A method for preparing chimeric antigen receptor (CAR) expressing cells

Abstract

Present invention discloses a new method for manufacturing genetically engineered T cells expressing a chimeric antigen receptor (CAR) that provides several improvements over conventional manufacturing methods, thereby enabling production of a robust supply of clinically useful CAR T-cell therapies with higher percentage of CAR positive cells with improved naive and memory cells in a short timeframe and in a cost-effective manner. Further similar method can be used to manufacture NK-CAR, TIL and other immune effector cells useful for immunotherapy. This new method treats the patient / human body as a bioreactor wherein the CAR-T cells expand on encountering the target antigen in their natural environment thus making them more efficient in terms of persistence and efficacy in neutralizing the tumours.

Description

A METHOD FOR PREPARING CHIMERIC ANTIGEN RECEPTOR (CAR) EXPRESSING CELLSCROSS-REFERENCE TO RELATED APPLICATIONThis application claims priority to Indian Provisional Application 202441019888 filed Mar. 18, 2024, the contents of which are incorporated herein by reference in their entireties.FIELD OF THE INVENTION

[0001] The present invention relates to a method for preparing chimeric antigen receptor (CAR) expressing immune cells with improved percent CAR expression within a shorter time compared to the conventional method with improved survival, expansion, and persistence in vivo and compositions comprising the same. Also disclosed are methods of using such compositions for treating a disease, e.g., cancer, autoimmune conditions, HIV, infectious disease, inflammatory disease, immune-oncology and immunodeficiency in a subject.BACKGROUND OF THE INVENTION

[0002] Chimeric Antigen Receptor T (CAR-T) cell therapy is a form of immune effector therapy wherein the patients / donors T cells are engineered to express a receptor that targets them exclusively to antigens associated with cancer cells. This field has evolved further wherein various immune cells other than the T cells are used to fight cancers. The process to manufacture these CAR-X (X stands for various immune cells like T, NK, Macrophages) ranges from 8-12 days depending on the process itself, and dosage requirements as directed by the clinical team. Once these cells are infused into the patient, they expand on encountering the antigen on the tumour cells thus killing most of the cancerous cells over time. In the earliest treated patients, these CAR-T cells were found in circulation even 10 years after infusion.

[0003] The manufacturing process of chimeric antigen receptor (CAR) T cells remains one of the most challenging steps of this emerging therapy.

[0004] Conventionally, the manufacturing process of CAR-X cells starts with the patient’s or donor’s leukapheresis (aka apheresis) i.e. collecting peripheral blood mononuclear cells from cancer patients or donors and isolating their T cells. The required cells are then activated and subsequently subjected to genetic modification by transduction with viral vector for the CAR gene delivery. Post this, the modified CAR cells are expanded in culture in vitro till the required number of cells are achieved. After this step the cells are subjected to a battery of tests to assess their safety and efficacy before being administered to the patient where they will mediate the elimination of the cancer cells. This entire process of harvest from patient or donor, to back to the patient ranges from 21-28 days which is time-consuming, expensive and often unsuitable for the treatment of high-risk patients.

[0005] It has been observed that in CD19-directed CAR-T cell clinical trials, about 20-30% of candidates were not infused with CAR-T cells due to manufacturing failures and / or disease progression during manufacturing because the target dose with sufficient CAR-T cells was not achieved. The manufacturing time for conventional CAR-T (C-CAR-T) cells is currently 8-14 days, and the vein-to-vein time ranges from 3-4 weeks. The longer manufacturing period and therefore wait time may allow existing malignancies to progress, which can result in worse outcomes and more adverse events. The extensive ex vivo culture needed to produce CD19-directed CAR-T cells may result in a significant loss of T cell sternness, which limits engraftment and persistence following adoptive transfer and decreases therapeutic effectiveness. The ex-vivo cell culture milieu, conditions, and duration, can cause loss of T cell sternness, exhaustion and oxidative stress which can result in CAR-T cells with compromised potency during their manufacturing process.

[0006] In a recent study, Ghassemi et al. (Ghassemi, Saba, et al. "Rapid manufacturing of non-activated potent CAR T cells. " Nature biomedical engineering 6.2 (2022): 118-128) have developed a rapid protocol for CAR T cell manufacturing from non-activated T cells within 24 hours from the time of T cell collection. T cell activation was a pre-requisite for effective transduction however the authors describe a process by overcoming the barriers to transduction present in quiescent T cells. This process is henceforth identified as “UPenn Process” in thisdocument. CAR T cells generated through this rapid expansion protocol exhibited higher antitumor activity in vivo than C-CAR T cells manufactured through the conventional or standard protocol (Ghassemi et al., 2022). However, their process requires lentiviral transduction for 20-24 hrs in the presence of GMP grade dNTPs, which increases the cost of production considerably. Further, the MOI (multiplicity of infection) of lentivirus used for transduction in this method was similar to the MOIs used in conventional CAR-T manufacturing process which increases the cost of production. The Lentivirus cost is around 20-30% of the manufacturing cost depending on the process. Accordingly, cost of manufacturing is directly modulated by the required MOI value.

[0007] Gracell Biotechnologies has developed a FasTCAR platform, which is able to concurrently activate and transduce resting T cells into a single “concurrent activation-transduction” step using XLenti vectors and producing CAR cells in 22- 36 hours, (https: / / www.gracellbio.com / fastcar / ) However, this process involves the steps of PBMC isolation and T-cell activation which further increases the cost and time. The percentage transduction of naive cells and other parameters that improve the CAR-T persistence has been disclosed.

[0008] WO2024081167 discloses novel and efficient methods and lentiviral vectors for manufacturing a population of immune cells engineered to express a Chimeric Antigen Receptor (CAR), an engineered T cell receptor (TCR), and / or a nucleic acid sequence encoding a polypeptide that enhances the immune cell function, or a functional derivative thereof in less than 72 hours; engineered cells generated by the methods, compositions comprising said cells and methods of treating a disease or condition using said cells. WO2024081167 employs electroporation where the electric pulses used can cause significant cell stress and damage, leading to lower cell viability and survival rates. Electroporation relies on creating temporary pores in the cell membrane and not provide stable and long-term gene expression due to viral integration. While electroporation is a rapid method for gene delivery, it has limitations in terms of stability, cell viability, efficiency, and applicability to different cell types.

[0009] There is therefore a need to provide a method for generation of CAR expressing immune cells which is simple, convenient, economical and fast with enhanced percentage CAR, improved sternness and reduced MOI. In summary, enhancing CAR-T cell persistence involves a multifaceted approach, from cell source selection to genetic manipulations. The present inventors have surprisingly developed a method which ameliorates the aforesaid shortcomings of the prior art.OBJECTS OF THE INVENTION

[0010] It is an object of the present invention to overcome the drawbacks of the prior art.

[0011] It is another object of the present invention to provide a method for preparing / manufacturing chimeric antigen receptor (CAR)-expressing cells with improved percentage CAR expression in the shorter duration with improved naive and central memory phenotype.

[0012] It is another object of the present invention to provide a method for manufacturing CAR-expressing cells within a shorter manufacturing time and enhance the tenacity and phenotypic attributes of the CAR T-cell product.

[0013] It is yet another objective to achieve effective transduction while minimizing the MOI without compromising transduction efficacy or increasing manufacturing cost.

[0014] It is another object of the present invention to provide a method for preparing CAR-expressing cells which is simple, convenient, and economical with improved CAR expression.

[0015] It is another object of the present invention to provide a method for preparing CAR-expressing cells in 1-4 days.SUMMARY OF THE INVENTION

[0016] The present disclosure relates to a methods of manufacturing immune effector cells (for example, T cells or NK cells, or macrophages) engineered to express a Chimeric Antigen Receptor (CAR-X), (X stands for various immune effector cells like T, NK, Macrophages, or thereof) and compositions generatedusing such methods. To provide CAR-T cell therapy with improved potency and persistence, as well as methods for making the same in 1-4 days. It further relates to a robust and reproducible method of manufacturing CAR-X (e.g. CAR-T) cells comprising promotional more CAR- expressing naive and central memory cell population within a shorter timeframe.

[0017] This also provides a method which ensures faster access to immunotherapy for patients, particularly those with rapidly advancing diseases conditions, at a reasonable cost. It further aims to minimize donor-to-donor variability inherent to the cellular starting material and to generate a consistent CAR X (e.g. CAR-T) cell product. The CAR-T cell therapy provided by the present invention represents a transformative advancement in cancer treatment, offering personalized, targeted therapy with rapid turnaround times. The manufacturing process for generating CAR-X cells in this invention not only shortens the production time to 4 days but also significantly reduces costs compared to the conventional method of producing CAR T cells.

[0018] In an embodiment, the method for manufacturing chimeric antigen receptor (CAR) T cells (e.g. anti-CD19 CAR, anti-CD20 CAR, anti- CD307e CAR, antiCD 19-CD20 CAR or anti- BCMA-CD307e bispecific CAR and the likes) comprising: (a) enriching a population of T cells (b) activating and culturing the population of T cells; (c) transducing the T cells with a viral vector comprising a polynucleotide encoding a CAR to recognize specific antigen present in the surface of cancer cells; (d) culturing the transduced T cells. The activation step (b) wherein the activation is performed in the presence of an activation agent such as CD3 and / or CD28 receptor using antibodies against them immobilized on polymer beads, or antibodies like CD3 itself or using Lentiviral vectors with pseudo typed envelopes co-expressing the CD3 and / or CD28 activating domains thereof. Further the transducing step (c) are performed in the presence of transduction enhancers that improves the gene transduction; and the cells are cultured in a medium supplemented with inosine and in absence of glucose. The method also involves minimizing the use of MOI without compromising transduction efficacy thuskeeping a check on the manufacturing costs. The method is performed in equal or less than 96 hours, less than 72 hours, less than 48 hours, or less than 24 hours.

[0019] The manufactured T cell compositions are used in the treatment or prevention of numerous conditions including, but not limited to cancer, infectious disease, autoimmune disease, immune-oncology, inflammatory disease, and immunodeficiency.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and other aspects, features, and advantages of certain exemplary embodiments of the present invention will be more apparent from the following description taken in conjunction with the accompanying drawings wherein:

[0022] Figure 1 : Schematic overview illustrating the CAR-T manufacturing process of the present invention and the conventional process (prior art). Both the present and conventional CAR-T processes involve the same quality control (QC) and in-process quality control (IPQC) measures. In the conventional CAR-T process, cells can be further expanded from day 3 onwards. While CAR-T cells produced using the present invention can also be expanded further if required, the focus of the current invention is on generating CAR-T cells comprising proportional more CAR expressing naive and memory cells in a shorter time frame economically.

[0023] Figure 2 (A to C): Shows the CAR percentage for samples from three healthy donors, tested under various conditions: a) concurrent activation and virus addition, b) 8-hour activation, and c) 12-hour activation before virus addition. Results indicate that 12-hour activation followed by virus addition leads to the highest T cell transduction efficiency.

[0024] Figure 3 (A-C): Illustrates the percentage of CAR expression and cell viability across various cell densities. Seeding densities ranging from 1 x 10A6 to 5 x 10A6 cells / mL were assessed for CAR expression, viability, and cell expansion over different time points. An increase in cell density from 1 million to 5 million cells / mL reduced CAR expression by 50% (3A). However, cell viability remained unaffected (3B), while cell proliferation decreased with higher cell densities (3C).

[0025] Figure 4 (A-D): Demonstrates the impact of seeding density at- scale in Gas Permeable Bags (GPB). Displays images of at-scale seeding densities of 0.5 x 10A6 cells / mL, 1 x 10A6 cells / mL, and 2 x 10A6 cells / mL in GPBs from three different healthy donors (4A). Shows CAR percentages on day 3, with un-transduced cells as a control, for the three different seeding densities (0.5 x 10A6 cells / mL, 1 x 10A6 cells / mL, and 2 x 10A6 cells / mL) from three healthy donors. Increasing the seeding density to 2 million cells / cmA2 reduced the CAR percentage from 47.22% to 26.24% (4B). Figure 4C: Illustrates cell viability and proliferation across different seeding densities, indicating that cell viability is unaffected by varying seeding densities, while cell proliferation decreases by increasing seeding density(4C). The phenotype of CAR-T cells on day 3, showing a significant decrease in cell phenotype at a seeding density of 2 million cells / cmA2 (4D).

