Rapid T-cell production

Abstract

The present disclosure provides methods of rapidly manufacturing genetically modified immune effector cells, such as T cells. The method allows fast (llt; 1 day) to produce genetically modified immune effector cells, such as CAR T cells.

Description

[0001] Related Applications

[0002] This patent application claims priority to U.S. Provisional Application Serial No. 63 / 292,843, filed on December 22, 2021, the entire disclosure of which is incorporated herein by reference.

[0003] Reference to a sequence listing submitted electronically

[0004] The sequence listing is submitted electronically with this application as an XML file named 2459S_005WO.xml, created on December 22, 2022, with a size of 13,000 bytes, which is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to a rapid T cell manufacturing workflow that enables the production of a genetically modified T cell product in less than 1 day using viral-mediated transduction, and also provides innovations in product release testing to help overcome barriers to the clinical use of this product. Background Art

[0006] T cell therapy has shown great potential in the treatment of diseases, especially cancer, infectious diseases, and autoimmune diseases. One approach to enhance T cell therapy is to genetically modify T cells using viral-mediated gene transfer to enhance their activity and / or specificity for desired target cells. For example, the use of lentiviruses and retroviruses to express chimeric antigen receptors (CARs) on T cells has shown great potential in cancer treatment. Autologous T cells expressing chimeric antigen receptors specifically targeting CD19 (CAR-T cells) have shown significant efficacy in patients with relapsed or refractory B-cell malignancies.

[0007] The remarkable clinical success of autologous CD19CAR-T cells in relapsed / refractory non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL) highlights the potential of genetically engineered T cells. If the disease remains sensitive to chemotherapy, high-dose treatment is used for aggressive, relapsed or refractory disease, followed by autologous stem cell rescue (1, 2). Unfortunately, up to 50% of patients relapse or become refractory, and relapsed / refractory patients have poor responses to traditional chemotherapy (e.g., complete remission rate (CR) of 7% and median overall survival (OS) (3) of 6.3 months). In contrast, CD19CAR-T therapy has shown significantly improved effects in these patients (e.g., CR>50%, OS of 1 year (4)).

[0008] Despite the FDA approval of commercial CD19 and BCC CAR-T cell products and regulatory approval in many other countries around the world, existing therapies remain cost-prohibitive for use in most parts of the world and pose a significant financial burden even in the United States. It is estimated that it currently costs pharmaceutical companies approximately $100,000 per patient to produce autologous CAR-T products (5). In addition to the cost, the manufacturing process is slow, resulting in undesirable delays in patient treatment. Current FDA-approved products take 3–6 weeks or longer to deliver to patients, resulting in a significant number of patients experiencing disease progression due to delayed treatment. Another challenge with current CAR-T therapies is that they are poorly effective against most malignancies other than B-cell malignancies, such as NHL, acute lymphoblastic leukemia, and multiple myeloma. Furthermore, even for a disease such as NHL, where initial results have been promising, many patients (approximately 50%) do not have a durable remission after 1 year (6, 7). Although the reasons for poor patient CAR-T treatment outcomes are likely multifactorial, one challenge is the poor persistence of infused CAR-T cells in patients. It has been reported that the persistence of CAR-T cells is associated with the differentiation state of the manufactured CAR-T product. In particular, more differentiated products are thought to have reduced in vivo persistence compared to products with more immature / naive cells (reviewed in (8)).

[0009] Traditional CAR-T manufacturing is a long, complex and expensive process that typically involves T cell isolation, T cell activation, and T cell transduction (often combined with strategies to improve transduction efficiency, such as spin inoculation, recombinant human fibronectin fragments, polybrene, etc.). T cell stimulation for efficient transduction typically includes stimulation with CD3 or CD3 / CD28 antibodies and cytokine stimulation (e.g., IL-2, IL-7, IL-21 and / or IL-15). In addition to utilizing complex and sophisticated manufacturing processes (which in many cases require expensive equipment), the process is also time-consuming and labor-intensive. For example, T cells are typically separated using magnetic beads, which involves a long and expensive process. The beads also typically need to be removed, adding another manufacturing step that requires time, specialized equipment, and expertise. Complex manufacturing almost always requires the use of specialized clean room facilities to ensure the sterility of the product.

[0010] In order to effectively transduce T cells with a virus (such as a lentivirus or a retrovirus), it has been previously reported and almost universally practiced in the field that T cells need to first be activated (such as stimulated with CD3 or CD3 / CD28) for 1-3 days in the presence of cytokines (such as IL-2, IL-7 and / or IL-15) prior to viral transduction. Therefore, traditional manufacturing involves an initial activation step of CD3 and / or CD3 / CD28 for 1-3 days before effective transduction can be performed. It is widely practiced that T cells are expanded for 1-2 weeks after activation. After viral transduction, traditional manufacturing partially utilizes a T cell expansion stage to produce enough T cells for patient infusion.

[0011] In most cases of clinical CAR-T cell manufacturing, T cells are isolated during the initial manufacturing phase (i.e., prior to viral transduction). In particular, the T cell isolation step is part of all previously reported rapid genetically engineered T cell manufacturing workflows (1 day or less) involving viral genetic engineering. In almost all cases of T cell isolation for clinical manufacturing, magnetic beads (usually CD3 / CD28 T cells are purified using magnetic beads (ThermoFisher Scientific, Waltham, MA) or CD4 / CD8 beads (Miltenyi Biotec, Bergisch Gladbach, Germany). These beads, which need to be removed in large quantities before infusion into patients, bind tightly to T cells and take several days to fall off (usually after internalization of the target surface antigen). For example, the manufacturer, ThermoFisher Scientific, reports that CD3 / CD28 beads take several days to fall off, and attempting to remove the beads before about 3 days will result in the loss of a significant portion of the T cells (due to binding to the beads). (assets.fishersci.com / TFS-assets / LSG / manuals / 1113lD_32D_61D.pdf).

[0012] MiltenyiBiotec reports that their magnetic beads do not release from bound cells within 2-3 days. In fact, it has been reported that MiltenyiBiotec beads may be internalized after several days rather than actually released, and therefore, they may have unknown effects on T cell products (thermofisher.com / us / en / home / life-science / cell-analysis / cell-isolation-and-expansion / cell-isolation / see-how-miltenyi-microbeads-interact-with-your-t-cells.html).

[0013] Due to the expense and treatment delays inherent in complex manufacturing workflows, as well as the benefits of reducing T cell culture duration to maintain naive populations (e.g., to enhance in vivo persistence), simple and rapid manufacturing workflows are needed. In particular, workflows of 1 day or less are particularly advantageous because the reduction in culture duration will significantly increase the naive T cell population and ultimately enable simple, closed system manufacturing outside of cleanroom facilities. In addition, processes that do not require separation of T cells would be advantageous for a variety of reasons, including increasing the simplicity of the process, requiring less cost and expertise, and reducing actual operating requirements, thereby improving scalability. In addition, by avoiding the T cell separation step, the product can contain additional cell types, such as NK cells, which also have known favorable therapeutic properties, such as the ability to lyse tumor or pathogen-infected cells. Surprisingly, our studies also revealed additional benefits of using mixed mononuclear cell populations (such as PBMCs or monocyte-depleted PBMCs) rather than isolated T cells for rapid CAR-T manufacturing. For example, the isolated T cells showed a highly desirable reduction in the naive T cell population in the manufactured product compared to the same manufacturing process using PBMCs without a T cell separation step.

[0014] It has been reported that current state-of-the-art clinical rapid CAR-T manufacturing workflows use viral gene delivery. The manufacturing method utilizes a T cell separation step, which adds additional unnecessary cost and complexity and may impair the ability to efficiently manufacture cells in the shortest possible time because the magnetic beads cannot be removed from the cells without significant cell loss.

