disease. Several cryoprotective agents are commonly used for the cryopreservation of human lymphocytes, including dimethyl sulfoxide (DMSO), which is widely used at concentrations of around 10% to prevent ice crystal formation. Glycerol, though more frequently used for red blood cells, can also be effective for certain lymphocytes. Ethylene glycol, known for its lower toxicity, is sometimes used with other agents. Trehalose, a non-permeating agent, stabilizes cell membranes, and serum or serum substitutes, like human serum albumin or fetal bovine serum (FBS), provide additional protection. These agents are often combined to enhance cell survival and viability post-thaw.

[0154] In yet some further embodiments, cell cry opreservation solution may be any buffer approved by the Ministry of Health for clinical use. More specifically, the cryopreservation may comprise according to some embodiments, the Cryostor™ (BioLife). In some embodiments of the disclosed methods, the at least one marker of circulating innate lymphoid cell precursor cells, of the enrichment step (a) of the disclosed methods, is at least one of the Cluster of Differentiation 62L (CD62L) and CCR7. More specifically, CD62L is a Functional and Phenotypic Marker for Circulating Innate Lymphoid Cell Precursors, which include naive T cells (TN), memory stem cells (TSCM) and central memory T cells (TCM). Accordingly, these cell populations can be described as TCM / SCM / N cells or TCM / SCM / N cell populations. CD62L / L-selectin is a marker found on naive T cells and further distinguishes central memory (TCM, CD62L+) from effector memory (TEM, CD62L-) T cells. The regulation of CD62L plays a pivotal role in controlling the traffic of T lymphocytes to and from peripheral lymph nodes.

[0155] The CD62L antigen is a 74 kDa glycoprotein and is a member of the selectin family of cell surface molecules, also referred to as L-selectin, LECAM-1, or LAM-1, HLHRC, LEU-8, LNHR, LSEL, LYAM-1 and PLNHR. CD62L binds a series of glycoproteins including CD34, GlyCAM-1, and MAdCAM-1. CD62L is important for homing of naive lymphocytes via the high endothelial venules to peripheral lymph nodes and Peyer's patches. The CD62L antigen also contributes to the recruitment of leukocytes from the blood to areas of inflammation. Most hematopoietic cells, including most peripheral blood B cells, T cells, monocytes, dendritic cells, granulocytes, and some myeloid cells from bone marrow and thymocytes, express CD62L. CD62L is continuously endoproteolytically cleaved from the cell surface of CD62L-expressing neutrophils and lymphocytes (shedding). Proteolysis is accelerated, e.g., after antigen activation of T cells. In some embodiments, CD62L as used herein refers to the human CD62L, as denoted by UNIPROT accession no P14151. In yet some further embodiments, CD62L is encoded by the nucleic acid sequence as denoted by Genebank accession Number NM_000655.5. In yet some further embodiments, CD62L comprises the amino acid sequence as denoted by SEQ ID NO: 1, and any variants or derivatives thereof. Still further, in some embodiments, the CCR7 molecule, can be used for the characterization and / or selection of the T naive / scM and TCMcells. The C-C chemokine receptor type 7 (CCR7) is a seven-transmembrane G protein-coupled receptor (GPCR) composed of approximately 378 amino acids, characterized by an extracellular N-terminus, seven hydrophobic a-helical transmembrane domains connected by alternating extracellular and intracellular loops, and an intracellular C-terminal tail. The CCR7 chemokine receptor protein is expressed on the surface of certain immune cells, including T cells and dendritic cells and plays a critical role in the immune system by directing the migration of these cells to lymphoid tissues in response to its ligands, CCL19 and CCL21. This receptor is crucial for the organization of immune responses, particularly in the trafficking of cells to lymph nodes where they can initiate or modulate immune reactions. In some embodiments, CCR7 as used herein refers to the human CCR7, as denoted by UNIPROT accession no. P32248. In yet some further embodiments, CCR7 is encoded by the nucleic acid sequence as denoted by Genebank accession Number NM_001301714.2. In yet some further embodiments, CCR7 comprises the amino acid sequence as denoted by SEQ ID NO: 2, and any variants or derivatives thereof.

[0156] As used herein, step (a) of the disclosed methods involves contacting population of cells obtained from peripheral blood with a compound that specifically selects for the marker, as discussed herein. As used herein, the expression “a compound that specifically selects for the marker” refers to a compound that exhibits specific recognition and binding affinity for a defined cellular marker (e.g., CD62L and / or CCR7), such that the compound is capable of distinguishing cells expressing the marker from cells that do not express it. The term encompasses compounds that, upon binding, enable the selective detection, capture, or isolation of cells bearing the marker, thereby permitting the separation of such cells from a mixed population. In particular, the compound is one that recognizes an epitope, determinant, or structural feature of the marker with sufficient affinity and selectivity to enable practical use in cell isolation techniques. Non-limiting examples of such compounds that may be useful in the disclosed methods include antibodies and antigen-binding fragments thereof (such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, single-chain variable fragments, Fab fragments, or nanobodies), as well as other binding molecules such as aptamers, engineered ligands, receptor fusion proteins, or small molecules that bind specifically to the marker (e.g., CD62L and / or CCR7). The term further encompasses compounds conjugated to solid supports, beads, magnetic particles, or other functional moieties, provided that such conjugation does not abolish the specific binding to the marker (e.g., CD62L and / or CCR7) and allows the use of the compound in methods for selecting for and isolating cells expressing the marker.

[0157] Thus, in some embodiments, the compound that specifically selects for the marker may comprise at least one antibody specific for each of the disclosed marker of circulating innate lymphoid cell precursor cells, specifically, CD62L and / or CCR7.

[0158] In some embodiments, antibodies against both markers are used as an antibody mixture to characterize T-cell differentiation profile and for selection purposes.

[0159] Still further, in some specific embodiments, it should be understood that the cells expressing the CD62L marker comprise central memory T cells (TCM) and memory stem T cells (TSCM). Accordingly, the disclosed methods provide the desired population of naive-like T cells, which comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM)). In some embodiments, the activation step (b) comprises contacting the cell population with: (i) Cluster of Differentiation 3 (CD3) and Cluster of Differentiation 28 (CD28); and (ii) a cytokine cocktail comprising at least one of Interleukin 7 (IL-7), Interleukin 15 (IL-15) and Interleukin 21 (IL-21). Such cytokine cocktail contains combinations that include binary mixtures of IL-7 and IL-15, IL-7 and IL-21, or IL-15 and IL-21, as well as the ternary mixture comprising IL-7, IL-15, and IL-21. The disclosed cytokine cocktail further covers use of these cytokines in any order or ratio, whether contacted with the cells simultaneously, sequentially, or separately.

[0160] More specifically, CD3 and CD28 are surface proteins found on T cells, each playing critical roles in T cell activation and function. CD3 is a complex of proteins associated with the T cell receptor (TCR) and is essential for signal transduction following antigen recognition, thereby initiating T cell activation. CD28 serves as a co-stimulatory receptor that, when engaged along with the TCR / CD3 complex, provides necessary secondary signals to fully activate T cells, promoting their proliferation, survival, and cytokine production. As for the cytokine cocktail used herein, the cocktail includes at least one of the cytokines IL-7, IL-15, and IL-21, each of which plays distinct roles in T cell biology. IL-7 is crucial for T cell survival and homeostasis, IL- 15 promotes the proliferation and activation of T cells and natural killer (NK) cells, and IL-21 enhances the function of T cells, B cells, and NK cells. In some embodiments, the cytokine cocktail may comprise any appropriate amount of the disclosed cytokines, for example, any amount ranging from about 0.1 pg / ml to about 10Omg / ml, specifically, about 0.1 ng / ml to about 10Ong / ml, more specifically, 0.1, 0.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24,

[0161] 24.5. 25. 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45,

[0162] 45.5. 46. 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66,

[0163] 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81, 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87,

[0164] 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 10Ong / ml. In some embodiments of the disclosed methods, the activation step (b) may be performed using CD3 / CD28 and a cocktail of IL7, specifically, in an amount of 10 ng / ml, IL15, specifically, in an amount of 10 ng / ml and IL21, specifically, in an amount of 20 ng / ml as demonstrated by the examples. It should be understood that the activation step further enriches the CD62 enriched population (that may include in addition to T cells also other mononuclear cells), for T cells. In some embodiments, the activator and / or agonist of CD3 and / or CD28 comprise a colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists.

[0165] Still further, in some particular and non-limiting embodiments, the CD3 / CD28 used for the activation step (a) of the disclosed methods may be provided using a TransAct BEADS, specifically T Cell TransAct™ from Miltenyi Biotec.

[0166] As indicated above, the disclosed methods use a population of cells may be originated from, or in some embodiments, may be obtained from a subject, specifically, a mammalian subject. As discussed above, such population of cells, may be peripheral blood cells, a leukapheresis cell product, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ. In some embodiments, the cell population used in the disclosed method is peripheral blood. In some embodiments, peripheral blood may be obtained from at least one donor by apheresis and / or magnetic isolation. More specifically, pheresis (or apheresis) is a procedure that involves the removal of blood from a patient or donor, separating it into its individual components (such as plasma, platelets, or white blood cells), and then returning the remaining components back to the patient or donor. Still further, magnetic isolation of blood refers to a technique used to selectively separate and purify specific cell types from a blood sample using magnetic particles. In this method, magnetic beads coated with antibodies or ligands specific to the target cells are mixed with the blood. The target cells bind to the magnetic beads, and when a magnetic field is applied, these cells are isolated from the rest of the blood components. Alternatively, or additionally, the cell population used I the disclosed methods may comprise leukapheresis cell product. As also demonstrated by the examples (e.g., Examples 10 and 12), the leukapheresis cell product may be cryopreserved, prior to the use in the disclosed methods.

[0167] In yet some further embodiments, the cells of the cell populations used in the disclosed methods (peripheral blood cells, leukapheresis cell product, cell population derived from infiltrating tissue microenvironment or from a primary or secondary lymphoid organ) are of one donor. In some embodiments, the population of cells comprise peripheral blood mononuclear cell(s) (PBMC). Peripheral blood mononuclear cells (PBMCs) are a heterogeneous group of blood cells characterized by having a round nucleus, which include lymphocytes (T cells, B cells, and natural killer cells) and monocytes. They are commonly isolated from whole blood using density gradient centrifugation.

[0168] In some embodiments, the cell populations used in the disclosed methods, e.g., PBMC cells (or any one of leukapheresis cell product, cell population derived from infiltrating tissue microenvironment or from a primary or secondary lymphoid organ) are of an autologous source, or in other words, the cell population used is autologous PBMCs. It should be noted however that in some alternative embodiments, the disclosed method may be also applicable for cells of an allogeneic source, e.g., allogeneic PBMC. According to such embodiments, such allogeneic cells may be derived from one or more allogeneic donor. It should be however noted that in case of using cells of allogeneic source, an additional step may be required. More specifically, naive T-cell subsets may be considered higher GVHD risk as they were not tolerized to host antigens. Thus, such applications in an allogeneic setting will either require an additional depletion step of the Naive T cells (by CD45RA depletion for example) from the naive-like T cell product or alternatively genetic engineering to reduce alloreactivity (e.g., TCR editing, HLA modification).

[0169] Still further, as noted above, the methods disclosed herein, comprise the step of genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, to obtain a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In some embodiments the exogeneous nucleic acid molecule encodes at least one receptor molecule or at least one antigen binding domain.

[0170] In some embodiments, the exogeneous nucleic acid molecule encodes at least one receptor molecule. In some embodiments, the receptor molecule being a chimeric antigen receptor (CAR) molecule and / or a T cell receptor (TCR) or any part and / or derivative thereof.

[0171] More specifically, Chimeric Antigen Receptor (CAR), as used herein, refers to a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain and an intracellular cytoplasmic signaling domain comprising a functional stimulatory domain. The receptors are chimeric because they couple between extracellular antigen-binding capabilities and intracellular T- or B-cell activating functions, in a single receptor molecule. CARs have been engineered to give the B or T cells they are expressed in the new ability to recognize a specific antigen of interest, thereby facilitating an immune reaction against it. For example, the technology is used in immunotherapy for specifically recognizing specific cancer cells' antigens of interest in order to more effectively direct the immune cells towards those target cells and destroy them. The term "chimeric protein” relates to proteins created through the joining / fusing of two or more genes that originally coded for separate proteins. Translation of this chimeric / fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant chimeric / fusion proteins are created artificially by recombinant DNA technology. Chimeric or chimera usually designate hybrid proteins made of polypeptides having different functions, sources or physico-chemical patterns.

[0172] CAR, as used herein, relates to artificial T cell receptors (also known as chimeric T cell receptors, chimeric immuno-receptors). These are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell.

[0173] The initial design (also referred to a first generation) joined an antibody-derived scFv to the CD3ζ intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains.

[0174] Second generation CARs added intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. More recently, third generation CARs combine multiple signaling domains, such as CD27, CD28, 4-1BB, ICOS, or 0X40, to augment potency.

[0175] It should be understood that the disclosed CAR molecules may be further improved by adding at least one additional signaling domain.

[0176] Still further, the CAR molecule provided herein comprises at least one target-binding domain, which may be in some embodiments, any target-recognition element, for example, at least one antibody or any antigen-binding fragments or domains thereof, as discussed herein above. In yet some further embodiments, the target-recognition element of the CAR molecule of the present disclosure comprises at least one antibody or any antigen-binding fragment / s, portion / s or chimera / s thereof.

[0177] Exemplary categories of antigen-binding domains that can be used in the context of the present invention include antibodies, antigen-binding portions of antibodies (e.g., single chain variable fragments (scFv)), peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand- binding portion of a receptor that specifically binds a particular antigen or antigen-binding scaffolds. The antigen binding domains in accordance with the present disclosure may recognize and bind a specific antigen or epitope. It should be therefore noted that the term “binding specificity”, ’’specifically binds to an antigen”, “specifically immuno-reactive with”, “specifically directed against” or “specifically recognizes”, when referring to an antigen or particular epitope, refers to a binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three- dimensional structural characteristics as well as specific charge characteristics. Still further, as indicated above, an "antigen-binding domain" can comprise or consist of an antibody or antigen- binding fragment of an antibody such as single chain variable fragments (scFv). The term "antibody" as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen or any epitope thereof. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0178] A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains. In humans, antibodies are encoded by three independent gene loci, namely the immunoglobulin heavy locus (IgH) on chromosome 14, containing the gene segments for the immunoglobulin heavy chain, the immunoglobulin kappa (K) locus (IgK) on chromosome 2, containing the gene segments for part of the immunoglobulin light chain and the immunoglobulin lambda (λ) locus (IgL) on chromosome 22, containing the gene segments for the immunoglobulin light chain.

[0179] The antibody and BCR heavy chains comprise 51 Variable (V) gene segments, 27 Diversity (D) gene segments, 6 Joining (J) gene segments. The antibody and BCR light chains comprise 40 VK, 31 Vλ, 5 Jκ, 4 Jλ gene segments.

[0180] Still further, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0181] Single domain antibodies also known as nanobodies (also known as Camelid single-domain antibodies or VHHs) have previously obtained by immunizing dromedaries, camels, llamas, alpacas, sharks, murine, rabbits and humans).

[0182] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen- binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0183] In yet some further embodiments of the CAR-molecule of the present disclosure, the antigen- binding fragment / s, portion / s or chimera / s of such antibody comprises at least one of a single chain variable fragment (scFv), and / or nanobody. As used herein, single chain variable fragments (scFv) comprise the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules. Nevertheless, scFv retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The antibody suitable for the invention may also be a bi-specific antibody (or a tri-specific antibody. As indicated above, the antibody suitable for the invention may also be a variable new antigen receptor antibody (V-NAR), as well as any humanized forms thereof.

[0184] Still further, in some embodiments, the nucleic acid molecule used for genetically modifying the disclosed enriched and activated cells encode at least one CAR molecule, as discussed in detail above. In some embodiments, the CAR molecule comprises at least one target-binding domain specific to at least one target molecule associated with and / or expressed by at least one pathologic disorder. In some embodiments, the CAR molecule is specific for at least one tumor associated antigen (TAA).

[0185] In some specific embodiments, the TAA is B cell maturation antigen (BCMA). Accordingly, the nucleic acid molecule used by the disclosed methods encodes an anti-BCMA CAR molecule. As indicated above, the T cells prepared by the methods of the present disclosure may be transfected or transduced by any exogeneous nucleic acid molecule that encodes at least one receptor molecule, for example, CAR molecules or TCR molecules. In some specific and non-limiting embodiments, the CAR T molecules useful in the methods disclosed herein may be CAR molecules that are specifically directed against that BCMA protein. B-cell maturation antigen (BCMA), also referred to as TNFRSF17 or CD269, is a member of the tumor necrosis factor receptor (TNFR) superfamily. Ligands for BCMA include B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), of which APRIL has a higher affinity for BCMA. BCMA is expressed preferentially by mature B lymphocytes, with minimal expression in hematopoietic stem cells or nonhematopoietic tissue and is essential for the survival of long-lived bone marrow plasma cells (PCs), but not overall B-cell homeostasis. Membrane-bound BCMA can undergo y- secretase-mediated shedding from the cell surface, leading to circulation of soluble BCMA (sBCMA) and reduced activation of surface BCMA by APRIL and BAFF. The overexpression and activation of BCMA are associated with MM. Moreover, the use of BCMA as a biomarker for MM is supported by its prognostic value. Still further, in some embodiments, BCMA as used herein refers to the human BCMA, that comprises the amino acid sequence as denoted by Q02223. In yet some further embodiments, BCMA is encoded by the nucleic acid sequence as denoted by Genebank accession Number Z14954.1. In yet some further embodiments, BCMA comprises the amino acid sequence as denoted by SEQ ID NO: 3, and any variants or derivatives thereof. Still further, in some specific and non-limiting embodiments, the genetically modifying step of the disclosed method comprises the step of genetically modifying the activated cells of express a CAR molecule that targets the BCMA antigen, designated H8BB CAR, that is also used herein as the HBI0101 CART. This CAR molecule comprises (i) a target binding domain specifically that recognizes and binds BCMA; (ii) a hinge and a transmembrane domain derived from the CD 8 a protein; (iii) an intracellular T cell signal transduction domain, comprising 4-1BB, and CD3 zeta chain domains. According to some specific embodiments, the CAR molecule comprises the amino acid sequence as denoted by SEQ ID NO: 23, or any variants thereof. In yet some further embodiments a variant of this CAR may be a variant with no leader sequence as denoted by SEQ ID NO: 24. It should be understood that the genetic modification step (c) of the disclosed methods may comprise in some embodiments transfecting and / or transducing the enriched cells with at least one exogeneous nucleic acid molecule, cassette or vector that comprise nucleic acid sequence encoding the desired receptor molecule (e.g., the CAR molecule). In some particular and non- limiting embodiments, this step may comprise contacting the cells with at least one transduction enhancer. Transduction enhancers are compounds that facilitate cell-virus contact, block or circumvent intrinsic cellular antiviral defenses, and / or modify the cell membrane to promote entry and uptake. These agents can be used alone or in combination to improve transduction efficiency. In certain embodiments, transduction enhancer formulations include, but are not limited to Vectofusin-1, RetroNectin and the like, as will be described in more detail herein after.

[0186] The present disclosure provides cells, specifically, of the T lineage that were enriched, activated and genetically engineered to express a desired exogeneous receptor molecule, for example, the CAR T molecules disclosed herein. It should be therefore noted that the target T cells of present disclosure are also referred to herein as host cell(s) comprising, transfected by, transformed by and / or engineered and / or edited by the nucleic acid sequence, cassette or vector disclosed herein. The term "host cell" includes a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. The skilled artisan upon reading this disclosure will understand that such terms refer not only to the particular subject cell but also is used to refer to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell". As used herein, a cell has been "traduced" or "transfected" by exogenous or heterologous DNA, e.g., the nucleic acid molecule / s encoding the desired receptor molecule, e.g., CAR, or any cassette, vector used by the methods of the present disclosure, when such DNA has been introduced inside the cell. The transducing / transfecting DNA may be integrated (covalently linked) into the genome of the cell. With respect to the present disclosure, a stably transfected or transduced cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transducing or transfecting DNA. It should be appreciated that in some embodiments, the host cells prepared by the disclosed methods may be genetically engineered T cells or any cell population comprising, at least in part, the T cells provided by the present disclosure. Still further, the invention further encompasses any population of cells comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9% or more, specifically, 100%) specifically, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9% or more, specifically, 100% of the host cells, specifically, the genetically engineered T cells prepared by the methods of the present disclosure.

[0187] As indicated above, the method of the present disclosure involves the use, and therefore, the provision of a nucleic acid molecule encoding any exogeneous receptor molecule (e.g., TCR or CAR), for example, the disclosed CAR (HBI0101), that are transduced or transfected to the enriched and activated T cells prepared by the disclosed methods. In some embodiments, such nucleic acid molecules may be, and / or comprised within, a cassette, which is used in the methods, cells, compositions and uses described in all aspects of the invention. The term "nucleic acid cassette" refers to a polynucleotide sequence comprising at least one regulatory sequence operably linked to a sequence encoding a nucleic acid sequence encoding for example, the CAR Ts or TCRs disclosed herein. All elements comprised within the cassette of the invention are operably linked together. The term "operably linked", as used in reference to a regulatory sequence and a structural nucleotide sequence, means that the nucleic acid sequences are linked in a manner that enables regulated expression of the linked structural nucleotide sequence. According to some embodiments, such nucleic acid cassettes may further comprise any control sequences that facilitate the transcription and / or translation of the receptor molecules, e.g., the CAR T molecules expressed by the T cells prepared by the methods of the preset disclosure. Such sequences include, as non-limiting examples, a promoter sequence, specifically, a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present disclosure, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various vectors of the present disclosure.