[0026] Figure 5 (A-F): The graph illustrates the impact of serum presence or absence in the medium on cell phenotype, CAR percentage, cell proliferation, and cell viability. Notably, the percentage of CAR-positive cells significantly increased in serum-free media (5A), while the naive cell population drastically decreased (5B). The cell numbers and viability with and without serum addition to the medium remains unaffected (5C). The CAR expression on day-3 is significantly enhanced when transduction is performed in serum- free media (5D). Cell numbers and viability which remain unchanged irrespective of serum addition to the medium (5E). Additionally, adding serum 24 hours post-transduction significantly boosted the percentage of naive cells (5F).

[0027] Figure 6 (A to C): Evaluation of the impact of adding serum at different time points (6, 12, and 24 hours) post-LVV addition. Graphs illustrate the CAR percentages and cell phenotype on day 3, measured with and without serum (Fig. 6A and 6B) and the levels of CD4 and CD8 remains unchanged (Fig. 6C) The results show there is not much difference in percentage CAR, cell phenotype by addition of serum at different time points.

[0028] Figure 7 (A to C): The graph shows the MOI titration conducted to determine the minimum MOI necessary for effective LVV transduction to achieve percentage CAR > 20%. MOIs ranging from 1 to 10 were tested using healthy donorT cells. After 48 to 60 hours of LVV addition, cell count, cell viability, CAR percentage, and phenotype were assessed. The results indicate that increasing the MOI from 1 to 5 led to an increased CAR percentage, whereas MOI above 5 to 7.5 did not show any significant increase in CAR percentage (7A), while cell proliferation decreased with MOI 10 (7B). Cell viability remained largely unaffected (7C).

[0029] Figure 8 (A-D): The graph illustrates the CAR percentages, cell count, cell viability, and cell phenotype in the presence of RetroNectin® (RN) concentrations ranging from 5 to 15 pg / cm2. RetroNectin concentration above 5 pg / cm2percentage did not have any impact on percentage CAR (8A). Overall, cell proliferation, percentage viability and phenotype were unaltered (8B, 8C and 8D).

[0030] Figure 9 (A-D): The graph depicts the CAR percentages, cell count, cell viability, and cell phenotype on day 3, in the presence of low RetroNectin concentrations ranging from 1 to 10 pg / cm2. The results show that increasing RetroNectin concentration above 5 pg / cm2decreased the percentage of the naive population, while a lower concentration (2.5 pg / cm2) significantly increased the CAR percentage without affecting the cell phenotype (9A and 9D). Across the RetroNectin concentrations, cell proliferation, percentage viability and phenotype were unaltered (9B and 9C).

[0031] Figure 10 (A-D): The graph illustrates the CAR percentages, cell count, cell viability, and cell phenotype in the presence of 2.5 pg / cm2RetroNectin concentrations, evaluated against different MOIs ranging from 1 to 10, 5 MOI without RetroNectin coating is used as control. The result shows a significant increase in CAR percentage when comparing MOI 5 to MOI 2.5 (10A). There is no change in cell numbers (10B), with no change in the variability (10C). RetroNectin concentration at 2.5 pg / cm2resulted in increased naive cell population(lOD).

[0032] Figure 11 (A-B): The graph shows the percentage of CAR expression with and without the use of LentiBOOST (LT). Transduction efficiency was analysed in the presence of Poloxamer 388 (LentiBOOST®) at concentrations ranging from 0.25 to 1 mg / mL, with varying MOIs from 1 to 5. The CAR percentages on day 3 are plotted in the graph (11A). The results indicate that all tested concentrations ofLentiBOOST showed similar CAR percentages. The Cell phenotype show no difference in the naive population across the different concentration of LenitBOOST® (11B).

[0033] Figure 12 (A to D): The graph shows the percentage of CAR expression using RectoNectin at a concentration 2.5 pg / mL, LentiBOOST at a lower concentration (ranging from 0.05 mg / mL to 0.25 mg / mL) or a combination of RectoNectin and LentiBOOST with only LLV (5 MOI). Transduction efficiencies were comparable between tested LentiBOOST concentrations. Addition of RetroNectin and LentiBOOST together did not significantly increase the percentage CAR (12A). Total number of cells and percentage viability was not affected by change in LentiBOOST concentrations (12 B and 12 C). The cell phenotype shows no difference in the percentage phenotype across the different concentrations of transduction enhancers (12D).

[0034] Figure 13 (A-D): The graph shows the percentage of CAR expression compared with three different transduction enhancers (LentiBOOST, RetroNectin and Synperonic® F108) using three healthy donor selected T cells. There is no significant difference in transduction efficiencies between LentiBOOST and Synperonic F108 between concentrations (13A). There was no impact on total number of cells (cell expansion) and percentage viability (13B and 13C). The cell phenotype shows no difference in the percentage phenotype across the different concentrations of transduction enhancers(13D).

[0035] Figure 14 (A-E): The graph illustrates the percentage of CAR expression compared with two different media with inosine supplementation (concentration varying from 15 to 25 mM) from two patient selected T cells. There is no difference in percentage CAR since there was no difference in the transduction efficiencies observed by varying inosine concentration(14A). There was no impact on total number of cells (cell expansion) (14B) and percentage viability (14C). The cell phenotype had an increase in percentage of naive population with increase in inosine concentration in the media (14D and 14E).

[0036] Figure 15 (A-D): The graph illustrates the titration of anti-CD3 antibody coating (concentration varying from 1 - 4 pg / cm2) compared to transact activationmethod. The % CAR on day 3 were plotted showing no change with increasing anti- CD3 concentration (15 A). There was no impact on total number of cells (cell expansion) and % viability (15B and 15C). The cell phenotype had an increase in percentage naive population with anti-CD3 coated wells compared to transact based activation (15D).

[0037] Figure 16 (A-F): The graph illustrates the comparison of different T cell selection beads for CART manufacturing. The experimental setup used to assess the effectiveness of CD3 and CD4 / CD8 T cell selection beads (16A). The percentage CAR on day 3 were plotted showing increase in transduction efficiencies with anti-CD3, using wells coated with RetroNectin and Synperonic F108 when compared to other conditions (16B). There was no impact on total number of cells (cell expansion) (16C) and % Viability (16D). The percentage activation is shown in Fig 16E. The cell phenotype had an increase in naive population with anti-CD3, RetroNectin and Synperonic F108 coated wells compared to transact based activation using Synperonic F108 (16F).

[0038] Figure 17 (A-E): The graph illustrates the usage of different media and activation conditions comparison for CART production. The percentage CAR on day 3 were plotted showing decrease in transduction efficiencies with LymphoONE™ media compared to TexMACS™ and TheraPEAK® (17A). There was no impact on total number of cells (cell expansion) (17B) and percentage viability(17C). The cell phenotype had an increase in percentage naive population with anti-CD3 coated wells in G-Rex compared to transact based activation (17D). There was no difference in type of activation methods used (17E).

[0039] Figure 18 (A D): The performance of TexMACS media was assessed against NutriT media using selected T cells isolated from three healthy donors and one patient sample. Data from day-3 CART samples indicate that NutriT media leads to a substantial increase in the percentage of CAR expression (18A), cell expansion (18B), and the proportion of naive T cell populations (18D) compared to cells cultured in TexMACS media, with no change in the viability (18C).

[0040] Figurel9 (A- J): The graph illustrates the comparison of different activation methods with and without Synperonic F108 as transduction enhancer: method 1:activation with anti-CD3 and RetroNectin, method 2: activation with anti-CD3 and RetroNectin and Synperonic F108 during LVV addition, method 3: activation with TracnsACT and Synperonic F108 during LVV addition. The percentage CAR on day 3 were plotted showing increase in transduction efficiencies with method 2, (anti-CD3 and RetroNectin coated wells with Synperonic F108 addition during transduction) compared to other activation methods (19A and 19B). There was no impact on total number of cells (cell expansion) (19C) and percentage viability (19D). There was no difference in percentage activation with type of activation methods used (19E). The cell phenotype had an increase in percentage naive population with method 2 (anti-CD3 and RetroNectin coated wells Synperonic F108 addition during transduction when compared to TransACT based activation) (19F). Percentage cytotoxicity by luciferase reporter assays and flow cytometrybased assays (19G 19H 191 and 19 J) showed no difference.

[0041] Figure 20 (A to E) The graph illustrates at-scale CART manufacturing in GPBs and comparison of different T cell selection beads for CART manufacturing. The percentage CAR from day 3 to day 10 were plotted (20A). Total number of cells (cell expansion) (20B) and percentage viability(20C) and percentage phenotype (20D) was plotted. Comparison of percentage cytotoxicity from day-3 vs day- 10 CART cells showed no significant difference (20E).

[0042] Figure 21 (A to D): depicts the MOI titration with and without Synperonic F108. T cells from three healthy donors were used to titrate MOI at levels of 0.5, 1, and 2.5, both with and without Synperonic F108. Day-3 evaluations showed that adding Synperonic Fl 08 significantly increased the CAR percentage (21 A), while cell proliferation (21B), viability (21C), and phenotype (21D) remained consistent regardless of Synperonic F108 presence.

[0043] Figure 22 (A-D): Depicts the large-scale CAR-T manufacturing in G-Rex. T cells from healthy donors and a CD 19 clinical trial patient were selected using the CytoSinct™ 1000 system. These cells were seeded in the G-Rex 100M-CS system, activated with TransACT, and transduced with CD19-LVV at 2.5 MOI plus Synperonic Fl 08. Media was added on day 3, with CAR percentage (22A), cell12expansion (22B), viability (22C), and phenotype (22D) measured on days 3, 6, 8, and 10.

[0044] Figure 23 (A-C): Large-scale CAR-T manufacturing in G-Rex. The G- RexlOOM-CS system, coated with anti-CD3 and RetroNectin, was used for large- scale CAR-T production. T cells from frozen apheresis samples of healthy donors, selected using the MARS BAR system, freshly selected T cells were seeded and transduced with CD19-LVV at 2.5 MOI and Synperonic F108. Media was added on day 3. CAR percentage, cell expansion, viability, (23A), and phenotype (23B) were measured on days 3, 6, and 8, with a comparison of cytotoxicity between day 3, and day 10 (23C).

[0045] Figure 24 (A-C): At-scale CAR-T manufacturing in G-Rex using activation bead CD3 and transduction enhancer RetroNectin. The G-RexlOOM-CS system, coated with anti-CD3 and RetroNectin, was utilized for large-scale CAR-T production. T cells, selected from frozen apheresis samples of healthy donors using the MARS BAR system, were seeded and transduced with CD19-LVV at 2.5 MOI in the presence of Synperonic Fl 08. Media was added on day 3. CAR percentage, cell expansion, viability, and phenotype were evaluated on days 3, 6, and 8., with a comparative analysis of cytotoxicity between day 3 and day 10.

[0046] Figure 25 A to D: RetroNectin titration with F108 (0.25 and 0.5 mg / mL) was performed to evaluate the effect of lower RetroNectin concentrations on CAR expression. RetroNectin concentrations ranged from 0.25 to 2.5 pg / cm2, with 0.25 and 0.5 mg / mL F108. The results indicated no significant increase in transduction efficiencies with the reduced RetroNectin concentrations in the presence of either F108 concentration during LVV addition. Additionally, no significant differences were observed in cell expansion, viability percentage, or phenotype.

[0047] Figure 26 A to J: Evaluation of conventional CAR-T process and method of present invention using patient- selected T Cells in a G-Rex System.

[0048] Figure 27 A to D: Comparative Analysis of CAR-T Expression Using Varying MOI in presence of synperonic F108 in Healthy Donor T Cells.13DETAILED DESCRIPTION OF THE INVENTION

[0049] The following description is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary.

[0050] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well- known functions and constructions are omitted for clarity and conciseness.

[0051] The terms and words used in the following description and claims are not limited to the bibliographical meanings but are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the scope of the invention as defined by the appended claims and their equivalents.

[0052] It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0053] Features that are described and / or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and / or in combination with or instead of the features of the other embodiments.

[0054] It should be emphasized that the term “comprises / comprising” when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof.

[0055] The present invention relates to a novel, faster, shorter, accelerated, and quick method for preparing higher levels of chimeric antigen receptor (CAR) expressing immune cells and compositions comprising the same. Also disclosed are methods of using such compositions for treating a disease, e.g., cancer, autoimmune diseases in a subject.14

[0056] Generally, most cancer patients have already undergone multiple rounds of pre-treatment, before undergoing immunotherapy, and their T cells often exhibit an exhausted phenotype, resulting in poor expansion and functionality of their CAR T cell products. In the present method additives or supplements are added to the T cell culture medium that can modulate or more particularly suppresses cellular programs that drive T cell terminal differentiation, senescence, or exhaustion in order to stimulate patients’ T cells and improve CAR T cell functionality.