[0015] In one prior art workflow, a <2 day production protocol reportedly involves approximately 24 hours of ex vivo culture (ashpublications.org / blood / article / 138 / Supplement%20124848 / 481328 / T-Cell-Stemness-with-a-Newel). The process reportedly involves a T cell separation step. The key differences are T cell separation, T cell activation methods, the use of cytokines, and the use of automation, which significantly increases the cost of the instrument (approximately $400,000) and the high cost of consumables. Since microbeads are used, the product may also include internalized microbeads.

[0016] In another prior art workflow, it is reported that it takes more than 1 day to use CD3 / CD28 Dynabeads and high dose IL-2 (300ul / ml) on isolated T cells. Due to the use of Dynabeads, if the cells are harvested at 1 day, the process will result in significant loss of T cells. In addition, the use of high doses of IL-2 can result in significant differentiation of T cells.

[0017] Due to the limitations of complex, lengthy, and expensive manufacturing workflows for gene-modified T cells, as well as the challenges of T cell differentiation in many current approaches, there is an unmet need to develop a rapid, cost-effective, and scalable CAR-T manufacturing platform to produce effective and affordable CAR-T products. The methods and systems herein address this long-standing need. Summary of the invention

[0018] According to the purposes and benefits described herein, in one aspect of the present disclosure, a method for rapidly manufacturing a genetically modified T cell population is provided, comprising obtaining a mixed mononuclear cell population and substantially activating a T cell population contained in the mixed mononuclear cell population at the same time, and exposing the mixed mononuclear cell population to a viral vector, wherein the viral vector is suitable for transducing at least the T cells contained in the mixed mononuclear cell population with exogenous nucleotides. No step of T cell separation is required. In an embodiment, the method comprises harvesting a mixed mononuclear cell population containing at least a genetically modified T cell population up to 24 hours from the step of simultaneously activating and exposing to at least one viral vector.

[0019] In embodiments, the activation step is performed by exposing the mixed mononuclear cells to one or more activators selected from the group of cytokines of IL-2, IL-7, IL-15 and IL21 and / or to an activator selected from one or more activators of CD3, CD28, OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). In embodiments, activation is performed by exposing the mixed mononuclear cells to one or more activators selected from IL-7 and IL-15. In other embodiments, the method includes a step of at least partial depletion before the step of activation and exposure to a viral vector, which is performed by adhering to a mononuclear cell population contained in a mononuclear cell population.

[0020] In an embodiment of the method, the steps of activation and exposure to a viral vector are preferably performed in the absence of any 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.

[0021] The mixed mononuclear cell population includes the genetically modified T cell population. The mixed mononuclear cell population can be obtained by apheresis or peripheral blood extraction.

[0022] In embodiments, the viral transduction vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus. In embodiments, a differential centrifugation step is provided after the steps of substantially simultaneous activation and exposure to the viral transduction vector to remove plasmid DNA from genomic DNA by DNA size selection.

[0023] In another aspect, the present disclosure provides genetically modified T cells produced by the above method.

[0024] In another aspect, the present disclosure provides a closed system kit for implementing a method for rapidly producing a genetically modified T cell population according to the above method. The closed system kit may include a first sterile container suitable for receiving a mixed mononuclear cell population, a second sterile container suitable for receiving a mixed mononuclear cell depleted of monocytes, a bead-free T cell activator, a viral vector suitable for transducing a T cell population with exogenous nucleotides, a suitable culture medium, and a suitable cell washing solution. In an embodiment, at least the first container is made of a material suitable for monocyte depletion in a mixed mononuclear cell population. The first and second containers may be suitable for aseptic introduction of the bead-free T cell activator, the viral transduction vector, the culture medium, and the cell washing solution.

[0025] The various elements / reagents of the closed system kit are substantially as described above.One or both of the first and second containers may be selected from a cell culture bag and a cell culture flask. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description, which illustrates an exemplary embodiment using the principles of the present invention, and the accompanying drawings:

[0027] Figure 1A Comparison of transduction efficiency of monocyte-depleted PBMCs with GFP lentiviral vectors using a 20-hour simultaneous transduction / activation process and different T cell activators is shown. After 20 hours, cells were washed to remove free virus / activators and lysed with lysis buffer. Reagents. GFP expression was determined by flow cytometry 3 days after transduction / activation. The workflow used is described in the section entitled "Detailed Example of Rapid Manufacturing Workflow". Figure A depicts the use of 3 commercial reagents, all of which involve CD3 and CD28 antibodies conjugated to a matrix or beads.

[0028] Figure 1B Shown as Figure 1A T cell production was performed as shown, except that soluble CD3 (100 ng / ml OKT3) or a combination of soluble CD3 (100 ng / ml) and CD28 (300 ng / ml) antibodies were used as activators. In addition, production was performed in the absence or presence of IL-7 and IL-15.

[0029] Figure 1C Shown as Figure 1B T cell production was performed as described, except that the manufacturer's recommended concentration was used. (including as T cell activators) and cytokines (IL-7 and IL-15) were used in all samples.

[0030] Figure 1D Shows the use of Figure 1A T cell production was performed using the workflow described in , except that T cell activation was performed using soluble CD3 (100 ng / ml OKT3), surface-bound CD3 (coated with 5 μg / ml OKT3), or CD69 expression was measured by flow cytometry at product harvest (20 h culture).

[0031] Figure 2 Simultaneous and sequential T cell activation is described: CD19CAR expression was assessed by flow cytometry using anti-FMC63 antibody (AcroBiosystems, Newark, DE). Cells were assayed for CAR expression on day 4 post-transduction. PBMCs were seeded and activated with T cell activators.

[0032] Figure 3 A comparison of various cytokines that modulate transduction efficiency using a rapid manufacturing workflow is shown. Using the methods described in the detailed examples section of the cytokine rapid manufacturing workflow, monocyte-depleted PBMCs were transduced using the specified cytokines using CD19CAR lentiviral vectors, and the expression of CD19CAR was assessed by flow cytometry 72 hours later using FMC63-specific antibodies (Acrobiosystems).

[0033] Figure 4Evaluation of different culture durations using the rapid manufacturing workflow is shown. Monocyte-depleted PBMCs were activated and transduced with CD19CAR vectors as described in the Detailed Examples section of the Rapid Manufacturing Workflow. Cells were washed to remove free virus and removed 6 or 17 hours after the start of the process. T cell activators. Cells were then kept in culture for 72 hours to allow assessment of CD19 CAR surface expression by flow cytometry.

[0034] Figure 5 The results show that UF-KURE19 cells showed increased in vivo efficacy compared to similar CAR-T cells manufactured for 6 days. NSG mice (n = 5-7 per group) were intravenously injected with RAJI luciferase cells (0.5×10 6 ), and then on day 7, the indicated number of UF-Kurel9CAR-T cells or CAR-T cells expressing the same CAR manufactured for 6 days were injected.

[0035] Figure 6 An efficient and rapid T cell manufacturing workflow is shown that does not require exogenous cytokines. Raji luciferase cells were injected into NSG mice and injected with the indicated CD19 CAR-T products or vectors 7 days later, followed by bioluminescence imaging on the indicated days.

[0036] Fig. 7A qPCR release testing for low fragment removal is shown. The data show the results of TaqMan-based qPCR detection of VSVG (replicating lentivirus) on DNA samples prepared from CD19CAR-T transduced cells and harvested 20 hours later using a rapid manufacturing workflow. DNA preparation was performed using the above protocol (using PacBio reagents) for low fragment removal. PCR detection was performed on samples with and without low fragment removal.

[0037] Figure 7B Data showing vector copy number results per transduced cell for T cells transduced with CD19 CAR lentiviral vector and cultured for 8 days at various MOIs. Expression of CD19 CAR was determined by flow cytometry using a CD19 CAR-specific antibody (AcroBiosystems), and copy number was determined by qPCR for GAG and PTPB2.

[0038] Figure 7C Data showing vector copy number per transduced cell and CD19 CAR surface expression for T cells manufactured using the rapid manufacturing workflow. Figure 7B The MOI of the vector described in was 10:1. Figure 7BAs shown, the vector copy number per transduced cell was determined by qPCR and flow cytometry. Low fragment size DNA was removed before PCR using the SRE kit (PacBio).

