Rapid T-cell production

Abstract

The present invention provides a method for rapid production of genetically modified immune effector cells, such as T-cells, which can be used to rapidly (<1 day) produce genetically modified immune effector cells, such as CAR-T cells.

Description

Related Applications

[0001] This application is a utility patent application claiming priority to U.S. Provisional Application No. 63 / 292,843, filed December 22, 2021, which is hereby incorporated by reference in its entirety. REFERENCE TO ELECTRONIC SUBMISSION SEQUENCE LISTING

[0002] The sequence listing electronically submitted together with this application as an XML file entitled 2459S_005WO.xml, having a creation date of December 22, 2022, and being 13,000 bytes in length, is hereby incorporated by reference in its entirety. [Technical field]

[0003] The present invention relates to a rapid T cell manufacturing process that allows for the production of a genetically modified T cell product in less than a day using viral-mediated transfer, and adds innovations in product release testing to overcome hurdles to clinical use of this product.

[0004] T cell therapy has great potential for the treatment of diseases, especially cancer, infectious diseases, and autoimmune diseases. One approach to improve and enhance T cell therapy involves genetically modifying T cells using virus-mediated gene transfer to enhance the activation and / or specificity of these cells for the desired target cells. For example, the use of lentiviruses and retroviruses to express kinematric antigen receptors (CARs) on T cells has shown great potential for cancer treatment. Autologous T cells expressing kinematric antigen receptors specifically directed against CD19 (CAR-T cells) have shown significant efficacy in patients with relapsed or refractory B cell lymphoma.

[0005] The potential of genetically modified T-cells has been highlighted by the remarkable clinical success of autologous CD19CAR-T cells against relapsed or refractory non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Aggressive, relapsed, or refractory disease is treated with high-dose therapy followed by autologous hepatocyte transplantation if chemotherapy remains effective (1, 2). Unfortunately, up to 50% of patients relapse or become refractory, and traditional chemotherapy has shown disappointing results in relapsed / refractory patients (e.g., complete remission rate (CR) is 7% and median overall survival (OS) is 6.3 months (3)). In contrast, CD19CAR-T treatment has shown meaningfully improved outcomes in these patients (e.g., >50% CR at 1 year, >50% OS (4)).

[0006] Although commercially available CD19 and BCMA CAR-T cell products are FDA approved in the United States and have regulatory approval in many other countries worldwide, existing therapies are cost-prohibitive for use in many countries around the world, and even in the United States, the economic hurdles are considerable. The current manufacturing cost of autologous CAR-T products at pharmaceutical companies is estimated to be ~100k USD per patient (5). In addition to the cost, the manufacturing process is slow, causing undesirable delays in patient treatment. Current FDA-approved products take ~3-6 weeks to reach patients, resulting in disease progression in a significant number of patients due to treatment delays. Another challenge with current CAR-T therapies is their poor efficacy against most malignancies other than B-cell malignancies such as NHL, acute lymphoma leukemia, and multiple myeloma. Furthermore, even for diseases such as NHL where initial results have been promising, many patients (~50%) do not have a sustained remission after one year (6, 7). There are several possible reasons why CAR-T therapy has not achieved optimal results in patients, but one challenge is the low persistence of CAR-T cells infused into patients. It has been reported that the persistence of CAR-T cells correlates with the differentiation state of the CAR-T preparation produced. In particular, more differentiated preparations are thought to have lower in vivo persistence compared to more immature cells, i.e., naive cells (reviewed in (8)).

[0007] Traditional CAR-T manufacturing is a long, complex and costly process involving T cell isolation, T cell activation and T cell transduction (often combined with methods to improve and enhance transduction efficiency such as rotational seeding, retronectin and polybrene). T cell stimulation to enable effective transduction generally involves stimulation with CD3 or CD3 / CD28 and cytokine stimulation (e.g. IL-2, IL-7, IL-21 and / or IL-15). Not only do they often utilize complex and sophisticated manufacturing processes that require costly equipment, but the process is also time and labor intensive. For example, T cells have been isolated using magnetic beads, which involves a long and costly process. Beads require an additional step for removal, which is time consuming and requires specialized equipment and specialized manufacturing techniques. Complex manufacturing requires the use of dedicated clean room facilities to ensure the sterility of the product.

[0008] As previously known and widely practiced, to efficiently transduce T cells with a virus (e.g., lentivirus or retrovirus), T cells must first be activated in the presence of cytokines (e.g., IL-2, IL-7, and IL-15) for 1-3 days before viral transduction. Thus, conventional production requires an initial activation step with CD3 and / or CD3 / CD28 for 1-3 days before efficient transduction can be performed. It is also widely practiced to expand T cells for 1-2 weeks after activation. After viral transduction, conventional methods include a partial T cell expansion phase to generate enough T cells for patient infusion.

[0009] In a large proportion of clinical CAR-T cell manufacturing cases, T cells are isolated at an early manufacturing stage (i.e., prior to viral transduction). In particular, the T cell isolation process accounts for a large proportion of the overall rapid genetically modified T cell manufacturing process (one day or less) reported to date involving virus-based genetic engineering. In nearly all cases of T cell isolation for clinical manufacturing purposes, T cell purification is performed using magnetic beads, typically either CD3 / CD28 Dynabeads® (ThermoFisher Scientific, Waltham, MA) or CD4 / CD8 magnetic beads (Miltenyi Biotec, Bergish Gladbach, Germany). These beads, which often must be removed prior to infusion into patients, bind tightly to T cells and require several days for shedding (which typically occurs after internalization of the target surface antigen). For example, ThermoFisher Scientific reports that CD3 / CD28 beads require several days for shedding, and as a result of bead shedding, a meaningful fraction of T cells will be lost (due to bound beads) unless bead removal is attempted ~3 days in advance (asset.fishersci.com / TFS-Assets / LSG / manuals / 11131D_32D_61D.pdf).

[0010] Miltenyi Biotech reports that their magnetic beads do not detach from the cells they are bound to within 2-3 days. In fact, Miltenyi Biotech beads may internalize after a few days instead of detaching, and therefore may have unknown effects on T cells (Tthermofisher.com / us / en / home / life-science / cell-analysis / cell-isolation-and-expansion / cell-isolation / see-how-miltenyi-microbeads-interact-with-your-t-cells.html).

[0011] Simple and rapid manufacturing processes are desirable due to the high costs and therapeutic delays inherent in complex manufacturing processes, and the potential benefits of shortening the culture time of T cells, thereby maintaining naive populations (e.g., for improved enhanced in vivo persistence). In particular, a process of one day or less would be advantageous, as it shortens culture time, meaningfully increases naive T cell populations, and ultimately allows for simple closed system manufacturing outside of clean room facilities. Furthermore, a process that does not require T cell isolation can achieve advantageous benefits for a number of reasons, including reduced costs due to increased process simplicity, reduced expertise, smaller personnel numbers, and increased scalability. Furthermore, because a T cell isolation step is not required, the product can include additional cell types, such as NK cells, which have known favorable therapeutic properties, such as tumor cells or cytolysis of pathogen-infected cells. Surprisingly, it has also been found that rapid CAR-T manufacturing can be accompanied by the benefit of utilizing a mixed population of mononuclear cells (e.g., PBMCs or monocyte-depleted PBMCs) as opposed to isolated T cells. For example, in the case of isolated T cells, the highly desirable naive T cell population was reduced in the manufactured product compared to the same manufacturing process carried out using PBMCs without the T cell separation step.

[0012] Conventional clinically rapid CAR-T manufacturing processes have been reported that utilize viral gene delivery, but these manufacturing methods use a T cell separation step that adds an extra layer of cost and complexity, and the inability to detach the magnetic beads from the cells without causing significant cell loss impedes the ability to efficiently manufacture cells in the shortest possible time.

[0013] One conventional process has been reported with a <2 day manufacturing protocol involving ~24 hours of ex vivo culture (aspublications.org / blood / article / 138 / Supplement%201 / 2848 / 481328 / Preservation-of-T-Cell-Stemness-with-a-Novel). This process reportedly involves a T cell isolation step. Key differences are the method of T cell isolation, T cell activation, use of cytokines, and use of automation, which significantly increase costs, both in terms of the equipment used (~400k) and high consumable costs. Due to the use of microbeads, the product will likely be accompanied by internalized microbeads.

[0014] Another conventional process that has been reported to be able to be performed for more than one day requires the use of CD3 / CD18 Dynabeads and a high dose of IL-2 (300 μl / ml) for the isolated T cells. The use of Dynabeads results in a significant loss of T cells if the cells are harvested in one day. Furthermore, the use of a high dose of IL-2 results in greater differentiation of the T cells.