[0057] The total manufacturing time varies depending on how much starting material (PBMCs or isolated T cells) is available and how fast patient-derived T cells grow. Reducing the manufacturing time is required in cases where the disease is rapidly progressing, and patient conditions is deteriorating. Studies have shown correlation of shortening the ex vivo culture period to that of improved CAR T cell functionality when compared to the long-term culture.

[0058] The present invention discloses a new shorter manufacturing processes for generating immune effector cells e.g., CAR-T cells in a short timeframe of 1 to 4 days, with improved one or more conditions of the manufacturing processes and improving the efficiency of manufacturing steps using one or more additives or supplements to improve the activation and transduction efficiently. Additives or supplements prevents cell-to-cell fratricide during manufacturing of genetically engineered T cells. Alternatively, or in addition, additive / supplements reduced exhaustion of the genetically engineered T cells improves the naive and central memory cell population.

[0059] The CAR-T cells generated by this method produced CAR T cells with increased percentage CAR expression, improved consistency, and transduction efficiency. The manufacturing method described herein result in decreased production times and costs, which enables an increased supply of CAR T-cell therapy at more affordable cost to patients. Further, the manufacturing processes reduced the variability of manufactured drug products by producing uniform, robust and higher percent of CAR T cells. Most importantly, the manufacturing method disclosed herein provide higher number of modified immune effector cells in short time with less differentiation and senescence and prolonged persistence with15enhanced phenotype and functionality in a cost-effective manner. The present invention provides a cost effective GMP scalable process.

[0060] The present process reduced the entire manufacturing time when compared to the conventional process. In an embodiment, present invention provides a novel accelerated method of manufacturing higher number and percent of genetically engineered T cells expressing CAR within a shorter timeframe of 48 hours to 72 hours, or 40 hours to 60 hours or 35 hours to 50 hours or 24 hours or 48 hours or 36 hours or 72 hours or 96 hours or less than 96 hours thereof.

[0061] The Chimeric Antigen Receptor T cells (CAR-T) are a specific type of genetically modified T cells used in immunotherapy. The CAR-T cells generated using the present method can expand in-vivo and maintain long term efficacy against target cells without compromising the functionality and the quality or viability of the cell.

[0062] In an embodiment, the population of immune cells is acquired from a blood sample from a subject. The population of immune cells comprises immune effector cells chosen from T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, myeloid-derived phagocytes, dendritic cells or a combination thereof.

[0063] In an embodiment, the population of immune cells comprises primary T cells or a subset of lymphocytes chosen from anergized T cells, naive T cells, T- regulatory cells, Th- 17 cells, stem T cells, or a combination thereof.

[0064] In the present invention, the T cell refers to one or more T cell subsets, including but not limited cytotoxic T cells, helper T cells, natural killer T cells, gamma-delta T cells, regulatory T cells, and memory T cells, preferably memory T cells.

[0065] In an embodiment, the non-limiting example of the T cells include peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours.

[0066] In some embodiments, after isolation of PBMC, both cytotoxic and helper T lymphocytes can be separated into naive, memory, and effector T cellsubpopulations either before or after activation, expansion, and / or genetic modification.

[0067] In autologous cell-based therapy, the CAR-T cell-manufacturing process starts from the collection of peripheral blood mononuclear cell from the patient, commonly achieved by a leukapheresis process. Collected apheresis products can be processed in various ways depending on the downstream procedures. The apheresis sample can be either fresh or frozen sample.

[0068] In an embodiment, the method of manufacturing of the present invention may use a closed system such as CliniMACS Prodigy, Lonza Cocoon, Cellares™ Cell Shuttle™ platform, or semi-integrated platforms including G-Rex, Gas Permeable Bags, Ambr 250, Adva X3, Aglaris, Replicell™ System or Quantum Cell Expansion System, MACS® GMP Cell Expansion Bags, EXP-Pak™ Cell Expansion Bio-Containers, VueLife™ bags, KryoSure™ bags, KryoVue™ bags, Lifecell® bags, PermaLife™ bags, X-Fold™ bags, Si-Culture™ bags, and VectraCell™ bags for manufacturing of the CAR-T cells.

[0069] In an embodiment, present invention uses Dynabeads, MACS Microbeads or MAGBeads, CytoSinct™ Nanobeads or similar known reagents in the art for the enrichment of specific subsets of T cells, such as CD3+ CD4+, CD8+, or CD45RA+, CD62L+ T cells.

[0070] In some embodiments, the present invention provides the method wherein the T cells are concurrently isolated / enriched, activated, and transduced using the viral vectors.

[0071] T cells for use in making the genetically engineered T cells may express one or more of the T cell markers, non-limiting examples include CD4+, CD8+, CD3+ or a combination thereof.

[0072] In an embodiment, the T cells are derived from cryopreserved T cells enriched from human blood cells.

[0073] In some embodiments, CD4+ T cells can be enriched from human blood cells.

[0074] In other embodiments, CD8+ T cells can be enriched.

[0075] In specific examples, both CD4+ and CD8+ T cells are purified from human blood cells.

[0076] In an embodiment the T cells enrichment is performed using CD3, CD4 / CD8 beads.

[0077] In an embodiment, the T cells are prepared by a process / method comprising: (i) obtaining blood cells from a human donor; and (ii) isolating and / or enriching CD4+ T cells and / or CD8+ T cells from the blood cells. In certain embodiments, step (ii) is performed using magnetic beads conjugated with anti-CD4 and / or anti- CD8 antibodies. In certain embodiments, the method further comprises of incubating the T cells from step (ii) in the presence of a T cell activating agent in a cell culture vessel to produce an activated population of T cells.

[0078] This process can be used in all types of T cells with minor modification which is obvious to the person skilled in the art.

[0079] In an embodiment the activating agent is selected from anti-CD3 antibodies or anti-CD3 / CD28 activation with TransACT™, recombinant human fibronectin fragment (rFN-CH-296), the commercial version of rFN-CH-296 is Rectronectin®, or a combination thereof. The activating agents are either coated to the culture system or added to the system.

[0080] In some embodiments the activating agent used is an anti-CD3 antibody at a concentration ranging from 1 to 4 pg / cm2.

[0081] In another embodiment the recombinant human fibronectin fragment or Rectronectin® is used as an activating agent in the concentration range of 0.25 to 15 pg / cm2, or 0.25 to 10 pg / cm2or 0.25 to 5 pg / cm2.

[0082] In some embodiments activating agent used is a combination of recombinant human fibronectin fragment or Rectronectin and CD3 antibody, wherein the combination of recombinant human fibronectin fragment or Rectronectin and CD3 antibody is coated to the culture system.

[0083] In some embodiments activating agent used is a combination of recombinant human fibronectin fragment or Rectronectin and CD3 antibody, wherein the recombinant human fibronectin fragment or Rectronectin is coated to the culture system while the anti-CD3 / CD28 is added to the culture system.

[0084] In an embodiment, the activated T cell is cultured in culture medium. The non-limiting examples of the culture medium is RPMI 1640, AIM-V™, ICM, TexMACS™, 88-581-CM Medium (CMM), X- VIVO- 15™, TheraPeak®, DMEM, IMDM, HPLM, OptiMEM, LymphoONE™, Nutri-T, or customized culture medium.

[0085] In an embodiment, the activated T cells are cultured in a glucose-free customized culture medium comprising inosine 5 to 25 mM. Inosine when used as an alternative to glucose promotes T cell persistence and anti-tumour activity.

[0086] In some embodiments, present invention provides a method where the transduction of T cells is performed concurrently with isolation and / or enrichment step.

[0087] In some embodiments, the enriched T cells are activated along with transduction simultaneously / concurrently at the time of seeding by using lentiviral vectors with pseudotyped envelopes co-expressing the CD3 and / or CD28 as activating domains.

[0088] In an embodiment, lentiviral vector particles are pseudotyped with an envelope protein selected from the group consisting of BaEV env, VSV-G, RD114- TR, NiV-G / F, Ampho-MLV env, GALV-TR, Cocal vesiculovirus envelope pseudotyped retroviral vectors and MV-H / F.

[0089] In some embodiments, the enriched T cells are activated along with transduction simultaneously using LentiSTIM vector and RetroSTIM vector technology.

[0090] In some embodiments, one or more nontoxic transduction enhancer is used for enhancing the lentiviral- and retroviral-mediated gene transduction by aiding the colocalization of target cells and viral particles. In certain aspects more than one viral particle may be used.

[0091] In some embodiments, the non-limiting examples of transduction enhancers include recombinant human fibronectin fragment or a commercial product referred to as Retronectin, non-toxic histidine-rich amphipathic peptide (Vectofusin®-1), cationic block co-polymer, non-ionic amphiphilic poloxamer 338, pluronic Fl 08 or synperonic F108, Poloxamer 338 (LentiBoost®), cationic additives such aspolybrene, non-ionic 9003-11-6, poloxamerF108, kolliphor P338, Akti-l / 2(Akt Inhibitor VIII), Cyclosporin A, Cyclosporin H, chloroquine diphosphate, dasatinib, daunorubicin hydrochloride, dexamethasone, 16,16-dimethyl prostaglandin E2, eeyarestatin I, etoposide, hydroxychloroquine sulfate, prostaglandin E2, rapamycin, rosuvastatin calcium, staurosporine, teniposide, other commercial available transduction enhancer ( e.g., TransPlus®, ExpressMag®) or a combination thereof.

[0092] Transduction involves using Multiplicity of Infection (MOI) to determine the optimal ratio of viral particles to target cells, ensuring efficient gene transfer without excessive cytotoxicity. By precisely titrating the MOI, the present invention have achieved high levels of CAR expression while maintaining cell viability. Additionally, the use of transduction enhancers further boosts the efficiency of the process. These enhancers facilitate the binding and entry of viral vectors into the target cells, leading to improved transduction rates and better overall performance of the CAR-T cells.

[0093] In an embodiment, the concentration range of the transduction enhancer can be 0.001 to 0.25 mg / mL (1 to 250 pg / cm2).

[0094] In an embodiment, the concentration range of the transduction enhancer is from 0.05 to 5 mg / mL.

[0095] In an embodiment, the concentration range of the transduction enhancer is from 0.1 to 3 mg / mL.

[0096] In an embodiment, the concentration range of the transduction enhancer is from 0.5 to 3 mg / mL.

[0097] In an embodiment, the concentration range of the transduction enhancer is from 0.25 to 3 mg / mL.

[0098] In an embodiment, the concentration range of the transduction enhancer is from 2 to 8 mg / mL.

[0099] In an embodiment, the concentration range of the transduction enhancer is from 0.0025 mg / mL (2.5 pg / cm2) or 0.25mg / mL or 0.5mg / mL or Img / mL.

[0100] In an embodiment, the present invention further focuses on lowering the amount of lentiviral vector (LVV) by using a transduction enhancer to minimize20the risk of unintended transduction of non-target cells, thereby enhancing the safety profile of CAR-T therapy.

[0101] In some embodiments, the non-limiting example of transduction enhancer and the concentration are selected from recombinant human fibronectin fragment (RetroNectin®), or synperonic F108 or a combination thereof.

[0102] Multiplicity of Infection (MOI) titration: MOI titration is crucial in the CAR-T cell manufacturing process as it ensures efficient transduction by determining the optimal ratio of viral particles to target cells. This process enhances the yield of functional CAR-T cells, minimizes the risk of cytotoxic effects by preventing cell overload with viral particles, and ensures the safety of the final product. Accurate MOI titration also promotes consistency across different batches, maintaining product quality and reproducibility, while optimizing the use of viral vectors, thereby reducing costs.

[0103] In some embodiments, the culture plate or flask or bag, bioreactor, G-Rex systems, or any platform know in the art for culturing of cells can be coated with the transduction enhancer before seeding the cells.

[0104] In an embodiment, the activated T cells (CD4+, CD8+, CD3+, Gamma Delta) population is incubated with at least one viral vector to produce T cells, wherein the viral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the use of such viral vector for transduction to insert a nucleic acid encoding a CAR. The non-limiting examples of the viral vector include lentivirus, adenovirus, adeno-associated virus and thereof.