[0039] Fig.7D Results of DNA gel electrophoresis are shown, indicating that low fragment removal does not significantly affect the amount or size distribution of genomic DNA. Genomic DNA from two different T cell samples transduced with CD19CAR vector lentivirus and harvested 20 hours later was prepared using the DNeasy blood and tissue kit. Agarose gel electrophoresis was performed directly with genomic DNA samples or after low input depletion using the PacBio 10kb SRE kit. Lane 1: DNA marker; Lane 2: Total genomic DNA of sample 1; Lane 3: Total genomic DNA after SRE kit treatment; Lane 4: Total genomic DNA of sample 2; Lane 5: Total genomic DNA of sample 2 after SRE kit treatment.

[0040] Although the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the accompanying drawings and are described in detail below. However, it should be understood that the description of specific embodiments is not intended to limit the present disclosure, which covers all modifications, equivalents and alternatives falling within the spirit and scope of the present disclosure defined by the appended claims. DETAILED DESCRIPTION

[0041] This document sets forth the details of one or more embodiments of the subject matter currently disclosed. Modifications to the embodiments described in this document and other embodiments will be apparent to those of ordinary skill in the art after studying the information provided in this document. The information provided in this document, particularly the specific details of the exemplary embodiments described, are provided primarily for clear understanding and should not be construed as unnecessary limitations. In the event of a conflict, the specification (including definitions) of this document shall prevail.

[0042] In order to address the above-summarized and other shortcomings of the prior art methods, and to develop a robust, economical, simple and scalable ultrafast T cell manufacturing method (i.e., 1 day or less), we have developed a method for manufacturing CAR-T cells without the use of magnetic beads or in some cases even without the need for T cell separation or the use of cytokines. In this case, we unexpectedly discovered a method that can achieve high transduction efficiency of T cells (starting from mixed PBMCs, monocyte-removed PBMCs or isolated T cells) using T cell activation and viral transduction simultaneously without the need for any enhancers (such as polybrene, spin inoculation, recombinant human fibronectin fragments). Surprisingly, the method can also be performed in the absence of any cytokines (such as IL-2, IL-7 and / or IL-15) or in the presence of cytokines. Contrary to previous suggestions, it was found that the transduction efficiency of T cells using this method was as high as the transduction efficiency using more traditional T cell activation and then viral transduction 1-3 days after activation. It is worth noting that another advantage of using PBMCs as a starting source compared to isolated T cells is the ability to utilize CD3 activation alone without the need for CD28 co-stimulation due to the presence of other mononuclear cells that provide the stimulatory signal. It is known that when performing T cell activation, CD3 stimulation alone without the need for the use of CD3 / CD28 activators can preserve more of the desired central memory T cells (9). Therefore, compared to previously reported methods, the developed T cell manufacturing workflow involves a highly simplified process that can significantly reduce costs and improve efficiency.

[0043] An ultrafast preparation that can use PBMCs, monocyte-depleted PBMCs, or isolated T cells as starting products Development of manufacturing technology

[0044] According to the presently disclosed methods, manufacturing can be performed with or without a T cell pre-isolation step. By not performing a T cell pre-isolation step, the manufacturing workflow can be performed faster and at lower cost, and surprisingly also produces a product with a higher percentage of desired naive T cells. If a single blood or peripheral blood sample is used without direct isolation of T cells, T cells can be enriched by monocyte depletion, which is performed by simply adhering to a solid surface (e.g., a tissue culture flask / plate or bag). Monocyte depletion can be performed on an adherent surface such as a plate or bag, or in a closed system. Monocyte depletion is a simple, inexpensive, and rapid method for partially purifying a product.

[0045] On the other hand, by avoiding the T cell isolation step, the final product can include not only genetically engineered T cells, but also other cell types such as NK cells that may exhibit beneficial therapeutic properties.

[0046] According to the present disclosure, in one aspect, T cells are activated simultaneously using a bead-free activator 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 a known cytokine that drives T cell differentiation, and cells cultured in the presence of IL-2 are known to show reduced preservation of their naive / undifferentiated phenotype. This is particularly true when high doses (e.g., 300 IU / ml) are used (11-12).

[0047] In another aspect, surprisingly, it has been found that a workflow involving simultaneous activation and transduction of T cells in the presence of IL-7 or IL-15 is highly efficient and does not require the presence of both cytokines. Thus, the workflow of manufacture can be performed using IL-7 alone or IL-15 alone.

[0048] In another aspect, surprisingly, the workflow involving simultaneous activation and transduction of T cells is highly efficient without the addition of exogenous cytokines, including IL-2, IL-7 or IL-15. Thus, the manufacturing workflow can be performed with the addition of any exogenous cytokines.

[0049] In another aspect, the genetically modified T cell product can be produced within 24 hours, preferably including a culture time of about 17-20 hours.

[0050] In this workflow, no pre-activation step is required prior to viral transduction or T cell isolation, both of which save critical manufacturing time and costs. It has not been previously reported that simultaneous transduction and activation using unpurified mononuclear cells (e.g., PBMCs) or monocyte-depleted PBMCs (as opposed to purified T cells) can be effective for rapid CAR-T manufacturing.

[0051] Cell activation can be performed using various agents that activate T cells through CD3 or CD3 and CD28, including Transact (MiltenyiBiotec), Cloudz T cell activators (Biotechne), soluble or surface-bound CD3 and / or CD28 antibodies, or custom microvesicles with bound CD3 and / or CD3 / CD28 antibodies. In particular, Cloudz T cell activators were found to be able to achieve a high and unexpected ability to simultaneously activate and virally transduce T cells compared to Transact. In addition, soluble or surface-bound CD3 (with or without CD28) will also play a role in the less than 1 day workflow described herein.

[0052] Cell activation can also be performed using agents that activate T cells through CD3 and other co-stimulatory molecules other than (or in addition to) CD28, such as OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0053] After cell activation and transduction, the cells can be harvested within less than a day (e.g., 17-20 hr) after transduction and can be used directly for therapeutic purposes or cryopreserved for later use. Although the expression of the genes transduced into the T cells is not fully expressed when the cells are harvested and infused into the recipient, this manufacturing method results in T cells that exhibit high therapeutic efficacy.

[0054] Therefore, this method enables rapid manufacturing of cell therapy products in less than 1 day.

[0055] In another component of the approach, the T cell manufacturing process can be performed in a fully closed system using either manual or automated processes. In another component, manufacturing can be performed in a fully closed system outside a cleanroom, allowing manufacturing to be performed in many centers that do not have dedicated cleanroom infrastructure.

[0056] Another component of the method is a method for eliminating false positive reactivity in the release test required for viral transduction of cell therapy products in rapid manufacturing. In the first few days of manufacturing, cell transfection (such as 293 or 293T cells) is present in the cell therapy product to produce residual plasmid DNA of slow virus or retrovirus. The plasmid DNA shows false positive results in the vector copy number and replicating virus test qPCR analysis. In this workflow, the release test is integrated into the workflow and includes a size separation step based on differential centrifugation to eliminate the plasmid DNA and enable the T cell products of rapid manufacturing to be used clinically, which is usually required for product release.

[0057] The presently disclosed subject matter is further illustrated by the following specific but non-limiting examples.The following examples may include a compilation of data representing data collected at various times during the development and experimentation related to the present invention.