[0188] In some embodiments, promoters applicable in the present disclosure may be either inducible or constitutive. In yet some further embodiments, minimal promoter may be used, still further, endogenous promoter or heterologous promoter are also applicable in the cassettes disclosed herein. In some embodiments, cassettes useful for the methods of the present disclosure may further comprise a Signal peptide leader, for example, a signal peptide leader and any necessary component. In yet some further embodiments, the nucleic acid molecules encoding the CAR provided by the present disclosure may further comprise at least one degron sequence, at least one 2A peptide sequence or a CHYSEL site, at least one mRNA stabilizing sequence, at least one stop codon (or termination codon), at least one 3-frame stop codon sequence, at least one protein stabilizing sequence, at least one polyadenylation sequence at least one transcription enhancer, splice donor and / or splice acceptor sites, and any transcription and / or translation element / s.

[0189] The present disclosure provides thus nucleic acid molecules, that encodes a desired receptor. The term “nucleic acid”, “nucleic acid sequence”, or "polynucleotide" and “nucleic acid molecule” refers to polymers of nucleotides, and includes but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA / RNA hybrids including polynucleotide chains of regularly and / or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and — H, then an —OH, then an — H, and so on at the 2" position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides. Preparation of nucleic acids is well known in the art. Still further, it should be understood that the present disclosure encompasses any vector or vehicle that comprises any of the nucleic acid molecule / s or any cassettes described herein for transducing or transfecting to the T cells prepared by the methods of the present disclosure.

[0190] Still further, in some embodiments, the nucleic acid molecule / s of or any cassette used by the methods of the present disclosure may be comprised within a nucleic acid vector. In more specific embodiments, such vector may be any one of a viral vector, a non-viral vector and a naked DNA vector.

[0191] Vectors, as used herein, are nucleic acid molecules of particular sequence can be incorporated into a vehicle that is then introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression. Many vectors, e.g., plasmids, cosmids, minicircles, phage, viruses, etc., useful for transferring nucleic acids into target cells may be applicable in the present disclosure. The vectors comprising the nucleic acid(s) may be maintained episomally, e.g., as plasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g., retrovirus-derived vectors such as AAV, MMLV, HIV-1, ALV, etc. Vectors may be provided directly to the subject cells. In other words, the cells are contacted with vectors comprising the nucleic acid molecules, and / or cassettes of the invention that comprise the nucleic acid sequence encoding the encoding the immune effector of interest and the engineered CAR T disclosed herein such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. DNA can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV). More specifically, in some embodiments, the vector may be a viral vector. In yet some particular embodiments, such viral vector may be any one of recombinant adeno associated vectors (rAAV), single stranded AAV (ssAAV), self- complementary rAAV (scAAV), Simian vacuolating virus 40 (SV40) vector, Adenovirus vector, helper-dependent Adenoviral vector, retroviral vector and rientiviral vector. As indicated above, in some embodiments, viral vectors may be applicable in the present invention. The term "viral vector" refers to a replication competent or replication-deficient viral particle which are capable of transferring nucleic acid molecules into a host. The term "virus" refers to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism. The viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane. Examples of viruses useful in the practice of the present invention include baculoviridiae, parvoviridiae, picornoviridiae, herepesviridiae, poxviridiae, adeno viridiae, picotmaviridiae. The term recombinant virus includes chimeric (or even multimeric) viruses, i.e., vectors constructed using complementary coding sequences from more than one viral subtype.

[0192] In some embodiments, the nucleic acid molecules, and / or cassette used by the methods of the present disclosure, may be comprised within a retroviral vector. A retroviral vector, as used herein consists of proviral sequences that can accommodate the nucleic acid molecule encoding the engineered receptor molecule (e.g., CAR T disclosed herein), to allow incorporation of both into the target T cells prepared by the methods of the present disclosure. The vector may also contain viral and cellular gene promoters, to enhance expression of the nucleic acid molecule encoding the exogeneous receptor of interest (CAR T). Retroviral vectors stably integrate into the dividing target cell genome so that the introduced gene is passed on and expressed in all daughter cells. They contain a reverse transcriptase that allows integration into the host genome.

[0193] In some specific and non-limiting embodiments, the pMSGVl retroviral vector may be used for the CAR T molecule to modify the T cells prepared by the methods of the present disclosure. In some alternative embodiments, the nucleic acid molecules, and / or cassette of the invention may be comprised within an Adeno-associated virus (AAV). The term "adenovirus" is synonymous with the term "adenoviral vector". AAV is a single-stranded DNA virus with a small (~20nm) protein capsule that belongs to the family of parvoviridae and specifically refers to viruses of the genus adenoviridiae. The term adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (AdllA and Ad IIP), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Due to its inability to replicate in the absence of helpervirus coinfections (typically Adenovirus or Herpesvirus infections) AAV is often referred to as dependovirus. AAV infections produce only mild immune responses and are considered to be nonpathogenic, a fact that is also reflected by lowered biosafety level requirements for the work with recombinant A A Vs (rAAV) compared to other popular viral vector systems. Due to its low immunogenicity and the absence of cytotoxic responses AAV-based expression systems offer the possibility to express nucleic acid sequences encoding the receptor molecules, e.g., CAR T disclosed herein for months in quiescent cells. Production systems for rAAV vectors typically consist of a DNA-based vector containing a transgene expression cassette, which is flanked by inverted terminal repeats. Construct sizes are limited to approximately 4.7-5.0 kb, which corresponds to the length of the wild- type AAV genome. rAAVs are produced in cell lines. The expression vector is co-transfected with a helper plasmid that mediates expression of the AAV rep genes which are important for virus replication and cap genes that encode the proteins forming the capsid. Recombinant adeno-associated viral vectors can transduce dividing and non- dividing cells, and different rAAV serotypes may transduce diverse cell types. These single- stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous Homologous Recombination without causing double strand DNA breaks in the host genome.

[0194] It is believed that a rate-limiting step for the AAV-mediated expression of transgenes is the formation of double-stranded DNA. Recent reports demonstrated the usage of rAAV constructs with a self-complementing structure (scAAV) in which the two halves of the single-stranded AAV genome can form an intra-molecular double-strand. This approach reduces the effective genome size usable for gene delivery to about 2.3kB but leads to significantly shortened onsets of expression in comparison with conventional single-stranded AAV expression constructs (ssAAV). Thus, in some embodiments, ssAAV may be applicable as a viral vector by the methods of the invention.

[0195] In yet some further embodiments, HDAd vectors may be suitable for the methods of the present disclosure. The Helper-Dependent Adenoviral (HDAd) vectors HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. HDAds are constructed by removing all viral sequences from the adenoviral vector genome except the packaging sequence and inverted terminal repeats, thereby eliminating the issue of residual viral gene expression associated with early generation adenoviral vectors. HDAds can mediate high efficiency transduction, do not integrate in the host genome, and have a large cloning capacity of up to 37 kb, which allows for the delivery of multiple transgenes or entire genomic loci, or large cis-acting elements to enhance or regulate tissue-specific transgene expression. One of the most attractive features of HDAd vectors is the long-term expression of the transgene. Still further, in some embodiments, SV40 may be used as a suitable vector by the present disclosure. SV40 vectors (SV40) are vectors originating from modifications brought to Simian virus-40 an icosahedral papovavirus. Recombinant SV40 vectors are good candidates for gene transfer, as they display some unique features: SV40 is a well-known virus, non-replicative vectors are easy-to-make, and can be produced in titers of 10(12) lU / ml. They also efficiently transduce both resting and dividing cells, deliver persistent transgene expression to a wide range of cell types, and are non-immunogenic. Present disadvantages of rSV40 vectors for gene therapy are a small cloning capacity and the possible risks related to random integration of the viral genome into the host genome. In yet some alternative embodiments, as also exemplified by the present disclosure, lentiviral vectors may be used in the present disclosure. Lentiviral vectors are derived from lenti viruses which are a subclass of Retroviruses. Commonly used retroviral vectors are "defective", i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising the nucleic acid molecules, vectors and / or cassette in accordance with the invention, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing the retroviral vectors comprising the nucleic acid molecules, vectors and / or cassette of the invention that contains the nucleic acids sequence encoding the receptor molecules, e.g., the disclosed CAT-T, into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nonviral vectors, in accordance with the invention, refer to all the physical and chemical systems except viral systems and generally include either chemical methods, such as cationic liposomes and polymers, or physical methods, such as gene gun, electroporation, particle bombardment, ultrasound utilization, and magnetofection. Efficiency of this system is less than viral systems in gene transduction, but their cost- effectiveness, availability, and more importantly reduced induction of immune system and no limitation in size of transgenic DNA compared with viral system have made them attractive also for gene delivery.

[0196] For example, physical methods applied for in vitro and in vivo gene delivery are based on making transient penetration in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNA entrance into the targeted cells is facilitated.

[0197] In more specific embodiments, the vector may be a naked DNA vector. More specifically, such vector may be for example, a plasmid, minicircle or linear DNA. Naked DNA alone may facilitate transfer of a gene (2-19 kb) into skin, thymus, cardiac muscle, and especially skeletal muscle and liver cells when directly injected. It enables also long-term expression. Although naked DNA injection is a safe and simple method, its efficiency for gene delivery is quite low.

[0198] Minicircles are modified plasmid in which a bacterial origin of replication (ori) was removed, and therefore they cannot replicate in bacteria. Linear DNA or Doggybone™ are double-stranded, linear DNA construct that solely encodes an antigen expression cassette, comprising antigen, promoter, polyA tail and telomeric ends. It should be appreciated that all DNA vectors disclosed herein, may be also applicable for all nucleic acid molecules, vectors and / or cassettes used in the methods and compositions of the invention, as described herein. Still further, it must be appreciated that the present disclosure further uses any vectors or vehicles that comprise any of the nucleic acid molecules, vectors and / or nucleic acid cassettes disclosed herein, as well as any host cell expressing the nucleic acid molecules, and / or nucleic acid cassettes disclosed by the present disclosure.

[0199] In some embodiments, all method steps are performed in a low serum medium containing 0.5 to 3% serum. More specifically, all method steps are performed in a medium containing about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0% serum. In some specific embodiments, all method steps are performed in a medium containing no more than 1% serum. In yet some particular embodiments, all method steps are performed in a medium containing 1% serum.

[0200] Still further, the serum used in the medium for all method step comprises human AB serum.

[0201] More specifically, starting material for Human AB Serum is collected from healthy male donors with an AB blood type. A single gendered pool is used to reduce variation in the final product. The AB blood type means that donors have both A and B antigens on their red blood cells and therefore lack blood group antibodies. This guarantees that the serum will not react with other blood types, further minimizing immunoreactivity for researchers. Typical lots of Human AB Serum are produced from 150-250 donors and pooled together, but custom batches with fewer donors are available. Human AB Serum can be produced through two different methods: plasma-derived and off-the-clot. Plasma-derived Human AB Serum is, as the name implies, generated from donor source plasma. Plasma is converted into serum by the defibrination process. Defibrination is typically achieved chemically using thrombin from either a human or bovine source to convert fibrogen into fibrin, thus turning the plasma into serum.

[0202] Off-the-clot Human AB Serum is produced from donor whole blood, wherein the blood is allowed to clot naturally, and the serum is extracted from the resultant fractionation. This allows the product to retain various growth factors and compounds found in whole blood without additional processing compounds, making it the closest thing to normal human material.

[0203] It should be appreciated that the present disclosure encompasses the use of any appropriate medium for all steps of the disclosed methods. A cell culture medium, as used herein, is a formulated composition of nutrients and growth factors designed to support the growth, proliferation, and maintenance of various cell types, in vitro. It replicates the physiological conditions cells encounter in their natural environment. The base solution typically consists of deionized or distilled water, which serves as the primary solvent, along with essential salts like sodium chloride, potassium chloride, and calcium chloride to maintain osmotic balance and provide necessary ions. Nutrient components include amino acids, such as glutamine, glycine, and tryptophan, which are vital for protein synthesis, as well as vitamins like ascorbic acid (Vitamin C), biotin, and riboflavin (Vitamin B2) required for metabolic processes. Carbohydrates, primarily glucose, serve as an energy source. Growth factors and hormones, either derived from serum components like fetal bovine serum or as synthetic additives like epidermal growth factor (EGF) and insulin, are included to promote cell proliferation and differentiation. Buffering agents such as sodium bicarbonate and HEPES help maintain the medium's pH, especially in CO2 incubators or environments with limited CO2 control. Optional components may include antibiotics like penicillin-streptomycin to prevent bacterial contamination and antimycotics like amphotericin B to inhibit fungal growth. Additionally, phenol red is often added as a pH indicator, changing color to signal shifts in the medium's pH, which aids in monitoring cell culture conditions. To name but few, cell culture media applicable in the present disclosure may include but are not limited to any cell culture media specifically formulated for human cells, particularly those of hematopoietic origin, including RPMI 1640, which is widely used for the culture of human lymphocytes, T cells, and hybridoma cells, and is often supplemented with fetal bovine serum (FBS) and growth factors like interleukin-2. Another example is X-VIVO media, a serum-free, chemically defined medium ideal for the expansion of human T cells and dendritic cells, commonly used in immunotherapy research, including CAR-T cell development. Additionally, Iscove’s Modified Dulbecco’s Medium (IMDM) is frequently used for human hematopoietic progenitor cells, supporting their growth and differentiation with a rich nutrient profile and additional cytokines or growth factors. In some particular and non-limiting embodiments, all steps of the disclosed methods are performed in a CTS AIM-V and / or a DEME, medium.

[0204] More specifically, CTS AIM-V Medium is a chemically defined, serum- free medium specifically designed for the culture and expansion of human lymphocytes, including T cells and dendritic cells, optimized for clinical purpose, particularly in immunotherapy. This medium includes a basal nutrient solution with essential inorganic salts and buffering agents, a comprehensive mix of essential and non-essential amino acids for protein synthesis and metabolism, water-soluble vitamins to support cellular functions, glucose as the primary energy source, and essential lipids for cell membrane integrity. Growth factors may be added as needed, although the medium is optimized for serum-free culture, and pH is maintained with sodium bicarbonate or HEPES buffers. The xeno-free formulation, containing no animal-origin components, makes CTS AIM-V suitable for clinical-grade applications. Similarly, DMEM (Dulbecco’s Modified Eagle Medium) is a widely used basal medium rich in nutrients, supporting the growth and maintenance of various mammalian cells, including human cell lines. It contains inorganic salts for osmotic balance, essential and non-essential amino acids, vitamins such as biotin and riboflavin, glucose for energy, and buffering agents like sodium bicarbonate to maintain pH stability. Often supplemented with 5-10% fetal bovine serum (FBS) and sometimes antibiotics or additional growth factors.

[0205] Still further, in some embodiments, the disclosed methods may be performed in at least one of the x-vivo, Optimizer, TexMACS, and / or ImmunoCult media.

[0206] X-VIVO Media, OpTmizer™ T-Cell Expansion SFM, TexMACS™ Medium, and ImmunoCult™-XF T Cell Expansion Medium are all specialized, serum-free, and chemically defined media designed for the culture, activation, and expansion of human immune cells, particularly lymphocytes such as T cells and dendritic cells. X-VIVO Media is optimized for clinical applications like CAR-T cell production, minimizing variability and contamination risk by excluding serum and undefined components. OpTmizer™ T-Cell Expansion SFM is tailored for the activation and expansion of human T cells in adoptive therapies, incorporating essential inorganic salts, amino acids, vitamins, glucose, lipids, and often supplemented with cytokines like IL-2. TexMACS™ Medium is a GMP-grade, serum-free medium ideal for T cell activation and expansion, particularly in conjunction with magnetic cell separation technologies, ensuring clinical compatibility by excluding animal-derived components. ImmunoCult™-XF T Cell Expansion Medium, a xeno-free formulation, is specifically developed to support robust T cell proliferation without serum, making it suitable for both research and clinical immunotherapy applications. All these media include essential components such as inorganic salts for osmotic balance, amino acids for protein synthesis, vitamins, glucose as the primary energy source, lipids for membrane integrity, and pH buffers like sodium bicarbonate or HEPES, ensuring stable conditions for cell growth and function in advanced immunotherapy research and clinical settings.

[0207] Still further, in some embodiments, the population of T cells prepared by the methods of the present disclosure is characterized by a CD4+T cell to CD8+T cell ratio that is greater than one. As shown by the present disclosure, the CD4+:CD8+ ratio for the present disclosure is 4.8±2.9, specifically, the ratio ranges between 4 to 10, between 4.8 to about 7.9, as discussed herein after. It should be understood that the CD4+:CD8+ ratio provided by the methods of the present disclosure is clearly increased, for example, as compared to the ratio obtained using other prior art methods, that provide a ratio that is below 1, for example, 0.9+0.5.

[0208] Specifically, a CD4+ T cell to CD8+ T cell ratio greater than one as referred to herein in connection with the resulting T cell population of the disclosed methods, indicates that the number of CD4+ T cells (helper T cells) is higher than the number of CD8+ T cells (cytotoxic T cells) in a given sample. This ratio is often used as an indicator of immune system health, with a normal ratio typically ranging between 1.0 and 4.0 in healthy individuals. A ratio greater than one suggests that the immune system has a sufficient number of helper T cells to coordinate immune responses, which is important for effectively managing infections and maintaining overall immune function. According to some embodiments, a CD4+T cell to CD8+T cell ratio that is greater than one may be any ratio between 1.1 to 10, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.10, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or more. In yet some further embodiments, a CD4+:CD8+ ratio of 4.8 to 7.7 or more.

[0209] It should be understood that although the naive-like T cell population obtained by the disclosed methods is enriched in CD4+cells and contains a comparatively smaller proportion of CD8+cells, this subpopulation, while less pronounced, nonetheless exhibits significant functional activity. As demonstrated in the experimental data (see, for example, Figure 16), the CD8+cells, despite representing a minor fraction of the overall population, are effective and potent. Thus, the disclosed methods enable preservation of the population (as exemplified by their transcriptomic signature in Figures 25 to 28), which is capable of persisting under conditions of chronic antigen stimulation. Still further, in some embodiments, the population of naive-like T cells prepared by the disclosed methods display a decreased proportion of senescent-like (CD27- CD28-) T-cells. More specifically, senescent-like (CD27-CD28-) T-cells, as used herein, refer to a subset of T-cells that have lost the expression of the surface markers Cluster of Differentiation 27 (CD27) and Cluster of Differentiation 28 (CD28). These cells are typically associated with aging or chronic immune activation and are characterized by a senescent phenotype. Senescent T-cells are less responsive to antigenic stimulation, have reduced proliferative capacity, and often exhibit increased secretion of inflammatory cytokines. The loss of CD27 and CD28 is indicative of a terminal differentiation state, where the T-cells have undergone multiple rounds of division and have lost the ability to proliferate further. It should be therefore understood that the cell population produced by the disclosed methods display increased responsiveness to antigenic stimulation and increased proliferative capacity, as compared with population of cells not produced by the disclosed methods, and specifically, to cells produced by the traditional procedure.

[0210] More specifically, as shown by the present disclosure the disclosed methods provide improved naive-like populations that display increased proportion of CD27+CD28+ cells. For example, the resulting population is composed of about 50% to 99% cells that are CD27+CD28+ cells, specifically, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 67.1, 67.2%, 67.3, 67.4, 67.5%, 67.6%, 67.7%, 67.8%, 67.9%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. More specifically, about 67.4%±3.9 of the cells obtained by the disclosed methods are cells expressing the CD27+CD28+ markers, specifically, about 67.4% to about 71.3% of the cells. It is interesting to note that the cell populations produced by the prior art methods display much lower proportion of the CD27+CD28+ cells, for example, only about 13.9%±16.1 of the cell population. It means that the methods of the present disclosure produce cell population with increased ratio of cells of the desired phenotype, with an increase of between about 2.2 to about 5.1 or more folds, as compared with control cell population, that may be in some embodiments, cells that were not prepared by the disclosed methods, and / or cell populations prepared by methods other than the disclosed methods, and / or population of cells obtained by the prior art methods, and / or cell populations prepared by the traditional method disclosed herein as TM.

[0211] More importantly, the naive-like T cells prepared by the disclosed methods display a decreased proportion of senescent-like (CD27- CD28-) T-cells. For example, cells obtained by the methods of the present disclosure display almost none (only between 0%) to about 8%, senescent-like (CD27- CD28-) T-cells, specifically, 3.4%±3.9, or in other words, between 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5% or less senescent-like cells. It should be note that cell populations produced by the prior art methos display about 47.3%±28.2, specifically, between about 47% to 75.5% senescent-like cells. It means that the prior art methods result in preparation of cell population where the majority of the cells are senescent-like cells. In contrast, the cell population prepared by the methods of the present disclosure display decreased proportion of senescent-like (CD27- CD28-) T-cells, for example, between 3.8% to about 7.5%. Therefore, a decreased proportion is in some embodiments, a decrease of about 6.7 folds to about 19.3 folds reduction of the proportion of senescent-like (CD27- CD28-) T-cells. More specifically, a decreased proportion of senescent-like (CD27- CD28-) T-cells, is of about 2, 3, 4, 5, 6, 7., 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 folds or more, as compared with the proportion of the senescent-like (CD27- CD28-) T-cells in a cell population prepared by prior art methods, or cells obtained from the subject.