[0015] In light of the challenges faced by many current approaches to T cell differentiation due to limitations in the complex, lengthy, and costly manufacturing process of genetically modified T cells, there is a great need to develop a rapid, cost-effective, and scalable CAR-T manufacturing platform that can produce potent and affordable CAR-T products. The methods and systems described herein address this long-felt need. [Prior art documents] [Non-patent literature]

[0016] [Non-Patent Document 1] 1. Rodriguez J, Caballero MD, Gutierrez A, Solano C, Arranz R, LahuertaJJ, Sierra J, Gandarillas M, Perez-Simon JA, Zuazu J, Lopez-Guillermo A, Sureda A, Carreras E, Garcia-Larana J, Marin J, Garcia JC, Fernan Varelaz, J, Fernand Arela, J Jarque I, AlboC, Leon A, SanMiguel J, Conde E. Autologous stem-cell transplantation in diffuse large B-cell non-Hodgkin's lymphoma not achieving complete response after induction chemotherapy: theGEL / TAMO experience. Ann Uncle. 2004;15(10): 1504-9. Epub 2004 / 09 / 16. doi:10.1093 / annonc / mdh391. PubMed PMID: 15367411.

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Summary of the Invention

[0017] In accordance with the objects and advantages of the present invention, one embodiment of the present invention relates to a method for the rapid production of a genetically modified T cell population, comprising the steps of obtaining a mixed mononuclear cell population, activating a T cell population contained within the mixed mononuclear cell population at about the same time, and exposing the mixed mononuclear T cell population to a viral vector to introduce an exogenous nucleotide into at least a T cell population contained within the mixed mononuclear T cell population. No step of isolating T cells is required. In an embodiment of the method of the present invention, the mixed mononuclear cell population having at least a genetically modified population is harvested within 24 hours of the simultaneous activation and exposure to at least one viral vector.

[0018] In some embodiments, the activation step is performed by exposing the mixed mononuclear cells to an activating agent selected from one or more of the group of cytokines consisting of IL-2, IL-7, IL-15, and IL-21 and / or an activating agent directed to one or more of CD3, CD28, OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In some embodiments, the activation step is performed by exposing the mixed mononuclear cells to an activating agent selected from one or more of the group consisting of IL-7 and IL-15. In other embodiments, the activation step and the exposure step to the viral vector include a step of at least partially depleting by adherence a monocyte population included in the mononuclear cell population.

[0019] In some embodiments of the methods, the activation step and the exposure step to the viral vector are preferably performed in the absence of exogenous cytokines. The activation step can be performed by exposing the mixed mononuclear cell population to one or more of a CD3 activator, a CD28 activator, a soluble or surface-bound CD3 antibody, or a soluble or surface-bound CD28 antibody.

[0020] The mixed mononuclear cell population comprises a genetically modified T cell population. The mixed mononuclear cell population can be obtained by apheresis or by peripheral blood sampling.

[0021] In some embodiments, the viral transfer vector is selected from the group consisting of lentivirus, retrovirus, and adenovirus. In some embodiments, the substantially simultaneous activation and exposure to the viral transfer vector steps are followed by a differential centrifugation step to remove plasmid DNA from genomic DNA by DNA size selection.

[0022] Another aspect of the invention relates to genetically modified T cells produced by the above method.

[0023] Yet another aspect of the invention relates to a closed system kit for carrying out the method for rapid production of genetically modified T-cell populations according to the above method. The closed system kit of the invention may comprise a first sterile container for receiving a mixed mononuclear cell population, a second sterile container for receiving a monocyte-depleted mixed mononuclear cell population, a bead-free T-cell activator, a viral vector for introducing an exogenous nucleotide into the T-cell population, a suitable medium, and a suitable cell washing solution. In some embodiments, at least the first container is formed from a material suitable for depleting monocytes from a mixed mononuclear cell population. For the first and second containers, containers may be used that allow the introduction of the bead-free T-cell activator, the viral introduction vector, the medium, and the cell washing solution under sterile conditions.

[0024] Otherwise, the various components / reagents of the closed system kit are substantially the same as described above. Each of the first and second containers, or both, may be selected from the group consisting of a cell culture bag and a cell culture flask. [Brief description of the drawings]

[0025] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the invention are applied. In the accompanying drawings:

[0026] Figure 1A shows a comparative example of transduction efficiency using a 20 hour co-transduction / activation step and GFP lentiviral vector with different T cell activation reagents in monocyte-depleted PBMCs. After 20 hours, cells were washed to remove free virus / activation reagents and lysis buffer was used to remove Cloudz® reagent. GFP expression was determined by flow cytometry 3 days after transduction / activation. The steps used are described in the section entitled "Detailed Example of Rapid Manufacturing Steps". Panel A shows an example of the use of three different reagents with CD3 and CD28 conjugated to matrices or beads.

[0027] Figure 1B shows T cell production using a similar process to that of Figure 1A, except that soluble CD3 (100 ng / ml OKT3) or a combination of CD3 (100 ng / ml) and CD28 (300 ng / ml) was used as the activating agent, and production was performed in the presence and absence of IL-7 and IL-15.

[0028] FIG. 1C shows T cells produced according to the process shown in FIG. 1B, except that Immunocult® was used as the T cell activation reagent at the manufacturer's recommended concentration, and cytokines (IL-7 and IL-15) were used in all samples.

[0029] FIG. 1D shows an example of T cell production performed according to the steps shown in FIG. 1A, except that T cell activation was performed using soluble CD3 (100 ng / ml OKT3), surface-bound CD3 (OKT3-coated surface at 5 μg / ml), or TransAct® (used at the manufacturer's recommended concentration).

[0030] The relationship between simultaneous (table left) and sequential (table right) T cell activation is shown in Figure 2. CD19CAR expression was assessed by flow cytometry using anti-FMC63 antibody (AcroBiosystems, Newark, Delaware). Cells were examined for CAR expression 4 days after transduction. PBMCs were cultured either simultaneously with lentivirus addition or 24 hours prior to virus addition and activated with Cloudz® T cell activation reagent.

[0031] A comparative example of the use of various cytokines in modulating transduction efficiency using the rapid manufacturing process is shown in Figure 3. Monocyte-depleted PBMCs were transduced with CD19CAR lentivectors using the process described in the "Detailed Example of the Rapid Manufacturing Process" section using the indicated cytokines, and CD19CAR expression was assessed after 72 hours by flow cytometry using the FMC63 specific antibody (Acrobiosystems).

[0032] Figure 4 shows the evaluation of different culture times using the rapid manufacturing process. Monocyte-depleted PBMCs were activated and transduced with CD19CAR vector as described in the "Detailed Example of the Rapid Manufacturing Process" section. 6 or 17 hours after the start of the process, cells were washed to remove free virus and to remove CloudZ® T cell activation reagent. Cells were then maintained in culture for 72 hours and CD19CAR surface expression was assessed by flow cytometry.

[0033] Figure 5 shows that UF-KURE19 cells have improved in vivo efficacy over similar CAR-T cells produced for 6 days. NSG mice (n=5-7 / group) were inoculated with RAJI-luciferase cells (0.5×10 6 On day 7, the recommended number of UF-Kure19 CAR-T cells or CAR-T cells expressed the same CAR as when produced over 6 days.

[0034] No exogenous cytokines are required for an effective rapid T cell manufacturing process, as shown in Figure 6. NSG mice were injected with Raji-luciferase cells and 7 days later with the recommended CD19CAR-T product or vehicle, with bioluminescence imaging performed on the recommended days.

[0035] Figure 7A shows qPCR release testing with low fragment removal. Data shows TaqMan-based qPCR testing of VSVG (a replication-competent lentivirus) on DNA samples generated from CD19CAR-T transduced cells using the rapid manufacturing process and harvested after 20 hours. DNA generation was performed using the protocol described above (using PacBio reagents) for low fragment removal. PCR testing was performed on samples with low fragment removal or no fragment removal.

[0036] Figure 7B shows the data of vector copy number per transduced cell of T cells transduced with CD19CAR lentiviral vector and cultured for 8 days at various MOIs. CD19CAR expression was determined by flow cytometry using a CD19CAR-specific antibody (AcroBiosystems), and copy number was determined by qPCR targeting GAG and PTPB2.

[0037] Figure 7C shows data on vector copy number per transduced cell and CD19CAR surface expression on T cells produced using the rapid manufacturing process with an MOI of 10:1 for the vector shown in Figure 7B. Vector copy number per transduced cell was determined by qPCR and flow cytometry as shown in Figure 7B. Low fragment size DNA was removed prior to PCR using the SRE kit (PacBio).