[0105] In an embodiment present invention provides a method for producing chimeric antigen receptors comprises the steps of designing and synthesizing a chimeric antigen receptor construct, wherein the construct includes an antigenbinding domain, a transmembrane domain, and an intracellular signaling domain. This method is not limited to any specific target antigen and can be adapted for manufacturing CARs targeting a variety of antigens, including but not limited to CD19, BCMA, CD307e, bispecific and dual CAR such as CD19-CD20, BCMA- CD307e and CD20. The method includes the following steps: designing of antigen -binding domain by engineering an antibody fragment that specially binds to the21desired target antigen exemplified; construct assembly by linking antigen -biding domain to a transmembrane domain and more or more intracellular signaling domains to form the CAR construct; cloning the CAR construct into an appropriate expression vector for delivery into T cells; using a viral or non-viral method to transduce T cells under conditions that promote their expansion and activation and assessing the functionality, specificity and safety of the CART cells through various in vitro an in vivo assays.

[0106] Non-limiting examples of chimeric antigen receptor construct targeting different antigen are also provided for in WO2021 / 023721(BCMA), WO / 2024 / 047558(CD307e), W02016100232A1(CD19-CD20) or such others CAR-T cells in the art.

[0107] In an embodiment, suitable amount of any of the viral vectors, which encodes a CAR construct disclosed herein (e.g., an anti-CD19 CAR or an anti- CD20 CAR, or an anti-CD307e CAR, or anti-CD19-anti-CD20 dual CAR, anti- CD307e-anti-BCMA bispecific CAR, anti BCMA CAR or any immune-targeting antigen antibody may be incubated with a suitable amount of immune effector cells (e.g. T cells), for a suitable period to allow for entry of the viral vector into the T cells. The transduction process may involve the use of a range of selected LV dose or multiplicity of infection (MOI) that increases percentages of CAR T cells.

[0108] In an embodiment, present method can be used for co-transduction using two viral particles to enhances the gene delivery efficiency and allows for simultaneous introduction of multiple genetic elements.

[0109] In an embodiment, the method of the present invention can be used for generating single CAR, dual CAR, bicistronic CAR or bispecific CAR or other CAR design thereof.

[0110] In an embodiment, the method of the current invention can be used for generating CAR sequence that encoded the anti-CD19, anti-CD20, anti-CD- 19 / anti-CD20 dual, anti CD307e or anti-BCMA or any other known CARs.

[0111] In an embodiment the viral vector is lentiviral vector comprising a nucleic acid enclosing a chimeric antigen receptor (CAR), wherein the CAR contains abinding domain, a hinge, a transmembrane and a signaling domain together with one or more costimulatory domain.

[0112] In certain embodiments, an anti CD 19 CAR exemplified herein has an amino acid sequence encoded by a nucleic acid comprising a nucleotide sequence as set forth in any one of the SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 thereof.

[0113] In certain embodiments, an anti CD 19 CAR exemplified herein has an amino acid sequence encoded by a sequence as set forth in any one of the SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 thereof.

[0114] In some embodiment, the MOI during the incubation may range from about 0.1 to about 10.

[0115] In some other embodiment, the MOI during the incubation may range from about 1.0 to 5, preferably 1 to 2.5.

[0116] In some embodiment, the MOI during the incubation is 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

[0117] In some embodiment, the MOI during the incubation is less than 10.

[0118] In some embodiment, the MOI during the incubation is less than 5.

[0119] In some embodiment, the MOI during the incubation is less than 2.

[0120] In an embodiment, an equal ratio of virus and cells is used to get at least more than 50% transduction. In some embodiment, 0.5-2 million cells in 0.1-5 ml complete media to which an MOI of 0.5-10 of virus along with one or more transduction enhancers are added to improve the transduction efficiency.

[0121] In an embodiment, the transduced T cells is expanded for about 48 to 72 hrs.

[0122] The T cells can be expanded beyond 72 hours if required. In certain aspects optional it can be expanded for 10 to 12 days. This method provides flexibility in the duration of the expansion phase, accommodating the variability in cell growth rates and other process parameters.

[0123] The transduced T cells comprise a nucleic acid encoding a CAR and an inducible gene expression system. When an induction agent is administered to a cell being transduced with the inducible gene expression system, the gene expression system is induced, and the CAR is expressed on the surface of the transduced cell. The CAR has an antigen binding domain that binds specifically to an antigen expressed on the surface of malignant cells of the patient. In an embodiment, T Cell expressing CAR produced by the present manufacturing method consists of less differentiated naive and central memory T cells. These cells exhibit superior proliferation, persistence, and antitumor responses. In some embodiments, the T cell populations manufactured using the present invention29exhibit a variety of specific phenotypic properties predominately consisting of naive and central memory T-cell population.

[0124] In various embodiments, expanded T cell populations comprise one or more of the following phenotypic markers of naive T cells including CD62L, CCR7, CD45RA, CD27, CD28, and T cells characterized by the expression of phenotypic markers of central memory T cells including CD45RO, CD62L, CCR7, CD28, CD27, CXCR3 and CD95.

[0125] In some embodiments, T cells produced using the present invention are characterized by the expression of phenotypic markers of naive T cells including CD62L, CD127, CD197, CD45RA.

[0126] In another embodiment, T cells characterized by the expression of phenotypic markers of central memory T cells including CD45RO, CD62L, CCR7, CD28, CD27CXCR3 and CD95.

[0127] In an embodiment, characterization of the Effector Memory cells (EM) are double negative for CD62L and CD45RA.

[0128] In embodiment characterization of Terminal Differentiated Effector Memory T cells (TEMRA) are positive for CD45RA, CD95, but negative for CD62L.

[0129] The naive T cells, central memory cells, EM and TEMRA are functionally different from each other, and the potency of CAR-T therapy depends on their population in the drug product. In certain aspect present method can provide a C AR- T product with 25%, to 90% of naive and central memory cells or at least more than 25% of naive and central memory cells or at least more than 30% of naive and central memory cells or at least more than 40% of naive and central memory cells or at least more than 50% of naive and central memory cells or at least more than 60% of naive and central memory cells, or at least more than 70% of naive and central memory cells, or at least more than 80% of naive and central memory cells, at least more than 90% of naive and central memory cells.

[0130] In some embodiments the present method provides a CAR-T product comprising of proportional a greater number of naive and memory cells. In aspecific embodiment, the CAR-T product preferably comprises at least >50% of total cell population as naive and central memory cells.

[0131] In some embodiments, the method further comprises harvesting the population of transduced modified T cells for storage (e.g., reformulating the population of immune cells in cryopreservation media) or administration.

[0132] The cells produced using present invention can be expanded beyond the initial 3-4 days, similar to conventional processes, without any limitations. This flexibility allows for further cell proliferation and optimization as needed. In an embodiment, the enriched T cell population are stimulated or activated for about 0 to 24 hours, or 4 to 24 hours, or for about 12 to 18 hours or for 6 hours, or for 10 hours or for 12 hours or for 15 hours, or for 16 hours or for 17 hours or for 18 hours with anti-human CD3 or anti-CD28 coated beads and cultured in a customized medium supplemented with inosine 5-20 mM.

[0133] The T cells were seeded in bags coated with transduction enhancers.

[0134] In an embodiment the transduction enhances is a recombinant human fibronectin fragment example RetroNectin®.

[0135] In some embodiments, the RetroNectin concentration range from 0.25- 20pg / cm2and anti-CD3 antibody concentration: l-10pg / cm2. The activated cells are incubated with viral vector particles for examples lentiviral vector (LVVs) at a multiplicity of infection (MOI) of about 0.1 to 10 or of about 1.0 to 2.5 for about 24 hours to 72 hours. During the first 6 to 10 hours of the transduction the cell are serum starved and after 6 to 10 hours of LVV addition the culture medium is supplemented with serum to enhance the naive cell population.

[0136] In Ghassemi et al. (2022), they have reported that serum starvation of nonactivated T cells for 3h-6h and addition of 50 pM deoxynucleosides (dNs) during transduction is important for increasing % CAR transduction in non-activated T cells. In the present process addition of deoxynucleosides is not required. Further the present process is effective with and without serum. Without wishing to be bound by theory, it is believed that in the present process that the serum starvation alone can increase expression of the low-density lipoprotein receptor (LDL-R) such31that it is not required to add dNs during transduction, to further increase % CAR transduction in non-activated T cells.

[0137] The transduced cells are cultured for about 24 to 96 hours or 48 to 96 hours or 48 to 72 hours or 48 to 56 hours or 48 hours or 36 hours or 24 hours.

[0138] In an embodiment the manufacturing process reduces the 8 - 12 days manufacturing time to 1-4 days / 24 - 96 hours or 24 to 36 hours or 24 to 72 hours or 24 to 48 hours or 24 hours, or less than 36 hours, or less than 48 hours, or less than 72 hours or less than 96 hours with quality attributes similar to the cells made by the 8 - 12 days process. The impact of this modified process gives a significant reduction in time of manufacturing and cost associated with the same. Furthermore, the expansion process can alter the proportion of cell phenotypes, whereas the current process with minimal expansion maintains more of naive and memory cells.

[0139] In certain embodiments, the methods and compositions of the present disclosure relate to cells having a tumor specific chimeric antigen receptor whose expression is under the control of atmospheric conditions with reduced oxygen content compared to ambient air. This relative hypoxia may be achieved by reducing the atmospheric oxygen exposed to the medium.

[0140] In an embodiment, the present method can be performed under hypoxic condition (0 to 10% oxygen) for enhancing the sternness of the CAR-T cells.

[0141] In certain embodiments, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, about 5%, about 4%, about 3%, about 2%, or about 1% atmospheric oxygen content may be used.

[0142] In another embodiment, the present invention provides method and means for efficient integration of CAR polynucleotide sequences into the T cell genome by improving the transduction efficiency.

[0143] In an embodiment, the present invention provides a method for generating CAR-T cells with enhanced proliferation, sternness, persistence, cytotoxicity, with less differentiation.

[0144] In an embodiment, present invention improves transduction efficiency with reduced use of viral vector while preserving or improving the sternness.

[0145] In an embodiment, present method aims to generate consistent robust supply of clinically useful CAR T-cell therapies.

[0146] In an embodiment, the disclosed method for producing CAR-T cells involves the use of one or more additives or supplements to the culture media and or by processing the modified T cells in the presence of such additives or supplements or a combination thereof. Wherein the non-limiting examples of these additives which non-limiting and selected from a group comprising of cytokines, epigenetic modulators, AKT pathway inhibitors, PI3K inhibitors, activation agents, transduction agents or a combination thereof.

[0147] In an embodiment, the non-limiting examples of the cytokines are interleukin, chemokines, interferons, tumor necrosis factor, colony- stimulating factors. Preferably interleukin such as IL-2, IL-7, IL-10, IL-15, IL-18, IL-21 and others. In some embodiments, the culture media is customized or supplemented with interleukins. In specific embodiments, the culture media is supplemented with IL-7 and IL- 15.

[0148] In an embodiment, the present method discloses use of one or more AKT pathway inhibitors, and / or one or more PI3K inhibitors which targets different components of the PI3K / AKT / mTOR signalling pathway, to improve the cell potency, growth, and survival of the CAR-T cells.

[0149] The present process described herein has several advantages namely reduction in cost of production by eliminating the need for T cell ex-vivo expansion. Secondly, this method reduces the time between apheresis and drug administration which is extremely crucial for patients suffering from - cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. Thirdly, the morphology and phenotype of CAR-T cells produced will be directly in response to in-vivo conditions as the expansion of CAR-T cells will happen when the CAR- T cells are administered in-vivo.

[0150] According to another aspect of the present disclosure there is provided a method of treating a disease or condition in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amountof: CAR-T product / composition as manufactured using the present invention or the methods described herein.

[0151] The transduced T cell are formulated into a composition along with at least one pharmaceutically acceptable excipient for cryopreservation or administration. The pharmaceutically acceptable excipient are selected from a group known in the art and include, e.g., phosphate buffered saline, saline, Ringer's solution, dimethyl sulfoxide (DMSO), carboxymethylcellulose sodium, and glycerol, buffered isotonic solution, a buffered solution, a culture medium, serum (e.g., human serum).

[0152] In some embodiments, the disease or condition is selected from the group consisting of viral infection, a bacterial infection, a parasitic infection, a cancer, a malignancy, a non- cancerous condition, an autoimmune disease, a fibrotic disease, Alzheimer’s disease, protein deficiency conditions, and factor VIII deficiency.