[0058] Example

[0059] Development of rapid gene-modified T cell preparation technology

[0060] To date, the aforementioned rapid (e.g., 1 day or less) viral-based gene-modified T cell manufacturing processes involve an initial T cell isolation step. Here, we developed a rapid manufacturing process that can utilize PBMCs or monocyte-depleted PBMCs in addition to isolated T cells. If PBMCs are used as a starting source, an optional initial step is to perform monocyte depletion, as in some cases it is desirable to partially deplete monocytes from the product. For example, such depletion would allow the virus to preferentially infect specific cell types that are more desirable for the final product, including T cells and, in some cases, NK cells. However, the depletion is not complete, which would allow monocytes (e.g., CD80 / CD86) to provide stimulatory signals to T cells (e.g., exogenous CD3 stimulation alone to activate T cells without the need for co-stimulation with exogenous CD28 or other stimuli). In general, as shown in Figure 1, the depletion method described below resulted in a reduction of approximately 50% in monocytes in the starting blood product, starting 2 hours after inoculation. Additional incubation time after 2 hours had no significant effect on depletion using this method. In addition, the cell density of the inoculated cells (5×10 6 cells / ml and 2×10 6 cells / ml) showed equivalent results. Use tissue culture plates or flasks, or use bags in a closed system, such as those manufactured by Saint Gobain (Paris, France). Depletion of the “AC” series of bags showed similar results.

[0061] Table 1. Depletion of monocytes in PBMCs.

[0062] 1 hour 2 hours 3 hours 4 hours Cell concentration for inoculation MO% MO% MO% MO% <![CDATA[2x10 6 / ml]]> 21.46 13.77 14.86 14.95 <![CDATA[5x10 6 / ml]]> 20.87 11.25 10.97 11.09

[0063] Mononuclear cells from peripheral blood samples were incubated in 6-well tissue culture plates in a tissue culture incubator at 37°C for the indicated time points. Drew Scientific, Miami Lakes, FL) was used to measure the percentage of monocytes in non-adherent cells at the following time points. The starting population consisted of a starting monocyte percentage of 25.02%. MO% = monocyte percentage.

[0064] If isolated T cells are to be used, they can be isolated directly from whole blood, aliquots, or other T cell sources. Any available method can be used to isolate T cells. For example, magnetic bead methods such as CD3, CD4, or CD8 magnetic beads (e.g., Miltenyi Biotec microbeads or ThermoFisher Scientific ). CD3 microvesicles, CD3 / CD28 or microvesicles conjugated with CD3 and any other T cell co-stimulatory ligands can also be used to effectively and quickly separate T cells. In particular, microvesicles, especially lipid microvesicles, provide a fast and effective separation method while also providing activation signals.

[0065] T cell activation can be achieved in a variety of ways. If using PBMCs or mononuclear PBMCs, soluble activators or surface-bound (e.g., plates / bags) are preferred over magnetic bead-based methods for rapid manufacturing workflows. One effective activation method is the use of CD3 / CD28 soluble microspheres (from Biotechne / R&D Systems (Minneapolis, Minnesota)). Human T cell activator). This reagent consists of an alginate copolymer that dissolves within minutes, so magnetic beads are not required and will not lead to product contamination. Alternative activators can also be used, such as CD3 / CD28 activator (Stem Cell Technologies, Vancouver, Canada). Alternative activators that can be used include CD3 or CD3 / CD28 magnetic beads (e.g. ThermoFisher Scientific) or (Miltenyi Biotec). Based on magnetic beads and The reagents are not considered optimal because the beads require increased labor and the removal step is challenging due to the tight binding of the beads early on. This can result in excessive cell loss. Compared to other products, The activator is a “milder, slower” activator that produces an optimal viral transduction window at a later time point. 1-2 days of T cell activation prior to viral transduction is required for optimal viral transduction. If it is washed off before 2-3 days after addition, it will hinder T cell proliferation (miltenyibiotec.com / upload / assets / IM0017348.PDF). Therefore, for production protocols that involve simultaneous activation / transduction and harvesting of product in less than a day, Not the preferred activator.

[0066] In addition to matrix-associated activators, soluble or surface-coated CD3 antibodies (e.g., OKT3 antibodies or other CD3 antibodies) alone or in combination with soluble CD28 antibodies or other T cell co-stimulatory stimulants are another effective activation strategy that is fully compatible with the rapid preparation workflow. In particular, this method benefits from a PBMC-based approach that is able to activate T cells without the need for exogenous CD28 or other reagents for co-stimulation. In addition, compared to other activation stimuli, this method of using CD3 antibodies such as OKT3 significantly reduces costs and minimizes the risk that scaffolds (e.g., alginate, magnetic beads, etc.) present in the manufactured product may have unknown side effects on patients.

[0067] It has been found that specific T cell activators can be more effectively used using the rapid manufacturing workflow described herein in less than 1 day. To compare specific T cell activators, monocyte-depleted PBMCs (200,000 cells in 100 μl) were plated in 1% paraformaldehyde (10 ng / ml) containing IL-7 (10 ng / ml) and IL-15 (5 ng / ml). (Miltenyi Biotec) 3% in culture medium 96-well plates containing Immune Cell Serum Replacement (ThermoFisher Scientific). GFP lentiviral vector and the indicated activators (using the concentrations recommended by the manufacturer) were added and the cells were cultured for 4 days. The cells were washed and the medium was changed after 20 hours to remove free virus and free activators. The cells were lysed with the reagents and the lysis buffer was also used to remove the activating agent. The expression of GFP was measured by flow cytometry. Figure 2 As shown in A, and CtsCD3 / CD28 compared to, Human T cell activators are more efficient in maximizing transduction efficiency using a rapid manufacturing workflow. Of note, for most figures, the amount of viral vector used was kept low to easily facilitate differentiation between conditions, rather than using excess virus to maximize transduction efficiency. Figure 2 The ability of a rapid manufacturing workflow to result in high transduction efficiencies was observed in the literature using high levels of lentiviral vector (while still maintaining vector copies per transduced cell at less than 5). Figure 2 As shown in B, the rapid manufacturing workflow is also applicable to soluble CD3 and / or soluble CD3 and CD28 antibodies ( Figure 2 C). For example, the widely used CD3 antibody OKT3 can be used in this workflow ( Figure 2C) Manufacturing process using monocyte-depleted PBMCs, CD3 alone has similar function in terms of transduction efficiency compared to the addition of CD3 and CD28 antibodies. Of note, in this manufacturing workflow, OKT3CD3 antibody stimulation has a greater effect than CD28 in terms of transduction efficiency. More effective ( Figure 2 C). Using a rapid manufacturing workflow, both surface-bound (e.g., plate or flask) CD3 antibodies and soluble CD3 antibodies resulted in similar activation of CD4 and CD8 T cells, as measured by CD69 expression at product harvest (20 hours after culture initiation). Notably, compared to the group treated with soluble / surface-bound CD3, This led to a reduction in CD69 upregulation in CD4 T cells. This finding suggests that soluble CD3 or CD3 bound to culture vessels can be effectively used for T cell activation using a rapid workflow. Furthermore, these activators appear to be superior to other agents, such as

[0068] It is generally believed in the art that one needs to activate T cells sequentially and then perform viral transduction to maximize viral transduction efficiency. However, the results here show that, at least using our specific manufacturing workflow, performing T cell activation and viral transduction simultaneously results in transduction efficiencies that are essentially the same as those performed sequentially. For example, using T cell activators activated monocyte-depleted PBMCs and added The cells were transduced with a lentiviral CD19 chimeric antigen receptor (CAR) vector at the same time as the cells were transduced, or 24 hours later using the manufacturing process described in the Detailed Examples section of the Rapid Manufacturing Workflow. Figure 2 As shown, there was no significant difference in CAR expression between simultaneous or sequential viral transduction using the described manufacturing workflow.

[0069] Conventional and previously reported rapid T cell manufacturing methods all use culture conditions that almost universally use cytokines such as IL-2, IL-15, and / or IL-7. To evaluate the optimal cytokines for use in the rapid manufacturing workflow, the manufacturing process described in the Detailed Examples section of the rapid manufacturing workflow was performed using IL-2 (300u / ml), IL-15 (5ng / ml), IL-7 (10ng / ml), a combination of IL-15 and IL-7, or the complete absence of exogenous cytokines ( Figure 4). When comparing CD19CAR expression in T cells 72 hours after transduction, no differences in transduction efficiency were observed between the different cytokine groups. Surprisingly, there was no difference in transduction efficiency in the complete absence of cytokines. Therefore, the manufacturing process can be further simplified to be performed in the absence of any exogenous cytokines to further reduce costs and prevent stimulation of T cells that may lead to unwanted differentiation / activation. Figure 1B As shown, rapid T cell manufacturing was also almost equally effective in resulting in GFP transduction of T cells when comparing manufacturing with and without cytokines.