[0212] Still further, as was also demonstrated by Figure 20, in some embodiments, the cell population prepared by the disclosed methods may be characterized by reduced expression of at least one of the following inhibitory receptors, including PD-1, TIM-3, LAG-3, TIGIT, CTLA-4, as compared with cells prepared by traditional procedures. More specifically, the expression of inhibitory receptors such as PD-1, TIM-3, LAG-3, TIGIT, and CTLA-4 on T-cells is associated with immune regulation and exhaustion of the cells and thus, reduced expression of the inhibitory receptors is a good indicator for the quality of the cell population produced by the disclosed methods. More specifically, PD-1 (Programmed Cell Death Protein 1), is an inhibitory receptor expressed on T- cells after activation. It plays a crucial role in preventing autoimmunity by downregulating immune responses. TIM-3 (T-cell Immunoglobulin and Mucin-domain containing-3), is another inhibitory receptor expressed on T-cells and other immune cells. It is often co-expressed with PD-1 on exhausted T-cells. TIM-3 expression leads to the suppression of immune responses, particularly in the tumor microenvironment, by promoting T-cell dysfunction and inhibiting the production of pro-inflammatory cytokines. Still further, LAG-3 (Lymphocyte-activation Gene 3), is an inhibitory receptor structurally similar to CD4. It binds to MHC class II molecules and negatively regulates T-cell proliferation and function. LAG-3 is often upregulated in exhausted T-cells, working synergistically with PD-1 to suppress T-cell activity. TIGIT (T-cell Immunoreceptor with Ig and ITIM domains), as used herein, is an inhibitory receptor expressed on T-cells, natural killer (NK) cells, and other immune cells. It competes with co-stimulatory receptors like CD226 for binding to the same ligands, such as CD155, leading to reduced T-cell activation and promoting an immunosuppressive environment. CTLA-4 (Cytotoxic T-Lymphocyte- Associated Protein 4), is an inhibitory receptor that competes with CD28 for binding to B7 molecules on antigen- presenting cells (APCs). By outcompeting CD28, CTLA-4 effectively downregulates T-cell activation and proliferation, maintaining immune homeostasis.

[0213] As indicated above, the co-expression of these inhibitory receptors is often indicative of T-cell exhaustion, which is a state of dysfunction marked by a diminished capacity to proliferate, produce cytokines, and effectively clear infections or tumors. Thus, reduction in the expression of at least one of these inhibitory is negatively correlated with the quality of the T cell population produced by the disclosed methods. More specifically, reduction of about 5% to about 95%, as compared with the expression of the inhibitory receptors in cells produced by the traditional procedure. More specifically, reduced expression as indicated herein relates to a decrease, decline, lowering, diminishment, or lessening of the expression of at least one of the inhibitory receptors specified herein in about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.999, as compared with the expression of these inhibitory receptors in cells produced by the traditional procedures.

[0214] The present disclosure further envisaged, as also shown by Figure 21, that in some embodiments, the population of naive-like T cells prepared after step (a) or after step (d) display an increased proportion of cells expressing CD25. In some specific embodiments, the percent cells within the population of naive-like T cells prepared after step (a) or after step (d) that display expressing of CD25 is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0215] Still further, in some embodiments, the naive-like cells prepared by the disclosed methods comprise TNaive / SCMcells expressing CD45RA+CCR7+ (and / or CD45RA+CD62L+), and / or CD45RO-CCR7+ (and / or CD45RO-CD62L+). Additionally, the disclosed cells comprise cells expressing the TCM, CD45RA-CCR7+ (and / or CD45RA-CD62L+), and / or CD45RO+CCR7+ (and / or CD45RO+CD62L+).

[0216] In yet some further embodiments, the population of T cells produced by the disclosed method is characterized by reduction of differentiated cells, specifically, reduction in TEMcells expressing the CD45RA-CCR7- and TEMRA cells expressing the CD45RA+CCR7-.

[0217] Still further, in some embodiments, the population of naive-like T cells prepared by the methods of the present disclosure is characterized by at least one of:

[0218] (i) a higher average expression level of at least one gene associated with naive and stem cell memory T cell phenotypes in the naive-like T cells population, as compared with an average expression level of said gene in a control population and / or (ii) an increased proportion of T cells expressing the at least one gene associated with naive and stem cell memory T cell phenotypes in said naive-like T cells population, as compared with a control population.

[0219] In some embodiments, the gene associated with naive and stem cell memory expressed by the naive-like T cells prepared by the methods of the present disclosure is any gene, provided that said gene is not C-X-C chemokine receptor type 3 (CXCR3), Fas cell surface death receptor (FAS), or interleukin-7 receptor (IL7R).

[0220] In more specific embodiments, the at least one gene associated with naive and stem cell memory T cell phenotypes is selected from the group consisting of: marker of proliferation Ki-67 (MKI67), interleukin-2 receptor subunit beta (IL2RB), cluster of differentiation 28 (CD28), cluster of differentiation 27 (CD27), lymphoid enhancer-binding factor 1 (LEF1), L-selectin (SELL), C-C chemokine receptor type 7 (CCR7), and transcription factor 7 (TCF7). More specifically, in some embodiments, when referred to herein, Proliferation marker protein Ki-67 (MKI67), refers to the human MKI67, having the amino acid sequence as disclosed in P46013, and encoded b the nucleic acid sequence as denoted by Gene ID: 4288. In yet some further specific embodiments, MKI67 applicable in the cells prepared by the disclosed method, is the human MKI67, that comprises the amino acid sequence as denoted by SEQ ID NO: 4. In yet some embodiments, IL2RB, as used herein, is the human interleukin 2 receptor subunit beta (IL2RB), that comprises the amino acid as denoted by UniProt P14784, and encoded by Gene ID: 3560. In more specific embodiments, the IL2RB is the human IL2RB that comprises the amino acid sequence as denoted by SEQ ID NO: 5. Still further, in some embodiments, when referred to herein, CD28, is the human T-cell-specific surface glycoprotein CD28, that comprises the amino acid sequence as denoted by P10747, and encoded by the nucleic acid sequence of Gene ID: 940. In more specific embodiments, the CD28 is the human CD28 that comprises the amino acid sequence as denoted by SEQ ID NO: 6. In some embodiments, when referred to herein, CD27, is the human T-cell-specific surface glycoprotein CD27, that comprises the amino acid sequence as denoted by P26842, encoded by the nucleic acid sequence of Gene ID: 939. In more specific embodiments, the CD27 is the human CD27 that comprises the amino acid sequence as denoted by SEQ ID NO: 7. In some embodiments, when referred to herein, LEF1, is the human Lymphoid enhancer-binding factor 1 (LEF1), that comprises the amino acid sequence as denoted by Q9UJU2, encoded by the nucleic acid sequence of Gene ID: 51176. In more specific embodiments, the LEF1 is the human LEF1 that comprises the amino acid sequence as denoted by SEQ ID NO: 8. In some embodiments, when referred to herein, SELL, is the human L-selectin (SELL), that comprises the amino acid sequence as denoted by P14151, and encoded by the nucleic acid sequence of Gene ID: 6402. In more specific embodiments, the SELL is the human SELL that comprises the amino acid sequence as denoted by SEQ ID NO: 9. In some embodiments, when referred to herein, TCF7, is the human Transcription factor 7 (TCF7), that comprises the amino acid sequence as denoted by P36402, encoded by the nucleic acid sequence of Gene ID: 6932. In more specific embodiments, the TCF7 is the human TCF7 that comprises the amino acid sequence as denoted by SEQ ID NO: 10.

[0221] In yet some further embodiments, the population of naive-like T cells prepared by the methods of the present disclosure is characterized by at least one of:

[0222] (i) a lower average expression level of at least one effector-associated gene in the naive-like T cells population, as compared with an average expression level of the effector-associated gene in a control population; and / or

[0223] (ii) a decreased proportion of T cells expressing at least one effector-associated gene, in the naive- like T cells population, as compared with a control population.

[0224] In more specific embodiments, such at least one effector-associated gene is selected from: thymocyte selection-associated high mobility group box protein (TOX), hepatitis A virus cellular receptor 2 (H VCR2), lymphocyte activation gene 3 protein (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), eomesodermin (EOMES), T-box transcription factor TBX21 (TBX21), killer cell lectin-like receptor subfamily D member 1 (KLRD1), granulysin (GNLY), granzyme A (GZMA), granzyme B (GZMB), granzyme H (GZMH), and granzyme K (GZMK). In some embodiments, when referred to herein, TOX, is the human Thymocyte selection- associated high mobility group box protein (TOX), that comprises the amino acid sequence as denoted by 094900, encoded by the nucleic acid sequence of Gene ID: 9760. In more specific embodiments, the TOX is the human TOX that comprises the amino acid sequence as denoted by SEQ ID NO:11. In some embodiments, when referred to herein, HAVCR2, is the human Hepatitis A virus cellular receptor 2 (HAVCR2), that comprises the amino acid sequence as denoted by Q8TDQ0, and encoded by the nucleic acid sequence of Gene ID: 84868. In more specific embodiments, the HAVCR2 is the human HAVCR2 that comprises the amino acid sequence as denoted by SEQ ID NO: 12.

[0225] In some embodiments, when referred to herein, LAG3, is the human Lymphocyte activation gene 3 protein (LAG3), that comprises the amino acid sequence as denoted by Pl 8627, encoded by the nucleic acid sequence of Gene ID: 3902. In more specific embodiments, the LAG3 is the human LAG3 that comprises the amino acid sequence as denoted by SEQ ID NO: 13. In some embodiments, when referred to herein, TIGIT, is the human T-cell immunoreceptor with Ig and ITIM domains (TIGIT), that comprises the amino acid sequence as denoted by Q495A1, encoded by the nucleic acid sequence of Gene ID: 201633. In more specific embodiments, the TIGIT is the human TIGIT that comprises the amino acid sequence as denoted by SEQ ID NO: 14. In some embodiments, when referred to herein, EOMES, is the human Eomesodermin homolog (EOMES), that comprises the amino acid sequence as denoted by 095936, encoded by the nucleic acid sequence of Gene ID: 8320. In more specific embodiments, the EOMES is the human EOMES that comprises the amino acid sequence as denoted by SEQ ID NO: 15. In some embodiments, when referred to herein, TBX21, is the human I box transcription factor TBX21, that comprises the amino acid sequence as denoted by Q9UL17, encoded by the nucleic acid sequence of Gene ID: 30009. In more specific embodiments, the TBX21 is the human TBX21 that comprises the amino acid sequence as denoted by SEQ ID NO: 16. In some embodiments, when referred to herein, KLRD1, is the human Natural killer cells antigen CD94 (KLRD1), that comprises the amino acid sequence as denoted by Q13241, encoded by the nucleic acid sequence of Gene ID: 3824. In more specific embodiments, the KLRD1 is the human KLRD1 that comprises the amino acid sequence as denoted by SEQ ID NO: 17. In some embodiments, when referred to herein, GNLY, is the human Granulysin (GNLY), that comprises the amino acid sequence as denoted by P22749, encoded by the nucleic acid sequence of Gene ID: 10578. In more specific embodiments, the GNLY is the human GNLY that comprises the amino acid sequence as denoted by SEQ ID NO: 18. In some embodiments, when referred to herein, GZMA, is the human T Granzyme A (GZMA), that comprises the amino acid sequence as denoted by P12544, encoded by the nucleic acid sequence of Gene ID: 3001. In more specific embodiments, the GZMA is the human GZMA that comprises the amino acid sequence as denoted by SEQ ID NO: 19. In some embodiments, when referred to herein, GZMB, is the human Granzyme B (GZMB), that comprises the amino acid sequence as denoted by P10144, encoded by the nucleic acid sequence of Gene ID: 3002. In more specific embodiments, the GZMB is the human GZMB that comprises the amino acid sequence as denoted by SEQ ID NO: 20. In some embodiments, when referred to herein, GZMH, is the human Granzyme H (GZMH), that comprises the amino acid sequence as denoted by P20718, encoded by the nucleic acid sequence of Gene ID: 2999. In more specific embodiments, the GZMH is the human GZMH that comprises the amino acid sequence as denoted by SEQ ID NO: 21. In some embodiments, when referred to herein, GZMK, is the human Granzyme K (GZMK), that comprises the amino acid sequence as denoted by P49863, and encoded by the nucleic acid sequence of Gene ID: 3003. In more specific embodiments, the GZMK is the human GZMK that comprises the amino acid sequence as denoted by SEQ ID NO: 22.

[0226] In some embodiments, a control population, may be any population of cells that were not prepared by the methods of the present disclosure. In yet some further embodiments, the control population may be any population of T cells prepared by any method other than the method of the present disclosure. In more specific embodiments, the control population is a cell population prepared by the traditional method (TM) as discussed herein.

[0227] Thus, in some embodiments, the methods of the present disclosure comprise the following steps. In step (a), contacting a population of peripheral blood mononuclear cell(s) (PBMC) with a compound that specifically selects for cells expressing the CD62L marker, thereby obtaining an enriched CD62L+ T cell population. Step (b) involves subjecting the enriched CD62L T cell population obtained in step (a), to T-cell activation by contacting the enriched cell population with (i) CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL- 15) and Interleukin 21 (IL-21). The next step (c) involves genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule encoding at least one CAR molecule, in the presence of at least one transduction enhancer; thereby obtaining a cell population comprising T cells harboring said CAR molecule, e.g., the anti-BCMA CAR disclosed herein. Step (d) involves expanding the transduced or transfected cell population obtained in step (c); and in step (e), harvesting the expanded cell population obtained in step (d); thereby preparing a population of naive-like T cells. In some embodiments, steps (a) to (e) are performed in a single container or compartment, in low serum medium containing 1 % or less human AB serum. In some embodiments, the duration of the disclosed method is up to 6 days. It should be understood that it is further envisaged by the present disclosure that the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days, such that the duration of the disclosed method is up to 7 days, or 8 days. As indicated above, this extended expansion is used only when the number of cells is not sufficient. Moreover, the characterizing features of the cells as defined in the present disclosure are maintained also after the additional one or two days of expansion. In some embodiments, the PBMC used in the disclosed methods may be cryopreserved PBMCs or cryopreserved leukapheresis cell products.

[0228] In some aspects thereof, the present disclosure provides methods for preparing a population of naive-like T cells. More specifically, the naive-like T cells comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM)). The disclosed method comprising the following steps. In step (a), contacting a population of peripheral blood mononuclear cell(s) (PBMC) with a compound that specifically selects for cells expressing the CD62L marker, thereby obtaining an enriched CD62L+ T cell population. Step (b) involves subjecting the enriched CD62L T cell population obtained in step (a), to T-cell activation by contacting the cell population with (i) CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL- 15) and Interleukin 21 (IL-21). The next step (c) involves genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule encoding at least one CAR molecule, in the presence of at least one transduction enhancer; thereby obtaining a cell population comprising T cells harboring said CAR molecule, e.g., the anti-BCMA CAR disclosed herein. Step (d) involves expanding the transduced or transfected cell population obtained in step (c); and in step (e), harvesting the expanded cell population obtained in step (d); thereby preparing a population of naive-like T cells. In some embodiments, steps (a) to (e) are performed in a single container or compartment, in low serum medium containing 1 % or less human AB serum. It should be noted that the duration of the method is up to 6 days, but can be extended up to 8 days by adding one or two days to the expansion step, without compromising the desired naive-like phenotype.

[0229] In some particular embodiments, the activation step of the disclosed methods comprises the use of about 5-15 ng / ml Interleukin 7 (IL-7), 5-15 ng / ml Interleukin 15 (IL- 15) and 10-50 ng / ml Interleukin 21 (IL-21).

[0230] In addition to increased potency, proliferation capacity, and reduced exhaustion, the naive-like T cells obtained by the disclosed methods may exhibit enhanced trafficking capacity. As used herein, the term “trafficking” refers to the ability of T cells to migrate, home to, and localize within specific anatomical sites, including but not limited to secondary lymphoid organs such as lymph nodes, spleen, and bone marrow. Enhanced trafficking capacity denotes an improved ability of the T cells to reach and accumulate in such sites, thereby supporting their survival, expansion, and functional activity in vivo. This property provides a significant therapeutic advantage in the treatment of hematologic malignancies, including but not limited to lymphoma, leukemia, and multiple myeloma, where localization to lymphoid tissues is critical for therapeutic efficacy.

[0231] A further aspect of the present disclosure relates to a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject, the method comprising the step of administering to the subject an effective amount of a cell population comprising cells of the T lineage, or a composition comprising the same. The cells of the T lineage comprise naive-like T cells. In some embodiments, the naive-like T cells administered to the subject comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). It should be further understood that the cell population is prepared by a method comprising the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ), with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. It should be understood that it is further envisaged by the present disclosure that the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days, such that the duration of the disclosed method is up to 7 days, or 8 days. As indicated above, this extended expansion is used only when the number of cells is not sufficient. Moreover, the characterizing features of the cells as defined in the present disclosure are maintained also after the additional one or two days of expansion.

[0232] The present disclosure thus provides in some embodiments thereof, a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject. The method comprising the step of (I), preparing a population of naive-like T cells. In some embodiments, the naive-like T cells administered to the subject comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM); and (II) administering to the subject an effective amount of the cell population obtained by step (II), or a composition comprising the same. It should be further understood that the cell population is prepared in step (I), by a method comprising the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ) with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. In some embodiments, the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days, such that the duration of the disclosed method is up to 7 days, or 8 days.

[0233] In some embodiments, the cell population used by the disclosed therapeutic method is prepared by a method as defined by the present disclosure.

[0234] In some embodiments, the population of T cells prepared by the disclosed method are characterized by a CD4+T cell to CD8+T cell ratio that is greater than one.

[0235] In some embodiments, the population of T cells comprising naive T cells (TN) and stem-cell like and central memory T cells prepared by the disclosed method display decreased proportion of senescent-like (CD27-CD28-) T-cells, as detailed herein above.

[0236] In some embodiments, the population of cells used for the disclosed methods may be cell population of an autologous or allogeneic source.

[0237] In some embodiments, the cell population or a composition thereof is administered to the treated subject by adoptive transfer. The term “adoptive transfer” as herein defined applies to all the therapies that consist of the transfer of components of the immune system, specifically cells that are already capable of mounting a specific immune response. In such option, the insertion of the nucleic acid sequence encoding any exogeneous receptor, e.g., the CAR T disclosed herein, is performed in cells of an autologous or allogeneic source, that are then administered to the subject, specifically, by adoptive transfer.

[0238] In some non-limiting embodiments the steps include preconditioning the recipient, the administration process and post- administration care. More specifically, the stage of preconditioning the recipient may include (i) Lymphodepletion, specifically, before the administration of the adoptive cells, the recipient may undergo lymphodepletion, which is a process to reduce their existing lymphocytes. This can be achieved in some embodiments, through chemotherapy, radiation, or a combination of both. The purpose is to create a more favorable environment for the infused cells to expand and function effectively. The next step (ii) may involve supporting medications, specifically, patients may receive medications to support their immune system and manage potential side effects, such as cytokine release syndrome. The next step may be (iii), the administration process that may include, intravenous infusion. More specifically, the most common method of administering adoptive transfer cells is through intravenous (IV) infusion. This allows the cells to circulate through the bloodstream and reach various tissues. Infusion Procedure is where the cells are infused over a period, which can range from a few minutes to several hours, depending on the volume and concentration of the cell product. In some embodiments, the adoptive transfer is followed by monitoring the subject. The term "autologous" when relating to the source of cells, refers to cells derived or transferred from the same subject that is to be treated by the methods of the invention. The term "allogenic" when relating to the source of cells, refers to cells derived or transferred from a different subject, referred to herein as a donor, of the same species. Of particular interest are cells of autologous source, or in other words, autologous cells, that are obtained from the subject (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ of the subject), prior to the administration using adoptive transfer. Accordingly, in some embodiments, the cells that are prepared by the disclosed methods and further express, comprise, transduced or transfected with the nucleic acid molecule / s or any cassette provided and used by the methods disclosed herein may be cells of an autologous source.

[0239] In some embodiments, the disclosed therapeutic methods are particularly applicable for treating a pathologic disorder that may be at least one of: a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease, a deposition disorder and / or a neurodegenerative disorder.

[0240] In some particular embodiments, the disclosed methods are useful for treating at least one proliferative disorder, for example, at least one malignant neoplastic disorder.

[0241] In more particular embodiments, the neoplastic disorder is at least one hematological malignancy, and / or at least one solid tumor.

[0242] Still further, in some embodiments, the disclosed therapeutic methods may be applicable for treating at least one hematological malignancy, for example, at least one B cell malignancy.

[0243] As indicated above, the T cells prepared by the methods of the present disclosure, specifically, cells of the T lineage genetically engineered to express the CAR molecules provided by the preset disclosure, may be applicable for any disorder that involves B cell.

[0244] Thus, in some specific embodiments, the methods of the present disclosure are applicable for treating proliferative disorder, specifically, any B cell malignancy.

[0245] In some embodiments, B cell malignancies include myeloma, specifically, multiple myeloma, as well as any type of lymphoma, including non-Hodgkin lymphomas as well as Hodgkin lymphomas.