[0153] In an embodiment, the autoimmune disease is selected from the group comprising of systemic lupus erythematosus (SLE), systemic sclerosis (Scleroderma), idiopathic inflammatory myositis, Rheumatoid Arthritis, multiple sclerosis, inflammatory bowel disease (IBD), type 1 diabetes, hashimoto's thyroiditis, graves' disease, psoriasis or others thereof.

[0154] In some embodiments, the cancer is selected from the group consisting of breast cancer, triple-negative breast cancer, prostate cancer, ovarian cancer, glioma, glioblastoma, renal cell carcinoma, kidney cancer, mesothelioma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, lung adenocarcinoma, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, hepatocellular carcinoma, esophagus cancer, brain cancer, melanoma, Hodgkin's lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, blood cancers, lymphoma, leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myelogenous leukemia, B-cell acute lymphoblastic leukemia (ALL), pre-B ALL, and any combination thereof.

[0155] In some embodiments, the subject is a human.

[0156] Both the foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the disclosure, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.

[0157] EXAMPLESThe following examples describes the method of preparing / manufacturing chimeric antigen receptor (CAR) expressing immune cells comprising the various steps all within a short production / manufacturing timeframe.

[0158] Manufacturing Process:

[0159] Figure 1 illustrates a detailed process flow chart for the CAR-X (e.g., CAR- T) manufacturing protocol, which is completed within 3-4 days according to the present invention. The process involves receiving apheresis or buffy coat, selecting T cells, seeding, followed by activation, transduction, serum addition, and finally, CAR-T cell harvesting and cryopreservation. The flow chart also shows the conventional CAR-T manufacturing process.

[0160] Example 1:

[0161] Stepl: Isolation and enrichment of selected T cells:

[0162] In an embodiment CAR-T cells are manufactured from T-cells which are enriched from a pool for PBMCs isolated from a patient fresh sample or a cryopreserved PBMCs.

[0163] In one embodiment, the buffy coat sample is diluted with IxPBS at a 1:1 ratio. Peripheral blood mononuclear cells (PBMCs) are then isolated using a Ficoll- Paque density gradient centrifugation (Buffy coat: Ficoll; 3:4). Following centrifugation, the lymphocyte layer is separated and washed twice with CliniMACS-EDTA buffer containing HSA. CD4 and CD8 cells are subsequently selected using LS columns. Additionally, buffy coat samples can be processed using kits specifically designed for direct use with buffy coat, resulting in the selection of T cells.

[0164] In one embodiment, T-cells are enriched using magnetic beads conjugated with antibodies that specifically bind to T-cells. The markers CD3, which identifies all T-cells, along with CD4 and CD8, which distinguish between the subsets of T- cells, are employed to isolate T-cells for use in CAR-T cell therapy.

[0165] The present invention encompasses various alternative and non-limiting methods for the isolation and enrichment of selected T cells, including but not limited to: magnetic bead separation, flow cytometry and cell sorting, density gradient centrifugation, the Activation-Induced Markers (AIM) method, and or the use of cell strainers and media. These methods and materials provide flexibility in the isolation and enrichment of T cells, allowing for various applications and optimizations as required by the specific needs of the invention.

[0166] In an embodiment a predetermined seeding density is evaluated and established for proper activation as well as transduction of T cells.

[0167] Step 2: Seeding of enriched T cells for activation and transduction:

[0168] In an embodiment the cells are seeded in Gas Permeable Bags (GPBs) or G- Rex or CliniMACS Prodigy or Lonza Cocoon, or flasks at a density ranging from O.lxlO6to 10xl06cells per ml per cm2.

[0169] In an embodiment, the cell concentration is about 0.5xl06cells / mL to about 5xl06cells / mL or is about IxlO6cells / mL to about 4xl06cells / mL. In certain embodiment, the cell concentration is about 0.5xl06or IxlO6cells / mL or 2xl06cells / mL or 2.5xl06cells / mL or 3 x 106cells / mL, or 5 x 106cells / mL.

[0170] Evaluation of Seeding density and CAR expression:

[0171] For enhance CAR expression, various seeding densities ranging from 1 x 10A6 to 5 x 10A6 cells / mL using 24 well, 12 well or 6 well plates and at scale in gas permeable bags (GPB) was assessed. The percentage of CAR, cell viability and cell number at different time points (across different days) was evaluated (Figure 3 and 4). As a reference, un-transduced cells served as a control, illustrating the CAR percentages on day 3 (see figure 3A and 4B). Furthermore, figure 3B, 3C, 4C and 4D visually represents cell number, cell viability and the phenotype of CAR-T cells on the same day (day 3). These set of data demonstrate that a T cell density ranging 1.0 - 2.5 xlO6cells / ml yields high transduction efficiency, an increase in celldensity from 1 million to 5 million cells / mL resulted in a 50% reduction in CAR expression (Fig. 3A). The T-cell density of 1 x 106cells / ml provided highest transduction (Fig. 4B), while a cell density of 0.5 - l x 106helps in maintaining the naive population of T-cells (Fig 4D).

[0172] Example 2: Seeding density evaluation using 6 or 12 well plates: In an embodiment, the effect of seeding density on the CAR percentage, cell number and percentage viability were evaluated by seeding the cells at different seeding densities (1, 2.5 and 5 million cells / mL) in TexMACS medium supplemented with IL-7 and IL- 15. The CAR percentage, cell number and percentage viability are shown in the figure 3 A to 3C.

[0173] The results from the figure 3A-3C shows increasing cell seeding density from 1 million cells / mL to 5 million cells / mL reduced the CAR percentage by half. There was no impact on cell viability. The other observation was a decrease in cell proliferation with an increase in seeding density. From the above study and data, further experiments were carried out using Gas Permeable Bags (GPB) wherein the seeding density in Gas Permeable Bags was selected from 0.5 million cells / cm2to 2 million cells / cm2range. The seeding density of cells is a critical initial parameter in the manufacturing process, impacting both CAR expression and cell proliferation.

[0174] Example 3: Large-Scale Seeding Density Selection using GPBs: The seeding density evaluation using PL 70-2G bags for GPBs. Healthy donor- selected T cells were seeded at different densities in GPBs. Based on the surface area of the PL 70-2G bag (172 cm2), cells were seeded at 0.5, 1, and 2 million cells / cm2in three GPBs. These GPBs were then used for CAR-T manufacturing with CD 19 LVV in short time frame of 48 - 60 hours. After 48-60 hours of transduction, CAR percentage, cell expansion, percentage viability, and cell phenotype were analysed, and the results are depicted in Figure 4A to 4D. Based on our studies, we have determined that seeding density is a critical factor that significantly affects both CAR expression and cell proliferation during the production of CAR-T cells. In an embodiment, data obtained from the above two experiments, a seeding density of 1million cells / cm2was chosen to achieve at least more than 35 to 40 % CAR expression and over 90% cell viability.

[0175] Activation of T cell population using activation reagent / agents:

[0176] In an embodiment the enriched T cells are activated before transduction.

[0177] In some embodiments, T cell receptor activation is based on CD3 and CD28 (co-stimulatory domain) or CD3 alone. In certain aspects, TransACT beads are used to activate T cells (CD3 and CD28 based activation). TransACT activates T cells and helps in expansion in-vitro or ex-vivo.

[0178] In some embodiments T cell activation reagent is anti-CD3 monoclonal antibody (OKT3). This recognizes CD3, a human glycoprotein and member of the Ig superfamily-primarily expressed on T cells, some NKT cells, and thymocytes during T-cell differentiation. Binding of an anti-CD3 monoclonal antibody to CD3 stimulates T-cell activation. Anti-CD3 antibodies (the OKT3 clone in particular) are used to expand T cells in culture.

[0179] In some embodiment, activation reagent anti-CD3 antibody concentration ranges from 1 to 10 pg / cm2.

[0180] After T cells are activated, the cells are cultured to proliferate. T cells may be cultured for at least 1, 2, 3, or 4, days or beyond, one of the focuses of the present invention is to reduce the time required for manufacturing the T cells.

[0181] In an embodiment, the T cell collected from three different donors are tested to evaluate the activation condition required for more than 10% CAR-T expression in 1-4 days using the current manufacturing process. The following activation conditions are tested: (a) concurrent activation and LVV addition; (b) activation for 8 h following by LVV addition; and (c) activation for 12 h following by LVV addition (Figure 2).

[0182] Example 4 Cell Activation at different time: The cell activation and lentiviral vector (LVV) addition process at different time points to assess chimeric antigen receptor (CAR) expression, results are as depicted in Figure 2. Specifically: In Group A, cell activation and LVV addition occurred simultaneously. In Group B, LVV addition followed 8 hours after cell activation. In Group C, LVV additionoccurred 12 hours after cell activation. The outcomes of these interventions are presented in the Figure 2.

[0183] Experimental results from T cells selected from three healthy donors above indicate that Group C, characterized by 12-hour activation followed by LVV addition, yields the optimal T cell activation and transduction.

[0184] In an embodiment, the invention involves an activation process lasting 12 hours, followed by the addition of lentiviral vector (LVV), which results in achieving a CAR percentage exceeding 10% by day 3. This method demonstrates enhanced efficiency in the generation of CAR-T cells, ensuring a robust and effective transduction process.

[0185] Example 5 : Twelve-well cell culture plates were coated with varying concentrations (1, 2, 3, and 4 pg / cm2) of the anti-CD3 antibody, and the cells were seeded and compared to TransACT. Following 4-5 hours of activation, CD 19 LVV was introduced to the cells. Cell count, viability, and cell phenotype were estimated on day 3 post-transduction (figure 15 A to D). The CAR percentage on day 3 was comparable between cells activated with anti-CD3 and those activated with TransACT methods.

[0186] No significant difference in the percentage of CAR expression was observed with anti-CD3 antibody coating concentrations ranging from 1-4 pg / cm2compared to Trans ACT-activated cells (figure 15 A). However, anti-CD3 coating increased the naive T cell population compared to TransACT activation (figure 15D). The anti-CD3 -based activation is milder compared to TransACT. To further improve the present CAR-T process, anti-CD3 and RetroNectin were coated onto G-Rex systems.

[0187] Comparison between CD3 and CD4 / CD8 selection to evaluate the effectiveness and functionality of T cells selection based on these markers. To reduce the manufacturing cost a comparative evaluation was performed using CD3 nanobeads, CD4 / CD8 beads in T cell isolation.

[0188] Example 6: In an embodiment, additional experiments were conducted to analyze the impact of using the selection agents anti-CD3 and CD4 / CD8, combined with the activation agents (CD3 and TransACT), and transduction enhancers39(RetroNectin and Synperonic F108), Figure 16A illustrates the experimental layout. Two healthy donors' huffy coats were processed using either CD3 or CD4 / CD8 selection beads. The selected T cells were seeded for CAR-T generation with the activation agents CD3 and TransACT and the transduction enhancers RetroNectin and Fl 08. T-cell transduction was performed after 12 hours of activation by adding CD 19 LVV at 2.5 MOI with or without Fl 08, followed by serum addition after 6 hours of transduction. After 3 days of LVV addition, cell number, viability, and percentage CAR were measured. No significant differences in %CAR, viability, activation status, and cell phenotype were observed on day 3, as shown in Figures 16A-F. The results indicate that using either CD3 or CD4 / CD8 beads for isolating / selecting CD3 cells from apheresis or PBMC does not impact the functionality of CAR-T cells.

[0189] Example 6A: Experiments were conducted to analyze the impact of using activation agents anti-CD3 and TransACT with transduction enhancers (e.g., RetroNectin and Synperonic F108) in different culture media, with and without serum supplementation. T cells from healthy donors were seeded for CAR-T generation with these agents in G-Rex 24-well plates and compared with cells seeded in 12-well plates, using three different culture media: TheraPEAK, TexMACS, and LymphoONE. Plates were coated with anti-CD3 and RetroNectin for 3-5 hours before seeding. T-cell transduction was performed after 12 - 16 hours of activation by adding CD19 LVV at 2.5 MOI with Synperonic F108, followed by serum addition 6 hours post-transduction. After 3 days, cell number, viability, phenotype, activation status, and CAR percentage were measured (Figure 17). Results showed no significant differences in CAR percentage, viability, cell number, or cell phenotype on day 3. The data indicated that G-Rex plates, when coated with T cell activation reagents, produced activated T cells with similar activation status to TransACT beads. LymphoONE media resulted in a decreased CAR percentage from all three donors, while TheraPEAK and TexMACS performed well in terms of CAR percentage (figure 17A), cell expansion (figure 17C), viability (figure 17B), activation status (Figure 17E), and phenotype (figure17 D). The G-Rex container yielded slightly better CAR percentages and cell phenotypes compared to standard cell culture plates.