[0038] Figure 7D shows the results of DNA gel electrophoresis. The results indicate that low fragment removal rates do not significantly affect the amount or size distribution of genomic DNA. Genomic DNA was generated from two different T cell samples harvested 20 hours after transfection with the lentiviral CD19CAR vector using the DNeasy Blood and Tissue Kit. Genomic DNA samples were subjected to agarose gel electrophoresis either directly or after low-input deletion using the PacBio 10kb SRE Kit. Lane 1: DNA marker; Lane 2: Sample 1 total genomic DNA; Lane 3: Total genomic DNA after SRE kit treatment; Lane 4: Sample 2 total genomic DNA; Lane 5: Sample 2 total genomic DNA after SRE kit treatment.

[0039] While the present invention can be modified in various ways, specific embodiments of the present invention are shown for illustrative purposes only and are described in detail below. Note that the description of the specific embodiments does not limit the present invention, and all modifications and equivalents that fall within the spirit and scope of the invention described in the claims are intended to be included. Detailed Description

[0040] One or more embodiments embodying the subject matter of the present invention will be described in detail below. Other embodiments, including modifications to the embodiments disclosed herein, will be apparent to those skilled in the art in light of the information disclosed herein. The information disclosed herein, particularly the specific details of the exemplary embodiments, is intended primarily for ease of understanding, and no unnecessary limitations should be read therefrom. In the event of any discrepancy, the present specification, including definitions, will take precedence.

[0041] To address the challenges and other shortcomings of conventional methods, and to develop a reliable, cost-effective, simple and scalable ultrafast T cell production method (one day or less), we developed a method for producing CAR-T cells that does not require the use of magnetic beads, and potentially neither T cell isolation nor cytokines. The inventors have surprisingly identified a method that can achieve high T cell transduction efficiency (starting from either mixed PBMCs, monocyte-depleted PBMCs or isolated T cells) using simultaneous T cell activation and viral transduction without the use of any enhancers (such as polybrene, spin inoculation or retronectin). Surprisingly, this method can be performed in the absence or presence of cytokines (such as IL-2, IL-7 and / or IL-15). Contrary to previous suggestions, T cell transduction efficiency using this method can be as high as that achieved by more conventional T cell activation followed by viral transduction 1-3 days later. The use of PBMCs as a starting source rather than isolated T cells has additional benefits. That is, CD3 activation can be used alone without the need for CD-28 costimulation due to the presence of other mononuclear cells that provide costimulatory signals. It is known that T cell activation by CD3 stimulation alone, without the need for CD3 / CD28 activating reagents, can preserve more of the desired central memory T cells (9). Therefore, the developed T cell process can highly simplify the process, significantly reduce costs compared to conventional methods, and enhance and improve efficiency. Ultrafast production using PBMCs, monocyte-depleted PBMCs or isolated T cells as starting material Manufacturing process development

[0042] In the method of the present invention, production can be performed with or without a preliminary separation step of T cells. Without the preliminary separation of T cells, the production process can be performed more quickly, at a lower cost, and, more surprisingly, at a higher yield of the desired naive T cells. When using blood apheresis or peripheral blood samples without direct T cell separation, T cells can be enriched by monocyte depletion by simply attaching them to a solid surface (such as tissue culture flasks / plates or bags). Monocyte depletion can be performed on an attached surface such as a plate or bag, or in a closed system. Monocyte depletion is a simple, low-cost, and rapid method to partially generate the product.

[0043] In another embodiment, the T cell separation step is not performed, so that the final product can contain not only engineered T cells, but also other cell types such as NK cells that have beneficial therapeutic effects.

[0044] In the present invention, in one embodiment, T cells are simultaneously activated using a bead-free activation reagent and transduced in the presence of low concentrations of IL-7 and IL-15 (e.g., 5-10 ng / ml or less) and, importantly, in the absence of IL-2. IL-2 is known to promote T cell differentiation, and cells cultured in the presence of IL-2 are known to have poor preservation of the naive / undifferentiated phenotype, especially when larger doses (e.g., 300 IU / ml) are used (11-12).

[0045] In another embodiment, the process of simultaneously activating and transducing T cells is surprisingly more efficient in the presence of IL-7 or IL-15, without the need for either cytokine, and thus the production process can be carried out in the presence of IL-7 alone or in the presence of IL-15 alone.

[0046] In yet another embodiment, it has been surprisingly found that the process of simultaneous activation and transduction of T cells is highly efficient in the complete absence of added exogenous cytokines, including IL-2, IL-7 or IL-15, and thus the production process can be carried out with the addition of any exogenous cytokine.

[0047] In another embodiment, the genetically modified T cell product can be produced in less than 24 hours, preferably including approximately 17-20 hours.

[0048] This process does not require a pre-activation step prior to viral transduction or T cell isolation, both of which can reduce manufacturing time and costs.To date, the use of unpurified mononuclear cells (e.g., PBMCs) or co-transfection and activation of monocyte-depleted PBMCs (as opposed to purified T cells) has not been shown to be effective for rapid CAR-T manufacturing.

[0049] Cell activation can be achieved using a variety of reagents that activate T cells via CD3 or CD3 and CD28, such as Transact (MiltenyiBiotec), Cloudz T cell activator (Biotechne), soluble or surface-bound CD3 and / or CD28 antibodies, and custom microbubbles conjugated with CD3 and / or CD3 / CD28 antibodies. Notably, Cloudz T cell activator has an unexpectedly higher ability to activate and transduce viruses into T cells than Transact. In addition, soluble or surface-bound CD3 with or without CD28 also requires less than one day of processing time.

[0050] Cell activation can also be performed using reagents that activate T cells via other costimulatory molecules other than (or in addition to) CD3 and CD28, such as OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0051] After activation and transduction of the cells, the cells can be harvested in less than a day (e.g., 17 to 20 hours) and may be used directly for therapeutic purposes or cryopreserved for later use. T cells obtained by this method exhibit high therapeutic efficacy despite the fact that expression of the gene introduced into the T cells is insufficient not only at the time of cell harvest but also at the time of infusion into the recipient.

[0052] Thus, the methods of the present invention allow for rapid manufacturing protocols for cell therapy products, with implementation times of less than one day.

[0053] In another embodiment of the method of the present invention, the T cell manufacturing process can be carried out in a completely closed system using manual or automated processes. In yet another embodiment, manufacturing can be carried out in a completely closed system outside of a clean room, allowing manufacturing to be carried out in multiple centers that do not have a dedicated clean room infrastructure.

[0054] Yet another aspect of the process of the present invention is a method to eliminate false positive reactivity in release testing required for virus-transduced cell therapy products generated during rapid manufacturing processes. Residual plasmid DNA from cell transfection (e.g. 293 and 293T cells) producing lentivirus or retrovirus is present in the cell therapy product for the first few days of manufacturing. This plasmid DNA leads to false positive results when performing virus testing qPCR assessment of vector copy number and replication competence. The process incorporates a release test, combined with a differential centrifugation-based size separation step to eliminate this plasmid DNA, enabling clinical use of rapid manufactured T cell products with high release requirements.

[0055] The subject matter of the present invention is further illustrated by the following non-limiting specific examples, which include a compilation of data representative of data collected at various times during the course of development and testing of the invention. EXAMPLES

[0056] Development of a rapid genetically modified T cell manufacturing process

[0057] In conventional methods, the rapid (e.g., 1 day or less) viral-based genetically modified T cell production process described above has an initial T cell isolation step. The rapid production process developed here can utilize PBMCs or monocyte-depleted PBMCs in addition to isolated T cells. When PBMCs are used as the starting source, monocyte depletion is performed when it is desired to partially deplete monocytes from the product in an optional early step. This deletion allows the virus to preferentially infect certain cell types that are more desirable for the final product, including, for example, T cells and possibly NK cells. The deletion is not complete, allowing monocytes to send stimulatory signals (e.g., CD80 / CD86) to T cells (e.g., T cell activation is possible using only exogenous CD3 stimulation, without the need for co-stimulation with exogenous CD28 or other stimuli). In general, application of the depletion method described below results in approximately 50% monocyte depletion from the starting blood product beginning after 2 hours of culture, as shown in Figure 1. Continuation of culture after 2 hours did not significantly affect the depletion using this method. Furthermore, the cell densities at which the cell cultures were run (5x106 cells / ml and 2x106 cells / ml) showed the same results, whether in tissue culture plates or flasks or in a closed system such as the VueLife® "AC" series bags manufactured by Saint Gobain (Paris, France).

[0058] Table 1: Monocyte depletion from PBMCs TIFF2024546145000002.tif50119

[0059] Mononuclear cells from peripheral blood apheresis samples were cultured in 6-well tissue culture plates in a tissue culture incubator. The incubation was carried out at a temperature of 37° C. for the indicated times. Monocyte percentage was determined among adherent cells at the following time points using a hematology analyzer (Hemavet®, Drew Scientific, Miami Lakes, Florida). The starting monocyte percentage of the starting population was 25.02%. MO%=percent monocyte.

