High-speed T cell production

Abstract

The present disclosure provides a method for rapid production of genetically modified immune effector cells, such as T cells, from a mixed mononuclear cell population. The method includes activating a T cell population contained in the mixed mononuclear cell population, and, following a post-activation period of up to three hours, exposing the mixed mononuclear cells containing the activated T cell population to at least one viral vector employed to transduce at least the T cell population contained in the mixed mononuclear cell population with an exogenous nucleotide. The method allows for rapid production of genetically modified immune effector cells, such as CAR T cells.

Description

[Technical Field]

[0001] Related Applications This application is a continuation-in-part utility patent application that claims the benefit of priority to International Patent Application No. PCT / US22 / 53815, filed December 22, 2022, which in turn claims the benefit of priority to U.S. Provisional Application No. 63 / 292,843, filed December 22, 2021, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Reference to an electronically submitted sequence listing The Sequence Listing, which was submitted electronically with this application as an XML file named 2459S_005WO.xml, created on December 22, 2022, and having a size of 13,000 bytes, is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to a rapid T cell manufacturing workflow that uses viral-mediated transduction to enable the production of genetically engineered T cell products in less than one day, and further provides innovations in product release testing to reduce obstacles to clinical utilization of the product. [Background technology]

[0004] T cell therapy has shown great potential in the treatment of diseases, particularly cancer, infectious diseases, and autoimmune diseases. One way 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, expression of chimeric antigen receptors (CARs) on T cells using lentiviruses and retroviruses has shown great potential in cancer therapy. Autologous T cells expressing chimeric antigen receptors (CAR-T cells), particularly those directed against CD19, have shown remarkable efficacy in patients with relapsed or refractory B cell malignancies.

[0005] The potential of genetically modified T cells has been highlighted by the remarkable clinical success of autologous CD19 CAR-T cells in relapsed / 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 stem cell rescue if the disease is still sensitive to chemotherapy (1, 2). Unfortunately, up to 50% of patients relapse or become refractory, and the prognosis for relapsed / refractory patients is poor with conventional chemotherapy (e.g., complete response rate (CR) is 7% and median overall survival (OS) is 6.3 months (3)). In contrast, CD19 CAR-T therapy has demonstrated significantly improved outcomes in these patients (e.g., CR >50% and 1-year OS >50% (4)).

[0006] Although commercial CD19 and BCMA CAR-T cell products have been approved by the FDA in the United States and have received regulatory approval in many other countries globally, existing therapies remain expensive for use in most parts of the world, posing a significant economic burden in the United States. The current cost of manufacturing autologous CAR-T products at pharmaceutical companies is estimated at ~100,000 USD per patient (5). In addition to the expense, the manufacturing process is slow, resulting in undesirable delays in patient treatment. Current FDA-approved products take ~3–6 weeks or longer to reach patients, resulting in treatment delays and disease progression in a significant number of patients. Another challenge with current CAR-T therapies is their poor efficacy in most malignancies, with the exception of B-cell malignancies such as NHL, acute lymphoblastic leukemia, and multiple myeloma. Furthermore, even in diseases such as NHL, where initial responses are promising, many patients (~50%) do not achieve sustained remission after one year (6, 7). Although the causes of suboptimal outcomes of CAR-T therapy in patients are likely multifactorial, one challenge is the poor persistence of CAR-T cells infused into patients. It has been reported that CAR-T cell persistence correlates with the differentiation stage of the manufactured CAR-T product. In particular, more differentiated products are thought to have reduced in vivo persistence compared to products made with more immature / naive cells (reviewed in (8)).

[0007] Traditional CAR-T manufacturing is typically a long, complex, and expensive process involving T cell isolation, T cell activation, and T cell transduction (often combined with strategies to enhance transduction efficiency, such as spin inoculation, retronectin, or polybrene). T cell stimulation, which enables efficient transduction, typically involves 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 that often require expensive equipment, the process is time-consuming and labor-intensive. For example, T cells are typically isolated using magnetic beads, which involves a lengthy and expensive process. The beads also typically need to be removed, which adds another step to manufacturing that requires time, specialized equipment, and specialized knowledge. Complex manufacturing also almost always requires the use of specialized cleanroom facilities to ensure product sterility.