[0190] Example 7: MOI titration: Lentivirus is the key raw material for CAR-T manufacturing process. MOI titration is performed to evaluate the right MOI for LVV transduction which results in high CAR percentage of at least (10%) without affecting the phenotype of CAR-T cells. 1 to 10: namely MOI 1, MOI 2.5, MOI 5, MOI 7.5 and MOI 10 were validated with healthy donor selected T cells. One million cells per mL T cells were plated in a 24 well plate and activated by TransACT. After 12 to 16 h of activation virus is added. Post 48-60 h of LVV addition on day 3 cell numbers, percentage viability, percentage CAR (%CAR) and phenotype were measured and is shown in figure 7 A to C.

[0191] The study shows that, higher multiplicity of infection (MOI) during LVV transduction leads to an increase in the percentage of CART cells. However, when the MOI exceeds 5, cell proliferation decreases (7B). In some embodiments the MOI range from 1-10 preferably the MOI range from 1 to 5. Considering the cost of LVV manufacturing, and the safety, an MOI of 0.5 to 2.5 was chosen for subsequent experiments, along with the utilization of transduction enhancers. The statistical analysis employed was an ordinary one-way ANOVA test.

[0192] Comparative analysis of CAR-T expression using varying MOI in healthy donor T cells. Three T cell samples from healthy donors were transduced with multiplicities of infection (MOI) of 0.01, 0.1, and 1, respectively in presence of transduction enhancer Fl 08. The average CAR expression percentage for the 1 MOI group was 46.6%. In contrast, CAR expression percentages were less than 0.4% for the 0.01 MOI group and less than 4% for the 0.1 MOI group. No significant differences were observed in cell expansion, viability, or phenotype across the three donor samples (Ref. Figure 27A-D).

[0193] Evaluating Lentiviral Transduction using Transduction Enhancers:

[0194] RetroNectin®: To increase the LVV transduction in present CAR-T process, recombinant human fibronectin fragment (rFN-CH-296) contains three functional domains: the cell -binding domain, the heparin-binding domain, and the CS-1 sequence is evaluated. RetroNectin® (RN) a commercial availablerecombinant human fibronectin fragment, know to enhances lentiviral- and retroviral-mediated gene transduction by aiding the colocalization of target cells and viral particles. Specifically, virus particles bind RetroNectin reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptor VLA-5 and VLA-4 with the fibronectin C-domain and CS-1 site, respectively in T cells, B cells, monocytes, NK cells, eosinophils, bone marrow monocytic cells, and lymphoid progenitors. Thus, by facilitating close physical proximity, RetroNectin® enhances viral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and / or VLA-5. The T-cell population expanded in the presence of RetroNectin® contains a high proportion of naive T cells.

[0195] Example 8 : RetroNectin® titration was performed ranging from 5 pg / cm2, 10 pg / cm2, 15 pg / cm2 in 12 well plate. RetroNectin® reagent was coated on plates for 4 to 5 h in IxPBS and incubated at 37 °C incubator with 5 % CO2 and cells were seeded after removing excess RetroNectin® reagent post incubation. Post 48- 60 h of LVV addition, cell numbers, percentage viability, percentage CAR and phenotype were evaluated and plotted in Figure 8 A to D.

[0196] The above data suggests that 5 pg / cm2concentration of RetroNectin® significantly increased the percentage CAR without compromising the phenotype of CART cells. However, concentration above 5 % shows a slow decrease in the percentage of naive population. The cell numbers and viability remained unaffected. Since coating with 5 pg / cm2and above concentration of RetroNectin® already showed saturation with respect to CAR expression, titrate with lower concentrations to range of 0.25 to 2.5 pg / cm2were evaluated.

[0197] In specific embodiments, the RetroNectin concentration can be 2.5 pg / cm2.

[0198] Example 9 : Further experiments were conducted to evaluate the effect of using low concentrations of RetroNectin® for LVV transduction. For experiment, RetroNectin® was coated on 12-well plates at concentrations ranging from 1 to 10 pg / cm2. Percentage CAR was analysed from Day 3 samples. It was observed that RetroNectin® concentrations above 2.5 pg / cm2did not show any difference in CAR percentages. However, there was a significant increase in %CAR from 1 to 2.5pg / cm2of RetroNectin® compared to wells without RetroNectin® (figure 9A). Cell expansion (figure 9B), percentage viability (figure 9C), and phenotype (figure 9D) remained unchanged with varying concentrations of RetroNectin®. Based on the data, 2.5 pg / cm2of RetroNectin® is recommended for coating the cell culture plates.

[0199] MOI titration with constant RetroNectin: Testing constant RetroNectin® concentrations with varying Multiplicity of Infection (MOI) is crucial for optimizing transduction efficiency in gene therapy applications. By maintaining a fixed concentration of RetroNectin® and adjusting the MOI, was performed to check the required viral load that ensures efficient gene transfer while minimizing cytotoxicity. This approach helps in identifying the right balance between RetroNectin® coating and viral particle concentration, leading to enhanced CAR expression and improved overall performance of the transduced cells. This data is essential for refining manufacturing process to achieve consistent and reproducible results in the generation of CAR-T cells.

[0200] Example 10: The experiment maintained a consistent RetroNectin® concentration of 2.5 pg / cm2while varying the MOI from 1 to 10 (figure 10A-D). A control well used 5 MOI without RetroNectin® coating. On day 3, the CAR percentage indicated a significant increase in CAR-expressing cells in wells coated with RetroNectin® at 2.5 and 2.5 MOI compared to the control well with 5 MOI and no RetroNectin® coating. These findings indicate that a 2.5 MOI is sufficient to achieve desired CAR percentages (above 20%) with RetroNectin® coating, as shown in figure 10A, resulting in a 2-3-fold reduction in the cost of lentiviral vector (LVV) manufacturing and improving the safety. Additionally, the percentage population of naive cells was optimal with RetroNectin® at 2.5 and 2.5 MOI.

[0201] LentiBOOST® (LB) or poloxamer-based transduction enhancer or (Poloxamer 338 / synperonic Fl 08 and polybrene) is an effective, non-cytotoxic transduction enhancer designed to improve the efficiency of lentiviral vector (LVV) transduction. It acts as a universal, receptor-independent adjuvant, facilitating the fusion of lentivirus with the cell membrane and significantly improves lentivirus transduction efficiency by increasing the permeability of cells, facilitating the LVparticles into the cells. This technology significantly enhances transduction efficiency, even at lower multiplicity of infection levels, and is applicable to a wide range of cell types, including primary T-cells and hematopoietic stem cells (HSCs).

[0202] Example 11: Evaluating the efficiency of lentiviral transduction with various concentrations of LentiBOOST®.

[0203] In a small-scale experiment, T cells isolated from both healthy donors and patient samples were used to assess the efficiency of LentiBOOST titration (figure 11 A and B). During the transduction process, LentiBOOST was added to the T cells along with lentiviral vectors (LVV) at concentrations ranging from 0.05-1 mg / mL, with varying multiplicity of infection (MOI). Post-transduction analysis was conducted at 48-60 hours (day-3) to evaluate the percentage of CAR expression and cell phenotype.

[0204] Experiments evaluated the efficiency of lentiviral vector (LVV) transduction with GMP grade LentiBOOST, using three different concentrations (0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL) and varying multiplicities of infection (MOIs), as shown in Figures 11A and 11B. Figure 11A illustrates the impact of LentiBOOST on CAR expression percentages on day 3 post-transduction, comparing outcomes with and without LentiBOOST for MOIs 1 through 5. Figure 1 IB demonstrates the effect of LentiBOOST on naive T cell transduction efficiency across increasing MOIs (1 to 5). The data indicate that LentiBOOST generates high CAR expression compared to LVV alone across various concentrations and MOIs. Given that all tested concentrations of LentiBOOST® resulted in similar CAR percentages, a lower concentration (0.05 mg / mL, 0.125 mg / mL, 0.25 mg / mL) was tested with a constant MOI of 2.5 (Figure 12). Additionally, LentiBOOST® combined with RetroNectin® was assessed to determine if using two transduction enhancers would significantly increase the CAR percentage (Figure 12A). Posttransduction analysis, conducted on day 3, included cell numbers (Figure 12B), viability percentage (Figure 12C), CAR percentage, and cell phenotype (Figure 12D). The results show that there was no significant increase in CAR percentage at the various tested concentrations of LentiBOOST or when combined withRetroNectin. Additionally, there were no notable changes in cell proliferation, cell viability, or cell phenotype.

[0205] Another example of a poloxamer-based transduction enhancer is Synperonic Fl 08, a non-ionic surfactant also known as poloxamer 108. It is utilized as an adjuvant to improve the efficiency of lentiviral vector (LVV) transduction.

[0206] Example-11: MOI titration with or without Synperonic F108: T cells selected from three healthy donors were used for MOI titration at 0.5, 1, and 2.5, with and without Synperonic Fl 08. Evaluation of CAR percentage on day 3 revealed that the addition of Synperonic F108 significantly increased the CAR percentage (figure 21 A). Cell proliferation (figure 2 IB), viability percentage (figure 21C), and cell phenotype (Figure 2 ID) remained unchanged, regardless of the presence of Synperonic Fl 08.Table 1

[0207] Comparing different transduction enhancers, including Synperonic F108, LentiBOOST®, and RetroNectin®, provides a comprehensive evaluation of their effectiveness in enhancing LVV transduction efficiency. LentiBOOST®, another poloxamer-based enhancer, and RetroNectin®, which aids viral vector binding and cell transduction, have both demonstrated significant improvements in transduction efficiency. By assessing key parameters such as CAR expression, cell viability, and phenotypic markers post-transduction. This evaluation ultimately informs the selection of the best transduction enhancer, thereby improving the overall efficiency.

[0208] Further experiments were conducted to evaluate the transduction efficacy of different transduction enhancer for the present invention. Comparing different transduction enhancers, such as LentiBOOST®, RetroNectin®, and Synperonic Fl 08 enables the identification of the most effective agents for improving lentiviral vector efficiency and CAR expression. This evaluation helps in refining the developing process by selecting enhancers that maximize transduction efficiency without compromising cell viability or phenotype.

[0209] Example 12: To evaluate the efficiency of three different transduction enhancers — RetroNectin, LentiBOOST (LB), and Synperonic F108 — T cells from three healthy donors were isolated. Twelve-well plates were coated with varying concentrations of RetroNectin (2.5 to 15 pg / cm2) for 4-5 hours before seeding the cells. LentiBOOST (LB) at concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, and Synperonic F108 at concentrations of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL were added during the addition of lentiviral vectors (LVV) to the cells. Transduction efficiency was measured by assessing the CAR percentage on day 3 post-transduction Both LB and Synperonic F108 enhancers resulted in comparable CAR percentages on day 3 (figure 13A), with no significant differences in cell numbers (figure 13B), viability percentage (figure 13C), or cell phenotype (figure 13D), among the different enhancers. Among all tested transduction enhancers, Synperonic F108 showed comparable transduction efficacy to RetroNectin and LentiBOOST at lower concentrations ranging from 0.25 mg / mL to 1 mg / mL.

[0210] Comparison of T cell activation and selection using anti-CD3 and TransACT beads with RetroNectin, and with and without Synperonic F108. This study compares the effectiveness of anti-CD3 and TransACT beads, both with and without the addition of Synperonic F108, in combination with RetroNectin, for T cell activation and selection. The objective is to evaluate the impact of these enhancers on lentiviral vector (LVV) transduction efficiency in T cells, focusing on parameters such as CAR expression, cell viability, and phenotype.