[0070] Another critical attribute of the manufacturing process is the duration of culture. Since one goal of the manufacturing workflow is to limit its duration to preserve the naive T cell fraction, manufacturing processes using 6 h and 17 h culture durations were compared. At the end of both time points, the lysis buffer was removed Reagents were added, cells were washed to remove virus, and suspended in fresh medium without virus or activating agents for a total of 72 hours to measure CAR expression. Figure 4 As shown, a 6-hour incubation time of the lentiviral vector and T cell activator was insufficient to result in significant CD19CAR expression, as measured by flow cytometry on day 3. Intermediate durations greater than 15 hours and less than about 15 hours were not considered preferred due to limitations in being able to perform manufacturing within typical workday time.

[0071] Since the manufacturing platform can use PBMCs or monocyte-depleted PBMCs as a starting source and the product culture is less than one day, the final product will be composed of additional mononuclear cells in addition to T cells. To evaluate the composition of the product, three manufacturing processes were performed using the methods described in the detailed examples section of the rapid manufacturing workflow. As shown in Table 2, T cells are the largest portion of the product, but the product also contains small amounts of B cells, NK cells, and monocytes. Since the preferred method of the workflow involves the production of cryopreserved products, this testing was performed after the product was thawed. Since granulocytes are a small portion of the starting apheresis product and are highly sensitive to freeze / thaw, almost no granulocytes were detected in this product.

[0072] Table 2. Product composition of 3 manufacturing runs performed on thawed UF-KURE19 product (manufacturing 17 hours).

[0073]

[0074]

[0075] Product composition was determined by flow cytometry, and viability was assessed using antibodies to CD3, CD4, CD8, CD19, CD56, and CD14, as well as 7-AAD.

[0076] Since significant expression of proteins such as CAR in T cells does not occur within 17-20 hours after lentiviral transduction, to evaluate the activity of transduced CD19CAR-T products in vitro, we manufactured CD19CAR-T cells from monocyte-depleted PBMCs using the manufacturing workflow described in the Detailed Examples section of the Rapid Manufacturing Workflow. After 20 hours of activation / transduction, free virus was washed from the cells and removed. T cell activators. The cells were then cultured for a total culture time of 3 days before evaluating cytotoxic activity against target RAJI human lymphoma cells and measuring CAR surface expression. As shown in Table 3, the rapidly manufactured CAR-T cells were able to effectively lyse RAJI tumor cells.

[0077] Table 3. Results of in vitro testing of the cytotoxic activity of rapidly manufactured CD19 CAR-T cells after 3 days of culture.

[0078]

[0079] Monocyte-depleted PBMCs were transduced with CD19CAR lentiviral vector and then T cell activators activate T cells, as described in the detailed examples section of the rapid manufacturing workflow. After 20 hours, free viruses are removed and T cell activators are dissolved, and cell culture is continued for 3 days. The cytotoxic activity of CD19CAR-T cells against RAJI tumor cells is assessed by flow cytometry to measure the loss of calcium AM dye in tumor cells after 4 hours of co-culture with CAR-T cells. The expression of CD19CAR is measured by flow cytometry using FMC63-specific antibodies (AcroBiosystems).

[0080] When the product was harvested at 17-20 hours in culture, the CD19CAR protein was not significantly expressed on the surface of T cells using standard flow cytometry methods, so the activity of T cells was also evaluated in a mouse model to demonstrate the efficacy of the product. In this way, the full expression of CAR occurs in vivo, subsequently enabling T cells to obtain their cytotoxic activity against cells expressing human CD19. In order to demonstrate the improved efficacy of rapidly manufactured CAR-T products, this in vivo study used cryopreserved cells that were manufactured for 17 hours from monocyte-depleted PBMCs and transduced with CD19 CAR lentiviral vectors according to the workflow described in the detailed example section of the rapid manufacturing workflow. This product expressing CD19 CAR is called UF-KURE19 cells. Cells manufactured using the same workflow but kept in culture for 6 days instead of 17 hours are called Kure19. Cells were tested in a circulating mouse model of human lymphoma, which included intravenous injection of human RAJI tumor cells into immunodeficient mice (NSG), followed by intravenous injection of a single dose of CAR-T products on the 7th day after tumor cell injection. Although traditionally, approximately 5 million CAR T cells have been used in this model to demonstrate significant efficacy, lower doses have been used because of the expected increased potency of rapidly manufactured products. In this case, for the UF-Kure19 product, doses were 2 million and 4 million CAR-positive T cells for the UF-KURE19 cohort and 2 million CAR-negative T cells for the 6-day manufactured product. Figure 5 As shown, UF-KURE19 cells showed significant efficacy at both low and high dose levels. In contrast, although the 6-day cultured product showed reduced tumor progression compared to vehicle-treated mice, its efficacy was significantly reduced compared to the UF-KURE19 product. Similar to the mouse studies using Kure19, the UF-KURE19 injection group tolerated the treatment well (monitored by weight change, feeding, appearance and behavior) with no obvious signs of toxicity.

[0081] CD19 CAR-T cells have been found to persist for months or even years (as measured by detection of the transgene). For example, the Tisa-cel product has been found to persist for at least 2 years in some patients with good clinical outcomes (10). The proliferation of human T cells in the blood of RAJI lymphoma-bearing NSG mice was measured by flow cytometry. In Figure 9, the average number of human T cells detected per microliter of mouse peripheral blood at the indicated time points (n=3-5 mice measured per time point) after injection of RAJI tumor cells (500,000 intravenously) into female NSG mice is shown. It is worth noting that these measured blood samples were derived from the above-mentioned mouse efficacy studies. As shown in Table 4, the efficacy of UF-KURE19 cells was significantly higher than that of 6-day manufactured KURE19 CAR-T cells, which correlates with human T cell expansion.

[0082] Table 4. T cells per μL of mouse blood detected by flow cytometry

[0083] UF-Kure19 4x106 cells UF-Kure19 2x106 cells 6 days CAR-T2x106 cells Day 32 126.7 60.1 2.7 Day 39 410.1 ND 7.3 Day 46 969.1 995.6 14.9

[0084] ND = Not Determined

[0085] As mentioned above, the use of cytokines in the rapid manufacturing workflow is not necessary to achieve significant transduction efficiencies. Therefore, the effect of cytokine use on the memory / differentiation state of T cells at the time of harvest (20 hours) was assessed by flow cytometry. In addition, this study employed the use of different cytokines or no cytokines during the manufacturing process as shown in Figure 10 to better evaluate the impact of cytokines on the product. Finally, this study also compared monocyte-depleted PBMCs and the use of isolated T cells as a starting source. In addition to using purified T cells as the starting source for a subset of samples, the manufacturing workflow described in the Detailed Examples section of the Rapid Manufacturing Workflow was used. Using Human T cell enrichment kit (StemCell Technologies) purifies T cells from peripheral blood. As shown in Table 5, in CD8+T cells, when the product manufactured from isolated T cells is compared with monocyte-depleted PBMC, it is observed that the CD8+T cell components of the product are not the same. In particular, when starting from PBMC, the level of effector memory T cells is slightly lower and the percentage of TEMRA cells is slightly higher. The role of CD8+TERA cells has not been fully elucidated, but they are believed to have increased cytotoxic activity, which may be beneficial. In the CD8+T cell compartment, the level of highly beneficial naive T cells related to CAR-T efficacy is similar when starting with PBMC or from isolated T cells, however, the percentage of beneficial central memory T cells in PBMC is higher, and the level of effector memory cells with higher degree of differentiation and less ideal is lower than that of isolated T lymphocytes. In the CD4+T cell compartment, when starting with PBMC, the percentage of highly beneficial naive T cells increases compared to isolated T cells. Therefore, based on the phenotypic analysis of the product, it was found that the use of a PBMC starting workflow conferred a more preferred workflow that could be predicted to result in improved clinical outcomes and greater persistence of CAR-T cells in vivo. In addition, this study investigated the use of different cytokines (IL2, IL7, IL-15) or no cytokines during the manufacturing process. As shown in Table 5, starting with PBMCs, there were no significant differences in T cell memory / differentiation phenotypes under any conditions at the 20-hour workflow, further suggesting that cytokines are not a necessary component of the manufacturing workflow when starting with monocyte-depleted PBMCs. Interestingly, when starting with isolated T cells, a reduction in naive T cells was noted when no cytokines were used, suggesting that the use of cytokines may be more important for manufacturing when using isolated T lymphocytes as a starting source. PBMCs may provide an endogenous source of cytokines.