[0008] It has been previously reported, and is almost universally practiced in the art, that to efficiently transduce T cells with viruses (e.g., lentiviruses or retroviruses), T cells must first be activated (e.g., by CD3 or CD3 / CD28 stimulation) in the presence of cytokines (e.g., IL-2, IL-7, and / or IL-15) for a period of 1-3 days prior to viral transduction. Therefore, conventional manufacturing involves an initial activation step with CD3 and / or CD3 / CD28, followed by efficient transduction 1-3 days later. It is common practice to expand T cells for 1-2 weeks after activation. After viral transduction, conventional manufacturing utilizes, in part, a T cell expansion step to generate enough T cells for infusion into patients.

[0009] In a large percentage of clinical CAR-T cell manufacturing cases, T cells are isolated at an early manufacturing stage (i.e., before viral transduction). Notably, the T cell isolation step is part of all high-speed genetically modified T cell manufacturing workflows (one day or less) reported to date that involve viral-based genetic modification. In virtually all cases where T cells are isolated for clinical manufacturing, the T cells are attached to magnetic beads (typically CD3 / CD28 Dynabeads). (登録商標) These T cells are purified using either magnetic beads (ThermoFisher Scientific, Waltham, MA) or CD4 / CD8 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). These beads, which must be removed before infusion into patients, are tightly bound to T cells and require several days to be removed (typically occurring after internalization of the target surface antigen). For example, manufacturer ThermoFisher Scientific reports that if bead removal is attempted more than three days in advance, it takes several days for CD3 / CD28 beads to be removed, and that bead removal will result in the loss of a significant percentage of T cells (due to the bound beads) (assets.fishersci.com / TFS-Assets / LSG / manuals / 11131D_32D_61D.pdf).

[0010] Miltenyi Biotec reports that their magnetic beads do not release from bound cells for 2-3 days. In fact, it has been reported that Miltenyi Biotec's beads may not actually release but may be internalized after a few days, thus potentially having 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).

[0011] Due to the costs and therapeutic delays inherent in complex manufacturing workflows, as well as the benefits of shortening T cell culture periods and maintaining naive populations (e.g., increasing in vivo persistence), simple and rapid manufacturing workflows are desirable. A workflow of one day or less would be particularly advantageous, as shortening the culture period would significantly increase naive T cell populations and ultimately enable manufacturing in a simple, closed system outside of a cleanroom facility. Furthermore, a process that does not require T cell isolation would be advantageous for many reasons, including increased process simplicity, reduced cost and expertise, reduced manpower requirements, and increased scalability. Furthermore, avoiding the T cell isolation step could allow the product to contain additional cell types, such as NK cells, which are also known to have desirable therapeutic properties, such as the ability to lyse tumor or pathogen-infected cells. Surprisingly, our studies also revealed the additional benefit of utilizing a mixed population of mononuclear cells (e.g., PBMCs or monocyte-depleted PBMCs) as opposed to isolated T cells for rapid CAR-T manufacturing. For example, isolated T cells show a highly desirable reduction in naive T cell populations in the product when compared to the same manufacturing process performed using PBMCs without the T cell isolation step.

[0012] Prior art clinical high-speed CAR-T manufacturing workflows have been reported to use viral gene transfer, which utilizes a T cell isolation step that adds unnecessary cost and complexity, and the inability to remove magnetic beads from cells without significant cell loss can potentially impair the ability to efficiently manufacture cells in the shortest possible time.

[0013] In one prior art workflow, a <2-day manufacturing protocol is reported with ~24 hours of ex vivo culture (ashpublications.org / blood / article / 138 / Supplement%201 / 2848 / 481328 / Preservation-of-T-Cell-Stemness-with-a-Novel). This process is reported to involve a T cell isolation step. Key differences include the T cell isolation, T cell activation method, use of cytokines, and use of automation, which add significant cost for both equipment (~400k) and high consumable costs. Due to the use of microbeads, this product likely also contains internalized microbeads.

[0014] Another prior art workflow reportedly capable of being performed in more than one day requires the use of CD3 / CD28 Dynabeads and high doses of IL-2 (300 μl / ml) on isolated T cells. Due to the use of Dynabeads, this process would suffer from significant T cell loss if cells were harvested in one day. Furthermore, the use of high doses of IL-2 can lead to significant T cell differentiation.