[0211] Example 13: To compare three test conditions: (a) activation and transduction with anti-CD3 and RetroNectin, (b) activation and tranduction withanti-CD3, RetroNectin, and Synperonic F108, and (c) activation and transduction with TransACT and Synperonic F108, the following procedure was conducted. First, 12-well plates were coated with anti-CD3 and RetroNectin. Then, cells from three heathy donors were seeded for CAR-T generation. After a 12-hour activation period, CD19 LVV was added at an MOI of 2.5, with or without Synperonic F108. Serum was added six hours post-transduction. After LVV addition, cell number, viability percentage, phenotype, and CAR percentage were measured on day 3 and the results are plotted in figure 19 A to J. The results indicate a significant increase in CAR percentage (>80% with highest observed value being 94.42%.) in test condition (b) (figure 19A and 19B). However, there are no notable differences in cell expansion (figure 19C), viability (figure 19D), activation status (figure 19E), cytotoxic potential of CAR-T cells (figure 19G, 19H, 191 and 19J), or cell phenotype (figure 19F).Table 2

[0212] Example 14: Evaluate CAR-T manufacturing using TexMACS and NutriT medium by isolating T cells from three healthy donors and one patient sample. On day 3, measure CAR percentage, cell expansion, and percentage of the naive population (Figure 18). The data show that NutriT media significantly increases CAR percentage (figure 18A), cell expansion (figure 18B), viability (figure 18C) and the percentage of the naive population (figure 18D) compared to cells grown in TexMACS media.

[0213] Customization of culture medium for culturing the activated and transduced cells. In an embodiment culture medium is customized for improving the naive cell population with robust anti-tumor activity. The media plays an important role in cell phenotype, cell growth and cell proliferation. Different media are tested for customizing the media for improving the cell growth and phenotype.

[0214] In an embodiment, the TexMACS, medium having glucose is supplemented with inosine as primary carbon source for the cell at concentration ranging from 5 to 25 mM to improve the naive population as well as improve the anti-tumour effect of CAR-T cells manufactured.

[0215] In another embodiment a glucose free AIM-V medium is supplemented with inosine as primary carbon source for the cell. The AIM-V medium is supplemented with inosine at concentration ranging from 5 to 20 mM, as the primary carbon source to improve the naive population as well as improve the anti-tumour effect of CAR-T cells manufactured.

[0216] Example 15: To evaluate cellular phenotypes using different media with an alternative carbon source. Inosine is used as an alternative source in CAR-T manufacturing to evaluate its effect on the sternness of T cells. This experiment two types of media were used a) AIMV (without glucose) and b) TexMACS with glucose (see Figure 14). In the TexMACS medium, inosine is supplemented at a concentration of 15 to 25 mM in addition to glucose, and glucose is replaced with inosine at the same concentrations in another set. The addition of inosine does not affect the percentage of CAR-positive cells but significantly increases the percentage of naive CAR-gated cells (Figure 14E) compared to total cells (figure 14D). This increase in the naive population is directly correlated with the enhanced in vivo persistence of CAR-T cells, indicating a potential improvement in therapeutic efficacy. The percentage CAR expression on day 3 is shown in figure 14A while, figure 14 B and C shows the cell viability and cell number.

[0217] Effect of serum addition in transduction efficiency and phenotype: In an embodiment, to evaluate the increase in the transduction efficiency and the naive T cell population, cells were cultured till transduction without serum. Serum was added after 6 hrs, 12 hrs and 24 hrs post transduction.

[0218] The effect of serum on transduction efficiency and on the cell phenotype, where assessed by culturing the cells in TexMACS with and without serum and the result of the respective percentage CAR, phenotype and viability of cells on day-3 is plotted in figure 5.

[0219] Example 16: In this experiment, T cells are seeded in TexMACS medium containing IL-7 and IL- 15, with or without serum. After 12-16 hours of activation, lentiviral transduction with CD 19 LVV is performed. Post 48 hours of transduction, CAR percentage, cell count, viability, and cell phenotype are analyzed and plotted, as shown in Figure 5 (A to C). Results indicate that media without serum significantly increases the CAR percentage (fig. 5A) but decreases the naive population percentage (fig.5B). Serum addition 24 hours post-transduction significantly increases the naive population percentage.

[0220] Example 17: CAR-T manufacturing was tested for 4 different conditions: Condition 1 (TexMACS with serum): T cells cultured in TexMACS Medium supplemented with serum. Condition 2 (TexMACS without serum): T cells cultured in serum-free TexMACS Medium. Condition 3 (TheraPEAK without serum): T cells cultured in serum- free TheraPEAK® Medium. Condition 4 (TexMACS, serum addition post 24 h of transduction): T cells initially cultured in serum-free TexMACS Medium, followed by serum addition 24 hours after transduction. These conditions were evaluated to check the impact of different media formulations on CAR-T cell production in short time frame. For the experiment seed selected T cells in 24- well cell culture plates with and without 3% serum and activate them. Perform virus addition after 12 to 16 hours of activation. After 24 hours of transduction, add 3% AB serum to the respective wells. Measure the CAR percentage, cell proliferation, viability percentage, and cell phenotype (figure 5). The graph shows that serum deprivation before transduction significantly increases the CAR percentage (figure 5D-F). Additionally, serum addition after transduction significantly increases the naive cell population (fig.5F). Therefore, serum deprivation enhances the CAR percentage (fig 5D), while serum addition posttransduction boosts the naive population in culture (fig.5F).49

[0221] Based on the above experiments and data, it appears that using TexMACS medium with serum supplementation after 24 hours of transduction resulted in CAR percentages comparable to those observed with TheraPEAK-containing media. However, cells cultured in TexMACS medium, with or without serum, exhibited significantly higher CAR percentages. Additionally, the use of TexMACS medium with serum supplementation after 24 hours led to a notable increase in the percentage of naive cell population compared to other conditions. The next step involves evaluating the timing of serum addition after transduction to improve the naive population and CAR percentage.

[0222] Example 18: Further to determine the serum addition timepoint, different serum addition timings were evaluated using T cells selected from healthy donors. Briefly, 1 million T cells were seeded in 12-well plates and activated using TransACT. After 12 to 16 hours of activation, lentiviral transduction was performed. 3% AB serum was added at various timepoints post-transduction (6, 12, and 24 hours post-LVV addition). The results indicate that the addition of serum at different time points does not significantly affect CAR percentage (figure 6A) or cell phenotype (figure 6B and 6C).

[0223] Example 19: In an embodiment, the process of present invention is being applied at-scale to evaluate its effect on CAR-T production. A 215 cm2bag is coated with 2 pg / cm2anti-CD3 and 2.5 pg / cm2RetroNectin. After a coating period of 3-5 hours, selected T cells are seeded into the bag and allowed to activate for 12 to 16 hours. Lentiviral vector (LVV) is then added along with 0.5 mg / mL Synperonic F108, and the cells are expanded until day 10. On days 3 and 10, CAR-T cells are frozen and their cytotoxic potential is compared and plotted in Figure 20 A to E. This process achieves a CAR percentage above 80% from day 3 to day 10 (figure 20A), without affecting cell phenotype or functionality. The comparison of day 3 and day 10 CAR-T cells shows no difference in cytotoxicity percentage (figure 20E).

[0224] The lack of difference in cytotoxicity percentage on day 3 and day 10 implies that the CAR-T cells maintain their functional potency over this short manufacturing period. This suggests that the process developed for short-durationmanufacturing is effective in producing CAR-T cells that are functionally stable and ready for therapeutic use within a shorter timeframe. The stability in cytotoxicity percentage indicates that the cells retain their ability to target and kill cancer cells effectively, which is crucial for their therapeutic efficacy.

[0225] Example 20 In an at- scale CAR-T manufacturing process using the gas permeable rapid expansion (model No. G-RexlOOM-CS) culture system, frozen apheresis samples from healthy donors and a patient from the CD 19 clinical trial were used for T cell selection via the Cytosinct 1000 system. One hundred million selected T cells were seeded in the G-RexlOOM-CS and activated with TransACT. Following transduction with CD19-LVV at 2.5 MOI with Synperonic F108, media was added on day 3. The CAR percentage, cell expansion, viability percentage, and cell phenotype were measured on days 3, 6, 8, and 10. This process achieves the CAR percentages above 45% in healthy donors and above 30% in patient. Above 40-fold and above 28-fold cell expansion was achieved. The percentage viability of the patient cells was significantly improved from day-3 to day-6 with this process. The same process can be used in conventional CAR-T production by a person skilled in the art with or without slight modifications.

[0226] Example 21: Further experiments at- scale CAR-T manufacturing are conducted using the process of the present invention. The G-RexlOOM-CS culture system is utilized for this purpose. The G-RexlOOM-CS is coated with 2 pg / cm2anti-CD3 and 2.5 pg / cm2RetroNectin. Frozen apheresis samples from healthy51donors are used for T cell selection via the MARS BAR system. One hundred million selected T cells are freshly seeded into the G-RexlOOM-CS. Following transduction with CD19-LVV at 2.5 MOI with Synperonic F108, media is added to the G-Rex on day 3. The CAR percentage, cell expansion, viability percentage, and cell phenotype are measured on days 3, 6, and 8(figure 23 A and B. The cytotoxicity between day 3 and day 10 showed no significant difference in the cytotoxic potential, (figure 23C). This process achieves the CAR percentages above 69 % in healthy donor. Above 39-fold cell expansion was achieved respectively.

[0227] Example 22: In the at- scale CAR-T manufacturing process for quick and conventional CAR-T production using the G-RexlOOM-CS system. The G- RexlOOM-CS is coated with 2 pg / cm2anti-CD3 and 2.5 pg / cm2RetroNectin. Frozen apheresis samples from healthy donors are used for T cell selection via the MARS BAR system. One hundred million selected T cells, after being frozen and thawed, are seeded into the G-RexlOOM-CS. Following transduction with CD19- LVV at 2.5 MOI with Synperonic F108, media is added on day 3. The CAR percentage, cell expansion, viability percentage, and cell phenotype are measured on days 3, 6, and 8 (figure 24). Cytotoxicity is analysed by comparing day 3 and day 10 data. This process achieves the CAR percentages above 50% in healthy donor. Above 32-fold cell expansion was achieved respectively. This process can also be used for frozen or fresh selected T cells.Table 5Table 6:

[0228] Example 23: Evaluation of Conventional CART Process vs. Method of the present Invention Using Patient-Selected T Cells: Figure 26 (A- J): This scale-down study was conducted to compare the conventional CAR-T process with the method of the present invention using patient-selected T cells, with frozen healthy donor- selected T cells as controls. Cells were seeded in a G-Rex 6M plate with each well having a 10 cm2surface area and containing 10 million cells. The conventional CAR-T process utilized MOI-5 and MOI- 12, while the method of the present invention used lesser MOI (MOI-2.5 and MOI-5) with synperonic F108 as a transduction enhancer. Notably, there was a significant increase in % CAR using the method of the present invention.

[0229] Based on the results, the current method significantly enhances the CAR-T percentage compared to the conventional method for both patient and healthy donor T cells.

[0230] For patient T cells, the conventional method a MOI of 5 and 12 resulted in 3.14% and 5.59% CAR, respectively. In contrast, the present method using 5 MOI with F108 and EVV-OS (outsourced EVV) yielded a notable increase to 39.69% CAR. Similarly, using 2.5 MOI in combination with F108 EEV-PD (in-house EVV), the CAR percentage was 28.18%, and with 5 MOI combined with F108 and LVV-PD, the percentage was 29.53%.

[0231] For healthy donor T cells, the conventional method at 5 MOI and 12 MOI produced CAR percentages of 25.52% and 32.65%, respectively. However, the present method using 5 MOI with F108 and LVV-OS achieved a substantial increase to 72.12% CAR. Using 2.5 MOI with F108 and LLV-PD, the CAR percentage was 53.73%, and with 5 MOI combined with F108 and LVV-PD, it was 55.72%.

[0232] These results indicate that the present method, utilizing specific combinations of MOI, transduction enhancer, and different LVV sources, significantly enhances CAR expression in both patient and healthy donor T cells, thereby providing a more efficient and effective approach to CAR-T cell manufacturing.

[0233] In conclusion, the results demonstrate that the present method, which incorporates F108 as a transduction enhancer, significantly improves the CAR-T percentage for both patient and healthy donor T cells compared to the conventional method. This indicates a promising enhancement in the efficacy of the CART process when using the current method.

[0234] Example 23: Generation of CART cells using the manufacturing process of present invention.

[0235] Media and Buffer preparation used in the process:

[0236] TexMACS or NutriT media supplemented with IL-7 and IL-15

[0237] CliniMACS-EDTA buffer.

[0238] Isolation of PBMCs and selection of T cells:

[0239] The buffy coat sample was diluted with IxPBS (1:1). PBMCs were isolated using Ficoll paque (Buffy coat: Ficoll; 3:4) density gradient centrifugation. After centrifugation, the lymphocyte later is separated and washed with cliniMACS- EDTA with HSA buffer twice, followed by CD4 and CD8 selection using LS columns. The Buffy coat samples can also be processed using straight from buffy coat kits which yields selected T cells.