[0060] When using isolated T cells, the T cells can be isolated directly from whole blood, apheresis samples, or another source of T cells. The T cells can be isolated by any available method. For example, a magnetic bead approach can be used using CD3, CD4, or CD8 magnetic beads (e.g., Miltenyi Biotec microbeads or ThermoFisher Scientific dynabeads®). The T cells can also be efficiently and rapidly isolated using CD3 microbubbles, CD3 / CD28, or microbubbles conjugated with CD3 or other T cell costimulatory ligands. Specifically, lipid microbubbles can be used to provide a rapid and efficient isolation while simultaneously delivering an activation signal.

[0061] T cells can be activated by several approaches. When using PBMCs or mononuclear PBMCs, soluble activation reagents or surface binding (e.g., plates / bags) are preferred over magnetic bead approaches for rapid manufacturing processes. One effective activation approach uses CD3 / CD28 soluble microspheres (Clouds® Human T Cell Activation Reagent, Biotechne / R&D Systems, Minneapolis, Minnesota). This reagent is composed of an alginate copolymer that dissolves within minutes, does not require magnetic beads, and does not cause product contamination. Other activation reagents can be used, such as Immunocult® CD3 / CD28 Activator (Stem Cell Technologies, Vancouver Canada). Other activation agents that can be used include CD3 or CD3 / CD28 magnetic beads (e.g., Dynabeads® ThermoFisher Scientific) or TransAct® (Miltenyi Biotech). Magnetic bead-based and TransAct agents are not optimal. Magnetic beads are labor intensive and require a problematic removal step due to their strong initial binding. This results in excessive cell loss. TransAct® activation reagent has been reported to be a "soft and slow" activation reagent compared to other products, with optimal virus transduction windows reported at later times, and to require 1-2 days of T cell activation prior to virus transduction to allow optimal virus transduction. Furthermore, TransAct® has been reported to inhibit T cell proliferation if washed out 2-3 days after addition (miltenyibiotec.com / upload / assets / IM0017348.PDF). Thus, TransAct® is not a preferred activation reagent for manufacturing protocols that involve simultaneous activation and transduction and product harvest in less than one day.

[0062] In addition to matrix-compatible activation reagents, the use of soluble or surface-coated CD3 antibodies (e.g., OKT3 antibody, other CD3 antibodies, etc.) alone or in combination with soluble CD28 antibodies or other costimulatory agents is another effective activation method that is well suited to the rapid manufacturing process of the present invention. Notably, this method benefits from the PBMC-based approach to activate T cells without the need for exogenous CD28 or other reagents for costimulation. In addition, the application of this method using CD3 antibodies such as OKT3 can significantly reduce costs compared to other activation stimuli, while minimizing the risks associated with scaffolds (e.g., alginate, magnetic beads, etc.) present in the manufactured product that may cause unknown side effects to patients.

[0063] Specific T cell activation reagents have been found to work more effectively in the rapid manufacturing process of less than one day described above. To compare specific T cell activation reagents, monocyte-depleted PBMS (200,000 cells in 100 μl) were cultured in 96-well plates in 3% CTS® immune cell serum replacement (ThermoFisher Scientific) in TexMACs® (Miltenyi Biotec) medium containing IL-7 (10 ng / ml) and IL-15 (5 ng / ml). GFP lentiviral vector and activation reagent (using the manufacturer's recommended concentration) were added and cells were cultured for 4 days. Cells were washed and medium was changed after 20 hours to remove free virus and free activation reagent. For Cloudz®-containing cells, activation reagent was removed using lysis buffer. GFP expression was measured by flow cytometry. As shown in Figure 2A, Cloudz® human T cell activation reagent was more efficient than TransAct® and CTS CD3 / CD28 Dynabeads® in maximizing transduction efficiency using the rapid manufacturing process. It should be noted that in many of the accompanying figures, the amount of viral vector used was kept low to facilitate differentiation between conditions, as opposed to using an excess amount to maximize transduction efficiency. In Figure 2, the ability of the rapid manufacturing process to maintain high transduction efficiency when high levels of lentiviral vector are used (while maintaining vector copies below 5 per transduced cell) is seen. As also shown in Figure 2B, the rapid manufacturing process is also applicable to soluble CD3 and / or soluble CD3 and CD28 antibodies (Figure 2C). For example, the use of the widely used CD3 antibody, OKT3, is applicable to this process (Figure 2C). When monocyte depletion is used in conjunction with this manufacturing process, the use of CD3 alone functions similarly in terms of transduction efficiency to CD3 and CD28 antibody supplementation. It should be noted that in this manufacturing process, OKT3CD3 antibody stimulation was more efficient in terms of transduction efficiency than Immunocult® (Figure 2C).Using the rapid manufacturing process of the present invention, both surface-bound (e.g., plate or flask) and soluble CD3 antibodies activate CD4 and CD8 T cells similarly, as measured by CD69 expression at the time of product harvest (20 hours after the start of culture). It is noteworthy that Transact® was observed to reduce the level of CD69 upregulation in CD4 T cells compared to the group treated with soluble / surface-bound CD3. This fact indicates that soluble CD3 or CD3 bound to culture vessels can be used efficiently for T cell activation using the rapid process of the present invention. Furthermore, it is also observed that these activation reagents are superior to other reagents, such as TransAct®.

[0064] It is generally believed in the art that sequential T cell activation followed by viral transduction is necessary to maximize viral transduction efficiency. However, the above results show that, using at least one embodiment of the manufacturing process of the present invention, simultaneous T cell activation and viral transduction methods provide essentially equivalent transduction efficiency to sequential methods. For example, monocyte-depleted PBMCs were activated using Cloudz T cell activation reagent and transduced with lentiviral CD19 Chimeric Antigen Receptor (CAR) vectors either simultaneously with or 24 hours after the addition of Cloudz® using the manufacturing process described in the "Detailed Example of Rapid Manufacturing Process" section. As can be seen from Figure 2, there is no meaningful difference between CAR expression using simultaneous transduction and sequential viral transduction using the manufacturing process described above.

[0065] Both conventional and the above rapid T cell manufacturing approaches almost uniformly employ culture conditions using cytokines such as IL-2, IL-15 and / or IL-7. To evaluate the optimal cytokine to employ in the rapid manufacturing process, the manufacturing process described in the "Detailed Example of Rapid Manufacturing Process" section was performed using IL-2 (300 μ / ml), IL-15 (5 ng / ml), IL-7 (10 ng / ml), a combination of IL-15 and IL-7, or in the complete absence of exogenous cytokines (Figure 4). When comparing CD19CAR expression in T cells 72 hours after transduction between the different cytokine groups, no difference in transduction efficiency was observed. Surprisingly, no difference in transduction efficiency was observed even in the complete absence of cytokines. Thus, the manufacturing process can be simplified to be performed in the absence of exogenous cytokines, further reducing costs and reducing stimulation of T cells that leads to undesired differentiation / activation. As can be seen from Figure 1B, the rapid T cell manufacturing process was almost as efficient as GFP transduction of T cells when compared to production performed in the presence and absence of cytokines.

[0066] Another key attribute of the manufacturing process is the culture period. Since one objective of the manufacturing process is to limit the culture period and to preserve the fraction of naive T cells, we compared manufacturing processes using culture periods of 6 and 17 hours. At the end of both culture periods, the Cloudz® reagent was removed using lysis buffer, the cells were washed to remove virus, and suspended in fresh medium without virus or activation reagents for a total of 72 hours of culture and CAR expression was measured. As shown in Figure 4, a 6-hour culture period of lentiviral vector and T cell activation reagent was insufficient to yield meaningful CD19 CAR expression by flow cytometry at day 3 of culture period. Intermediate periods of more than a few hours but less than approximately 15 hours are generally not preferred due to the constraint of having to perform manufacturing during working hours.

[0067] The manufacturing process uses PBMC or monocyte-depleted PBMC as the starting source, with less than one day of culture time, so the final product consists of T cells plus incidental mononuclear cells. To evaluate the composition of the product, the manufacturing process was performed in triplicate using the method described in the "Detailed Example of the Rapid Manufacturing Process" section. As shown in Table 2, T cells are the largest fraction of the product, but the product contains small numbers of B cells, NK cells, and monocytes. Since the preferred manufacturing method produces a frozen product, testing was performed after the product was thawed. Granulocytes were barely detectable in the product, as they are not only present in small amounts in the starting apheresis sample, but are also extremely sensitive to freeze / thaw.

[0068] Table 2. Product composition obtained in three manufacturing runs using thawed UF-KURE19 (17 manufacturing hours) product. TIFF2024546145000003.tif44147

[0069] Product composition was determined and viability was assessed using flow cytometry for CD3, CD4, CD8, CD19, CD56 and CD14 antibodies as well as 7-AAD.