[0015] Due to limitations in the complex, lengthy, and expensive manufacturing workflow for genetically modified T cells, and challenges with T cell differentiation with many current approaches, there is a significant unmet need to develop a fast, cost-effective, and scalable CAR-T manufacturing platform that results in an effective, affordable CAR-T product. The methods and systems herein fulfill this long-felt need. Summary of the Invention

[0016] In accordance with the objects and advantages described herein, one aspect of the present disclosure provides a method for rapid production of a genetically modified T cell population, comprising obtaining a mixed mononuclear cell population and substantially simultaneously activating a T cell population contained within the mixed mononuclear cell population and exposing the mixed mononuclear cell population to a viral vector employed to transduce at least the T cell population contained within the mixed mononuclear cell population with an exogenous nucleotide. A T cell isolation step is not required. In one embodiment, the method comprises recovering the mixed mononuclear cell population comprising at least the genetically modified T cell population up to 24 hours after the activation and simultaneous exposure to at least one viral vector steps.

[0017] In one embodiment, the activation step is carried out by exposing the mixed mononuclear cells to an activator selected from one or more of the group of cytokines consisting of IL-2, IL-7, IL-15, and IL21, and / or an activator selected from one or more of the following activators: CD3, CD28, OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the activation step is carried out by exposing the mixed mononuclear cells to an activator selected from one or more of the group consisting of IL-7 and IL-15. In other embodiments, the method includes at least partially removing a monocyte population contained in the mononuclear cell population by adhesion or other suitable method, such as, but not limited to, elutriation, prior to the activation and viral vector exposure steps.

[0018] In certain embodiments of the method, the steps of activating and exposing to the viral vector are preferably performed in the absence of exogenous cytokines. The activation step may 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.

[0019] Mixed mononuclear cell population comprising a genetically modified T cell population. The mixed mononuclear cell population may be obtained by apheresis or peripheral blood collection.

[0020] In some embodiments, the viral transduction vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus. In some embodiments, following the steps of activating and substantially simultaneously exposing to the viral transduction vector, a step of differential centrifugation is provided to remove plasmid DNA from genomic DNA by DNA size selection.

[0021] In another aspect, the present disclosure provides a genetically modified T cell produced by the above method.

[0022] In yet another aspect, the present disclosure provides a kit for carrying out the method for rapid production of genetically modified T cell populations according to the above-described method. The closed system kit may include a first sterile container adapted to receive the mixed mononuclear cell population, a second sterile container adapted to receive the monocyte-depleted mixed mononuclear cell population, a bead-free T cell activator, a viral vector adapted to transduce the T cell population with an exogenous nucleotide, a suitable medium, and a suitable cell washing solution. In some embodiments, at least the first container is made of a material suitable for depleting monocytes from the mixed mononuclear cell population. The first and second containers may be adapted for the sterile introduction of the bead-free T cell activator, the viral transduction vector, the medium, and the cell washing solution. Alternatively, in some embodiments, a closed system is not required and a single suitable container may be included.

[0023] The various elements / reagents of the kit may otherwise be substantially as described above. One or both of the first and second containers may be selected from the group consisting of a cell culture bag and a cell culture flask.

[0024] In an alternative embodiment, monocyte removal is accomplished by an alternative suitable method, such as the use of an elutriation system, and only a single container is required for the kit, as described above. In yet another alternative embodiment, monocyte removal is not required, and thus only a single container is required for the closed system kit. [Brief explanation 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 present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0026] [Figure 1A] Figure 1A shows a 20-hour simultaneous transduction / activation process and a comparison of transduction efficiency using different T cell activation reagents on monocyte-depleted PBMCs using a GFP lentiviral vector. After 20 hours, cells were washed to remove free virus / activation reagent, and Cloudz® reagent was removed with lysis buffer. GFP expression was determined by flow cytometry 3 days after transduction / activation. The workflow utilized is described in the section titled "Detailed Example of a Rapid Manufacturing Workflow." Panel A shows the use of three commercially available reagents, all with CD3 and CD28 antibodies conjugated to substrates or beads.

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

[0028] [Figure 1C]FIG. 1C shows T cell production performed as described in FIG. 1B, except that Immunocult® was included as the T cell activation reagent at the manufacturer's recommended concentrations, and cytokines were used in all samples (IL-7 and IL-15).

[0029] [Figure 1D] Figure ID shows T cell production performed using the workflow described in Figure IA, except that T cell activation was performed using soluble CD3 (100 ng / ml OKT3), surface-bound CD3 (surface coated with 5 μg / ml OKT3), or TransAct® (at the manufacturer's recommended concentration). CD69 expression was measured by flow cytometry at product harvest (20 hours of culture).