[0240] Seeding and activation of selected T cells

[0241] The CAR-T cells were generated by seeding them at a density of 1 million cells / mL, equivalent to 0.5 million cells / cm2. The culture surface is coated with54anti-CD3 at a concentration of 2 pg / cm2and RetroNectin at 2.5 |ig / cm2. Cells are cultured in TexMACS medium, supplemented with IL-7 and IL- 15, each at a concentration of 12.5 ng / mL.

[0242] Transduction

[0243] CD19-CAR lentiviral vector (LVV) is added at a multiplicity of infection (MOI) of 2.5, along with Synperonic F108 at a concentration of 0.5 mg / mL during LVV addition. Serum is added 6 hours after LVV transduction.

[0244] Synperonic F 108 preparation: Synperonic F 108 was prepared in sterile Water For Injection (WFI) as 100 mg / mL stock. Briefly, 100 mg of Synperonic F 108 is weighed and added to 1 mL of sterile WFI in 15 mL flacon tube. This is incubated at RT for overnight and gently mixed without bubbles. Stock solution is sterile filtered with 0.22 pM filter and stored at 4 °C refrigerator for one month.

[0245] Phenotype analysis: Cell phenotype is analyzed with the help of flow cytometer. Antibodies such as CD62L or CCR7 and CD45RA are used to analyze the cell phenotype. The phenotypic characterization is carried out as follows:Table 7

[0246] Cytotoxicity assay: Cytotoxicity assay is performed by co-culturing T cells with target cells in different ratios (4:1, 2:1, 1:1, 1:2 and 1:4). Effector cells are thawed CART or UT cells which are frozen on day-3. Based on the CART cells, target cells are chosen.Table 8

[0247] Harvest: Cells were washed with cliniMACS-EDTA-HSA buffer and checked for cell count, viability, CAR percentage and cell phenotype. Functional competency of CART cells was tested with cytotoxicity assay using target cells coculture with CART cells. The rest of the CART cells were cryopreserved using freezing media.

[0248] Freezing media: For long-term stability of CART cells, the following buffers were utilized for formulation.Table 9Mix buffer 1 and 2 equally to prepare the freezing medium.

[0249] The CAR-T process of present invention was able to achieve CAR percentages above 80 % from day-3 to day- 10 without affecting the cell phenotype and functionality. The comparison of day-3 vs day- 10 CART cells showed no difference in % cytotoxicity.

[0250] The process exemplified with CD 19 CAR can also be applied to the production of other CAR-T cells, such as CD20, CD22, BCMA, CD307e, FcRF5, GPRC5D and others. This description is not intended to limit the scope of application, as the process can be adapted to target various CAR cells for different therapeutic markets.

[0251] This method can be readily applied to various types of chimeric antigen receptor cells, including T cells, NK-CAR, TIE gamma delta and hematopoietic cells or other immune effector cells, with appropriate modifications made by a skilled professional in the field. Additionally, the same process allows for cell expansion over a period exceeding 3 or 4 days, similar to conventional methods.

[0252] The present method for manufacturing CAR-T cells significantly enhances CAR expression and cytotoxicity by increasing the proportion of CAR-expressingnaive and memory cell populations. The process involves the following key steps to achieve at least more than 50% CAR expression: Firstly, a seeding density of 0.5 to 1 million cells / cm2is selected to optimize cell growth. Secondly, the culture G- REX system is coated with 2 pg / cm2of anti-CD3 activating agent and 0.2 to 2.5 pg / cm2of recombinant human fibronectin fragment for 3 to 5 hours before adding the T cells, to ensure effective activation and cell adhesion. Thirdly, after 12 to 16 hours of activation, LVV is added at a concentration of 2.5 MOI in the presence of synperonic F108 at 0.25 to 0.5 mg / mL, enhancing transduction efficiency. Fourthly, serum is added post a 6-hour serum starvation and 24 hours post-transduction to improve the proportion of naive and memory cells. Finally, the activated and transduced cells are cultured in NutriT or TexMACS media supplemented with 5 to 20 mM inosine and cytokines, providing an optimal environment for cell proliferation and function.

[0253] The unique features of the CAR-T manufacturing process of the present invention includes:1. Rapid 4-Day Process: The manufacturing process is completed in just four days, from cell isolation to harvest. This is significantly faster than conventional methods.2. In-Vivo Expansion: CAR-T cell expansion occurs primarily within the patient’s body (in-vivo), eliminating the need for extended culture time outside the body (ex-vzvo). This reduces the overall cost of CAR-T manufacturing.3. Enhanced Oxygen Environment: The use of Gas Permeable bags or G-Rex systems provides a better dissolved oxygen environment, which helps preserve or increase T cell sternness (the ability to self-renew and mature). This results in a product with greater proliferative potential and fewer exhausted T cells after infusion, requiring a lower dose compared to conventional CAR-T processes.4. Unique Coating on G-Rex: The process uniquely involves anti-CD3 and RetroNectin coating on G-Rex systems. This coating does not affect cellproliferation and viability, unlike tissue culture-treated plates or Gas Permeable bags. Transduction Enhancer: The use of the transduction enhancer Synperonic F 108, in conjunction with anti-CD3 and RetroNectin-based activation, results in a higher CAR percentage with a lower lentiviral vector (LVV) multiplicity of infection (MOI) of 2.5. Most importantly the cost for CAR-T production using the present invention reduced the cost by 1.676-fold compared to conventional CAR-T production. The present cost-effective method for manufacturing CAR-T cells, which ensures that this advanced therapy remains accessible to patients in need. By optimizing the production process, we aim to make these innovative treatments more affordable and widely available, ultimately improving patient outcomes.58

Claims

CLAIMS:

1. A method for generating a population of chimeric antigen receptor (CAR)- expressing immune effector cells, the method comprising: a. obtaining a population of cells containing immune effector cells from an apheresis sample; b. isolating / selecting the T cells from the population of immune effector cells from step (a) using CD4, CD8 beads or a combination thereof; c. seeding the isolated T cells in a culture system with a seeding density from 0.5 x 10A6 to 5 x 10A6 cells / mL; d. activating the isolated T cells by contacting them with activation agent for 12 to 16 hours to obtain activated T cells; e. performing transduction on the activated T cells obtained at step (d) with a lentiviral vector encoding a chimeric antigen receptor at a multiplicity of infection (MOI) of 0.1-10 to obtain transduced T cells, in presence of a transduction enhancer; and f. harvesting the transduced T cells expressing chimeric antigen receptor; wherein activation (step c) and transduction (step d) are performed together; and wherein the harvested cell population comprises at least more than 50% of naive cell and central memory cell population.

2. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein after step (e) the population of the transduced T cells are not expanded, or expanded for less than 4 days, less than 3 days, less than 2 days or less than 1 day.

3. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said MOI is preferably in the range 1 to 5.

4. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said59transduced T cells expressing chimeric antigen receptor is cryopreserved or administered.

5. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said activation agent is either added or coated in the culture system.

6. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 5, wherein said activation agent is selected from CD3 and / or CD28 receptor antibodies against CD3 and / or CD28 immobilized on polymer beads, or antibodies like CD3 itself or lentiviral vectors with pseudo typed envelopes co-expressing the CD3 and / or CD28 activating domains thereof.

7. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said activation agent is added at a concentration 1-4 pg / cm2.

8. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said transduction enhancer is selected from recombinant human fibronectin fragment, cationic polymer-based transduction enhancer, non-ionic amphiphilic poloxamer 338 or synperonic F108, cationic additives such as polybrene, non-ionic 9003-11-6, poloxamer F108, kolliphor P338, akti- l / 2(Akt Inhibitor VIII), cyclosporin A, cyclosporin H, chloroquine diphosphate, dasatinib, daunorubicin hydrochloride, dexamethasone, 16,16- dimethyl prostaglandin E2, eeyarestatin I, etoposide, hydroxychloroquine sulfate, prostaglandin E2, rapamycin, rosuvastatin calcium, staurosporine, teniposide or a combination thereof.

9. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said the concentration range of said transduction enhancer is in the range 0.001 to 0.25 mg / mL.

10. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein said culture system is a gas permeable rapid expansion -system.

11. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 10, wherein said gas permeable rapid expansion culture system is coated with a combination of recombinant human fibronectin fragment and anti-CD3 antibody.

12. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 10, wherein said gas permeable rapid expansion culture system is coated with recombinant human fibronectin fragment along with addition of anti- CD3 / CD28 beads.

13. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 12, wherein said CD3 antibody is present at a concentration ranging from 1 to 4 pg / cm2and said human fibronectin fragment in the range of from 0.25 to 15 pg / cm2.

14. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein in the transducing step (e) the cells are cultured in a medium supplemented with inosine in presence or absence of glucose.

15. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 14, wherein said inosine is provided in an amount 5 to 25 mM.

16. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein the transducing step (e) is performed in absence of serum.

17. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 1, wherein the transducing step (e) comprises first 6 to 10 hours of transducing the cell inabsence of serum followed by serum supplement post the 6 to 10 hours to enhance the naive cell population.

18. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in claim 14, wherein said serum is added at a concentration post 24h of transduction to enhance the naive cell population.

19. The method for generating a population of chimeric antigen receptor (CAR)-expressing immune effector cells as claimed in any of the preceding claims, wherein said chimeric antigen receptor (CAR) T cells is selected from anti-CD19 CAR, anti-CD20 CAR, anti- CD307e CAR, anti- CD 19- CD20 CAR or anti- BCMA-CD307e bispecific CAR and the like.

20. A process for generation of CAR-T cell, said process comprising the steps of : a. coating culture system with 0.25 to 2 pg / cm2of recombinant human fibronectin fragment and either coating with 1-4 pg / cm2of Anti- CD3 or adding Anti-CD3 / CD28 beads as activating agent; b. seeding isolated T cells in a G-REX system with a seeding density of 0.5 to 1 million cells / cm2; c. adding LVV at a concentration of 1 to 5 MOI to the T cells in the presence of synperonic F108 at 0.125 to 2 mg / mL after 12 to 16 hours of activation; and d. culturing the activated and transduced cells in media supplemented with or without 5 to 20 mM inosine and cytokine (IL15 and IL7); wherein steps (1 a to 1 d) take place within 1-4 days.

21. The process as claimed in claim 20, wherein said process is performed in absence or in absence of serum.

22. The process as claimed in claim 20, wherein said process comprises first 6 to 10 hours of transducing the cell in absence of serum followed by serum supplement post the 6 to 10 hours to enhance the naive cell population.

23. The process as claimed in claim 20, wherein said process comprises serum addition 24 hours after transduction.

24. The process as claimed in claim 20, wherein said process further comprises harvesting the population of transduced T cells and formulating with at least one pharmaceutically acceptable excipient for cryopreservation or administration, wherein at the time of harvesting the percentage of naive and memory cells is greater than 50% amount in the total T cell population.

25. The process as claimed in claim 24, wherein said CAR T cell population comprises 25%, to 90% of naive and central memory cells.

26. A method of treating a disease or condition in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising CAR-T cells prepared by the method as claimed in claimed 1 to 25.

27. The method as claimed in claim 26, wherein said disease or condition is selected from the group consisting of viral infection, a bacterial infection, a parasitic infection, a cancer, a malignancy, a non- cancerous condition, an autoimmune disease, a fibrotic disease, Alzheimer’s disease, protein deficiency conditions, and factor VIII deficiency.

28. The method as claimed in claim 27, wherein said cancer is selected from the group consisting of breast cancer, triple-negative breast cancer, prostate cancer, ovarian cancer, glioma, glioblastoma, renal cell carcinoma, kidney cancer, mesothelioma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, lung cancer, lung adenocarcinoma, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, hepatocellular carcinoma, esophagus cancer, brain cancer, melanoma, Hodgkin's lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, blood cancers, lymphoma, leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myelogenous leukemia, B-cell acute lymphoblastic leukemia (ALL), pre-B ALL, and any combination thereof.

29. The method as claimed in claim 27, wherein said autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), systemic sclerosis (Scleroderma), idiopathic inflammatory myositis, Rheumatoid Arthritis, multiple sclerosis, inflammatory bowel disease (IBD), type 1 diabetes, hashimoto's thyroiditis, graves' disease, psoriasis and thereof.

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