[0246] More specifically, B-cell lymphomas make up most of the non-Hodgkin lymphomas (NHL). The methods of the present disclosure are therefore applicable for any type of lymphoma, specifically affecting B lymphocytes. The most common types of B-cell lymphomas applicable in the present disclosure, include, but are not limited to Diffuse large B-cell lymphoma (DLBCL), as well as to any subtype thereof (primary mediastinal B-cell lymphoma), Follicular lymphoma, diffuse large B-cell lymphoma, Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphomas, Extranodal marginal zone B-cell lymphoma, also known as mucosa-associated lymphoid tissue (MALT) lymphoma, Nodal marginal zone B-cell lymphoma, Splenic marginal zone B-cell lymphoma, Burkitt lymphoma, Lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), Hairy cell leukemia (also called Chronic Lymphocytic Leukemia), Primary central nervous system (CNS) lymphoma, and Primary intraocular lymphoma (lymphoma of the eye).

[0247] Still further, in some embodiments, B cell-mediated disorder applicable in the present disclosure may be Hodgkin's lymphoma. As used herein, Hodgkin's lymphoma (formerly known as Hodgkin's disease) is a cancer of the immune system that is marked by the presence of a multinucleated cell type called Reed- Sternberg cells. The two major types of Hodgkin's lymphoma include classical Hodgkin's lymphoma and nodular lymphocyte-predominant Hodgkin's lymphoma and are both treatable by the compositions and methods disclosed herein.

[0248] In more specific embodiments, the B cell malignancy is multiple myeloma (MM) and any related conditions. Thus, the therapeutic methods of the present disclosure may be applicable in some embodiments thereof, for a B cell malignancy such as multiple myeloma (MM) and any related conditions. Multiple myeloma (MM), also known as plasma cell myeloma and simple myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, bleeding, frequent infections, and anemia may occur. Complications may include amyloidosis. The cause of multiple myeloma is unknown. Risk factors include obesity, radiation exposure, family history, and certain chemicals. Multiple myeloma may develop from monoclonal gammopathy of undetermined significance that progresses to smoldering myeloma. The abnormal plasma cells produce abnormal antibodies, which can cause kidney problems and overly thick blood. The plasma cells can also form a mass in the bone marrow or soft tissue. When only one tumor is present, it is called a plasmacytoma; more than one is called multiple myeloma. Multiple myeloma is diagnosed based on blood or urine tests finding abnormal antibodies, bone marrow biopsy finding cancerous plasma cells, and medical imaging finding bone lesions. Another common finding is high blood calcium levels. Because many organs can be affected by myeloma, the symptoms and signs vary greatly. The common symptoms of multiple myeloma are indicated as CRAB: C = calcium (elevated), R = renal failure, A = anemia, B = bone lesions. Myeloma has many other possible symptoms, including opportunistic infections (e.g., pneumonia) and weight loss. Multiple myeloma is considered treatable, but generally incurable. Monoclonal gammopathy of undetermined significance (MGUS) increases the risk of developing multiple myeloma. MGUS transforms to multiple myeloma at the rate of 1% to 2% per year, and almost all cases of multiple myeloma are preceded by MGUS. Smoldering multiple myeloma increases the risk of developing multiple myeloma. Individuals diagnosed with this premalignant disorder develop multiple myeloma at a rate of 10% per year for the first 5 years, 3% per year for the next 5 years, and then 1% per year. Obesity is related to multiple myeloma with each increase of body mass index by five increasing the risk by 11%. Studies have reported a familial predisposition to myeloma. Hyperphosphorylation of a number of proteins, the paratarg proteins, a tendency that is inherited in an autosomal dominant manner, appears a common mechanism in these families. This tendency is more common in African American with myeloma and may contribute to the higher rates of myeloma in this group. Rarely, Epstein-Barr virus (EBV) is associated with multiple myeloma, particularly in individuals who have an immunodeficiency due to e.g. HIV / AIDS, organ transplantation, or a chronic inflammatory condition such as rheumatoid arthritis. EBV-positive multiple myeloma is classified by the World Health Organization as one form of the Epstein-Barr virus-associated lymphoproliferative diseases and termed Epstein-Barr virus-associated plasma cell myeloma. EBV-positive disease is more common in the plasmacytoma rather than multiple myeloma form of plasma cell cancer. Tissues involved in EBV+ disease typically show foci of EBV+ cells with the appearance of rapidly proliferating immature or poorly differentiated plasma cells. The cells express products of EBV genes such as EBER1 and EBER2. While the EBV contributes to the development and / or progression of most Epstein-Barr virus-associated lymphoproliferative diseases, its role in multiple myeloma is not known. However, people who are EBV-positive with localized plasmacytoma(s) are more likely to progress to multiple myeloma compared to people with EBV-negative plasmacytoma(s). This suggest that EBV may have a role in the progression of plasmacytomas to systemic multiple myeloma. It should be thus understood that the methods of the present disclosure may be applicable for any type or stage of MM, or any stage, background, source or type as disclosed herein.

[0249] Still further, in some embodiments, the disclosed methods may be applicable for treating at least one protein misfolding disorder or deposition disorder, also named proteopathy. The present disclosure provides in some embodiments thereof, therapeutic methods applicable for subjects suffering from any proteopathy, specifically, amyloidosis, and any related conditions.

[0250] Proteopathy refers to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a gain of toxic function), or they can lose their normal function. The proteopathies (also known as proteinopathies, protein conformational disorders, or protein misfolding diseases) may further include such diseases as Creutzfeldt-Jakob disease and other prion diseases, Alzheimer's disease, Parkinson's disease, amyloidosis, multiple system atrophy, and a wide range of other disorders. In some specific embodiments, the proteopathy or protein-misfolding disorder may be Amyloidosis. Thus, in some embodiments, the therapeutic methods of the present disclosure may be applicable for treating amyloidosis, and any related conditions.

[0251] Specifically, Amyloidosis is a group of diseases in which abnormal proteins, known as amyloid fibrils, build up in tissue. Symptoms depend on the type and are often variable. They may include diarrhea, weight loss, feeling tired, enlargement of the tongue, bleeding, numbness, feeling faint with standing, swelling of the legs, or enlargement of the spleen.

[0252] There are about 30 different types of amyloidosis, each due to a specific protein misfolding. Some are genetic while others are acquired. They are grouped into localized and systemic forms. The four most common types of systemic disease are light chain (AL), inflammation (AA), dialysis (AP2M), and hereditary and old age (ATTR). It should be understood that the CAR molecules, nucleic acid molecules, cells, gene editing system / s, compositions and methods of the present disclosure, may be applicable for any type of amyloidosis, specifically, any type discussed in the present disclosure.

[0253] Additional examples of protein misfolding diseases relevant to the methods of the present disclosure may include any disorder that involves directly or indirectly BCMA expression, specifically, overexpression. Such disorders, include but are not limited to Alzheimer's disease, Cerebral P-amyloid angiopathy, Retinal ganglion cell degeneration in glaucoma, Prion diseases (multiple), Parkinson's disease and other synucleinopathies (multiple), Tauopathies (multiple) Frontotemporal lobar degeneration (FTLD), Amyotrophic lateral sclerosis (ALS), Huntington's disease and other trinucleotide repeat disorders (multiple), Familial British dementia, Familial Danish dementia, Hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHWA-I), Alexander disease, Pelizaeus-Merzbacher disease, Seipinopathies, Familial amyloidotic neuropathy, Senile systemic amyloidosis, Serpinopathies (multiple), AL (light chain) amyloidosis (primary systemic amyloidosis), AH (heavy chain) amyloidosis, A A (secondary) amyloidosis, Type II diabetes, Aortic medial amyloidosis, Apo Al amyloidosis, Apo All amyloidosis, Apo Al V amyloidosis, Familial amyloidosis of the Finnish type (FAF), Lysozyme amyloidosis, Fibrinogen amyloidosis, Dialysis amyloidosis, Inclusion body myositis / myopathy, Cataracts, Retinitis pigmentosa with rhodopsin mutations, Medullary thyroid carcinoma, Cardiac atrial amyloidosis, Pituitary prolactinoma, Hereditary lattice corneal dystrophy, Cutaneous lichen amyloidosis, Mallory bodies, Corneal lactoferrin amyloidosis, Pulmonary alveolar proteinosis, Odontogenic (Pindborg) tumor amyloid, Seminal vesicle amyloid, Apolipoprotein C2 amyloidosis, Apolipoprotein C3 amyloidosis, Lect2 amyloidosis, Insulin amyloidosis, Galectin-7 amyloidosis (primary localized cutaneous amyloidosis), Corneodesmosin amyloidosis, Enfuvirtide amyloidosis, Cystic fibrosis, Sickle cell disease.

[0254] In yet some further embodiments, since amyloidosis is also classified as a deposition disorder, the methods of the invention may be also applicable for any deposition disorder. Deposition disorder, as used herein is any disorder involving or characterized by deposition of insoluble extracellular protein fragments, or any other metabolite, that have been rendered resistant to digestion, thereby interfering and impairing tissue or organ function and may lead to organ failure.

[0255] Still further, in some embodiments, the disclosed methods may be also applicable for treating at least one plasma-cell pathology. Plasma-cell pathology refers herein to any disorder which involves plasma cells. For example, multiple myeloma, myelomatosis and medullary plasmacytoma are bone marrow-based, malignant disorders of postgerminal center B -cells that is characterized by a clonal proliferation of plasma cells, with associated serum and / or urine monoclonal proteins.

[0256] In some embodiments, the plasm-cell pathology may comprise any one of Multiple myeloma (MM), amyloidosis (AL), Monoclonal gammopathy of undetermined significance (MGUS), Plasmacytoma (PL) and / or Waldenstrom macroglobulinemia (WDS).

[0257] Still further, Monoclonal gammopathy of undetermined significance (MGUS), as used herein, is an asymptomatic preneoplastic plasma cell disorder that is characterized by serum M-protein less than 30 g / L, bone marrow clonal plasma cells less than 10 percent, absence of plasma cell myeloma-related end-organ damage (hypercalcemia, renal insufficiency).

[0258] In some embodiments, Plasmacytoma (PL), is a plasma cell dyscrasia in which a plasma cell tumour grows within soft tissue or within the axial skeleton.

[0259] Waldenstrom macroglobulinemia (WDS), as used herein, is a neoplastic disorder affecting lymphoplasmacytoid cells and plasma cells. It is characterized by having high levels of a circulating antibody, immunoglobulin M (IgM), which is made and secreted by the cells involved in the disease. WDS is an "indolent lymphoma" (characterized with slow growth and spread) and a type of lymphoproliferative disease which shares clinical characteristics with the indolent non- Hodgkin lymphomas. It is commonly classified as a form of plasma cell dyscrasia, similar to other plasma cell dyscrasias that, for example, lead to multiple myeloma.

[0260] In some embodiments, specifically when T cells that express the HBI0101 molecule disclosed herein are used (e.g., SEQ ID NO: 23 and SEQ ID NO: 24), the therapeutic methods of the present disclosure may be applicable for any disorder that is associated with the expression (e.g., modulated expression, specifically, overexpression) of the BCMA protein. Examples for such disorders include neoplastic disorders of B cells as disclosed above. However, it should be appreciated that in some embodiments, the disorder may be any proliferative disorder, or any neoplastic disorder. Particularly, where the exogeneous nucleic acid molecule encodes a receptor molecule, e.g., a CAR molecule directed against at least one TAA specific for the malignancy. The methods of the present disclosure may be applicable in some embodiments for any neoplasms, either benign neoplasms, in situ neoplasms, or malignant neoplasms. As used herein to describe the present disclosure, “proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non- solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods, compositions and kits of the present invention may be applicable for a patient suffering from any one of non-solid and solid tumors.

[0261] Malignancy, as contemplated in the present invention may be any one of lymphomas, leukemia, myeloma, carcinomas, melanomas and sarcomas. Therefore, in some embodiments any of the methods of the present disclosure (provided that they involve, directly or indirectly, B cells and / or expression of BCMA, or any other TAA), systems and compositions disclosed herein, may be applicable for any of the malignancies disclosed by the present disclosure.

[0262] More specifically, myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. Hypercalcemia (high calcium levels) is often encountered.

[0263] Lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma. Non limiting examples for lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.

[0264] Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic). Still further, carcinoma as used herein, refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.

[0265] Melanoma as used herein, is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin but are also found in other parts of the body, including the bowel and the eye. Melanoma can occur in any part of the body that contains melanocytes.

[0266] Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas.

[0267] It should be understood that the cells, compositions and methods of the present disclosure are applicable for any type and / or stage and / or grade of any of the malignant disorders discussed herein or any metastasis thereof. Still further, it must be appreciated that the methods, compositions and systems of the invention may be applicable for invasive as well as non-invasive cancers. When referring to "non-invasive" cancer it should be noted as a cancer that do not grow into or invade normal tissues within or beyond the primary location. When referring to "invasive cancers" it should be noted as cancer that invades and grows in normal, healthy adjacent tissues.

[0268] Still further, in some embodiments, the cells, compositions and methods of the present disclosure are applicable for any type and / or stage and / or grade of any metastasis, metastatic cancer or status of any of the cancerous conditions disclosed herein.

[0269] As used herein the term "metastatic cancer" or "metastatic status" refers to a cancer that has spread from the place where it first started (primary cancer) to another place in the body. A tumor formed by metastatic cancer cells originated from primary tumors or other metastatic tumors, that spread using the blood and / or lymph systems, is referred to herein as a metastatic tumor or a metastasis. Thus, in some embodiments, malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas / carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non- Hodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone / Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic / Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia / Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer). Still further, as discussed herein above, according to some embodiments, the methods of the present disclosure may be used for the treatment of a patient suffering from any autoimmune disorder. In some specific embodiments, the methods of the invention may be used for treating an autoimmune disease such as for example, but not limited to, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, fatty liver disease, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behcet's syndrome, Indeterminate colitis, rheumatoid arthritis, Graft versus Host Disease (GvHD), Eaton-Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM) and NIDDM, multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, scleroderma, thrombocytopenia, thyroiditis e.g. Hashimoto's disease, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, arthritis, alopecia areata, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, inflammatory arthritis, including juvenile idiopathic arthritis, gout and pseudo gout, as well as arthritis associated with colitis or psoriasis, Pernicious anemia, some types of myopathy and Lyme disease (Late).

[0270] In some embodiments, the cell population used by the disclosed therapeutic methods may comprise T cells, specifically, naive-like T cells (that comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM), harboring an exogeneous nucleic acid molecule encoding at least one CAR molecule and / or a TCR or any part and / or derivative thereof. In some embodiments, the CAR or TCR comprise at least one target-binding domain specific for at least one target molecule associated with and / or expressed by the at least one pathologic disorder.

[0271] In some specific embodiments, the CAR or TCR expressed by the T cells used in the disclosed therapeutic methods comprise at least one target-binding domain specific for at least one tumor associated antigen (TAA).

[0272] In some embodiments, the T cell / s used by the disclosed methods harbor an exogeneous nucleic acid molecule encoding at least one CAR molecule comprising:

[0273] (a), at least one target-binding domain; wherein at least one of said target binding domain specifically recognizes and binds B cell maturation antigen (BCMA); (b), at least one hinge and at least one transmembrane domain derived from the Cluster of Differentiation 8 a (CD8a) protein; and (c), at least one intracellular T cell signal transduction domain. In some embodiments, such domain may comprise at least one domain of tumor necrosis factor (TNF) receptor family member (e.g., 4-1BB), and optionally, at least one domain of a T cell receptor (TCR) molecule (e.g., CD3 zeta chain domains).

[0274] In certain embodiments, the cell population used by the disclosed therapeutic methods is of an autologous source. Specifically, the disclosed method is based on autologous cells. Accordingly, the disclosed method comprises:

[0275] In step (a), contacting a population of PBMC cells of the subject (or alternatively, of an allogeneic source), with a compound that specifically selects for cells expressing the CD62L marker, thereby obtaining an enriched CD62L T cell population. In step (b), subjecting the enriched CD62L T cell population obtained in step (a) to T-cell activation by contacting the cell population with (i) CD3 and CD28; and (ii) a cytokine cocktail comprising IL-7, IL-15 and IL-21. In step (c), genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule encoding at least one CAR and / or TCR molecule, in the presence of at least one transduction enhancer; thereby obtaining a cell population comprising T cells harboring said CAR and / or TCR molecule. In step (d), expanding the transduced or transfected cell population obtained in step (c). Step (e) involves harvesting and / or formulating the expanded cell population obtained in step (d), thereby preparing a cell population comprising naive T cells. In some embodiments, the naive-like T cells administered to the subject comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM).The naive-like T cells used by the disclosed methods (and prepared by the disclosed preparation methods) are characterized by CD4+T cell to CD8+T cell ratio that is greater than one. Finally, step (f) comprises administering to the subject an effective amount of the harvested and / or formulated cell population obtained in step (e), or any composition thereof. It should be noted that steps (a) to (e) are performed in the same cell culture vessel, in a low serum medium containing 1 % or less human AB serum. In yet some further embodiments, the duration of the method is up to 6 days (but the expansion step can be extended up to additional 2 days, for the total of up to 8 days without compromising the required phenotype).

[0276] In some embodiments, the cytokine cocktail may comprise any appropriate amount of the disclosed cytokines, for example, any amount ranging from about 0.1 pg / ml to about 10Omg / ml, specifically, about 0.1 ng / ml to about 10Ong / ml. In some embodiments of the disclosed methods, the activation step (b) may be performed using CD3 / CD28 and a cocktail of IL7, specifically, in an amount of 10 ng / ml, IL15, specifically, in an amount of 10 ng / ml and IL21, specifically, in an amount of 20 ng / ml as demonstrated by the examples. It should be understood that the genetically modifying step (c) of the disclosed methods may comprise in some embodiments transfecting and / or transducing the enriched cells with at least one exogeneous nucleic acid molecule. In some particular and non-limiting embodiments, this step comprises contacting the cells with at least one transduction enhancer. In certain embodiments, transduction enhancer formulations include compounds that facilitate cell-virus contact, compounds that block cellular antiviral defenses, and / or compounds that modify the cell membrane. Examples of transduction enhancers are cationic amphipathic peptides, peptide nanofibrils and the like. Commercial examples of transduction enhancers are Vectofusin-1®, RetroNectin, LentiBOOST™, NATE™, and the like.

[0277] A further aspect of the present disclosure relates to an effective amount of a cell population comprising cells of the T lineage, or a composition comprising the same, for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject. The cells of the T lineage comprise naive-like T cells. In some embodiments, the naive-like T cells used herein comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). Still further, the cell population used herein is prepared by a method comprising the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organjwith a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. In specific embodiments, the expansion of the cells in step (d) may be continued for a period of up to two additional days, such that the duration of the entire process is performed for a period of up to 8 days, allowing the cells to achieve the required cell number. Still further, in some embodiments of the present aspect, the present disclosure provides an effective amount of a cell population comprising cells of the T lineage, specifically, the disclosed naive-like cells, or a composition comprising the same, for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject. In some embodiments, the naive-like T cells administered to the subject comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). Still further, the method comprising (I) preparing the cell population from cells of the subject or from cells of at least one allogeneic source; and (II) administering the obtained naive-like cells to the subject. More specifically, the cell population used herein is prepared in step (I), by a method comprising the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organjwith a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. In specific embodiments, the expansion of the cells in step (d) may be continued for a period of up to two additional days, such that the duration of the entire process is performed for a period of up to 8 days, allowing the cells to achieve the required cell number.

[0278] In some embodiments, the effective amount of a cell population comprising cells of the T lineage for use according to the present disclosure, wherein the cell population is prepared by a method as defined by the present disclosure.

[0279] Still further, the effective amount of the cell population comprising cells of the T lineage for use in accordance with the present disclosure is of an autologous or allogeneic source. Thus, the cells used herein are in some embodiments autologous naive-like T cells. Alternatively, the cells used herein are in some embodiments allogeneic naive-like T cells.

[0280] In some embodiments, the effective amount of a cell population comprising cells of the T lineage for use according to the present disclosure is applicable for treating a pathologic disorder that may be at least one of: a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and / or a neurodegenerative disorder.

[0281] As described herein above, the present disclosure provides in some aspects thereof therapeutic and prophylactic methods and uses of the T cell population prepared by the disclosed methods. It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease.

[0282] The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, an immune-related condition and illness, immune-related symptoms or undesired side effects or immune-related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and / or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms. It should be appreciated that the terms "inhibition", "moderation", “reduction”, "decrease" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more. With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. The term "amelioration" as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the compositions and methods according to the invention, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the immune-related disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state. The term "inhibits" and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with. The term "eliminate" relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the invention described herein. The terms "delay" , "delaying the onset", "retard" and all variations thereof are intended to encompass the slowing of the progress and / or exacerbation of a disorder associated with the immune-related disorders and their symptoms slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the invention. As indicated above, the methods and compositions provided by the present invention may be used for the treatment of a “pathological disorder”, specifically, immune-related disorders as specified by the invention, which refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein. It should be appreciated that any of the methods and compositions described by the invention may be applicable for treating and / or ameliorating any of the disorders disclosed herein or any condition associated therewith. It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co- exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, “disease”, “disorder”, “condition”, “pathology” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. The present invention relates to the treatment of subjects or patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the therapeutic and prophylactic methods herein described are desired, including any vertebrate, specifically mammals such as humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and rodents, specifically, murine subjects. More specifically, the methods of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, livestock, equine, canine, and feline subjects, most specifically humans.