[0030] [Figure 2A] Figure 2A shows a comparison of simultaneous and sequential T cell activation: CD19 CAR expression was assessed by flow cytometry using anti-FMC63 antibody (AcroBiosytems, Newark, Delaware). Cells were tested for CAR expression 4 days after transduction. PBMCs were seeded and activated with Cloudz® T cell activation reagent either simultaneously with addition of the lentiviral vector or 24 hours prior to virus addition.

[0031] [Figure 2B] Figure 2B shows a comparison of simultaneous (conventional) and sequential (ultrafast) T cell activation using both CD19 and BCMA CARs. Surface-bound CD3 and CD28 antibodies were used for activation in the absence of cytokines. Detection was performed by flow cytometry 3 days after transduction using anti-FMC63 or anti-BCMA detection reagents (AcroBiosytems, Newark, Delaware).

[0032] [Figure 3]Figure 3 shows a comparison of various cytokines modulating transduction efficiency when using the rapid manufacturing workflow. Monocyte-depleted PBMCs were transduced with the CD19 CAR lentiviral vector using the indicated cytokines and the process described in the Detailed Example of the Rapid Manufacturing Workflow section, and 72 hours later, CD19 CAR expression was assessed by flow cytometry using the FMC63-specific antibody (Acrobiosystems).

[0033] [Figure 4] Figure 4 shows the evaluation of different culture periods when using the rapid manufacturing workflow. Monocyte-depleted PBMCs were activated and transduced with the CD19 CAR vector as described in the detailed example of the rapid manufacturing workflow section. Cells were washed to remove free virus, and the CloudZ® T cell activation reagent was removed 6 or 17 hours after the start of the process. Cells were then maintained in culture medium for 72 hours to assess CD19 CAR surface expression by flow cytometry.

[0034] [Figure 5] Figure 5 shows that UF-Kure19 cells demonstrated increased in vivo efficacy over similar CAR-T cells engineered in 6 days. NSG mice (n=5-7 per group) were intravenously injected with RAJI-luciferase cells (0.5x106), followed on day 7 by the indicated number of UF-Kure19 CAR-T cells or CAR-T cells expressing the same CAR engineered in 6 days.

[0035] [Figure 6] Figure 6 demonstrates that an efficient, high-throughput T cell manufacturing workflow does not require exogenous cytokines. NSG mice were injected with Raji-luciferase cells and 7 days later with the indicated CD19 CAR-T product or vehicle, followed by bioluminescence imaging on the indicated days.

[0036] [Figure 7A]Figure 7A shows qPCR release testing with low fragment removal. The data show the results of TaqMan-based qPCR testing of VSVG (replication-competent lentivirus) on DNA samples prepared from CD19 CAR-T-transduced cells harvested after 20 hours using the fast manufacturing workflow. DNA preparation was performed using the protocol described above (using PacBio reagents) for low fragment removal. PCR testing was performed on samples with and without low fragment removal.

[0037] [Figure 7B] Figure 7B shows data showing the vector copy number per transduced cell for T cells transduced with a CD19 CAR lentiviral vector and cultured for 8 days at various MOIs. CD19 CAR expression 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] Figure 7C shows data showing vector copy number per transduced cell and CD19 CAR surface expression for T cells produced using the rapid manufacturing workflow with the vector described in Figure 7B at an MOI of 10:1. Vector copy number per transduced cell was determined by qPCR and flow cytometry as described in Figure 7B. Low-fragment size DNA was removed using an SRE kit (PacBio) prior to PCR.

[0039] [Figure 7D]Figure 7D shows DNA gel electrophoresis results demonstrating that low-input fragment removal does not significantly affect the amount or size distribution of genomic DNA. Genomic DNA from two different T cell samples, lentivirally transduced with a CD19 CAR vector and harvested 20 hours later, was prepared using the DNeasy Blood and Tissue Kit. Agarose gel electrophoresis was performed on the genomic DNA samples directly or after low-input removal using the PacBio 10kb SRE Kit. Lane 1: DNA marker; Lane 2: Sample 1 total genomic DNA; Lane 3: SRE kit-treated total genomic DNA; Lane 4: Sample 2 total genomic DNA; Lane 5: SRE kit-treated total genomic DNA.

[0040] [Figure 8] Figure 8 shows the dramatic reduction in free antibody bound to a high-throughput T cell product using Fast Blue treatment. Monocyte-depleted PBMCs were activated with CD3 and CD28 antibodies using the Fast Blue method (see, e.g., U.S. Pat. No. 4,654,299) or the same passive adsorption of antibodies in PBS without Fast Blue. After 20 hours of activation, the products were washed and cryopreserved. After thawing, the products were washed and stained with anti-mouse antibodies to detect mouse antibodies bound to the products (both CD3 and CD28 antibodies are mouse anti-human antibodies). The percentage of antibody-bound cells was determined by flow cytometry.