[0086] Table 5. Phenotypes of T cells generated using the rapid workflow.

[0087]

[0088]

[0089]

[0090] T cells were produced using the rapid workflow described in the Detailed Examples section of the Rapid Production Workflow or using the same workflow except using isolated T cells as the starting cell source. After 20 hours, the cells were evaluated for T cell phenotype by flow cytometry.

[0091] Since in vitro studies have shown that cytokines do not appear to be required for efficient T cell transduction or maintenance of a preferred memory / differentiation T cell phenotype when starting with PBMCs using a rapid manufacturing process, in vivo efficacy studies in mice were performed to further confirm these findings. CD19CAR-T cells were manufactured using a rapid manufacturing workflow, starting with monocyte-depleted PBMCs, with or without cytokines during culture. The same human lymphoma tumor model (RAJI) in NSG mice as described above was used. In this case, each mouse was injected with 1.2×10 6 CD19CAR positive T cells. Figure 6 As shown, CAR-T products produced in the complete absence of exogenous cytokines effectively controlled tumor progression in a manner similar to those produced with cytokines. All vector control mice died of disease progression before imaging on day 45. Figure 6 As shown in A, low dose 1.2×l0 6 CD19CAR-positive T cells showed high potency, further demonstrating the efficacy of rapid CAR-T products. This study also clearly demonstrated that rapid (less than 1 day) production of CAR-T products starting with PBMCs does not require exogenous cytokines.

[0092] In addition to similar efficacy, there was no reduction in circulating human T cells in mice when the cytokine-free product was used. Figure 6 Human T cells in the blood of mice from the experiments shown were quantified. No cytokine CAR-T group: 4495 human T cells per microliter of mouse blood. IL7 / IL15CAR-T group: 1618 human T cells per microliter of mouse blood.

[0093] Detailed example of a rapid manufacturing workflow.

[0094] Selected representative modifications to the workflow are shown in brackets. In addition to the relatively manual workflow described below, the entire workflow can be performed in a fully closed and / or semi-automated or automated manner. For example, automated equipment can be used for cell washing, harvesting, etc.

[0095] Manufacturing process of CAR-T products starting from monocyte-depleted PBMCs in less than 1 day:

[0096] A. Obtaining starting cells (Day -1 or Day 0)

[0097] Autologous PBMCs are obtained from the patient using leukapheresis (or peripheral blood draw). The cell product is processed immediately or stored overnight. (If desired, the cell product can also be cryopreserved and processed at a later time).

[0098] B. Single Sample Processing (Day 0)

[0099] The aliquots are washed by centrifugation to remove plasma and reduce the number of contaminating platelets. (If peripheral blood is used instead of aliquots, PBMCs are isolated by a ficoll-based separation method). Next, the aliquots are diluted in a medium such as TexMACS (Miltenyi Biotec) supplemented with CTS Immune Cell Serum Replacement (ThermoFisher Scientific), incubated in a tissue culture flask or cell culture bag, and placed in an incubator at 37°C (±2°C) to adhere to monocytes at a concentration of up to 5 million cells / ml. After the monocytes have adhered to the flask or bag for at least 2 hours, the non-adherent cells are transferred to a new tissue culture flask or bag. The volume is adjusted to 2 million cells / ml with medium containing IL7 (lOng / ml) and IL15 (5ng / ml). (The medium can also be prepared without the addition of any exogenous cytokines or with IL-7 alone, IL15 alone, or other cytokines).

[0100] Activation of CT cells (Day 0)

[0101] CD3 / CD28 T cell activator (R&D Systems / Biotechne) can be added to the flask containing the monocyte-depleted and processed apheresis product. (Other activators include soluble CD3 antibodies, soluble CD3 / CD28 antibodies, surface-bound CD3 antibodies (with or without soluble CD28 antibodies), (Stemcell Technologies) or any other T cell activator may be used).

[0102] D. Cell Transduction (Day 0)

[0103] Immediately after the addition of the T cell activator, the viral vector was added to the flask or bag containing the monocyte-depleted PBMCs, using a multiplicity of infection (MOI) determined to result in a copy number per cell of less than 5. The cells were cultured in a 5% (± 0.5%) CO2 incubator at 37°C (± 2°C) for 17-20 hours.

[0104] E. Cell Harvest (Day 1)

[0105] If you use If the cells are activated with a reagent, GMP grade 6X release buffer (R&D Systems / Biotechne) is added directly to the flask or bag containing the activated / transduced cells. Next, the cells are washed and resuspended in a freezing buffer, such as Plasmalyte-A, 5% HSA, and 5% DMSO in a freezing bag or vial.

[0106] Description of the method for vector copy number and replication-competent virus release testing using rapid engineered T cell products:

[0107] Since a key hurdle in the field of rapid T cell manufacturing workflows involving viral vectors is conducting the required product release testing, the following method was developed. Current release testing requirements for cell therapy products transduced with retroviruses or lentiviruses include assessment of replication-competent lentivirus and vector copy number. When using traditional qPCR-based assays, both assays have significant false-positive results, even when levels of free plasmid contamination are low. Residual free plasmid from 293 cell transfections is present in the viral vector. At early time points in manufacturing following viral transduction, residual plasmid remains in the vector, making false-positive results nearly impossible to eliminate using traditional detection methods. Such false-positive reactions have been a major hurdle and have limited the ability to rapidly produce patient-ready products.

[0108] Since the target DNA for both viral integration and replicating vectors is integrated viral DNA, the manufacturing workflow included here is combined with a method to remove free residual plasmid DNA, so that PCR reactions or other molecular tests for vector copy number and replicating lentivirus can be performed without such false positive reactions. In particular, free plasmid DNA is much smaller than genomic DNA. A centrifugation-based method is used to deplete small DNA while maintaining larger genomic DNA. Using this method, there are no false positive reactions for plasmid DNA, which overcomes the main obstacle to rapid manufacturing of CAR-T, while measuring DNA integrated into the genome is still feasible.

[0109] To remove free plasmid DNA from CAR-T products that have been amplified for a short period of time (e.g., 0-3 days), small-sized DNA (e.g., less than 10 kb) can be depleted while centrifugation (e.g., using a solution such as a salt / polymer solution to preferentially precipitate high molecular weight DNA) can be used to retain genomic DNA. For example, the PacBio Short Read Eliminator Kit (PacBio; Menlo Park, CA) can be used and is capable of performing this separation from a single centrifugation step (circulomics.com / store / short-read-eliminator-kit-pl31401036). In addition to this commercial reagent, another approach is to use 4% PVP360000, 1.2M KCL, 20mM Tris-HCL ph8 in place of the commercial buffer SRE in the protocol described below, as it has previously been shown to effectively deplete small-sized fragments (e.g., DNA <10 kb in total DNA) (11). The purified DNA can then be used directly in an assay (e.g., a qPCR assay). Although the kit is not designed for this specific indication, it functions well and provides a simple, cost-effective, and rapid approach.