[0283] A further aspect of the present disclosure relates to an isolated cell population comprising T cells, specifically, naive-like T cells. The naive-like T cells comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). The T cells are characterized by a CD4+T cell to CD8+T cell ratio that is greater than one.

[0284] In some embodiments, about 30% to about 99%, or more of the T cells in the cell population comprise TNaive / SCMcells expressing the CD45RA+CCR7+ and cells expressing the TCM, CD45RA- CCR7+ markers. It should be understood that the range "about 30% to about 99% " encompasses specific percentages that include about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and about 99%. In some specific embodiments, about 60% or more of the T cells in the cell population comprise TNaive / SCMcells expressing the CD45RA+CCR7+ and cells expressing the TCM, CD45RA-CCR7+ markers.

[0285] In some embodiments, the isolated cell population of the present disclosure is characterized by at least one of:

[0286] (i) a higher average expression level of at least one gene associated with naive and stem cell memory T cell phenotypes in the naive-like T cells population, as compared with an average expression level of such gene in a control population; and / or

[0287] (ii) an increased proportion of T cells expressing at least one gene associated with naive and stem cell memory T cell phenotypes in the naive-like T cells population, as compared with a control population. In more specific embodiments, at least one gene associated with naive and stem cell memory T cell phenotypes is selected from the group consisting of: MKI67, IL2RB, CD28, CD27, LEF1, SELL, CCR7, and TCF7.

[0288] In yet some further additional or alternative embodiments, the cell population of the present disclosure is characterized by at least one of: (i) a lower average expression level of at least one effector-associated gene in the naive-like T cells of the population, as compared with the average expression level in T cells of a control population; and / or

[0289] (ii) a decreased proportion of T cells expressing at least one effector-associated gene, in said naive- like T cells population, as compared with a control population. In more specific embodiments, the at least one effector-associated gene is selected from the group consisting of: TOX, HAVCR2, LAG3, TIGIT, EOMES, TBX21, KLRD1, GNLY, GZMA, GZMB, GZMH, and GZMK.

[0290] In some embodiments, the isolated cell population provided herein, is prepared by a method comprising following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ)with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), modifying, e.g., genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. In step (d), expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. In some embodiments, the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days, such that the duration of the disclosed method is up to 7 days, or 8 days. It should be noted that the characterizing features of the cells as defined in the present disclosure are maintained also after the additional one or two days of expansion.

[0291] In some embodiments, the T cells of the cell population prepared and provided herein display a decreased proportion of senescent-like (CD27-CD28-) T-cells.

[0292] In some embodiments, the isolated cell population according to the present disclosure prepared by a method as defined herein before.

[0293] In some embodiments, the isolated cell population prepared by the present disclosure, is of an autologous source. Specifically, the naive-like T cells of the present disclosure are autologous naive-like T cells.

[0294] In some embodiments, the isolated cell population provided by the present disclosure, is formulated in a composition adapted for adoptive transfer. In other words, the cells are formulated in a composition compatible with adoptive transfer procedure.

[0295] In some embodiments, the cell population disclosed herein, comprises T cell(s) harboring an exogeneous nucleic acid molecule encoding at least one CAR molecule and / or a TCR or any part and / or derivative thereof. The CAR or TCR comprise at least one target-binding domain specific for at least one target molecule associated with and / or expressed by the at least one pathologic disorder.

[0296] In some embodiments, the CAR molecule is specific for at least one tumor associated antigen (TAA).

[0297] In some embodiments, the disclosed T cell population expresses CAR molecules specific for an antigen expressed by cells of a B cell malignancy. Still further, in some embodiments, the malignancy is multiple myeloma (MM) and any related conditions.

[0298] In some embodiments, the pathologic disorder is a deposition disorder. In some embodiments, the deposition disorder is amyloidosis, and any related conditions. In some embodiments, the T cell(s) provided by the present disclosure harbor an exogeneous nucleic acid molecule encoding at least one CAR molecule comprising: (a), at least one target- binding domain; wherein at least one of said target binding domain specifically recognizes and binds B cell maturation antigen (BCMA). (b), at least one hinge and at least one transmembrane domain derived from the Cluster of Differentiation 8 a (CD8a) protein; and (c), at least one intracellular T cell signal transduction domain, the domain comprising at least one domain of tumor necrosis factor (TNF) receptor family member (e.g., 4-1BB), and optionally, at least one domain of a T cell receptor (TCR) molecule (e.g., CD3 zeta chain domains).

[0299] A further aspect of the present disclosure relates to a composition comprising the isolated cell population prepared by the methods as defined according to the present disclosure, and at least one of pharmaceutically acceptable carrier / s, diluent / s, excipient / s and additive / s.

[0300] The compositions of the present disclosure may comprise an effective amount of the T cell populations prepared by the methods of the present disclosure. The term "effective amount” relates to the amount of an active agent present in a composition, specifically, the cell populations as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual (e.g., the thymus or bone marrow) to be treated to give an anticipated physiological response when such composition is administered. The precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein. An “effective amount" of the genetically engineered T cells disclosed herein, or any composition or preparation thereof, can be administered in one administration, or through multiple administrations of an amount that total an effective amount, preferably within a 24-hour period. It can be determined using standard clinical procedures for determining appropriate amounts and timing of administration. It is understood that the "effective amount" can be the result of empirical and / or individualized (case-by-case) determination on the part of the treating health care professional and / or individual. Still further, administration and doses are determined by good medical practice of the attending physician and may depend on the age, sex, weight and general condition of the subject in need.

[0301] It should be appreciated that the effective amount as discussed herein is applicable for each and every embodiment of each and every aspect of the present disclosure, specifically, for any of the T cells (e.g. cells that may express in some embodiments, any exogeneous receptor, e.g., the CAR molecule disclosed herein), and / or for ex-vivo use, any nucleic acid sequence encoding the disclosed CAR molecule, any construct, or gene editing system comprising the same, any dosage forms thereof, dosage unit forms thereof, compositions, kits, uses and methods thereof.

[0302] In some embodiments, the active ingredient of the therapeutic composition, and / or dosage form disclosed herein, for example, comprising the T cells genetically engineered to express any receptor molecule, specifically, CAR molecule such as the HBI0101 CAR molecule (also designated herein as H8BB CAR molecule, that comprises the amino acid sequence as denoted by SEQ ID NO: 23, or 24), as disclosed by the present disclosure. Accordingly, various effective amounts of these cells can be used, based on previous clinical studies performed by the present inventors with this CAR molecule. In some embodiments, the effective amount of HBI0101 CAR T cells prepared by the methods of the present disclosure may range between about 10x106cells to about 10000 x106cells, specifically, about 50x106cells to about 1500x106cells, specifically, 10x 106, 15x106, 10 x 10106, 15 x 106, 20 x 106, 25 x 106, 30 x 106, 35 x 106, 40 x 106, 45 x 106, 50x106, 60 x106, 70 x106, 80 x106, 90 x106, 100 x106, 110 x106, 120 x106, 130 x106, 140 x106, 150 x106, 160 x 106, 170 x106, 180 x106, 190 x106, 200 x106, 210 x106, 220x106, 230x106, 240 x106, 250 x106, 260 x106, 270 x106, 280 x106, 290 x106, 300x106, 310 x106, 320 x106, 330 x106, 340 x106, 350 x106, 360 x106, 370 x106, 380x106, 390 x106, 400 x106, 410 x106, 420 x106, 430 x106, 440 x106, 450 x106, 460x106, 470 x106, 480 x106, 490 x106, 500 x106, 510 x106, 520 x106, 530 x106, 540 x106, 550 x106, 600 x106, 650 x106, 700 x106, 750 x106, 800 x106, 850 x106, 900 x106, 950 x106, 1000 x106, 1050 x106, 1100 x106, 1150 x106, 1200 x106, 1250 x106, 1300x106, 1350 x106, 1400 x106, 1450 x106, 1500x106 / HBI0101 CAR T cells / per dose. More specifically, the amount and / or number of HBI0101 CAR T cells may range between about 100 x106to about 1000 x106, about 90 x106to about 900 x106, about 150 x106to about 800 x106. Still further, in some embodiments, an effective amount and / or number of cells is 800 x106HBI0101 CAR T cells, per dose. In yet some other embodiments, an effective amount and / or number of cells is 450 x106' HBI0101 CAR T cells, per dose. Still further, in some embodiments, an effective amount and / or number of cells is 150 x106HBI0101 CAR T cells, per dose. Still further, in some embodiments, the effective amount and / or number of cells may range between about 0.5x106to about 50x106HBI0101 CAR T cells / per Kg of body weight. Specifically, about 1 to about 40, about 2 x106to about 30 x106, about 2 x106to about 25 x106, about 2 x106to about 20 x106, about 2 x106to about 15 x106, about 2 x106to about 14 x106, about 2 x106to about 13 x106, about 2 x106to about 12 x106, about 2 x106to about 11 x106, about 2 x106to about 10 x106HBI0101 CAR T cells / per Kg of body weight. In some specific and non-limiting embodiments, effective amount of the cells is l lx106HBI0101 CAR T cells / per Kg of body weight. In some other specific and non-limiting embodiments, effective amount of the cells is 10x106HBI0101 CAR T cells / per Kg of body weight. In some embodiments, an effective amount as used herein refers to a daily dose of 10- 10OOx106. The present disclosure further envisaged the use of 80 x106CAR T cells prepared by the disclosed methods per dose. In some alternative embodiments, the present disclosure further envisaged the use of 160 x106, CAR T cells prepared by the disclosed methods per dose. In some alternative embodiments, the present disclosure further envisaged the use of 240 x106, CAR T cells prepared by the disclosed methods per dose. Still further, in some embodiments, the construct encoding the CAR molecule used by the methods of the present disclosure, also referred to herein as HBI0101 (prepared in a clinical grade), may be generated by transiently transfecting Phoenix- ECO cells (ATCC) with the plasmid encoding the gamma-retroviral vector MSGV1-HBI0101 using JetPrime reagent (Tamar) and subsequently transducing PG13 cells (ATCC) with HBI0101- Phoenix-ECO cell-free vector supernatants. PG13 transduced population was subsequently sub- cloned by limiting dilution, and the PG13-HBI0101 expanded to generate a seed bank. The certified PG13 seed bank was sent to the Indiana University Vector Production Facility (IU-VPF) in Indianapolis that has generated a master cell bank (MCB) and a GMP-certified HBI0101 clinical grade retroviral supernatant for the transduction of MM patients' autologous T-cells.

[0303] Still further, for ex vivo transduction of the cells, after activation step (b), peripheral blood mononuclear cells from subjects’ leukapheresis (e.g., AL and / or multiple myeloma patients) were used. More specifically, blood was collected from patients with AL or / and multiple myeloma (0253-20-HMG) and processed, for example, using a Ficoll gradient to isolate peripheral blood mononuclear cells (PBMC).

[0304] More specifically, pharmaceutical compositions used to treat subjects in need thereof according to the invention, 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 formulations are prepared by uniformly and intimately bringing into association the active ingredients, specifically, the cells prepared by the methods of the present disclosure, with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. Still further, pharmaceutical preparations are compositions that include the cells prepared by the methods of the present disclosure in a pharmaceutically acceptable vehicle. "Pharmaceutically acceptable vehicles" may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term "vehicle", when referred to the compositions in the present aspect, refers to a diluent, adjuvant, excipient, or carrier with which the cell population of the invention is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. As such, administration of the cells of the invention or any engineered cells of the T-lineage, and compositions thereof, can be achieved in various ways, specifically, adoptive transfer. The cells may be administered by the use of regional administration, intra-tumoral administration, or use of an implant that acts to retain the active dose at the site of implantation.

[0305] In numerous embodiments, the compositions of the present disclosure that comprise the T cells prepared by the methods of the present disclosure, may be administered in a form of combination therapy, i.e. in combination with one or more additional therapeutic agents. Combination therapy may include administration of a single pharmaceutical dosage formulation comprising at least one composition of the invention and additional therapeutics agent(s); as well as administration of at least one composition of the invention and one or more additional agent(s) in its own separate pharmaceutical dosage formulation. Further, where separate dosage formulations are used, compositions of the invention and one or more additional agents can be administered concurrently or at separately staggered times, i.e. sequentially. Still further, the concurrent or separate administrations may be carried out by the same or different administration routes. Thus, in some further embodiments, the T cells prepared by the methods of the present disclosure may be applicable in boosting the immune response of a subject suffering from an immune-related disorder, specifically, any disorder involving B Cells, or B cell malignancies, and may be used in combined treatment with any therapeutic agent, for example, a chemotherapeutic agent.

[0306] As used herein, a “chemotherapeutic agent” or “chemotherapeutic drug” (also termed chemotherapy) as used herein refers to a drug treatment intended for eliminating or destructing (killing) cancer cells or cells of any other proliferative disorder. The mechanism underlying the activity of some chemotherapeutic drugs is based on destructing rapidly dividing cells, as many cancer cells grow and multiply more rapidly than normal cells. As a result of their mode of activity, chemotherapeutic agents also harm cells that rapidly divide under normal circumstances, for example bone marrow cells, digestive tract cells, and hair follicles. Insulting or damaging normal cells result in the common side-effects of chemotherapy: myelosuppression (decreased production of blood cells, hence also immuno-suppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss).

[0307] Various different types of chemotherapeutic drugs are available. A chemotherapeutic drug may be used alone or in combination with another chemotherapeutic drug or with other forms of cancer therapy, in addition to the CAR T cells of the present disclosure, for example, other biological drugs (antibodies, ligands, receptors), radiation therapy or surgery.

[0308] Chemotherapeutic drugs affect cell division or DNA synthesis and function and can be generally classified into several groups, based on their structure or biological function. More specifically, chemotherapeutic agents that are classified as alkylating agents, anti-metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents such as DNA-alkylating agents, anti-tumor antibiotic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple- helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial or exotoxic agents. It should be appreciated that any combination therapy disclosed herein, using any of the indicated compounds with the CAR T, DNA cassettes, systems and cells of the present disclosure, together with any of the therapeutic agents discussed above, is encompassed by the present disclosure.

[0309] Another aspect provided by the present disclosure relates to a kit comprising: (a), a compound that specifically selects for cells expressing the CD62L marker.

[0310] (b), T-cell activation mixture comprising: (i) a colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL-15) and Interleukin 21 (IL-21).

[0311] (c), at least one exogeneous nucleic acid molecule encoding at least one CAR molecule and at least one transduction enhancer, for example a histidine-rich amphipathic peptide; thereby obtaining a cell population comprising T cells harboring said CAR molecule; and

[0312] (d), a low serum medium containing 1% or less human AB serum.

[0313] It should be understood that the kit disclosed herein is adapted for operation in a single container or compartment for a period of up to 6 days. In some embodiments, the time period may be extended up to additional 2 days, to a total of 8 days.

[0314] In some embodiments the disclosed kits are adapted for performing the disclosed methods, specifically, a method for preparing a population of naive-like T cells. More specifically, the naive- like T cells prepared by the disclosed method, comprise naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). The disclosed method comprises the following steps. Step (a), involves contacting a population of cells comprising cells of the T lineage (e.g., PBMCs, leukapheresis cell products, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ) with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker. Step (b) involves subjecting the enriched cell population obtained in step (a) to T-cell activation. Next, in step (c), the activated cells obtained in step (b), are modified, e.g., genetically modified with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule. Step (d) involves expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the disclosed method is up to 6 days. Specifically, in some embodiments, the disclosed method may be performed for no more than six days or less, for example, one day, two days, three days, four days, five days or six days. In some embodiments, the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days, such that the duration of the disclosed method is up to 7 days, or 8 days.

[0315] A further aspect of the present disclosure relates to a method for preparing a population of naive- like T cells. The naive-like T cells comprise at least one of naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM). The disclosed method comprising: (a), contacting a population of cells comprising cells of the T lineage subsets with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing the at least one marker; (b), subjecting the enriched cell population obtained in step (a) to T-cell activation; and (c), expanding the activated cell population; thereby preparing a cell population comprising naive-like T cells. It should be noted that the duration of the method is up to 6 days, but can be extended up to 8 days by adding one or two days to the expansion step, without compromising the desired naive-like phenotype.

[0316] It should be understood that the naive-like T cells obtained by the disclosed methods may be generated to restore the T-cell compartment that has been destroyed by the myeloablative regimen until immune reconstitution takes place.

[0317] In addition, virus-specific T-cells can be isolated, enriched and expanded via the naive-like T-cell platform, thus providing long-term protection from virus-mediated infections for immune - suppressed patients.

[0318] Still further, infiltrated tissues contain heterogeneous T cell populations, most of which are terminally differentiated and exhausted. However, CD62L+ early memory T cells exist, though at low frequency. Enrichment and expansion of such subsets, while preserving this early memory phenotype combine the advantages of natural antigen specificity of infiltrated T cells with the persistence and expansion of early memory T cells.

[0319] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0320] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Thus, as used herein the term "about" refers to ± 10 %.

[0321] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, and / or parts, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0322] It should be noted that various embodiments of this invention may be 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 the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges 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 numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0323] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0324] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0325] Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0326] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0327] EXAMPLES

[0328] Experimental procedure

[0329] Reagents:

[0330] Table 1: antibodies and reagents used for T-cell differentiation, T-cell exhaustion and T-cell cellular subsets

[0331] Table 2: reagents used for Cytokines and Degranulation assay

[0332] Procedures

[0333] CART cell generation by retroviral transduction

[0334] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy or patient (multiple myeloma or light chain amyloidosis) leukapheresis products by density gradient centrifugation (Cytvia premium grade) and transduced with a clinically tested retroviral supernatant (HBI0101) encoding an anti-BCMA chimeric antigen receptor (CAR) (NCT04720313).

[0335] Traditionally manufactured CART cells (TM)

[0336] Cells were generated by stimulation of PBMCs in AIM-V CTS medium (Gibco) supplemented with 5% AB serum (Grifols Bio Supplies Inc.) and ImM GlutaMAX (Gibco) for two days in the presence of anti-CD3 soluble antibody (0KT3, Miltenyi) and 300IU / mL IL-2 (Proleukin, Novartis). On day 2, stimulated cells (2.5x106cells / well) were transduced by spinoculation (1000g, 10 mins) on RetroNectin® (10pg / mL, Takara Bio) - precoated non-tissue culture six- well plates on which retroviral supernatant was previously precipitated by centrifugation (2000g, 32°C, 2 hours). On day 3, transduced cells were suspended and expanded into a GREx10OM device (Wilson Wolf) for five to seven more days filled up to 1 liter expansion medium (AIM-V CTS, 5% AB serum, ImM GlutaMAX, 300IU / mL IL-2). Entire TM manufacturing process lasts between 8-10 days. Generation of “Nai've-like” CART cells

[0337] Generation of “Naive-like” CART cells is a 6-day process, which major steps include:

[0338] 1. CD62L+ PBMCs magnetic selection (optional: cryopreservation, CryoStor-10, BioLife)

[0339] 2. CD62L+ T-cell activation (TransAct beads, Miltenyi)

[0340] 3. CD62L+ T-cell retroviral transduction (Vectofusin-1®, Miltenyi)

[0341] 4. CART-cell short ex-vivo expansion (G-Rex)

[0342] 5. CART final product formulation and cryopreservation (CryoStor-5, BioLife).

[0343] All the above steps are processed in a single vessel, namely G-Rex (Wilson Wolf), in low-human serum media (AIM-V + 1% human serum), and in the presence of IL-7, IL-15 (10ng / mL each) and IL-21 (20ng / mL).

[0344] More specifically, for generating naive-like CART cells, CD62L+ PBMCs were first isolated by magnetic sorting (Miltenyi 200-070-236) using the CiiniMacs or Prodigy systems, as per the manufacturer’s instructions. Cells were then seeded at the concentration of Ix106PBMCs / mL in a GRex device, and stimulated with TransAct (i.e., CD3 / CD28 nanomatrix) reagent (Miltenyi, 200-076-202 or 200-076-204) in AIM-V CTS medium containing 1% AB serum, in the presence of IL-7, IL-15 (Miltenyi premium grade, 10ng / mL each, 170-076-184 and 170-076-186, respectively), and IL-21 (Miltenyi premium grade, 20ng / mL, 170-076-189) for two days. At day 2, stimulated cells were transduced by addition of a mixture of retroviral supernatant: Vectofusin- 1® (Miltenyi 170-076-165) (1 / 6) followed by an overnight incubation at 37oC. On day 3 of the process, expansion medium (AIM-V 1% AB serum IL7 / 15 / 21 cocktail) was topped above the cells (60-70mL). Cells were expanded for up to 3 days; naive-like CART cell manufacturing process lasts 6 days.

[0345] CART products were cryopreserved in CryoStor 5 (CS5, BioLife Solutions, 205102) in liquid nitrogen vapor phase.

[0346] Flow cytometry

[0347] CART-cell phenotypic characterization, T-cell differentiation and exhaustion profiles were assessed by labeling Ix105cells with antibody mixtures as detailed in Table 1.

[0348] Cells were incubated for 20 mins on ice, washed twice in FACS buffer consisting of PBS (Biological Industries) + 2% FBS (heat inactivated Gibco). Sytox (Invitrogen) was added before samples acquisition as per manufacturer’s instructions. Samples were acquired on a Dx-Flex cytometer (Beckman Coulter), and the data was processed with Kaluza Analysis software 2.1.