[0041] [Figure 9] Figure 9 shows that Fast Blue treatment does not interfere with T cell activation. Using the Fast Blue method described above, monocyte-depleted PBMCs were incubated with Fast Blue-conjugated CD3 / CD28 antibodies in tissue culture plates (Experiment 1 and Experiment 2) or in plates treated identically but without CD3 / CD28 antibodies (non-activated control). Approximately 16 hours after cell activation, cells were washed and stained with CD69 antibodies to determine their activation status.

[0042] [Figure 10] Figure 10 shows that a short post-activation step may provide a small benefit in transduction efficiency. Monocyte-depleted PBMCs were activated and transduced with lentiviral GFP using the method described in the "High-Speed ​​Manufacturing Workflow Detailed Example" section. The only difference was that virus was added either simultaneously with cell activation or at the indicated time points after activation. GFP expression was measured by flow cytometry in the products after 3 days.

[0043] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are hereinafter described in detail. It should be understood, however, that the description of the specific embodiments is not intended to limit the disclosure, which embraces all modifications, equivalents, and alternatives within the spirit and scope of the disclosure as defined by the appended claims. DETAILED DESCRIPTION OF THE INVENTION

[0044] Details of one or more embodiments of the subject matter disclosed herein are set forth in this document. Modifications of the embodiments described in this document, as well as 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, and the specific details of the exemplary embodiments particularly described, are provided primarily for clarity of understanding, and no unnecessary limitations should be understood therefrom. In the event of a conflict, the specification of this document, including definitions, will control.

[0045] To address the above-summarized and other shortcomings of prior art methodologies, and to develop a robust, cost-effective, simple, and scalable ultrafast T cell manufacturing method (i.e., one day or less), we developed a method for producing CAR-T cells that does not use magnetic beads, or in some cases, the need for T cell isolation, or even the use of cytokines. In this case, we surprisingly identified a method that allows for 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 need for any enhancers (e.g., polybrene, spin-inoculation, retronectin). Surprisingly, this process can be performed in the absence or presence of cytokines (e.g., IL-2, IL-7, and / or IL-15). Contrary to previous proposals, we found that T cell transduction efficiency using this method was comparable to that using more conventional T cell activation, in which viral transduction occurs 1–3 days after activation. Of note, another advantage of using PBMCs rather than isolated T cells as the starting material is the ability to utilize CD3 activation alone, without the need for CD28 costimulation, due to the presence of other mononuclear cells providing a simultaneous signal. It is known that desired central memory T cells are better preserved when T cell activation is performed via CD3 stimulation alone, without the need for CD3 / CD28 activating reagents (9). Therefore, the developed T cell manufacturing workflow involves a highly simplified process that significantly reduces costs and significantly improves efficiency compared to previously reported methods.

[0046] Development of an ultra-rapid manufacturing process that can utilize PBMCs, monocyte-depleted PBMCs, or isolated T cells as starting products The methods disclosed herein allow manufacturing to be performed with or without a T cell pre-isolation step. Without a T cell pre-isolation step, the manufacturing workflow can be performed faster, less expensive, and, surprisingly, can produce a product containing a higher percentage of desired naive T cells. When blood apheresis or peripheral blood samples are used without direct T cell isolation, T cells can be enriched via monocyte removal by simple adherence to a solid surface (e.g., tissue culture flasks / plates, or bags). Monocyte removal can be performed on an adherent surface such as a plate or bag, or in a closed system. Monocyte removal can be performed using a ThermoFisher (Waltham, MA) Rotea Counterflow Centrifugation System. (登録商標) It can also be done via any other method, including elutriation using automated equipment such as a TECHNICAL FIELD: DEPLETION OF MONOCYTES IS A SIMPLE, INexpensive, AND FAST METHOD TO PARTIALLY PUFF THE PRODUCT.

[0047] In another embodiment, by avoiding a T cell isolation step, the final product may contain not only genetically modified T cells, but also other cell types that may exhibit advantageous therapeutic properties, such as NK cells.

[0048] According to the present disclosure, in one embodiment, T cells are activated using a bead-free activation reagent and simultaneously 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 cytokine known to drive T cell differentiation, and cells cultured in the presence of IL-2 are known to exhibit reduced preservation of their naive / undifferentiated phenotype. This is particularly true when high doses (e.g., 300 IU / ml) are utilized (11-12).