[0070] Since meaningful expression of proteins such as CAR does not occur in T cells within 17-20 hours after lentiviral transduction, to evaluate the in vitro activity of the transduced CD19CAR-T product, CD19CAR-T cells were produced from monocyte-depleted PBMCs using the manufacturing process described in the "Detailed Example of Rapid Manufacturing Process" section. After 20 hours of activation / transduction, the cells were washed free of virus and CloudZ® T cell activation reagent was removed. The cells were then cultured for a total culture period of 3 days before being evaluated for cytotoxic activity against target RAJI human lymphoma cells and CAR surface expression. As can be seen from Table 3, the rapidly manufactured CAR-T cells were able to lyse RAJI tumor cells at a high rate.

[0071] Table 3. Results of in vitro testing of cytotoxic activity of rapidly manufactured CD19 CAR-T cells after 3 days of culture. TIFF2024546145000004.tif30158

[0072] Monocyte-depleted PBMCs were transduced with CD19CAR lentiviral vector as described in the "Detailed Example of Rapid Manufacturing Process" section, and T cells were activated with CloudZ® T cell activation reagent. Free virus was removed, T cell activation reagent was dissolved after 20 hours, and cell culture was continued for 3 days. Cytotoxic activity of CD19CAR-T cells against RAJI tumor cells was assessed by measuring calcein AM dye loss from tumor cells after 4 hours of co-culture with CAR-T cells by flow cytometry. CD19CAR expression was measured by flow cytometry using FMC63 specific antibody (AcroBiosystems).

[0073] Since the CD19CAR protein was not meaningfully expressed on the surface of T cells by standard flow cytometry methods when the product was harvested after 17-20 hours of culture, the efficacy of the product was evaluated by assessing T cell activity in a mouse model. In this way, full expression of the CAR occurs in vivo, and T cells can acquire cytotoxic activity against cells expressing human CD19. To demonstrate the improved efficacy of the rapid-manufactured CAR-1 product, this in vivo study utilized cryopreserved cells that were produced in 17 hours by transducing CD19CAR lentivirus using monocyte-depleted PBMC as starting material as described in the section "Detailed Example of the Rapid Manufacturing Process." The product expressing CD19CAR is referred to as UF-KURE19 cells. The cells using the same process but continued to be cultured for 6 days instead of 17 hours are referred to as Kure19. A circulating mouse model of human malignant lymphoma was tested in which human RAJI tumor cells were injected intravenously into immunodeficient mice (NSG), followed by intravenous injection of one dose of the CAR-T product 7 days after tumor cell injection. Previously, ~5 million CAR-T cells were used in this model to demonstrate meaningful efficacy, but the dose was lowered in anticipation of improved efficacy of the rapid manufacturing product of the present invention. For this UF-KURE19 product, the UF-KURE19 groups were administered 2 and 4 million CAR-positive T cells, while the 6-day manufactured product was administered 2 million CAR-positive T cells. As can be seen from Figure 5, UF-KURE19 cells show significant efficacy at low and high doses. In contrast, the 6-day culture product inhibits tumor progression compared to vehicle-treated mice, but with significantly reduced efficacy compared to the UF-KURE19 product. Similar to the mouse studies with Kure19, the UF-KURE19 injected groups responded well to treatment (as measured by weight change, food intake, appearance and behavior) with no obvious signs of toxicity.

[0074] CD19CAR-T cells have been shown to be effective (as measured by transgene detection) for months and in some cases years. For example, the Tisa-cel product has been shown to be effective for at least 2 years in some patients with favorable clinical outcomes (10). Human T cell proliferation in the blood of NSG mice bearing RAJI lymphoma tumors was measured by flow cytometry. Figure 9 shows the average number of human T cells detected per μl of mouse peripheral blood at given time points after injection of RAJI tumor cells (0.5 million iv) into female NSG mice (n=3–5 measurements per time point). It should be noted that the blood samples from which these measurements were taken were derived from the mouse efficacy study shown above. As shown in Table 4, the efficacy of UK-KURE19 cells was significantly higher than that of 6-day-old KURE19CAR-T cells, which correlates with human T cell proliferation.

[0075] Table 4: Number of cells detected by flow cytometry per μl of mouse blood TIFF2024546145000005.tif33163

[0076] As mentioned above, the use of cytokines during the rapid manufacturing process was not necessary to achieve meaningful transduction efficiency. Therefore, the effect of cytokine use on the memory / differentiation state of T cells at harvest (20 hours) was evaluated by flow cytometry as follows. Furthermore, this evaluation was performed with different cytokines or without cytokines in the manufacturing process shown in Figure 10 to examine the effect of cytokines on the product. Finally, a comparison was made between the use of monocyte-depleted PBMCs and isolated T cells as the starting source. The manufacturing process described in the section "Detailed Example of Rapid Manufacturing Process" was used, except that purified T cells were used as the starting source for a subset of samples. T cells were purified from peripheral blood using the Human T Cell Enrichment Kit from RosetteSep® (StemCell Technologies). As can be seen from Table 5, it is observed that the CD8+ T cell components of the products are not identical when comparing the products starting from isolated T cells and monocyte-depleted PBMCs. Notably, the PBMC starting source results in slightly lower levels of effector memory T cells and a slightly higher percentage of TEMRA cells. The role of CD8+TEMRA cells is not fully understood, but may result in stronger beneficial cytotoxicity. The levels of highly beneficial naive T cells, known to correlate with CAR-T efficacy, are similar when starting with PBMCs or isolated T cells in the CD8+ T cell compartment, but the percentage of beneficial central memory T cells is higher in PBMCs compared to isolated T cells and lower levels of more differentiated, less effector memory T cells. In the CD4+ T cell compartment, the percentage of highly beneficial naive T cells is higher when using PBMCs as a starting source than isolated T cells. Thus, based on product phenotyping, starting with a process using PBMCs appears more advantageous, resulting in improved clinical outcomes and improved in vivo persistence of CAR-T cells. Additionally, this study investigated the use of different cytokines (IL2, IL7, IL-15) or no cytokines during manufacturing.As can be seen from Table 5, when starting from PBMCs, there were no significant differences in terms of T cell memory / differentiation phenotypes at the 20 hour step for any condition, suggesting that cytokines are surprisingly not a necessary component of this manufacturing process when starting from monocyte-depleted PBMCs. Interestingly, when starting from isolated T cells and no cytokines were used, a decrease in naive T cells was observed, suggesting the importance of using cytokines during manufacturing when starting from isolated T cells, i.e. PBMCs can be an endogenous source of cytokines.

[0077] Table 5. Phenotypes of T cells produced using the rapid process CD8+T cells TIFF2024546145000006.tif86170 TIFF2024546145000007.tif211170

[0078] T cells were produced using the rapid process described in the "Detailed Example of the Rapid Manufacturing Process" section, or using the same process as this one except using isolated T cells as the starting cell source. After 20 hours, cells were assessed for T cell phenotype by flow cytometry.

[0079] Since the in vivo studies demonstrated that cytokines were not required for efficient T cell transduction or to maintain the preferred memory / differentiated T cell phenotype using a rapid manufacturing process starting from PBMCs, in vivo efficacy studies in mice were performed to further confirm these findings. CD19 CAR-T cells were manufactured using a rapid manufacturing process starting from monocyte-depleted PBMCs, with either IL-7 (10 ng / ml) or IL-15 (5 ng / ml) in culture, or without cytokines. The same human lymphoid tumor model (RAJI) was used in NSG mice as above. In this case, 1.2 × 10 per mouse were cultured 7 days after tumor cell injection. 61000 CD19CAR positive T cells were injected into the control mice. As can be seen from Figure 6, the CAR-T product produced in the complete absence of exogenous cytokines was able to suppress cancer progression as efficiently as the product produced with cytokines. All control mice died of cancer progression before imaging on day 45. It should be noted that the low dose of 1.2 × 10 6 CD19 CAR positive T cells from 1000 cells demonstrated high efficacy as shown in Figure 6A, further demonstrating the robustness of the rapid process CAR-T product. This study also clearly confirms that no exogenous cytokines are required to generate a rapid (<1 day) manufactured CAR-T product from a PBMC starting source.