[0110] An example of how to use this method is to use any commercial total DNA isolation kit that can isolate human genomic DNA (e.g., DNeasy blood and tissue kit, Qiagen, Hilden, Germany) to initially isolate total DNA. Next, the following method or similar methods can be used to remove low fragment DNA.

[0111] 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 commercial reagents, 4% PVP360000, 1.2M KCL, 20mM Tris-HCL ph8 can be added to the total DNA sample at a 1:1 volume ratio.

[0112] 2. Centrifuge at 10,000 × g for 30 minutes at room temperature.

[0113] 3. Remove the supernatant with a pipette.

[0114] 4. Add 70% ethanol to the test tube.

[0115] 5. Centrifuge the tube at 10,000 × g for approximately 2 minutes at room temperature.

[0116] 6. Remove the supernatant and repeat the wash with 70% ethanol.

[0117] 7. Add Buffer EB (or 10 mM Tris-HCl pH 8 if not using the PacBio kit) to the tube, incubate at 50°C for 10-30 minutes, and then resuspend the DNA pellet.

[0118] The low-fragment removal workflow was combined with a rapid manufacturing workflow to prove that this is an effective process for release testing of genetically engineered T cell products at early time points in culture (e.g., 17 hours to 3 days). For example, CAR-T products manufactured using a 17-20 hour workflow were harvested at 20 hours, and alternative aliquots were harvested at 72 hours. Total DNA was isolated using the DNeasy blood and tissue kit (Qiagen), and low-fragment depleted DNA was prepared using a short read eliminator kit (PacBio). Real-time PCR for VSV-G was used to measure the replicative lentivirus of low-fragment depleted DNA and total DNA samples, and real-time PCR detection of GAG and housekeeping gene PTBP2 was used to test vector integration. Primers and fluorescent probes used for PCR reactions can be found in Table 6.

[0119] Table 6. PCR primers and probes used for GAG, VSV-G and PTPBP2-qPCR assays.

[0120] GAG primer

[0121] 5'-GGAGCTAGAACGATT CGCAGT TA-3'

[0122] 5'GGT TGTAGC TGT CCCAGTATT TGT C-3'

[0123] GAG probe

[0124] 5'- / 56-FAM / ACA GCC TTC TGATGT TTC TAACAG GCCAGG 3'BHQ-l

[0125] PTPB2 primers

[0126] 5'-TCT CCATTC CCTATG TTCATG C-3'

[0127] 5'-GTT CCC GCAGAATGG TGA GGT G-3'

[0128] PTPB2 probe

[0129] 5'- / 5HEX / ATG TTC CTC / ZEN / GGA CCAACT TG 3iabfq 3'

[0130] VSVG Primers

[0131] 5'-AGG GAA CTG TGG GAT GAC TG

[0132] 5'-GAACAC CTGAGC CTT TGA GC

[0133] VSVG Probe

[0134] 5'-6FAM-GAACACCTGAGCTTTGAGC MGBNFQ-3'

[0135] PCR products were quantified using a standard probe consisting of a linearized plasmid engineered to express single copies of GAG, VSV-G, and PTBP2. Fig. 7A As shown, qPCR reactions performed with low-fragment depleted DNA did not detect VSV-G and were therefore negative for replicating lentiviruses, as expected on two different products manufactured for 20 hours. In contrast, when total genomic DNA without low-fragment depletion was used, both samples showed false-positive qPCR results for VSV-G, likely due to low-fragment free plasmid contamination. To evaluate whether alternative approaches, such as cell washing or culturing for 3 days (instead of 20 hours) could overcome the false-positive reactivity of replicating lentivirus detection, we performed further assays. In particular, we harvested lentiviral-transduced CD19CAR-T products at 20 and 72 hours and washed the cells extensively prior to genomic DNA isolation. In particular, for the 72-hour sample, the cells (5×10 6 cells) were washed 15 times in 50 ml PBS. For the 20-hour samples, cells (5×10 6 Cells were washed 5 times in 50 ml PBS volume. In all cases (20 h and 72 h samples for both products), there was a false positive reactivity using the VSV-G qPCR assay. Therefore, increasing the culture duration to 3 days and extensive cell washing was not sufficient to reduce this false positive reactivity.

[0136] Of note, for the replicating lentivirus assay, no positive assays have been reported to the U.S. Food and Drug Administration for clinical products in the past 10 years, reinforcing that the positive reactions consistently observed within 17–20 h and 72 h of rapid product manufacturing are due to false-positive reactions (fda.gov / media / 113790 / download). Furthermore, the VSV-G qPCR assay turned negative when the culture duration was extended to 8 days, indicating that free plasmid DNA was lost at this time point and that true replicating virus was not present.

[0137] Similar to the replication-competent lentiviral testing, another important release test for virally transduced cell therapy products is vector integration, which is typically measured by qPCR targeting the GAG ​​gene, for which free plasmid contamination is also present in the viral vector and can produce false-positive reactivity. Initially, GAG / PTPB2 qPCR was performed to determine the vector copy number on CD19CAR lentiviral-transduced T cells cultured for 8 days in order to assess the multiplicity of infection (MOI) of the vector and to assess the baseline for complete vector integration. Figure 7B As shown, an MOI of 10:1 resulted in a peak vector copy number of 1.5 per transduced T cell (i.e., expressing CD19CAR). Next, in addition to harvesting cells at 20 hours, 3 days, and 7 days, the vector copy number per transduced cell was determined using a rapid manufacturing workflow when the MOI of the CD19CAR vector was 10:1. In addition, the SRE kit (PacBio) was used to remove low-fragment DNA. As Figure 7C As shown, vector integration results were similar at all time points. Of note, CD19CAR surface expression was very low at 20 hours, although it was fully expressed around 72 hours.

[0138] Finally, gel electrophoresis of total DNA samples prepared from two 20-hour rapid-made T cell products before and after low-molecular-weight fragment removal demonstrated that genomic DNA was maintained as expected, such that only unwanted low-molecular-weight fragments were depleted ( Fig.7D ). In addition, use DNA concentration was quantified by spectrophotometry (ThermoFisher Scientific) and no measurable changes were observed in DNA concentration before and after low fragment removal, indicating that only trace amounts of DNA were removed.

[0139] As will be appreciated, the presently described CAR-T cell manufacturing workflow provides a significant improvement over prior art methods. In particular, without intending any limitation:

[0140] 1. Manufacturing is mainly performed using unfractionated cells from apheresis products or apheresis cells that have undergone monocyte depletion by adhering to a solid surface. In addition, peripheral blood mononuclear cells (PBMCs) directly isolated from peripheral blood or monocyte-depleted PBMCs can be utilized. Therefore, it does not require purification of T cells, which is considered to be very important for rapid CAR-T workflows involving viral transduction. To the best of our knowledge, this is the first 1-day or less protocol for rapid gene-modified T cell manufacturing using viral transduction using PBMCs rather than isolated T cells as a starting source.

[0141] 2. Using different methods for cell activation. It takes several days for magnetic beads to fall off the cells, so methods using such magnetic beads either delay the date when the product can be harvested and / or result in significantly reduced product yields due to loss of bead-bound cells. In addition, we unexpectedly observed that the use of specific non-magnetic bead-based T cell activators improves transduction efficiency. The currently described activation methods provide improved transduction efficiency while avoiding the increased cost and increased manufacturing complexity involved in utilizing magnetic beads as required in prior art methods.

[0142] 3. The manufacturing process currently described is the first workflow described for a rapid CAR-T manufacturing workflow that can be performed efficiently in the absence of exogenous cytokines. Cytokines in the manufacturing process significantly increase costs and may also cause changes in T cells. Since a major benefit of a rapid manufacturing workflow is to keep the starting population of T cells (such as naive T cells) as close as possible to the original cells collected from the patient, avoiding exogenous cytokines is seen as a major advantage.