[0049] In another aspect, it has been surprisingly found that workflows involving simultaneous activation and transduction of T cells are highly efficient in the presence of either IL-7 or IL-15, and do not require both cytokines, and thus the manufacturing workflow can be performed with IL-7 alone or IL-17 alone.

[0050] In another aspect, surprisingly, the workflow involving 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 manufacturing workflow can be performed with the addition of any exogenous cytokine.

[0051] In another embodiment, the genetically modified T cell product can be produced in less than 24 hours, preferably comprising a culture time of about 17-20 hours.

[0052] This workflow does not require a pre-activation step or a T cell isolation step prior to viral transduction, both of which save significant manufacturing time and cost. There have been no reports yet that high-speed CAR-T manufacturing can be efficiently performed using simultaneous transduction and activation of non-purified mononuclear cells (e.g., PBMCs) or monocyte-depleted PBMCs (not purified T cells).

[0053] In alternative embodiments of this workflow, an optional short post-activation delay may be employed in place of simultaneous cell activation and viral transduction prior to the addition of virus. For example, a period of 1 to 6 hours may be employed between the activation and transduction steps, allowing for product production in 24 hours or even less. In other embodiments, a post-activation delay of up to 3 hours may be provided. In still other embodiments, a post-activation delay of about 1 hour to about 3 hours may be provided. A small increase in transduction efficiency has been observed with a short post-activation step. While the small benefit of this post-activation step is observed only in certain cases, it can be implemented within the rapid manufacturing workflow described herein for additional benefits, as discussed below.

[0054] Cell activation can occur using a variety of reagents that activate T cells via CD3 or CD3 and CD28, including Transact (MiltenyiBiotec), Cloudz T Cell Activator (Biotech), soluble or surface-bound CD3 and / or CD28 antibodies, or custom microbubbles conjugated to CD3 and / or CD3 / CD28 antibodies. In particular, Cloudz T Cell Activator has been shown to enable a high and unexpected ability to simultaneously activate and virally transduce T cells compared to Transact. Furthermore, soluble or surface-bound CD3, with or without CD28, works well in the workflow described herein, which takes less than one day.

[0055] Cell activation can also occur using reagents that activate T cells through CD3 and other costimulatory 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).

[0056] After cell activation and transduction, cells can be harvested less than a day (e.g., ~17-20 hours) after transduction and used directly for therapeutic purposes or cryopreserved for later use. This production method results in T cells that exhibit high therapeutic efficacy despite the fact that the transduced gene expression is insufficient when the cells are harvested and when infused into a recipient.

[0057] Another element of this method provides a method that better enables the use of surface-bound (e.g., plate-, flask-, or bag-bound) antibodies (e.g., CD3 and / or CD28 antibodies) used for the activation step. Typically, antibodies are bound to surfaces by passive adsorption, a method that can lead to reversible binding of the antibody. Because CD3 and CD28 antibodies can bind to T cells with high affinity, the antibody can bind to the final product even when it is initially bound to a solid surface (e.g., a plate, flask, or bag). Because the presence of antibody bound to the product can be detrimental to the viability of the product in the recipient, methods can be employed to ensure that the antibody is more strongly bound to the solid surface (e.g., a plate, flask, or bag), thereby limiting the amount of antibody remaining in the product.

[0058] Therefore, this method allows for a rapid manufacturing protocol for cell therapy products that can be carried out in less than one day.

[0059] In another element of this approach, the T cell manufacturing process can be performed in a completely closed system using manual or automated processes. In another element, manufacturing can be performed in a completely closed system outside of a clean room, allowing manufacturing in many facilities that do not have specialized clean room facilities.

[0060] Another element of this approach is a method to eliminate false-positive reactivity required for release testing of virally transduced cell therapy products produced during rapid manufacturing. Residual plasmid DNA from cell transfection (e.g., 293 or 293T cells) to produce lentivirus or retrovirus is present in cell therapy products during the first few days of manufacturing. This plasmid DNA can give false-positive results in qPCR assays that test for vector copy number and replication-competent virus. In this workflow, release testing is integrated into the workflow, involving a differential centrifugation-based size separation step to remove this plasmid DNA, enabling clinical use of rapid-manufactured T cell products that often require release testing.