[0080] In addition to similar efficacy, the amount of human T cells circulating in the mice was not reduced when the cytokine-free product was used. Human T cells were quantified in mouse blood by flow cytometry analysis and in the experiment shown in Figure 6, which was performed 41 days after tumor cell injection using a human-specific CD3 antibody. No cytokine CAR-T group: 4495 human T cells / microliter mouse blood. IL7 / IL15 CAR-T group: 1618 human T cells / microliter mouse blood. Detailed Example of Rapid Manufacturing Process

[0081] Some representative application steps added to this process are shown in parentheses. In addition to the steps below that require relatively high manual operations, the entire process can be performed in a fully enclosed and / or semi-automated or automated state. For example, cell washing, cell collection, etc. can be performed using automated equipment. Manufacturing process starting from monocyte-depleted PBMCs to produce CAR-T products in less than one day A. Obtaining Starting Cells (Day 1 or Day 0) Autologous peripheral blood mononuclear cells were obtained from patients using leukapheresis (or peripheral blood draw). The cell product was either processed immediately or kept overnight (the cell product could be frozen and processed at a later date if desired). B. Processing of Apheresis Samples (Day Zero) Apheresis samples were washed by centrifugation to remove plasma and reduce contaminating platelet counts (PBMCs were isolated by, for example, a Ficoll-based separation method if peripheral blood draws were used instead of apheresis samples). Apheresis samples were then diluted in medium such as TexMACS (Miltenyi Biotec) supplemented with CTS immune cell serum preparation (ThermoFisher Scientific) and cultured in tissue culture flask(s) or cell culture bags in a 37°C (± 2°C) culture incubator for monocyte attachment at a concentration of up to 5 million cells per ml. Monocytes were allowed to adhere to the flask(s) or bag(s) for at least 2 hours, after which non-adherent cells were transferred to a new tissue culture flask or bag. The cell number was adjusted to 2 million per ml using medium containing IL7 (10 ng / ml) and IL15 (5 ng / ml) (medium can be prepared without the addition of any exogenous cytokines, or with IL-7, IL15 alone, or with other cytokines). C. Cell Activation (Day 0) Cloudz® CD3 / CD28 T cell activation reagent (R&D Systems / Biotechne) can be added to the flask containing the monocyte-depleted apheresis product (other activation reagents can also be used, including soluble CD3 antibody, soluble CD3 / CD28 antibody, surface-bound CD3 antibody with or without CD28 soluble antibody, Immunocult® (Stemcell Technologies), and other T cell activation reagents). D. Cell transduction (day zero) Viral vector was added to multiple flasks or bags containing monocyte-depleted PBMCs immediately after addition of T cell activation reagents, using multiple infections determined to result in copy numbers per cell below 5. Cells were cultured for 17-20 hours at 37°C (±2°C) in a 5% (±0.5%) CO2 incubator. E. Cell collection (1st day) If cells were cultured using Cloudz reagent, GMP-grade 6X Release Buffer (R&D Systems / Biotechne) was added directly to the flask or bag containing the activated / transduced cells. The cells were then washed and resuspended in a freezing buffer such as Plasmalyte-A, 5% HSA and 5% DMSO in the cryobag(s) or vial. Describe a methodology that allows for vector copy number and replication-competent virus release testing using rapid engineered T cell products

[0082] A key hurdle in the field of rapid T cell manufacturing processes involving viral vectors is the necessary product release testing, and the following methodology was adopted. Current release testing requirements for retroviral or lentiviral transduced cell therapy products include assessment of replication-competent lentivirus and vector copy number. Both assessments are problematic because traditional qPCR-based assessment methods produce false positive results even at low levels of free plasmid contamination. Residual free plasmid from 293 cell transfection is present in the viral vector. At the early manufacturing time point after viral transduction, plasmid from the vector remains, making it nearly impossible to eliminate false positive results using traditional testing methods. This false positive reactivity is a major hurdle and limits the ability to rapidly manufacture patient-ready products.

[0083] The manufacturing process used here, when combined with a method to remove free residual plasmid DNA, allows PCR and other molecular tests to be performed on both vector copy number and replication-competent lentiviruses without this false positive reaction, since the DNA of interest for both viral integration and replication-competent vectors is the integrated viral DNA. Notably, free plasmid DNA is much smaller than genomic DNA. Using a centrifugation-based method, the larger genomic DNA can be preserved while completely removing the small DNA. This method allows the measurement of the integrated DNA without the false positive reaction from plasmid DNA, which is a major obstacle in rapid CAR-T manufacturing.

[0084] To remove free plasmid DNA in the short-term (e.g., 0–3 days) expanded CRT-T product, centrifugation can be used (e.g., using a salt / polymer solution that preferentially precipitates high molecular weight DNA) while still retaining genomic DNA, after completely removing small sized DNA (e.g., <10 kb). For example, the PacBio Short Read Eliminator Kit (PacBio; Menlo Park, California) can be used to perform this separation in a single centrifugation step (circulomics.com / store / Short-Read-Eliminator-Kit-p131401036). In addition to this commercial reagent, an alternative method using 4% PVP360,000, 1.2 M KCL, 20 mM Tris-HCL ph8 in the protocol below can be used instead of the commercial buffer SRE, as it has previously been shown to effectively completely remove small sized fragments (e.g., <10 kb from total DNA) (11). The purified DNA can then be used directly in evaluation studies (e.g., qPCR evaluation). Although the kit was not designed for this purpose, it works well and is simple, cost effective, and quick to operate.

[0085] In one example use of this method, first isolate the total genome using any commercially available total genome isolation kit capable of isolating human genomic DNA (e.g., DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). Next, remove low-fragment DNA using the following procedure or a similar approach. 1. Using a wide-bore pipette tip, add Buffer SRE to the starting genomic DNA sample and mix thoroughly by pipetting. If not using PacBio or other commercially available reagents, 4% PVP 360,000, 1.2M KCL, 20mM Tris-HCL ph8 can be added to the total DNA sample in a 1:1 volume ratio. 2. Centrifuge at 10,000xg for 30 minutes at room temperature. 3. The supernatant was removed with a pipette. 4. 70% ethanol was added to the test tube. 5. Centrifuge at 10,000xg for approximately 2 minutes at room temperature. 6. The supernatant was removed and the sections were washed with 70% ethanol repeatedly. 7. Add Buffer EB (or use 10 mM Tris-HCL pH 8 if not using the PacBio kit) to the test tube and incubate at 50°C for 10 to 30 minutes to resuspend the DNA pellet.

[0086] The use of a low-fragment deletion step in conjunction with a rapid manufacturing process proved to be an effective process that allowed for release testing to be performed on genetically modified T-cell products at early time points in culture (e.g., 17 h to 3 days). For example, CAR-T products manufactured using the 17-20 h process were harvested at 20 h and surrogate aliquots were harvested at 72 h. Total DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) and low-fragment deleted DNA was prepared using the Short Read Eliminator Kit (PacBio). Total DNA samples were tested as well as low-fragment deleted DNA for replication-competent lentivirus using real-time PCR assessment directed against VSV-G and for vector integration using real-time PCR assessment directed against GAU and the housekeeping gene PTBP2. Primers and fluorescent probes used for PCR reactions are listed in Table 6.

[0087] Table 6. PCR primers and probes used for GAG, VSV-G and PTPBP2 qPCR evaluation TIFF2024546145000008.tif114147

[0088] PCR products were quantified using standard probes consisting of linearized plasmids expressing single copies of GAG, VSV-G and PTBP2. As can be seen in Figure 7A, qPCR reactions using low-fragment deletion DNA failed to detect VSV-G, i.e., the two different products produced at 20 h were negative for the expected replication-competent lentivirus. In contrast, when total genomic DNA without low-fragment deletion was used, both samples gave false-positive qPCR results for VSV-G, likely due to low-fragment-free plasmid contamination. Further evaluation was performed to see if alternative approaches such as washing the cells or culturing for 3 days (as opposed to 20 h) could solve the problem of false-positive reactivity with replication-competent lentivirus testing. Specifically, lentiviral-transduced CD19CAR-T products were harvested after 20 and 72 h, and the cells were washed extensively before genomic DNA isolation. Cells (5 × 10 cells) were cultured in 50 ml of PBS 15 times for the 72 h sample and 5 times for the 20 h sample. 6 ) were washed. VSV-G qPCR evaluation showed false-positive reactivity in all cases (20- and 72-h samples of both products), indicating that extending the incubation time to 3 days and the extent of cell washing were not sufficient to suppress this false-positive reactivity.

[0089] Of note with regard to replication-competent lentivirus testing, no clinical product has been reported to FDA in the past decade that reinforces the consistently positive reactivity seen with rapid products at 17-20 and 72 hours, suggesting that false positive reactivity is the cause (fda.gov / media / 13790 / download).Furthermore, when the incubation period was extended to 8 days, the VSV-G qPCR evaluation was negative, suggesting that free plasmid DNA was lost at this time point and that true replication-competent lentivirus was not present.