[0349] CART cell cytotoxicity assay

[0350] Assessment of CART cytotoxicity was performed by co-culturing CART cells with BCMA- expressing human myeloma cell line NCI-H929 (ATCC) genetically modified to express the firefly luciferase (referred as H929-luc), at different effector to target (E:T) ratios and for different incubation time periods, as detailed for each experiment. The bioluminescent signal from tumor cells was used as a surrogate marker for the tumor killing ability of CAR T cells. The traceability of remaining H929 tumor cells using bioluminescence measurement (GloMax, Promega) following addition of luciferin (Vivo Gio, Promega) to the cell co-culture, and an incubation of 7 mins at 37°C. The percent of H929 lysis was then calculated according to the following formula:

[0351] % H929 lysis = { 1 - bioluminescence in co-culture well } x 100% maximal bioluminescence in H929-luc alone well

[0352] Recursive antigen stimulation

[0353] To mimic the conditions of CART cell repetitive exposure to high tumor burden, a “stress test” was conducted by the inventors. To that end, coculture of H929-luc myeloma target cells and traditional or naive-like CAR T cells was initiated at effector : target (E:T) ratios ranging from 1 : 1 to 1:64. Briefly, Ix105CART cells were seeded in a 24-wells plate in culture media consisting of AIM-V+5% AB serum+1% GlutaMAX and no cytokines. CART cells were serially diluted to get 0.5x105, 0.25x105and 0.125x105CART / well respectively. Then, Ix105H929-luc were added to each well, mixed well, and incubated for the indicated time periods. CART cell and H929-luc alone wells serve as controls. An aliquot of the cell suspension was analyzed daily to monitor tumor cell eradication over time by bioluminescence as detailed above, where background levels (CART cell alone) indicate maximal tumor cell eradication. At the indicated time-points, additional aliquots of the co-culture cell suspension were analyzed for cell count (Countess, Invitrogen), and by flow cytometry for viable H929 tumor cells (SYTOX-CD138+CD3-) and viable CART cells (SYTOX-CD138-CD3+CAR+), T-cell differentiation and exhaustion statuses. Proliferation rate was calculated as the ratio of input viable CART cells at the beginning of each round versus viable CART cell numbers at the end of each round. Tumor load in each co-culture well was calculated as the ratio of residual viable H929 target cells after each round versus viable H929 cell numbers at the beginning of each round (Ix105cells).

[0354] After each round of stimulation, a new stimulation was re-initiated with the residual CART cells from the previous stimulation round and Ix105fresh H929-luc target cells.

[0355] Cytokines and Degranulation assay

[0356] Traditional or naive-like CART-cells were cryopreserved following four rounds of stimulation with H929 target cells at 1:1 E:T ratio (see above). For intracellular cytokine staining, CART-cells were thawed and then incubated with H929 target cells for 4-5 hours at 37°C in the presence of Brefeldin A (BD Biosciences) and anti-CD107a. Cells were subsequently stained with cell surface markers CD4-PC7, CD8-Krome Orange and CD3-AF700 and CD107a-BV605. After cell surface staining, cells were fixed and permeabilized with Fixation / Permeabilization Kit with GolgiStop Kit (BD Biosciences) according to the manufacturer’ s protocol. Cells were then stained with anti- IFN-y-BV650, anti-TNF-a-BV785, IL-2-APC / Fire, and Granzyme B-APC. After staining, cells were analyzed using the Dx-Flex flow cytometer (Beckman Coulter).

[0357] Single-cell RNA sequencing and transcriptomic analysis

[0358] To investigate the transcriptional profiles of CART cells generated using Naive versus traditional manufacturing (TM) platforms, single-cell RNA sequencing (scRNA-seq) was performed using the 10x Genomics Chromium Single Cell 3’ Gene Expression platform (v3 chemistry), following the manufacturer’s protocol. Cryopreserved Naive and TM CART cell products from three donors (a total of six samples) were thawed in AIM-V medium (GIBCO) supplemented with 5% human serum (Valley Biomedical) and 1% GlutaMax (GIBCO). Cells were washed, counted, and processed directly for single-cell encapsulation without prior selection or enrichment.

[0359] Approximately 10,000-20,000 total cells per sample were loaded onto the Chromium Controller to generate gel bead-in-emulsions (GEMs), enabling single-cell barcoding and reverse transcription. Complementary DNA (cDNA) synthesis and amplification were performed according to the manufacturer’s instructions, and sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit. Libraries were sequenced on an Illumina NovaSeq 6000 system, targeting a sequencing depth of approximately 35,000-65,000 reads per cell.

[0360] Raw NovaSeq output was converted to FASTQ files using Cell Ranger v8.0.1 (mkfastq). These were processed with the count command (— expect-cells=20000), using a custom reference genome comprising GRCh38 (Ensembl v106) and the MS6VA plasmid.

[0361] A total of 6 samples (BCMA#137 and BCMA#120; MM patients and PHE-011; Healthy Donor including both Naive and TM CART products) were analyzed. Filtering retained genes detected in >3 cells and cells with >2,000 genes and <5% mitochondrial reads, resulting in 9,993-18,183 cells and 27,669-32,347 genes.

[0362] All samples were normalized with SCTransform (Seurat v5.2.1) and subjected to PCA and UMAP based on the top 50 PCs. Integration across all samples used 3,000 features, yielding a unified object of 85,800 high-quality cells and 34,534 genes. PCA, UMAP, and clustering (resolution = 0.4) were recomputed, identifying 14 clusters. CAR-positive cells were identified based on expression of the CAR transgene and used for all downstream analyses, others were considered CAR-negative. CD8-positive cells were annotated based on detectable expression of CD8A, CD8B, or CD8B.

[0363] Analyses were performed in R v4.4.3 using Seurat v5.2.1, with dot plots generated via Seurat’s functions and refined using ggplot2.

[0364] For the pseudo-bulk heatmap, scaled average expression values from the DotPlot object were extracted for the six CAR-positive samples. The heatmap was generated using the pheatmap R package, with custom visualization via an in-house wrapper.

[0365] CAR-positive cells were identified in silico based on expression of the CAR transgene and used for all downstream analyses. Marker gene expression was assessed for T cell subsets of interest, including genes associated with naive and stem cell memory phenotypes (MKI67, IL2RB, CXCR3, CD28, CD27, FAS, LEF1, SELL, CCR7, IL7R, TCF7) and effector T cell states (TOX, HAVCR2, LAG3, TIGIT, EOMES, TBX21, KLRD1, GNLY, GZMA, GZMB, GZMH, GZMK). Gene expression was visualized using Dot plots, which report both average scaled expression (z- score) and the proportion of cells expressing each marker. To assess relative expression levels across samples, expression values were z-scored at the sample level and visualized by heatmap.

[0366] EXAMPLE 1

[0367] Developing a short and effective method for the preparation of “Naive-like” CART cells

[0368] As illustrated by Figure 1, upon antigen stimulation, Naive / stem-cell like memory T-cells (TNaive / SCM) differentiate into cytotoxic effector (TEM) and memory (TCM) T-cells, mediating tumor killing, before becoming functionally exhausted (TE or TEMRA). Traditional manufacture of CART cells depletes essential T-cell subsets from the CART final product, namely the naive and stem-cell like T-cells (TNaive / SCM), while enriching effector T-cells (TEMand TEMRA). This results in early relapse and thus reduced clinical outcome. T-cell sternness is associated with durable tumor control, whereas fully differentiated and senescent short-lived T-cells are associated with an increased risk of relapse.

[0369] This is even more critical in patients, where T-cells are often of poorer quality, due to aging, but also to the lympho-toxic regimen patients have endured.

[0370] Figure 2A-2B demonstrate the impact of disease presentation on T-cell differentiation status. Specifically, as shown in Figure 2A, peripheral blood mononuclear cells (PBMCs) derived from multiple myeloma (MM) and light chain amyloidosis (AL) patients, display an increased proportion of TEMand TEMRA cells as compared with cells obtained from healthy donors (HDs). Moreover, as shown by Figure 2B, T-cells from MM patients display increased proportions of senescent-like cells (defined as CD27-CD28-, gated on CD3+ cells).

[0371] Figure 3 illustrates a CART -cell traditional manufacturing platform. Conventional CART cells are generated through a complex and time-consuming (8-14 days) process. TM production is typically 8-10 days with the option to extend the production (e.g., for expanding the cells) up to 14 days. This is to ensure sufficient amount of CAR+ cells at the end of production, if not enough cells were generated by day 10.

[0372] Major steps related to this process include: Leukapheresis collection, PBMCs purification, T-cell activation with anti-CD3 (OKT3) and IL-2, T-cell retroviral transduction, CART cell expansion and CART cell harvest and formulation. This process requires the use of various vessels (e.g., flasks, plates, G-Rex), and multiple centrifugation steps. More importantly, at the end of this process, CART cells display mainly a T-cell effector phenotype. TNaive / SCMcells have the capacity to self-renew, they exhibit a high proliferative capacity, while they can also differentiate into T-cells with potent effector functions upon antigen stimulation. Preserving a pool of TNaive / SCMin the CART product, is essential to provide sufficient amount and highly performant CART-cells with long-lasting durability in vivo, even upon recursive stimulation with tumor cells. Such cells may even play a role in the case of relapse prevention. The inventors therefore developed a novel manufacturing platform for the generation of CART cells with stem- cell like features.

[0373] An effective CART-cell “naive” manufacturing platform was developed as illustrated in Figure 4. More specifically, “Naive-like” CART cells were generated through a streamlined process involving one single-vessel within 6-days. Naive-like production typically lasts 6 days. However, in cases where the cell number is low, the option to extend the process up to 8 days, is used. Adding up to two days for the cell expansion step (d), ensures sufficient amount of CAR+ cells at the end of production, if not enough cells were generated by day 6.1n this extended time interval of one or two days, the cell population maintain all characterizing feature, specifically, the CD4+ / CD8+ ratio, and the genetic signature discussed in Example 9.

[0374] Major steps of the method include: (1) Leukapheresis collection (with the option of cryopreserved leukapheresis); (2) CD62L+ cells purification (CD62L microbeads, Miltenyi) (with the option of cryopreserved CD62L+ PBMCs) ; (3) T-cell activation with TransAct beads (Miltenyi) and IL- 7 / 15 / 21 cytokines cocktail (Premium grade, Miltenyi); (4) T-cell retroviral transduction in the presence of Vectofusin-1 as enhancer; (5) CART cell short expansion in AIM-V 1% Human Serum and IL-7 / 15 / 21 cytokines cocktail (3 days); and (6-7) CART cell harvest and formulation (CryoStor-5). Figure 5A, 5B schematically illustrates the difference between the traditional and the naive procedures of the present disclosure, that not only shortens the traditional procedure, but also simplify the preparation as it is performed in a single container, as opposed to the use of various containers that require transferring the cells, thereby subjecting the cells to contamination and cell loss.

[0375] As indicated in Figures 4 and 5, an initial step in the platform disclosed herein is the enrichment of naive / stem-cell like memory and central memory T-cell. Figure 6 demonstrates the rational for enriching these cells, especially when the source material is in lymphocytes obtained from patients, as required when autologous preparation is used.

[0376] The figure shows the T-cell differentiation profile of leukapheresis product from a healthy donor (Fig. 6A) or multiple myeloma patient (Fig. 6B). T-cell sorting using CD45RA as selection marker would result in enrichment of TNaive / SCMbut of TEMRA cells as well. In the case of multiple myeloma patients in which the proportion of TNaive / SCMis often markedly reduced as a consequence of the lymphotoxic regimens endured by the patients, this strategy for T-cell sorting would result mainly in the enrichment of terminally differentiated T-cells. In contrast, as shown by the figure, T-cell selection using the CD62L marker, results in enrichment of both the naive / stem-cell like memory and central memory T-cell compartments. Both compartments showed increased proliferative capacity and long-term persistence when used for adoptive cell therapy in comparison with the effector T-cells.

[0377] Indeed, as demonstrated by Figure 7A, 7B and 7C, CD62L+ selection by magnetic sorting significantly increases the CD4 / CD8 ratio in the starting material (Fig. 7A), increases the proportions of naive and early memory T-cell (TNaive / SCM+ TCM) (Fig. 7B), and significantly decreases the proportion of senescent-like (CD27-CD28-) T-cells following CD62L+ enrichment (Fig. 7C).

[0378] EXAMPLE 2

[0379] Traditional versus “naive” manufacturing platform

[0380] Encouraged by the high-quality product, the inventors next compared the cell population resulting from the traditional with the newly developed “naive” procedure. As shown by Figure 8A, enrichment by CD62L+ magnetic sorting significantly increases the proportion of “naive-like” T- cells in the starting material. Moreover, as shown by Figure 8B, the proportion of “naive-like” CART-cells in the final product is significantly increased when using the “naive” platform in comparison with the traditional manufacture platform. A further characterization of the resulting enriched cells is presented by Figure 9. More specifically, the differentiation status of CD3+ T cells at leukapheresis collection (MM patient) before (Fig. 9A) and after (Fig. 9B) CD62L magnetic sorting. CD62L enrichment increases the proportion of CD4+ cells in the starting material, the proportion of CD27+CD28+ and the proportion of TNaive / SCMcells (CD45RA+CCR7+ or alternatively CD45RA+CD62L+).

[0381] Figure 10 presents the characterization of HBI0101 CAR-T final product (FP) of the naive platform of the present disclosure. More specifically, the differentiation status of CART cells from MM patient at Day 6 (Fig. 10A: “naive” platform) or Day 8 (Fig. 10B: TM platform) was next examined. As shown by Figure 10A, the final product generated via the “naive” platform mostly consists of CD4+ cells and displays an early memory phenotype (TNaive / SCM: CD45RA+CCR7+ / CD62L+ and TCM: CD45RA-CCR7+ / CD62L+) with a high percentage of CD27+CD28+ CART cells. In contrast, the final product generated via the TM platform presented in Figure 10B, shows similar proportions of CD4+ and CD8+, and is mainly consisting of effector cells (TEM: CD45RA-CCR7- / CD62L-). It should be noted that similar percentages of transduced cells (CAR+) are observed in both platforms.

[0382] Figure 11 demonstrates cellular composition of the CART final product using “Traditional” versus “naive” manufacturing platform. As clearly shown by the figure, both manufacturing platforms generate a CAR product mostly constituted of CD3+ cells. The feature that significantly differs between the two processes is the CD4 / CD8 ratio which is reversed: increased proportion of CD4 in the “naive” process; increase CD8 in the “traditional” process.

[0383] EXAMPLE 3

[0384] Naive-like CART cells are functionally superior to CART cells prepared by the traditional procedure

[0385] The inventors next compared the functionality of the Naive-like CART cells in myeloma eradication. More specifically, as shown by Figure 12, CART cells were generated via the traditional (TM; dotted line, squares) or “naive” (black line, circles) manufacturing platforms from mononuclear cells derived from a myeloma patient. The killing potency of these cells was assessed in co-culture experiment with myeloma cell line (NCI-H929) expressing the BCMA molecule, in different effector (CART) to target (Myeloma) cell ratios. As shown by the figure, CAR T cells prepared by the "naive" procedure of the present disclosure showed a clear functional advantage over CART cells prepared using the traditional procedures, in target cells eradication, even in a very low effector to target cell ratio. Interestingly, the advantageous effect was clear in the lower ratios of 1:32 and 1:64. EXAMPLE 4

[0386] Naive-like CART cells are functionally superior to CART cells prepared by the traditional procedure under "stress" conditions

[0387] Next, the inventors assessed the proliferative capacity and the functionality of naive-like CART cells upon recursive tumoral exposure. This “stress test” was aimed at recapitulating chronic stimulation by cancer cells, which is suspected to lead CART cells to exhaustion, and consequently to relapse. Specifically, the inventors evaluated the ex vivo expansion of the CART cells following several rounds of stimulation, as well as their capacity to kill myeloma cells over time. The T-cell maturation and exhaustion profiles were also assessed over the duration of the assay. Furthermore, the inventors sought to elucidate the mechanism by which naive-like CAR T cells kill myeloma cells.

[0388] For this aim, CART cells were generated from three independent patients (BCMA#120 (MM), BCMA#112 (MM) and BCMA#119 (AL)) and one healthy volunteer (HD, PHE-011) according to the traditional (TM) or novel “Naive” manufacturing processes. It is noteworthy that CART cells generated from BCMA#120 and BCMA#112 were used in the in-vivo studies described below (see EXAMPLE 11 and EXAMPLE 12, respectively), and that the CART cells of BCMA#112 were generated from frozen leukapheresis.

[0389] In this "stress test", CART cells (Naive-like and TM) were thawed and co-cultured with myeloma cell lines at different effector to target (E:T) ratio. After three to four days of co-culture, CART cells were counted and co-cultured with fresh myeloma cells at the specified E:T ratios. Following each stimulation round, cells were counted and assessed by flow cytometry for CAR-T and CD 138 plasma cell markers.

[0390] Naive-like CART cells derived from MM or HD leukapheresis exhibited a significantly greater proliferative capacity upon chronic exposure to tumor cells compared to traditional CART cells (Figures 13A-13B and 14A-14B). The reduced capacity of TM CART cells to retain their proliferating capacity over time upon antigen rechallenge results in tumor relapses as depicted by the increased percentage of tumor cells accumulating over the repetitive stimulation cycles (Figures 13C and 14C).

[0391] While CART cells generated from a healthy volunteer (HD TM) showed increased proliferative capacity in comparison with TM CART cells from myeloma patients in the first re-stimulation rounds, their proliferation started progressively to decline from the fourth stimulation (rechallenge- 4; RC-4) (H929 1:4) and sixth stimulation (RC-6) (MM1S 1:4) (Figures 15B and 15C, respectively). In contrast, naive-like CART cells from MM patients exhibited a proliferative ability comparable to that of naive-like CART cells derived from a HD, and this ability was sustained all along the rechallenge experiment. The NAIVE manufacturing platform has restored T -cell abilities that were deteriorating in MM patients. This observation was evidenced only when CART cells were co-cultured at a 1:4 E:T ratio and seems to appear only at a later point at a 1:1 E:T ratio (Figure 15A).

[0392] EXAMPLE 5

[0393] Dynamics of CD47CD8+composition in Naive CART ra. TM CART cells subjected to "stress" conditions and correlation with tumor burden

[0394] To investigate the phenotypic dynamics of CAR-T cells under chronic antigen exposure, naive- like and TM CAR-T cells were characterized following the in vitro "stress" conditions described in EXAMPLE 4. Increased tumor load was accompanied by a decrease in CD3+CAR+ T-cells (Figures 16A-16B and 17B-17B), and more specifically in the CD4+ T-cell subset (Figures 16C and 17C). This was more pronounced for co-culture with H929 at the E:T ratio 1:4 (Figure 16, right panels). It is noteworthy that naive-like CART cells which composition was significantly biased toward a CD4+ phenotype at the co-culture initiation, showed an increase in their CD8+ T- cell compartment at advanced time-points of the rechallenge experiment, indicating that even small proportions of these cells in the CART product is capable of persisting over antigen chronic stimulation.

[0395] The inventors also examined the relationship between the percentage of CD4+ cells (out of CART cells) and the tumor load (% of initially loaded tumor cells) in the co-culture of TM CART cells with H929 (Figure 18A-18B) or MM1S (Figure 18C) myeloma cells. Pearson’s correlation analysis showed a moderate negative correlation between the two variables in the co-culture with MM1S cells (Figure 18C) (Pearson I--0.6806. p=0.00l ). indicating that as the percentage of CD4+ cells decreases in the co-culture, tumor load tends to increase.

[0396] EXAMPLE 6

[0397] Differentiation profile of Naive CART ra. TM CART cells subjected to "stress" conditions

[0398] The inventors then evaluated the differentiation profile of the Naive and TM CART cells along repetitive exposure to BCMA-expressing tumor cells. Figure 19 show the expansion of the early- memory CART cell subsets (TNaive / SCMand TCM) upon stimulation with cancer cells, and their progression towards a more effector phenotype (TEMand TEMRA). In the case of the CART cells generated via the Naive platform (Fig. 19A), these early-memory subsets persist all along the rechallenge rounds, they almost completely vanish in the case of TM CART cells (Fig. 19B) (see absolute numbers in the right panels of Fig. 19A and 19B). This observation coincides with the loss-of-function of TM CART cells, which proliferation and functionality were deteriorating along with the antigen re-stimulations. This figure also provides evidence as to the capacity of the early- memory T-cell subsets to differentiate into effector cells when encountering their cognate antigen.

[0399] EXAMPLE 7

[0400] Exhaustion profile of Naive CART ra. TM CART cells subjected to "stress" conditions

[0401] The inventors next analyzed the exhaustion profile of Naive CART cells following repetitive exposure to cancer cells, compared to TM CART. Expression of PD-1, TIM-3, LAG-3, TIGIT and CTLA-4 were assessed weekly along tumor rechallenge rounds. Figure 20A shows the percent of CAR+ cells expressing the aforementioned activation / exhaustion markers. At the initiation of the co-culture, Naive CART cells significantly displayed higher percentages of TIM-3+ and CTLA- 4+ CAR+ cells. This probably reflects the activation status of the T-cells before transduction (TransAct vs. 0KT3), which is still maintained at cell harvest at D+6 for Naive CART cells, but which is attenuated in the case of TM CART which were harvested at >D+8. In the first week of repetitive stimulation with myeloma cells (two rounds of re-stimulation), CART cells are highly activated, as indicated by the high percentages of LAG-3+ and CTLA-4+ CART cells. However, while this status is reversible in the case of Naive CART cells, it persists towards an even more pronounced exhaustion phenotype in the case of TM CART, with half of the CART cells expressing TIM-3 and / or LAG-3 and / or TIGIT by RC-6. Figure 20B indicates that TM CART cells are more prone to exhaustion along this “stress test” than Naive CART cells, since the proportion of TM CART cells (co-) expressing at least one exhaustion marker is significantly higher than those of Naive CART cells. Moreover, TM CART cells accumulate much more exhaustion markers than their Naive counterparts.