[0061] The matter disclosed herein is further illustrated by the following specific, non-limiting examples, which may include compilations of data representative of data collected at various time points during the course of development and experimentation related to the present invention. [Example]

[0062] Development of a rapid genetically modified T cell manufacturing process Previously described rapid (e.g., one day or less) virus-based genetically modified T cell manufacturing processes initially involve a T cell isolation step. Here, we have developed a rapid manufacturing process that can utilize PBMCs or monocyte-depleted PBMCs in addition to isolated T cells. When using PBMCs as the starting material, an optional initial step is monocyte depletion, and in some cases, it is desirable to partially remove monocytes from the product. This depletion allows the virus to preferentially infect specific cell types more desirable in the final product, including, for example, T cells and, in some cases, NK cells. However, depletion is not complete, allowing monocytes to provide stimulatory signals (e.g., CD80 / CD86) to T cells (e.g., allowing T cell activation using only exogenous CD3 stimulation without the need for costimulation with exogenous CD28 or other stimuli). Generally, the depletion method described below begins 2 hours after seeding and results in approximately 50% monocyte depletion from the starting blood product, as seen in Figure 1. An additional 2-hour incubation period did not significantly affect depletion using this approach. Furthermore, regardless of the cell density (5 × 10 cells) seeded, 6 cells / ml, and 2 × 10 6 The results are comparable between tissue culture plates and flasks. (登録商標) Similar depletion results are observed in closed systems using bags such as the "AC" Series bags. Additionally, the ThermoFisher (Waltham, MA) Rotea Counterflow Centrifugation System, well known for its use in depleting monocytes from blood samples, is also used. (登録商標) Elutriation techniques, including

[0063] Table 1. Monocyte removal from PBMCs [Table 1]

[0064] Mononuclear cells from peripheral blood apheresis samples were incubated in 6-well tissue culture plates in a tissue culture incubator at 37°C for the indicated time points. (登録商標) The percentage of monocytes among non-adherent cells was determined at the following time points using a chromatographic assay (Drew Scientific, Miami Lakes, FL). The starting population consisted of a starting monocyte percentage of 25.02%. MO% = percentage of monocytes.

[0065] If isolated T cells are used, they can be isolated directly from whole blood, an apheresis sample, or another source of T cells. T cells can be isolated using any available method. For example, magnetic bead approaches such as CD3, CD4, or CD8 magnetic beads (e.g., Miltenyi Biotec microbeads, or ThermoFisher Scientific dynabeads) can be used. (登録商標) ) can be used. T cells can also be efficiently and rapidly isolated using CD3 microbubbles, CD3 / CD28, or CD3-conjugated microbubbles, and any other T cell costimulatory ligand. In particular, microbubbles, especially lipid microbubbles, offer a fast and efficient method of isolation while also providing an activation signal.

[0066] T cell activation can be achieved by multiple approaches. When utilizing PBMCs or mononuclear PBMCs, soluble activation reagents or surface binding (e.g., plate / bag) are preferred over magnetic bead-based approaches in high-speed manufacturing workflows. One effective activation approach is the use of CD3 / CD28 lytic microparticles (Cloudz® from Biotechne / R&D Systems, Minneapolis, MN). (登録商標) The first step is to use a human T cell activation reagent (Immunocult). This reagent is made of an alginate copolymer that dissolves within minutes, therefore does not require magnetic beads and does not lead to product contamination. (登録商標)Alternative activation reagents are also available, such as CD3 / CD28 Activator (Stem Cell Technologies, Vancouver, Canada). Alternative activators include CD3 or CD3 / CD28 magnetic beads (e.g., Dynabeads). (登録商標) ThermoFisher Scientific), or TransAct (登録商標) (Miltenyi Biotec), magnetic bead-based materials, and TransAct (登録商標) The material is not considered optimal because magnetic beads require increased labor and a difficult removal step due to the beads being strongly bound at the early stage, which may result in excessive cell loss. (登録商標) The activation reagent is also reported to be a "gentle and slow" activation reagent compared to other products, resulting in an optimal window for viral transduction at later time points. (登録商標) It has been reported that T cell activation prior to viral transduction by TransAct requires 1-2 days. (登録商標) It has been reported that TransAct can inhibit T cell proliferation if washed out within 2-3 days of application (miltenyibiotec.com / upload / assets / IM0017348.PDF). Therefore, for manufacturing protocols with simultaneous activation / transduction and product recovery in less than one day, TransAct is the preferred choice. (登録商標) is not a preferred activating reagent.