[0090] Similar to testing for replication-competent lentiviruses, another important release test for virus-transduced cell therapy products is vector integration, which is often measured by qPCR against the GAG ​​gene, where free plasmid contaminants are present in the viral vector and prone to induce false-positive reactivity. First, GAG / PTPB2 qPCR was performed to determine vector copy number in CD19CAR lentivirus-transduced T cells, and these cells were maintained in culture for 8 days to assess the vector multiplicity of infection (MOI) and to assess baseline complete vector integration. As can be seen in Figure 7B, at an MOI of 10:1, the vector copy number per transduced T cell reached a peak of 1.5 (i.e., CD19CAR was expressed). Next, the vector copy number per transduced T cell was determined by rapid manufacturing process using a CD19CAR vector MOI of 10:1, except when cells were harvested at 20 hours, 3 days, and 7 days. In addition, low-fragment DNA was removed using an SRE kit (PacBio). As can be seen in Figure 7C, vector integration results were similar across all time points. Of note is CD19CAR surface expression, which was very low at 20 hours, with full expression occurring by approximately 72 hours.

[0091] Finally, gel electrophoresis of the two 20-hour rapid manufactured T cell products before and after low fragment removal shows that genomic DNA was maintained as expected, with only unwanted low molecular weight fragments being deleted (Figure 7D). Furthermore, quantification of DNA concentration using a Nanodrop® spectrophotometer (ThermoFisher Scientific) showed no measurable change in DNA concentration before and after low fragment removal, indicating that only small amounts of DNA were removed.

[0092] As can be seen from the above, the CRT-T cell manufacturing process of the present invention can achieve significant improvements over conventional methods, including but not limited to the following: 1. Manufacturing can be performed using undifferentiated cells from apheresis products or apheresis cells that have been subjected to monocyte depletion mainly by attachment to solid surfaces. In addition, peripheral blood mononuclear cells (PBMCs) directly isolated from peripheral blood or monocyte-depleted PBMCs can also be used. Thus, purified T cells, which were considered to be important for rapid CAR-T processes using viral transduction, are not required. To the best of the inventor's knowledge, this is the first protocol for manufacturing in less than one day using PBMCs instead of isolated T cells as the starting source for rapid genetically modified T cell manufacturing using viral transduction. 2. Use a different method to activate cells. Magnetic beads require several days for cells to detach, so the method using these beads results in a delayed harvest date of the product and / or loss of bead-bound cells, resulting in a much lower yield. Furthermore, unexpectedly, the inventors have found that the use of certain non-magnetic bead-based T cell activation reagents improves transduction efficiency. The activation approach of the present invention not only improves transduction efficiency, but also avoids the increased cost and complicated manufacturing steps associated with the use of magnetic beads required in conventional methods. 3. The manufacturing process of the present invention primarily features the rapid CAR-T manufacturing process described above, which can be efficiently performed in the absence of exogenous cytokines. The use of cytokines during manufacturing not only significantly increases costs, but also alters T cells. A major advantage of the rapid manufacturing process is that it allows the starting population of T cells (e.g., naive T cells) to be maintained as close as possible to the cells originally collected from the patient. A major advantage is the elimination of the use of exogenous cytokines. 4. Using a fully enclosed system to produce T cell products using bags as described above provides a convenient kit-based product, i.e. virus / media / activation reagents etc. are contained and then mixed with the apheresis product. This method allows the product to be produced outside of a clean room, facilitating widespread public use of the product and simplifying product production, reducing costs and ensuring accessibility. 5. The method of the present invention overcomes a key hurdle in the field of performing necessary product release testing. Currently required retroviral and lentiviral transduced cell therapy products are evaluated for replication-competent lentivirus and vector copy number. In both evaluations, low levels of free plasmid contamination can result in meaningful false positive results when using traditional qPCR systems. Free plasmid from virus-generating cells such as 293 cells remains in the viral vector. Early in manufacturing after viral transduction, plasmid from the vector remains, making false positive results nearly impossible to eliminate using traditional testing methods. This false positive reactivity is a major obstacle for other groups as well, hindering the ability of rapid manufactured products to be used in patients. Because the DNA of interest for both viral integration and replication-competent vectors is the integrated viral DNA, the manufacturing process of the present invention can be combined with a method to remove free residual plasmid DNA, allowing qPCR to be performed on both vector copy number and replication-competent lentivirus without eliciting this false positive reaction. Notably, free plasmid DNA is much smaller than genomic DNA. Centrifugation-based methods can be used to delete the smaller DNA while maintaining the larger genomic DNA. This method overcomes a major hurdle in rapid CAR-T manufacturing by eliminating false-positive reactivity from plasmid DNA while still allowing viable measurement of DNA that is integrated into the genome.

[0093] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0094] All patent documents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials mentioned throughout this specification are hereby incorporated by reference unless specifically noted otherwise.

[0095] When referring to URLs or other identifiers or addresses, such identifiers are subject to change, and specific information travels around the Internet, but equivalent information can be found by searching the Internet. The above references are evidence of the pervasiveness of such information.

[0096] Abbreviations used herein to refer to any protecting groups, amino acids, or other compounds are conventional abbreviations, widely accepted abbreviations, or conform to the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726-1732), unless otherwise indicated.

[0097] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the representative methods, devices, and materials are described herein.

[0098] In some cases, the nucleotides and polypeptides disclosed herein are contained in publicly available databases, such as GENBANK® and SWISSPROT. The information contained in such publicly available databases, including sequences and other information regarding the nucleotides and polypeptides, is incorporated herein by reference. Unless otherwise indicated or understood, references to such publicly available databases are to the most current databases as of the filing date of this application.

[0099] Unless otherwise indicated, all characteristics, such as amounts of ingredients, reaction conditions, and the like, used in the specification and claims should be understood to be modified by the word "about." Accordingly, unless stated to the contrary, the numerical parameters set forth in the specification and claims are approximations and may vary depending upon the objective properties sought to be achieved by the present subject matter.

[0100] The use of "for example," "including," "having," and the like herein should be understood to be accompanied by the phrase "including but not limited to," although this is not expressly stated. Similarly, "example" or "exemplary" should be understood to be non-limiting. The modifier "substantially" is a term that permits deviations from the descriptor that do not impart a negative meaning to the intended purpose. A descriptive term should be understood to be modified by the term "substantially," even if "substantially" is not expressly stated. Thus, for example, "the lever extends vertically" means "the lever extends substantially vertically," inasmuch as precise vertical alignment is not necessary for the lever to perform its function.

[0101] The terms "comprising", "including", "having", "involving", "comprises", "includes", "has", "involves", etc. are interchangeable and have the same meaning. Specifically, each term, when defined, corresponds to the general U.S. patent law definition of "comprising" and should therefore be understood as an open term meaning "at least the following" and should not be construed to exclude additional features, limitations, embodiments, etc. Thus, for example, "a process having steps a, b, and c" means that the process has at least steps a, b, and c. Whenever there is a singular term, it means "one or more", even if such interpretation is not present in the context. The terms "comprise", "include", "have", "contain" (and variations thereof) are open-ended linking verbs that, when used in the claims, allow for the addition of other elements unless the context indicates otherwise.

[0102] Where in the claims or the specification the term "comprise" or "comprise" is used in the singular, it means one or more, unless the context dictates to the contrary.

[0103] As used herein, the term "about" when preceding a numerical value or quantity, such as mass, weight, time, volume, concentration, or percentage, means in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% of the stated value, as such variations are appropriate in the practice of the methods of the invention. When the term "or" is used in the claims, it means "and / or" unless otherwise expressly stated or unless these alternatives are mutually exclusive.

[0104] As used herein, a range can be expressed as a range from "about" one specific value to "about" another specific value. A number of values ​​are disclosed herein, and each value is given as being inclusive of "about" that value as well as the value itself. For example, if the value "10" is disclosed, then the value is "about 10". Also, between two specific values ​​are inclusive values. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0105] As used herein, "optionally" and "in some cases" mean that the described event or instance may or may not occur, i.e., it includes cases where the event occurs and cases where it does not occur. For example, the term "optionally" refers to both the variable portion and the non-variable portion.

[0106] It should be understood that various details of the subject matter of the present invention may be changed without departing from the scope of the subject matter disclosed herein. Moreover, the foregoing description is illustrative only and is not intended to be limiting. Obvious modifications and variations are possible in light of the teachings disclosed herein. All such modifications and variations are intended to be within the scope of the appended claims when interpreted in accordance with the breadth fairly, legally, and equitably permitted therein.