[0402] Figure 21A displays the activation profile of Naive and TM CART cells after an overnight co- culture with NCI-H929. CD25 expression in Naive CART cells was elevated even in unstimulated CART, or upon stimulation with BCMA-negative K562 cells. Exposure to BCMA-expressing myeloma cells, did not affect the mean fluorescence intensity of CD25, which seems to be already maximal. However, despite CD25 high expression, Naive CART cells remained reactive only to BCMA-expressing H929 cells, as indicated by the percent of CD25+CD69+ or CD25+4-1BB+ CART cells.

[0403] In parallel, Figure 23B indicates that IFN-y secretion following CART cell overnight co-culture with myeloma cell line is significantly lower in the Naive versus TM CART cells. Considering the CD4+-skewed CART product generated by the Naive platform (Figure 12), combined to the early- memory phenotype of these cells (Figure 9B, Figure 11 A), it may be not surprising that the Naive CART cells produce less IFN-y than their TM counterparts following one single stimulation. Further stimulations with tumor cells are required to drive the Naive CART cells into a more effector status.

[0404] Together, Figures 20 and 21 indicates that the high activation status of the Naive CART cells at cell harvest (CD25high) does not impede neither with their antitumoral function nor with their long-term persistence upon recursive culture with cancer cells, and that a longer / stronger stimulation with the tumor cells is required for them to acquire an effector phenotype, while preserving a more pronounced efficacy over time.

[0405] EXAMPLE 8

[0406] Cytokine secretion and degranulation of Naive CART ra. TM CART cells subjected to "stress" conditions

[0407] Next, the inventors evaluated the cytokine secretion and degranulation capacity of the remaining CART cells following four sequential stimulations (RC-4) with fresh myeloma cells, which were cryopreserved for subsequent analysis.

[0408] The RC-4 CART cells were subjected to a 5-hrs incubation either alone (unstimulated), or with K562 (negative control), or PM A ionomycin (positive control) or with fresh MM IS myeloma cells (E:T ratio: 1:1). Staining of intracellular IFN-y, TNFa, IL-2, GZM and mobilized CD107a was performed after cell fixation and permeabilization, and the percent of cytokine-expressing CAR+ cells was assessed by flow cytometry.

[0409] Both Naive and TM CART cells secreted IFN-y, TNF-a, and IL-2 in response to repeated antigen- specific stimulation. However, a significantly higher percentage of TNF-a and IL-2-positive cells was observed in the Naive group compared to the TM group. No significant difference was detected in the frequency of IFN-y -positive cells between the two groups. Although not statistically significant, the proportion of CART cells co-expressing all three cytokines was higher in the Naive group (Figure 22A).

[0410] Figure 22B shows that both Naive and TM CART cells contained intracellular granzyme B and were capable of degranulation upon specific stimulation. Notably, the absolute number of TM CART cells recovered after the fourth tumor cell stimulation was lower than that of the Naive CART cells. This suggests that the superior persistence and enhanced pro-inflammatory polyfunctionality of Naive CART cells may confer a stronger antitumor effect in vitro. Furthermore, Naive CART cells exhibited significantly lower expression of exhaustion markers PD-1 and TIM-3 compared to their TM counterparts, indicating a less exhausted phenotype (Figure 23). The prominent CD4+ phenotype of the Naive CART cells does not impede with their cytotoxic function, which seems rather enhanced in comparison to TM CART cells.

[0411] Figure 24 shows CD4 and CD8 CART cell subsets following four sequential stimulations with fresh myeloma.

[0412] In conclusion:

[0413] (i) Naive CART cells display an early-memory phenotype which confers them with a significantly higher proliferative capacity than corresponding TM CART cells;

[0414] (ii) Naive CART cells have the potential to differentiate into effector cells, and eradicate myeloma cells, rechallenge after rechallenge, without becoming exhausted (at least during the 35 days the experiment was running);

[0415] (iii) While Naive CART cells are mainly CD4+, they are effective in tumor eradication. They mostly produce TNFa and IL-2 and granzymes, with the potential of degranulating upon specific stimulation;

[0416] (iv) Throughout the RC rounds, a CD8+ subset reappeared in the co-culture, suggesting either that subset was always present but CD8 expression was down-regulated, or that their number were too low to allow detection by FACS, but at a certain point in the experiment, they start to proliferate and increase their number;

[0417] (v) Increased proportion of CD8+ cells was negatively correlated with tumor relapse in TM CAR T cultures. At D+35 the experiment was resumed, and yet Naive CART cells were functional at eradicating tumor cells;

[0418] (vi) Overall, the data presented in this study is highly consistent with the inventors' observations in vivo, where Naive CART cells demonstrate antitumoral superiority over TM CART cells (see EXAMPLES 10-12);

[0419] (vii) Most importantly, CAR T cells evaluated in this study were generated from one HD, two MM and one AL patients. Thus, the large umbrella of BCMA-related pathologies was covered to assess the benefit of this new platform on the potential clinical outcome;

[0420] (viii) Finally, this data indicates that, despite the broad spectrum in the starting materials from which Naive CART cells were derived, Naive CART products exhibit similar improved features. EXAMPLE 9

[0421] Molecular distinctions between traditional (TM) and Naive CART products

[0422] To define the molecular distinctions between the traditional CART (TM) and CD62L-enriched Naive CART products, single-cell RNA sequencing (scRNAseq) was performed using the 10x Genomics platform as detailed in the Experimental Procedure.

[0423] A total of six CAR-T cell samples were analyzed, including two derived from multiple myeloma (MM) patients (BCMA#137 and BCMA#120) and one from a healthy donor (PHE-011), with both naive-like and traditional memory (TM) CAR-T products represented. It is noteworthy that CART cells generated from BCMA#137 and BCMA#120 were used in the in-vivo studies described below (see EXAMPLES 10 and 11).

[0424] Transcriptomic analysis focused on CAR-positive cells revealed that genes associated with naive and stem cell memory T cell phenotypes (including MKI67, IL2RB, CD28, CD27, LEF1, SELL, CCR7, TCF7) exhibited higher average expression levels and a greater proportion of expressing cells in the Naive CART product compared to the TM product. Exceptions to this were CXCR3, FAS, and IL7R, which did not follow the same pattern (Figure 25 and Table 3).

[0425] Conversely, effector-associated genes (TOX, HAVCR2, LAG3, TIGIT, EOMES, TBX21, KLRD1, GNLY, GZMA / B / H / K) were more highly expressed in TM-derived CART cells, both in terms of expression intensity and the fraction of expressing cells (Figure 26 and Table 3).

[0426] Notably, even within the CAR-positive CD8+ subset (Figure 27), SELL, CCR7, LEF1, and TCF7 expression remained elevated in Naive CART cells, underscoring the distinct transcriptional program preserved in this product. To assess expression strength within each sample, the inventors calculated scaled gene expression (z-score) values, which are summarized in the heatmap (Figure 28).

[0427] Table 3. Percentage of cells expressing the selected markers and average expression per condition

[0428] (Naive and TM)

[0429] *Values were retrieved from the dot plots depicted in Figures 25 and 26.

[0430] These findings confirm that the Naive CART cell manufacturing platform maintains early memory transcriptional features while limiting effector differentiation, distinguishing it from conventional (TM) CART cell products at the molecular level. EXAMPLE 10

[0431] Naive-like CART cells (from a MM patient (BCMA#137)) are functionally superior to CART cells prepared by the traditional procedure in in vivo study

[0432] A preclinical evaluation of CART cell generated via the “naive” manufacturing platform was conducted in a NOD SCID gamma (NSG) xenograft model in vivo. This experiment compared side- by-side CART cell products generated via the traditional manufacture (TM) platform, with CART cell products generated via the “naive platform, at suboptimal doses. Under such “stress” conditions, when CART cells are outnumbered by the tumor cells, the inventors can assess the superiority of the “naive” CART cells over TM CART cells. Such experiments might also provide an estimation of the dosage of “naive” CART cells for future clinical investigation in a first-in-human trial.

[0433] The scope of this experiment was to compare the antitumoral efficacy of HBI0101 CART cells generated via the traditional manufacture (TM) or the “naive” platforms. The inventors referred to the disseminated MMls human multiple myeloma xenograft model which resembles more the human myeloma [PMID: 32123307]. In this experiment, tumor cells were injected in the mouse vein tail. This way, myeloma cells engraft in the bone marrow, as described for human myeloma.

[0434] This model was used to assess the advantage of the Naive CART cells over conventional CART resides, not only in their “sternness” (i.e., their high proliferative capacity and their potency to differentiate into effector and memory T-cell subtypes), but also on their trafficking potency, that the CD62L (L-selectin) or CCR7 receptors confer to these cells their ability to traffic through the secondary lymphoid organs (i.e., lymph nodes, spleen, bone marrow). This represents a huge advantage in the context of hematologic malignancies (i.e., lymphoma, leukemia, myeloma) [PMID: 37904019]. While the inventors did not look for a therapeutic effect in the treated mice, they anticipated that in the case Naive CART have any additional therapeutic potential over the TM CART cells, they will be able to unravel it in this model and in the case of effective therapeutic outcome, they might even be able to get an insight into the recommended CAR+ dosage these cells should be delivered in the clinic. To strengthen the differences between the CART cells generated via the TM or naive platforms, CART cells from a multiple myeloma (MM) patient (BCMA#137) was used (as per amended Helsinki’s protocol 0090-20-HMO). Disseminated myeloma xenografts were induced in compliance with the ethical approval HU-24-17759-5.In these experiments, disseminated human xenograft myeloma was induced by injecting NSG mice with lx10˄6 NCI- MMls-luc cells i.v. in the vein tail. Two weeks following tumor inoculation, CART cells generated from a multiple myeloma (MM) patient via the “naive” or the traditional manufacture (TM) platforms were infused i.v. (vein tail) at escalating doses ranging from 0.1- to lx10˄6 CAR+ cells. Non-transduced (NonT) T control cells were adjusted to the highest dose of CART total cells as per each platform. Mice were monitored twice weekly by IVIS camera for the bioluminescence intensity. Blood was analyzed by flow cytometry and qRT-PCR for CART detection at the indicated time-points. The experiment was terminated at around D+92 as per ethical protocol.

[0435] MM1S XENOGRAFT DISSEMIATED MODEL

[0436] Cells were washed twice and the pellet was suspended in 5mL PBSxl to bring the cells to the concentration of 5x10˄6 MMlS / mL. 24 NSG mice (11 females; 13 males) were injected in their vein tail with 0.2mL (lx10˄6 MMlS-LUC / mouse) of this cell suspension.

[0437] ASSESSMENT OF MYELOMA ENGRAFTMENT

[0438] At day 13 post MMls inoculation (D+13), tumor engraftment was visualized by In Vivo Imaging System (IVIS) upon luciferin i.p. injection (25mg / mL, O.lmL / mouse), and 7 minutes waiting time before mouse anesthesia by isoflurane inhalation, and imaging.

[0439] Mice were randomized based on their bioluminescence, and on their gender, so the groups will be as uniform as possible (Table 4).Blood from 3 females and 3 males were collected for D-l assessment (DNA, TCD (T Cell differentiation) and plasma).

[0440] Table 4: Averaged BLI per treatment group at D-l before CART cell infusion

[0441] CART CELL INFUSION

[0442] Two weeks following tumor inoculation, cryopreserved naive and TM CART and NonT cells derived from BCMA#137 batch were thawed in warm T-Cell Medium (5% Human Serum+1% GlutaMax).

[0443] Following 10 mins incubation at RT, cells were centrifuged at 180g for 10 mins. Then, cells were suspended in Saline+2.5% Human Serum Albumin (HSA), counted using NC- 200 and analyzed for %Td (percent transduction using the TCD (T Cell Differentiation panel by flow cytometry.

[0444] Cells were incubated at 37°C in TCM+5% HS until % of transduction was determined.

[0445] Table 5 shows QC release testing of Naive CART BCMA#137 batch.

[0446] In addition, Naive and TM CART final products were sent to scRNA seq analysis to deepen the understanding of the differences between the two products at the molecular level (see EXAMPLE 9).

[0447] Figure 29A, 29B, shows characterization of MM patient’s CART final products to assess the percent of transduced cells, in both, the naive (29 A), and traditional (29B) processes.

[0448] Table 5: QC release testing of Naive CART BCMA#137 batch

[0449] NSG mice were i.v. infused with 0.2mL of cell suspension in their vein tail and monitored for weight and bioluminescence twice weekly.

[0450] In addition, mice were bled once a week via the vein tail. Blood was processed for DNA (CART cell persistence in blood), T-cell differentiation status (flow cytometry). Remaining blood was processed into plasma and kept frozen at -20°C.

[0451] The experiment was conducted over 92 days post CART infusion.

[0452] Figure 30 shows the long-term anti-myeloma efficacy of the Naive CART cells over the TM CART cells and shed some light as to the dosage that should be used in the clinical setting. A significantly longer survival was achieved in NSG mice treated with Naive CART low dose (Naive-0.1) in comparison with the highest dose of TM CART cells (TM-1) (median survival TM- 1 vs. Naive 0.1: 35 days post CART vs. 59 days, respectively; p=0.0407 (Analysis by Gehan- Breslow-Wilcoxon test)

[0453] Figures 30-32 further suggest that at least a 10-fold reduction in Naive CART cell dosage in comparison with the TM CART dosage should be sufficient to achieve a durable remission.

[0454] CART cells were enumerated in the peripheral blood of treated mice in various timepoints throughout the experiment by either flow cytometry (Figure 33A) or qPCR (Figure 33B).

[0455] Both detection methods showed comparable results. TM and Naive CART cells infused with lx10˄6 / dose peak at D+8, Naive CART infused at 0.1x10˄6 / dose peak at D+15 and TM CART infused at 0.1x10˄6 / dose peak at D+22 (Figure 33). There were additional expansion peaks for the Naive- 1 group (Figure 33).

[0456] Maximal CART cell concentration (Cmax) and total CART expansion (AUC) were determined for each treatment group for the first peak following CART cell infusion, and statistical analysis using 2-way ANOVA was performed (Figure 34).

[0457] It is unclear why a dose-dependency could not be observed. It is possible that Cmax for the high dose groups was missed since the mice were bled only once weekly. It is also possible that CART cells in the high dose Naive group still express the CD62L and / or CCR7 markers and therefore are located in the lymphoid organs rather than in the peripheral blood. Analysis of T-cell immunophenotyping at Cmax in the peripheral blood of the evaluable mice groups (TM-1 and Naive-1), shows significant increased proportions of CAR+CD4+ subset in the Naive group in comparison with the TM group, the later showing dominantly CAR+ of the CD8+ subset (Figure 35).

[0458] Analysis of the T-cell differentiation profile at Cmax reveals a higher proportion of early memory T-cell subsets (TCM) in the Naive treatment groups, in contrast to TM CART cells displaying an effector memory (TEM) phenotype (Figure 36), and increased proportion of senescent-like (CD27- CD28-) T-cells (Figure 37).

[0459] More specifically, at termination, survivor mice (three in the Naive- 1 group, one in the TM-1 group) were sacrificed, and their organs (including heart, lungs, liver, kidney and spleen) were kept in formalin. Blood samples were analyzed for T-cell differentiation panel (including CAR+ cell detection and quantification) by flow cytometry (Figure 33A), and CAR+ cells / pg genomic DNA (qRT-PCR) (Figure 33B). In addition, about half of the spleen and both posterior femurs were dissociated on a 10Opm-mesh and analyzed by FACS and qRT-PCR. None of the mice, but mouse #2 (Naive- 1) were positive for CAR+ neither in the blood, nor in the bone marrow or the spleen. CAR+ cells were detected in Mouse#2’s spleen (5,381 CAR+ / pg DNA), in the bone marrow (270 CAR+ / pg / DNA) and in the blood (853 CAR+ / pg DNA). It is noteworthy, that survivor mice, except Mouse#2, emitted light following luciferin injection, indicating the presence of MMls-luc cells. However, no visible tumors were detected at biopsy. The inventors therefore concluded that in these mice, the tumors were contained to the marrow. It is also possible that CAR+ were present at the tumor site, and thus undetectable in the spleen / blood or femurs’ bone marrow.

[0460] This is the first striking demonstration of Naive CART cell superiority over TM CART cells. The use of the disseminated multiple myeloma model, which reproduces more accurately the clinical presentation of the disease in human patients, has probably contributed to the ability to highlight the fundamental differences between the two types of CART cells. Expression of the CD62L (L- selectin) and / or the CCR7 chemokine receptor, which characterize the early memory T-cell subtypes, direct the cells to the secondary lymphoid organs, and in the case of multiple myeloma, to the bone marrow.

[0461] Combined with their high proliferative ability (significantly higher Cmax and AUC), as demonstrated in Figure 34A, 34B, respectively, the Naive CART cells, can be infused ten-fold less (and possibly even lower doses, since the TM-1 group did not show therapeutic effect but in one mouse) than TM CART cells. A striking difference between the CART cells generated via the traditional or Naive platform, is the low proportion of CD8+ T-cells (Figure 29). In the Naive CART final product used in this experiment, there was less than 15% CD8+ T-cells (in comparison with the 44% CD8+ in the TM CART final product) (Figure 29A, 29B). Despite the low proportion of the CD8+ T-cell subset, yet CD8+ T-cells represent 27-68% of the Naive CART cells in the peripheral blood of Naive-1 and Naive 0.1 treated mice, respectively, at peak (Figure 35). Also, since Naive- 1 group displays the longest progression-free survival, it seems that the presence higher proportion of CD4+ at peak in this treatment group correlated with a better outcome.

[0462] This experiment was performed using CART cells generated from a MM patient, which makes the results even more impressive than if CART cells derived from a healthy donor were used. If one could extrapolate from this murine model to human, it seems that Naive CART cells can be administered at a ten-fold less (or maybe even lower) dose than TM CART cells.

[0463] EXAMPLE 11

[0464] Naive-like CAR-T cells derived from a second MM patient (BCMA#120) replicate functional superiority over traditionally prepared CAR-T cells in an in vivo study

[0465] In an additional experiment, the inventors replicated the experimental design described above, this time using CAR-T cells derived from a different multiple myeloma (MM) patient (BCMA#120). To rule out any sex-based effects, this study also included NSG mouse xenograft models of both genders (22 NSG mice: females, n= 11 / males, n=l l ). The experimental setup, using the disseminated MMlS-luc xenograft model, treatment groups, and follow-up schedule, was identical to the procedure described in Example 10, accordingly, similar results to those described in EXAMPLE 10 were obtained.

[0466] EXAMPLE 12

[0467] Naive-like CART cells (from frozen leukapheresis of a third MM patient (BCMA#112) replicate functional superiority over CART cells prepared by the traditional procedure in in vivo study

[0468] In this experiment, the infused CART cells were generated from a third MM patient’s FROZEN leukapheresis (BCM A# 112). The patient unfortunately died and this is the reason it was feasible to process cryopreserved leukapheresis material from an entire bag. Thus, beyond data reproducibility with cells derived from a third MM patient, an additional aim was to validate the feasibility of Naive CART cell generation from a cryopreserved starting material. This experiment therefore aimed at demonstrating the antitumoral superiority of Naive CART cells over TM CART cells, using cells generated from a cryopreserved source material.

[0469] As in the previous experiments, the inventors referred to the disseminated myeloma xenograft model in NSG mice. In this experiment, solely NSG females (DB 24 / 10 / 2024 - 04 / 11 / 2024; ~15- week-old) were used, since efficacy in both genders was already proven in the previous experiments.

[0470] At the day of tumor inoculation, MMlS-luc were collected and washed in Saline. Cells were brought to the concentration of 5x10˄6 / mL. Mice were i.v. infused with 0.2mL of cell suspension in their vein tail (lx10˄6 MMlS-luc / mouse).

[0471] CART CELL GENERATION AND INFUSION

[0472] Following Ficoll gradient to isolate the PBMCs from the leukapheresis bag, PBMCs were cultivated in AIM-V+1% HS supplemented with IL-7 (10ng / mL), IL- 15 (10ng / mL) and IL-21 (20ng / mL), overnight at 37°C.

[0473] Figure 38A-38C illustrates the downregulation of CD62L on T-cells following freeze / thaw cycle, as indicated by the decrease in the mean fluorescence intensity (MFI), followed by the full recovery of CD62L marker expression upon cultivation overnight in Naive CART media, as evidenced by the increase in the CD62L MFI.

[0474] The day afterwards, recovered PBMCs were labeled with CD62L microbeads (as per manufacturer’s instructions), and positive cells were isolated magnetically. Five million CD62L+ PBMCs were suspended in AIM_V+1% HS+IL7 / 15 / 21 in the presence of TransAct (research grade, 1:100), and transferred into a GREx6 device for two days at 37°C (Day 0). At D+2, cells were transduced in the GREx device by addition of the retroviral supernatant (1 / 12) in the presence of the transduction enhancer Vectofusin-1. From D+3 until D+6 (cell harvest) cells were expanded by toping up fresh media supplemented with fresh cytokines above the cells.