[0143] 4. The described use of a completely closed system to manufacture T cell products using bags allows for a convenient kit-based product where virus / media / activators etc. would be provided in a bag and then mixed with the apheresis product. The product can be manufactured outside a cleanroom using this approach to facilitate widespread use and manufacture of the product, which can reduce costs and increase availability.

[0144] 5. The currently described methods overcome a key obstacle in the field of required product release testing. Current release testing requirements for retroviral or lentiviral transduced cell therapy products include assessment of replicating lentivirus and vector copy number. Both of these tests have significant false positive results when using traditional qPCR-based assays, even when free plasmid contamination levels are low. Residual free plasmids from virus-producing cells such as 293 cells are present in viral vectors. At early time points in manufacturing after viral transduction, plasmids remain in the vector, making it almost impossible to eliminate false positive results using traditional detection methods. This false positive reaction has been a major obstacle for other groups and has limited the ability to quickly produce products that can be used in patients. Since the target DNA for both viral integration and replicating vectors is integrated viral DNA, the manufacturing workflow included herein is combined with methods to remove free residual plasmid DNA, so that qPCR reactions for vector copy number and replicating lentivirus can be performed without such false positive reactions. In particular, free plasmid DNA is much smaller than genomic DNA. A centrifugation-based method is used to deplete small DNA while retaining larger genomic DNA. Using this method, there is no false-positive reactivity of plasmid DNA, which overcomes the main obstacle to rapid manufacturing of CAR-T, while measuring DNA integrated into the genome is still feasible.

[0145] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0146] Unless otherwise indicated, all patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials mentioned throughout the disclosure herein are incorporated by reference in their entirety.

[0147] Where reference is made to a URL or other such identifier or address, it is understood that such identifiers may change and particular information on the Internet may come and go, but equivalent information may be found by searching the Internet. Reference is made to the availability and public dissemination of such information.

[0148] As used herein, unless otherwise indicated, any abbreviations for protecting groups, amino acids, and other compounds are in accordance with common usage, recognized abbreviations, or IUPAC-IUB biochemical nomenclature (see Biochem. (1972) 11(9): 1726-1732).

[0149] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.

[0150] In some cases, the nucleotides and polypeptides disclosed herein are contained in publicly available databases, such as and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides contained in such publicly available databases is expressly incorporated by reference. Unless otherwise stated or apparent, references to such publicly available databases are references to the most recent version of the database as of the filing date of this application.

[0151] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., reaction conditions), and so forth used in the specification and claims are to be understood as modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought by the presently disclosed subject matter.

[0152] Wherever any phrase "for example," "such as," "including," etc. is used, it should be understood that "and not limited to" follows it unless otherwise expressly stated. Similarly, "example," "exemplary," etc. are understood to be non-limiting. The term "substantially" allows deviations from the descriptor without adversely affecting the intended purpose. Even if the word "substantially" is not explicitly cited, the descriptive term is understood to be modified by the term "substantially." Thus, for example, the phrase "the lever extends vertically" means "the lever extends substantially vertically," as long as the lever does not require a precise vertical arrangement to perform its function.

[0153] The terms "comprising" and "including," as well as "having" and "involving" (similarly, "comprises," "includes," "has," "involves"), etc., are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "comprising" in U.S. patent law and is therefore interpreted as an open term meaning "at least the following," and is also interpreted as not excluding other features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it should be understood to mean "one or more" unless such an interpretation is meaningless in the context. The terms "comprise," "have," "include," and "contain" (and variations thereof) are open-ended linking verbs that allow for the addition of additional elements when used in a claim.

[0154] Unless the context dictates otherwise, the use of the words "a" or "an" when used with the term "comprising" in a claim or specification refers to one or more.

[0155] As used herein, when referring to a value or amount of mass, weight, time, volume, concentration, or percentage, the term "about" means in some embodiments including variations of ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some specific embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% compared to the specified amount, as long as such variations are suitable for performing the disclosed methods. The use of the word "or" in the claims means "and / or" unless it is expressly stated that only alternatives are referred to, or if the alternatives are mutually exclusive.

[0156] As used herein, ranges can be expressed as from "about" one particular value and / or to "about" another particular value. It is also understood that a number of values ​​are disclosed herein, and each value is also disclosed herein as "about" that particular value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0157] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally variant portion means that the portion is a variant or a non-variant.

[0158] It should be understood that the various details of the subject matter currently disclosed may be changed without departing from the scope of the subject matter disclosed herein. In addition, the above description is for illustrative purposes only, not for limiting purposes. In accordance with the above teachings, obvious modifications and variations are possible. When interpreted according to the broadness that the attached claims fairly, legally and equitably enjoy, all of these modifications and variations are within the scope of the attached claims.

[0159] References:

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Claims

1. A method for rapidly producing a genetically modified T cell population, comprising: Obtaining a mixed mononuclear cell population; At essentially the same time: activating a T cell population contained in the mixed mononuclear cell population; and The mixed mononuclear cell population is exposed to a viral vector suitable for transducing at least the T cell population contained in the mixed mononuclear cell population with exogenous nucleic acid.

2. The method of claim 1, comprising harvesting the mixed mononuclear cell population comprising at least a genetically modified T cell population at most 24 hours from the step of simultaneous activation and exposure to at least one viral vector.

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

5. The method according to claim 1, comprising a step of at least partial depletion of the monocyte population comprised in the mononuclear cell population by adhesion prior to the step of activation and exposure to the viral vector.

6. The method of claim 5, wherein the steps of activating and exposing to the viral vector are performed in the absence of any exogenous cytokines.

7. The method of claim 6, wherein the activation step is 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-linked CD28 antibody.

8. The method of claim 7, wherein the activation step is performed by exposing the mixed mononuclear cell population to one or both of a CD3 activator, or a soluble or surface-bound CD3 antibody.

9. The method of claim 1, wherein the activation and exposure to the viral vector steps are performed without prior T cell isolation or pre-activation steps.

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

11. The method of claim 1, comprising obtaining the mixed mononuclear cell population by apheresis or peripheral blood extraction.

12. The method of claim 1, comprising selecting the viral transduction vector from lentivirus, retrovirus and adenovirus.

13. The method of claim 1, further comprising the step of differential centrifugation performed after substantially simultaneous activation and exposure to the viral transduction vector to remove plasmid DNA from genomic DNA by DNA size selection.

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

15. A closed system kit for implementing the method for rapidly producing a genetically modified T cell population according to any one of claims 1 to 12, comprising: a first sterile container suitable for receiving a mixed population of mononuclear cells; a second sterile container adapted to receive the monocyte-depleted mixed mononuclear cell population; Bead-free T cell activator; Viral vectors suitable for transducing T cell populations with exogenous nucleic acids; a suitable culture medium; and Appropriate cell washing solution; wherein at least said first container is made of a material suitable for depleting monocytes from said mixed mononuclear cell population; Further wherein the first and second containers are suitable for aseptically introducing the bead-free T cell activator, the viral transduction vector, the culture medium and the cell washing solution.

16. A closed system kit according to claim 15, wherein the bead-free T cell activator is selected from one or more of a CD3 activator, a CD28 activator, an OX40 activator, a CD2 activator, a CD 27 activator, an ICAM-1 activator, an LFA-1 (CD11a / CD18) activator, an ICOS (CD278) activator, a 4-1BB (CD137) activator and a CD3 antibody.

17. The closed system kit according to claim 16, further comprising one or more cytokines selected from IL-2, IL-7 and IL-15.

18. The closed system kit according to claim 16, wherein the bead-free T cell activator is selected from one or more of a soluble or surface-bound CD3 antibody and a soluble or surface-bound CD28 antibody.

19. The closed system kit according to claim 18, wherein the bead-free T cell activator is selected from one or more soluble or surface-bound CD3 antibodies.

20. The closed system kit according to claim 15, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus.

21. The closed system kit according to claim 15, wherein one or both of the first container and the second container are selected from a cell culture bag and a cell culture flask.

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