[0107] literature 1. Rodriguez J, Caballero MD, Gutierrez A, Solano C, Arranz R, Lahuerta JJ, Sierra J, Gandarillas M, Perez-Simon JA, Zuazu J, Lopez-Guillermo A, Sureda A, Carreras E, Garcia-Larana J, Marin J, Garcia JC, Fernand Varelaz, J, Fernand Arela, J Jarque I, Albo C, Leon A, SanMiguel J, Conde E. Autologous stem-cell transplantation in diffuse large B-cell non-Hodgkin's lymphoma not achieving complete response after induction chemotherapy: the GEL / TAMO experience. Ann Uncle. 2004;15(10): 1504-9. Epub 2004 / 09 / 16. doi:10.1093 / annonc / mdh391. PubMed PMID: 15367411. 2. Vose JM, Zhang MJ, Rowlings PA, Lazarus HM, Bolwell BJ, Freytes CO, Pavlovsky S, Keating A, Yanes B, van Besien K, Armitage JO, Horowitz MM, Autologous B, Marrow Transplant Registry Lymphoma Working C. Autologous transplantation for diffuse aggressive non-Hodgkin's lymphoma in patients never achieving remission: a report from the Autologous Blood and Marrow Transplant Registry. J Clin Oncol. 2001;19(2):406-13. Epub 2001 / 02 / 24. doi: 10.1200 / JCO.2001.19.2.406. PubMed PMID: 11208832. 3. Crump M, Neelapu SS, Farooq U, Van Den Neste E, Kuruvilla J, Westin J, Link BK, Hay A, Cerhan JR, Zhu L, Boussetta S, Feng L, Maurer MJ, Navale L, Wiezorek J, Go WY, Gisselbrecht C. Outcomes in refractory diffuse large B-cell lymphoma: results from the international SCHOLAR-1 study. Blood. 2017;130(16): 1800-8. Epub 2017 / 08 / 05. doi: 10.1182 / blood-2017-03-769620. PubMed PMID: 28774879; PMCID: PMC5649550. 4. Westin JR, Kersten MJ, Salles G, Abramson JS, Schuster SJ, Locke FL, Andreadis C. Efficacy and Safety of CD19-Directed CAR-T Cell Therapies in Patients With Relapsed / Refractory Aggressive B-Cell Lymphomas: Observations From the JULIET, ZUMA-1, and TRANSCEND Trials. American journal of hematology. 2021. Epub 2021 / 07 / 27. doi: 10.1002 / ajh.26301. PubMed PMID: 34310745. 5. Harrison RP, Zylberberg E, Ellison S, Levine BL. Chimeric antigen receptor-T cell therapy manufacturing: modelling the effect of offshore production on aggregate cost of goods. Cytotherapy. 2019;21(2):224-33. Epub 2019 / 02 / 17. doi:10.1016 / j .jcyt.2019.01.003. PubMed PMID: 30770285. 6. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, Braunschweig I, Oluwole OO, Siddiqi T, Lin Y, Timmerman JM, Stiff P J, Friedberg JW, Flinn IW, Goy A, Hill BT, Smith MR, Deol A, Farooq U, McSweeney P, Munoz J, Avivi I, Castro JE, Westin JR, Chavez JC, Ghobadi A, Komanduri KV, Levy R, Jacobsen ED, Witzig TE, Reagan P, Bot A, Rossi J, Navale L, Jiang Y, Aycock J, Elias M, Chang D, Wiezorek J, Go WY. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med. 2017;377(26):2531-44. Epub 2017 / 12 / 12. doi: 10.1056 / NEJMoal 707447. PubMed PMID: 29226797; PMCID: PMC5882485. 7. Kersten MJ, Spanjaart AM, Thieblemont C. CD19-directed CAR T-cell therapy in B-cell NHL. Curr Opin Oncol. 2020;32(5):408-17. Epub 2020 / 08 / 03. doi: 10.1097 / CCO.0000000000000668. PubMed PMID: 32740094. 8. Zhang H, Zhao P, Huang H. Engineering better chimeric antigen receptor T cells. Exp Hematol Oncol. 2020;9(l):34. Epub 2020 / 12 / 10. doi: 10.1186 / s40164-020-00190-2. PubMed PMID: 33292660; PMCID: PMC7709221. 9. Li Y, Kurlander RJ. Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation. Journal of translational medicine. 2010;8: 104. Epub 2010 / 10 / 28. doi: 10.1186 / 1479-5876-8-104. PubMed PMID: 20977748; PMCID: PMC2987859. 10. Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, Jager U, Jaglowski S, Andreadis C, Westin JR, Fleury I, Bachanova V, Foley SR, Ho PJ, Mielke S, Magenau JM, Holte H, Pantano S, Pacaud LB, Awasthi R, Chu J, Anak O, Salles G, Maziarz RT, Investigators J. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B- Cell Lymphoma. The New England journal of medicine. 2019;380(l):45-56. Epub 2018 / 12 / 07. doi: 10.1056 / NEJMoal 804980. PubMed PMID: 30501490. 11. Jones A, Torkel C, Stanley D, Nasim J, Borevitz J, Schwessinger B. High- molecular weight DNA extraction, clean-up and size selection for long-read sequencing. PloS one. 2021;16(7):e0253830. Epub 2021 / 07 / 16. doi: 10.1371 / journal.pone.0253830. PubMed PMID: 34264958; PMCID: PMC8282028.

Claims

1. 1. A method for rapid production of a genetically modified T-cell population, comprising: Obtaining a mixed mononuclear cell population Substantially simultaneously with this acquisition, activating a T-cell population within said mixed mononuclear cell population; and exposing the mixed mononuclear cell population to a viral vector to introduce an exogenous nucleotide into at least the T-cell population within the mixed mononuclear cell population; A method for rapid production of genetically modified T-cell populations.

2. 10. The method of claim 1, wherein the mixed mononuclear cell population having at least genetically modified T-cells is harvested within 24 hours of the steps of simultaneously activating and exposing to at least one viral vector.

3. The method of claim 1, wherein the activation is achieved by exposing the mixed mononuclear cells to an activating agent selected from one or more of the group of cytokines consisting of IL-2, IL-7, IL-15, IL-12, IL-18, and IL21, and / or to an activating agent selected from one or more of the group of cytokines CD3, CD28, OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and wherein the activation is optionally achieved by exposing the mixed mononuclear cells to one or more of the group consisting of IL-7 and IL-15.

4. The method of claim 1, further comprising a step of at least partially depleting by adhesion a monocyte population contained in said mononuclear cell population prior to said activation and exposure to a viral vector.

5. The method of claim 4, wherein the activation and exposure to the viral vector steps are performed in the absence of exogenous cytokines.

6. The method of claim 5, wherein the activation step is carried out by exposing the mixed mononuclear cell population to one or more of a CD3 activator, a CD28 activator, a soluble or surface-bound CD3 antibody, or a soluble or surface-bound CD28 antibody, and wherein the activation step is optionally carried out by exposing the mixed mononuclear cell population to one or both of a CD3 activator or a soluble or surface-bound CD3 antibody.

7. 2. The method of claim 1, wherein the steps of activating and exposing to the viral vector are carried out without a pre-isolation or pre-activation step of T-cells.

8. 10. The method of claim 1, further comprising cryopreserving the mixed mononuclear cell population comprising the genetically modified T-cell population.

9. 10. The method of claim 1, wherein the step of obtaining the mixed mononuclear cell population is performed by apheresis or peripheral blood sampling.

10. The method described in claim 1, wherein the viral vector is selected from the group consisting of lentivirus, retrovirus and adenovirus.

11. 10. The method of claim 1, further comprising the step of performing a differential centrifugation step following the substantially simultaneous activation step and viral transfer vector step to remove plasmid DNA from genomic DNA by DNA size selection.

12. A genetically modified T-cell produced by the method of any one of claims 1 to 11.

13. A closed system kit for the rapid production method of a genetically modified T-cell population according to any one of claims 1 to 11, comprising: a first sterile container for receiving the mixed mononuclear cell population; a second sterile container for receiving the monocyte-depleted mixed mononuclear cell population; Bead-free T-cell activators, a viral vector that introduces an exogenous nucleotide into a T-cell population, optionally selected from the group consisting of lentivirus, retrovirus, and adenovirus; the appropriate medium, and Have the correct cell washing solution, comprising at least a first container with a suitable material for depleting monocytes from said mixed mononuclear cell population; The first container and the second container are sterilized to introduce the bead-free T-cell activator, the viral transfer vector, the culture medium, and the cell washing solution, and optionally, one or both of the first container and the second container are selected from the group consisting of a cell culture bag and a cell culture flask.

14. 14. The closed system kit of claim 13, wherein the bead-free T-cell activator is selected from one or more of the group consisting of a CD3 activator, a CD28 activator, an OX40 activator, a CD2 activator, a CD27 activator, an ICAM-1 activator, an LFA-1 (CD11a / CD18) activator, an ICOS (CD278) activator, and a 4-1BB (CD137) activator and a CD3 antibody; the bead-free T-cell activator is optionally selected from the group consisting of one or more soluble or surface-bound CD3 antibodies and soluble or surface-bound CD28 antibodies; and the bead-free T-cell activator is further optionally selected from the group consisting of one or more soluble or surface-bound CD3 antibodies.

15. 15. The closed system kit of claim 14, further comprising one or more cytokines selected from the group consisting of IL-2, IL-7, and IL-15.

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