[0475] At D+6, cells were harvested, washed with saline 1% HSA, and cryopreserved in CS-5 at the concentration of 10x10˄6 CAR+ / mL CS-5. Cells were stored at -80°C until infusion few days afterwards. Frozen aliquots of the FP were thawed to assess conformance to release criteria. Table 6 below summarizes the batch analysis of BCMA#112 Naive CART final product (FP).

[0476] TM CART cells were generated as per clinical CART product protocol. Due to low lactate concentration at D+8, cells were harvested at D+10, and processed to cryopreservation at -80°C until mice infusion. Table 6. QC release testing of Naive CART BCMA#112 batch CART doses corresponding to lx10˄6 CAR+ or 0.1x10˄6 CAR+ respectively, were formulated in saline+2.5% HSA according to the percent of transduction post thaw. NonT naive and TM cells were adjusted to the number of total cells in the highest doses.

[0477] At D-l before CART infusion, mice xenografts were assessed for myeloma engraftment, using the luciferase / luciferin reporter system. At D+14, two weeks following tumor inoculation, females were infused in their vein tail with 0.2mL of cell suspension (5x10˄6 CAR+ / mL for the lx10˄6 CAR+ groups; 0.5x10˄6 CAR+ / mL for the 0.1x10˄6 CAR+ groups). Mice group consisted of five females, randomized at the day of CART infusion. Females were weighted twice weekly, and evaluated for their motricity, posture, welfare. Tumor progression / regression was monitored using IVIS camera upon i.p. injection of luciferin (25mg / mL, 10OpL / mouse). Exclusion criteria are based on the bioluminescence intensity (10˄9- 10˄10) and the mice well-being, with sacrifice of the subjects showing a decrease of 20% from their initial weight at two consecutive evaluations, or limb paralysis, or any kind of distress. Sample mice (total of six mice, from different cages) were bled to assess background levels before CART infusion. All the mice were bled at D+3 following CART infusion, and then once weekly to assess the pharmacokinetic of the CART cells in the peripheral blood, and T-cell immunophenotyping. Plasma samples were kept at -80°C for further cytokine panel evaluation.This experiment was terminated at D+91 post CART cell infusion as per ethical protocol. A clear discrimination between TM and Naive treatment groups is observed, with Naive CART cells displaying superiority over TM CART cells. Figure 39A- 39C shows the anti-myeloma efficacy of the Naive CART cells, even at the low CAR+ dose.Figure 40 shows the CAR+ cell expansion following infusion in the different treatment groups, assessed by flow cytometry. Interestingly, CAR+ in the Naive-0.1 group expanded at a greater extent than in the Naive- 1 group (though the standard error mean is high, and thus this observation statistically insignificant). Such behavior was already observed in the experiment described in Example 10. It is possible that at the high dose, naive CAR+ cells do not have to expand that much to counteract the myeloma and thus keep their low differentiation status (high expression of CD62L / CCR7) and stay in the bone marrow / lymphoid organs. In contrast, at low / suboptimal CART dose, naive CAR+ cells do have to significantly expand to outnumber the myeloma cells. This proliferation drives their differentiation into effector cells and consequently they downregulate their CD62L / CCR7 markers, which release them from the lymphoid organs to the peripheral blood. It is also possible that when facing myeloma several times, naive CAR+ cells will have to expand in a way that resembles their behavior in the Naive-0.1 group. In that case, these cells might be released to the peripheral blood at later time-points, as depicted by the several expansion peaks observed at the different timepoints. The major difference between this experiment (EXAMPLE 12) and the previous ones (EXAMPLES 10 and 11), beyond the source of the CART cells from different MM patients, is the starting material those Naive / TM CART cells. While in EXAMPLES 10 and 11, Naive CART cells were generated from FRESH leukapheresis, directly processed into CD62L+ PBMCs via magnetic sorting, in EXAMPLE 12, Naive CART cells were derived from CRYOPRESERVED leukapheresis derived from a deceased MM patient. Due to proteolytic cleavage, CD62L expression is decreased following freeze / thaw cycle, consequently affecting CD62L+ cell enrichment. Therefore, a prerequisite incubation of the thawed leukapheresis cells in Naive T-cell medium is necessary to allow CD62L re-expression, and further magnetic sorting. Generation of Naive CART cells from cryopreserved leukapheresis starting material will be discussed in a different report.

[0478] In fact, this experiment (EXAMPLE 12) demonstrates the feasibility of generating Naive CART cells out of cryopreserved leukapheresis, and confirms the antitumoral superiority (i.e., higher proliferative capacity, long-term persistence, relapse prevention) of Naive CART cells over TM CART cells with source material derived from a third MM patient.

[0479] EXAMPLE 13

[0480] Clinical trial protocol summary

[0481] A first-in-human, open-label, phase la / lb dose-escalation and expansion trial is initiated to evaluate the safety, feasibility, and preliminary efficacy of a novel anti-BCMA C AR-T cell therapy derived from CD62L-enriched early memory T cells (“naive-like CAR-T”) in patients with relapsed or refractory multiple myeloma (RRMM).

[0482] The study protocol was reviewed and approved by the Institutional Review Board (IRB) in accordance with the Helsinki Declaration, and regulatory authorization has been obtained to proceed with clinical testing.

[0483] Phase la: Dose Escalation

[0484] In the dose-escalation phase, patients receive a single intravenous infusion of naive-like CAR-T cells following standard lymphodepleting chemo therapy. A 3+3 design is used to evaluate ascending CAR+ cell doses across three planned cohorts: In the case of over dosage in DL1 -infused patients, a de-escalation cohort (DL-1) is conducted and the dose is adjusted to a lowest dose, according to a risk / benefit assessment plan, and upon principal investigator’s (PI) discretion,

[0485] Primary endpoints include dose-limiting toxicides (DLTs), manufacturing feasibility, and CAR-T cell expansi on / persistence in peripheral blood.

[0486] Secondary endpoints include preliminary anti-myeloma activity (response rates per IMWG criteria), progression-free and overall survival (PFS). Correlative studies include serial monitoring of CAR-T phenotypes by flow cytometry, cytokine profiling, and bone marrow biopsy to assess CAR-T efficacy, infiltration and persistence.

[0487] Phase lb: Dose Expansion

[0488] Upon identification of a recommended dose, additional patients are enrolled in a dose-expansion cohort to further evaluate safety and efficacy at that dose level.

Claims

CLAIMS:

1. A method for preparing a population of naive-like T cells, wherein said naive-like T cells comprise at least one of: naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM), the method comprising:(a) contacting a population of cells comprising cells of the T lineage with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing said at least one marker;(b) subjecting the enriched cell population obtained in step (a) to T-cell activation;(c) genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule; and(d) expanding the activated genetically modified cell population; thereby preparing a cell population comprising naive-like T cells; wherein the duration of said method is up to 6 days.

2. The method according to claim 1, wherein the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days.

3. The method according to claims 1 or 2, wherein all the steps are performed in the same cell culture vessel.

4. The method according to any one of claims 1-3, further comprising the step of (e) harvesting the expanded cell population obtained in step (d).

5. The method according to claim 4, further comprising the step of (f) formulating the expanded cell population.

6. The method according to any one of claims 1 to 5, wherein the population of cells in step (a) comprises at least one of: peripheral blood cells, a leukapheresis cell product, a cell population derived from infiltrating tissue microenvironment and a primary or secondary lymphoid organ.

7. The method according to claim 6, wherein said population of cells from peripheral blood comprises peripheral blood mononuclear cell(s) (PBMC(s)); wherein said infiltrating tissuemicroenvironment is derived from at least one of inflamed tissues and tumor tissue; and wherein said primary or secondary lymphoid organ is bone marrow or lymph nodes.

8. The method according to any one of claims 1 to 7, wherein said at least one marker for circulating innate lymphoid cell precursor cells is at least one of Cluster of Differentiation 62L (CD62L) and CCR7.

9. The method according to any one of claims 1 to 8, wherein said activation step (b) comprises contacting the cell population with: (i) CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL-15) and Interleukin 21 (IL-21).

10. The method according to any one of claims 1 to 9, wherein said exogeneous nucleic acid molecule encodes at least one receptor molecule or at least one antigen binding domain.

11. The method according to claim 10, wherein said receptor molecule is a chimeric antigen receptor (CAR) molecule and / or a T cell receptor (TCR) or any part and / or derivative thereof.

12. The method according to any one of claims 1 or 11, wherein said genetically modifying step (c) comprises contacting the cells with at least one transduction enhancer.

13. The method according to any one of claims 1 to 12, wherein all method steps are performed in a low serum medium containing 0.5-3% serum.

14. The method according to claim 13, wherein said serum comprises human AB serum.

15. The method according to any one of claims 1 to 14, wherein the population of naive-like T cells prepared by said method is characterized by a CD4+T cell to CD8+T cell ratio that is greater than one.

16. The method according to claims 1 to 15, wherein the population of naive-like T cells prepared by said method displays a decreased proportion of senescent-like (CD27-CD28-) T-cells.

17. The method according to any one of claims 1 to 16, wherein the population of naive-like T cells prepared by said method is characterized by at least one of:(i) a higher average expression level of at least one gene associated with naive and stem cell memory T cell phenotypes in said naive-like T cells population, as compared with an average expression level of said gene in a control population; and(ii) an increased proportion of T cells expressing said at least one gene associated with naive and stem cell memory T cell phenotypes in said naive-like T cells population, as compared with a control population.

18. The method according to claim 17, wherein said at least one gene associated with naive and stem cell memory T cell phenotypes is selected from the group consisting of: marker of proliferation Ki-67 (MKI67), interleukin-2 receptor subunit beta (IL2RB), cluster of differentiation 28 (CD28), cluster of differentiation 27 (CD27), lymphoid enhancer-binding factor 1 (LEF1), L-selectin (SELL), C-C chemokine receptor type 7 (CCR7), and transcription factor 7 (TCF7).

19. The method according to any one of claims 1 to 18, wherein the population of naive-like T cells prepared by said method is characterized by at least one of:(i) a lower average expression level of at least one effector-associated gene in said naive-like T cells population, as compared with an average expression level of said effector-associated gene in a control population; and(ii) a decreased proportion of T cells expressing at least one effector-associated gene, in said naive- like T cells population, as compared with a control population.

20. The method according to claim 19, wherein said at least one effector-associated gene is selected from: thymocyte selection-associated high mobility group box protein (TOX), hepatitis A virus cellular receptor 2 (HAVCR2), lymphocyte activation gene 3 protein (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), eomesodermin (EOMES), T-box transcription factor TBX21 (TBX21), killer cell lectin-like receptor subfamily D member 1 (KLRD1), granulysin (GNLY), granzyme A (GZMA), granzyme B (GZMB), granzyme H (GZMH), and granzyme K (GZMK).

21. The method according to any one of claims 1 to 20, comprising:(a) contacting a population of PBMC(s) with a compound that specifically selects for cells expressing the CD62L marker, thereby obtaining an enriched CD62L+ T cell population;(b) subjecting the enriched CD62L T cell population obtained in step (a), to T-cell activation by contacting the cell population with (i) CD3 and CD28; and (ii) a cytokine cocktail comprising IL-7, IL- 15 and IL-21;(c) genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule encoding at least one CAR molecule, in the presence of at least one transduction enhancer; thereby obtaining a cell population comprising T cells harboring said CAR molecule;(d) expanding the transduced cell population obtained in step (c); and(e) harvesting the expanded cell population obtained in step (d); thereby preparing a population of naive-like T cells; wherein steps (a) to (e) are performed in a single container or compartment, in low serum medium containing 1% or less human AB serum, and wherein the duration of said method is up to 6 days.

22. A method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject, the method comprising the step of administering to said subject an effective amount of a population of naive-like T cells, wherein said naive-like T cells comprise at least one naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM), and wherein said cell population is prepared by a method comprising:(a) contacting a population of cells comprising cells of the T lineage with a compound that specifically selects for at least one marker for circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing said at least one marker;(b) subjecting the enriched cell population obtained in step (a) to T-cell activation;(c) genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule; and(d) expanding the activated cell population; and optionally,(e) harvesting and / or formulating the expanded cell population obtained in step (d); thereby preparing a cell population comprising naive-like T cells; wherein the duration of said preparation method is up to 6 days.

23. The method according to claim 22, wherein said naive-like T cell population is prepared by a method as defined in any one of claims 1 to 21.

24. The method according to any one of claims 22 and 23 wherein said naive-like T cell population is a cell population of autologous or allogeneic source.

25. The method according to any one of claims 22 to 24, wherein said naive-like T cell population or a composition thereof is administered to said subject as part of an adoptive transfer procedure.

26. The method according to any one of claims 22 to 25, wherein said pathologic disorder is at least one of: a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease, a deposition disorder and / or a neurodegenerative disorder.

27. The method according to claim 26, wherein said proliferative disorder is at least one malignant neoplastic disorder.

28. The method according to claim 27, wherein said malignant neoplastic disorder is at least one hematological malignancy, and / or at least one solid tumor.

29. The method according to claim 28, wherein said hematological malignancy is at least one B cell malignancy.

30. The method according to claim 29, wherein said B cell malignancy is multiple myeloma (MM) and any related conditions.

31. The method according to claim 30, wherein said deposition disorder is amyloidosis, and any related conditions.

32. The method according to any one of claims 22 to 31, wherein said cell population comprises naive-like T cell(s) harboring an exogeneous nucleic acid molecule encoding at least one CAR molecule and / or a TCR or any part and / or derivative thereof, and wherein said CAR or TCR comprise at least one target-binding domain specific for at least one target molecule associated with and / or expressed by said at least one pathologic disorder.

33. The method according to any one of claims 22 to 32, wherein the naive-like T cells harbor an exogeneous nucleic acid molecule encoding at least one CAR molecule comprising:(a) at least one target-binding domain; wherein at least one of said target binding domain specifically recognizes and binds B cell maturation antigen (BCMA);(b) at least one hinge and at least one transmembrane domain derived from the Cluster of Differentiation 8 a (CD8α) protein; and(c) at least one intracellular T cell signal transduction domain, said domain comprising at least one domain of tumor necrosis factor (TNF) receptor family member, and optionally, at least one domain of a T cell receptor (TCR) molecule.

34. The method according to any one of claims 22 to 33, wherein said cell population is autologous, the method comprising:(a) contacting a population of PBMC cells of said subject, with a compound that specifically selects for cells expressing the CD62L marker, thereby obtaining an enriched CD62L T cell population;(b) subjecting the enriched CD62L T cell population obtained in step (a) to T-cell activation by contacting the cell population with (i) CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL-15) and Interleukin 21 (IL-21);(c) genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule encoding at least one CAR and / or TCR molecule, in the presence of at least one transduction enhancer; thereby obtaining a cell population comprising T cells harboring said CAR and / or TCR molecule;(d) expanding the transduced or transfected cell population obtained in step (c);(e) harvesting and / or formulating the expanded cell population obtained in step (d), thereby preparing a cell population comprising naive-like T cells characterized by CD4+T cell to CD8+T cell ratio that is greater than one; and(f) administering to said subject an effective amount of the harvested and / or formulated cell population obtained in step (e), or any composition thereof; wherein steps (a) to (e) are performed in the same cell culture vessel, in a low serum medium containing 1 % or less human AB serum, and wherein the duration of said preparation method is up to 6 days.

35. An effective amount of a population of naive-like T cells, or a composition comprising the same, for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onsetof a pathologic disorder in a mammalian subject, wherein the naive-like T cells comprise at least one of naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM), and wherein said naive-like T cell population is prepared by a method comprising:(a) contacting a population of cells comprising cells of the T lineage with a compound that specifically selects for at least one marker for circulating innate lymphoid cell precursor cell(s), thereby enriching for cells expressing said at least one marker;(b) subjecting the enriched cell population obtained in step (a) to T-cell activation;(c) genetically modifying the activated cells obtained in step (b), with at least one exogeneous nucleic acid molecule, thereby obtaining a cell population comprising T cells harboring an exogeneous nucleic acid molecule; and(d) expanding the activated cell population; and optionally,(e) harvesting and / or formulating the expanded cell population obtained in step (d); thereby preparing a cell population comprising naive-like T cells; wherein the duration of said method is up to 6 days.

36. The effective amount of a population of naive-like T cells for use according to claim 35, wherein said cell population is prepared by a method as defined in any one of claims 1 to 21.

37. The effective amount of a population of naive-like T cells for use according to any one of claims 35 to 36, wherein said cell population is autologous or allogeneic.

38. The effective amount of a population of naive-like T cells for use according to any one of claims 35 to 37, wherein said pathologic disorder is at least one of: a proliferative disorder, an inflammatory disorder, an infectious disease caused by a pathogen, an autoimmune-disease, a cardiovascular disease and / or a neurodegenerative disorder.

39. An isolated population of naive-like T cells, said population comprise at least on of naive T cells (TN), stem-cell like memory T cells (TSCM) and central memory T cells (TCM), wherein the T cells are characterized by a CD4+T cell to CD8+T cell ratio that is greater than one.

40. The isolated cell population according to claim 39, wherein said cell population is characterized by at least one of:(i) a higher average expression level of at least one gene associated with naive and stem cell memory T cell phenotypes, in said naive-like T cells population, as compared with the average expression level of said gene in a control population; and(ii) an increased proportion of T cells expressing at least one gene associated with naive and stem cell memory T cell phenotypes, in said naive-like T cells population, as compared with a control population.

41. The isolated cell population according to claim 40, wherein said at least one gene associated with naive and stem cell memory T cell phenotypes is selected from the group consisting of: MKI67, IL2RB, CD28, CD27, LEF1, SELL, CCR7, and TCF7.

42. The isolated cell population according to any one of claims 39 to 41, wherein said cell population is characterized by at least one of:(i) a lower average expression level of at least one effector-associated gene in said naive-like T cells population, relative to the average expression level of said effector-associated gene in a control population; and(ii) a decreased proportion of T cells expressing at least one effector-associated gene, in said naive- like T cells population, as compared with a control population.

43. The isolated cell population according to claim 42, wherein said at least one effector- associated gene is selected from the group consisting of: TOX, HAVCR2, LAG3, TIGIT, EOMES, TBX21, KLRD1, GNLY, GZMA, GZMB, GZMH, and GZMK.

44. The isolated cell population according to any one of claims 39 to 43, prepared by a method as defined in any one of claims 1 to 21.

45. The isolated cell population according to any one of claims 39 to 44, wherein said cell population is autologous.

46. The isolated cell population according to any one of claims 39 to 45, wherein said cell population is formulated in a composition compatible with an adoptive transfer procedure.

47. The isolated cell population according to any one of claims 39 to 46, wherein the cell population comprises T cell(s) harboring an exogeneous nucleic acid molecule encoding at leastone CAR molecule and / or a TCR or any part and / or derivative thereof, and wherein said CAR or TCR comprise at least one target-binding domain specific for at least one target molecule associated with and / or expressed by said at least one pathologic disorder.

48. The isolated cell population according to claim 47, wherein said pathologic disorder is a B cell malignancy, and wherein said malignancy is multiple myeloma (MM) and any related conditions.

49. The isolated cell population according to claim 48, wherein said pathologic disorder is a deposition disorder, and wherein said deposition disorder is amyloidosis, and any related conditions.

50. The isolated cell population according to any one of claims 39 to 49, wherein the T cell(s) harbor an exogeneous nucleic acid molecule encoding at least one CAR molecule comprising:(a) at least one target-binding domain; wherein at least one of said target binding domain specifically recognizes and binds B cell maturation antigen (BCMA);(b) at least one hinge and at least one transmembrane domain derived from the Cluster of Differentiation 8 a (CD8α) protein; and(c) at least one intracellular T cell signal transduction domain, said domain comprising at least one domain of tumor necrosis factor (TNF) receptor family member, and optionally, at least one domain of a T cell receptor (TCR) molecule.

51. A composition comprising the isolated naive-like T cell population according to any one of claims 39 to 50, and at least one of pharmaceutically acceptable carrier / s, diluent / s, excipient / s and additive / s.

52. A kit comprising:(a) a compound that specifically selects for cells expressing the CD62L marker;(b) T-cell activation mixture comprising: (i) a colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28; and (ii) a cytokine cocktail comprising Interleukin 7 (IL-7), Interleukin 15 (IL- 15) and Interleukin 21 (IL-21);(c) at least one exogeneous nucleic acid molecule encoding at least one CAR molecule and at least one transduction enhancer; thereby obtaining a cell population comprising T cells harboring said CAR molecule; and(d) a low serum medium containing 1 % or less human AB serum; wherein the kit is adapted for operation in a single container or compartment for a period of up to 6 days.

53. The kit according to claim 52, for use in the method as defined in any one of claims 1 to 21.

54. A method for preparing a population of naive-like T cells, wherein said naive-like T cells comprise at least one of naive T cells (TN), stem-cell like memory T cells (TSCM), and central memory T cells (TCM), the method comprising:(a) contacting a population of cells comprising cells of the T lineage subsets with a compound that specifically selects for at least one marker of circulating innate lymphoid cell precursor cells, thereby enriching for cells expressing said at least one marker;(b) subjecting the enriched cell population obtained in step (a) to T-cell activation; and(c) expanding the activated cell population; thereby preparing a cell population comprising naive-like T cells; wherein the duration of said method is up to 6 days.

55. The method according to claim 54, wherein the method further comprises the step of expanding the cells obtained in step (d) up to additional 2 days.

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