[0067] In addition to the matrix associated with the activation reagent, soluble or surface-coated CD3 antibodies (e.g., OKT3 or other CD3 antibodies), alone or in combination with soluble CD28 antibodies or other T cell costimulators, offer another effective activation strategy that is fully compatible with the described rapid manufacturing workflow. This approach is particularly useful for PBMC-based approaches, enabling T cell activation without the need for exogenous CD28 or other costimulators. Furthermore, using CD3 antibodies such as OKT3 significantly reduces costs compared to other activation stimuli and minimizes the risk of scaffolds (e.g., alginate, magnetic beads, etc.) being present in the manufactured product, which may have unknown side effects for patients.

[0068] When employing surface-coating (e.g., plate, bag, flask) antibodies, it is advantageous to ensure that the antibodies adhere strongly, ideally irreversibly, to the surface. If the antibodies bind to the final cell product, there is a risk that they will be rapidly eliminated within the recipient. Indeed, OKT3, a CD3 antibody very commonly used for T cell activation, has been widely used in humans to rapidly eliminate circulating T cells (Todd PA, Brogden RN. Muromonab CD3. A review of its pharmacology and therapeutic potential. Drugs. 1989 Jun;37(6):871-99. doi: 10.2165 / 00003495-198937060-00004. Erratum in: Drugs 1989 Oct;38(4). PMID: 2503348). It has also been reported that human T cells bound to the OKT3 antibody are rapidly eliminated even in immunodeficient NSG mice (Wunderlich M, Brooks RA, Panchal R, Rhyasen GW, Danet-Desnoyers G, Mulloy JC. OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues. Blood. 2014 Jun 12;123(24):e134-44. doi: 10.1182 / blood-2014-02-556340. Epub 2014 Apr 28. PMID: 24778156; PMCID: PMC4055932).

[0069] When CD3 and / or CD28 antibodies are typically coated onto solid surfaces for T cell activation, they are typically incubated in a coating buffer, such as phosphate-buffered saline (PBS), Tris, or bicarbonate, to allow passive adsorption of the antibodies onto the surface. In some cases, the solid surface can also be washed with PBS, with or without detergent, such as 0.1% Tween 20, to remove free or loosely bound antibodies. While these methods allow antibodies to adhere to the solid surface and remove free antibodies, the interaction between the antibody and the surface is reversible. As seen in Figure 8, when monocyte-depleted PBMCs were incubated on an antibody-bound surface, a significant percentage of the cells were found to be bound to CD3 antibodies after removal from the culture medium (55.8% of the cells in this case). The high affinity of antibodies for cell surface antigens (e.g., CD3 or CD28) may cause some antibodies to detach from the solid surface due to their reversible binding when commonly employed antibody surface coating methods are used. Therefore, in a rapid manufacturing workflow, the product may still have high levels of bound antibody at the time of harvest. When antibodies bound to solid surfaces (e.g., plates, flasks, bags) are used in this rapid workflow, the workflow can incorporate an additional step not previously described for CAR-T manufacturing workflows. This additional step involves more strongly and optimally irreversibly attaching the antibody to the solid surface to minimize antibody binding to the product. Any of a number of methods for covalently and / or more strongly attaching antibodies to solid surfaces can be utilized for this purpose, compared to simple passive adsorption, as previously reported. For example, bisdiazonium compounds such as Fast Blue Salt B can be employed as previously described (expired U.S. Patent No. 4,654,299(A)). Additionally, numerous approaches have been described for chemically crosslinking antibodies to solid surfaces, such as the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, dimethyl pimelimidate, and glutaraldehyde.Other approaches for strong binding of antibodies to solid surfaces, which can also minimize contamination of the product antibody, include surface modifications that enhance antibody binding, such as modifying the surface with protein G, protein L, and / or protein A, as well as gamma irradiation (cobalt sterilization) to improve adsorptive binding. Covalent attachment of antibodies to fluorinated ethylene propylene (FEP), a surface commonly used for cell culture bags, has also been specifically reported. For example, high molecular weight polyvinyl alcohol (PVOH) treatment of FEP surfaces allows for antibody immobilization using various approaches, such as glutaraldehyde immobilization, photoactivatable linkers, and NHS-ester crosslinking (Pivetal J, Pereira FM, Barbosa AI, Castanheira AP, Reis NM, Edwards AD. Covalent immobilization of antibodies in Teflon-FEP microfluidic devices for the sensitive quantification of clinically relevant protein biomarkers. Analyst. 2017 Mar 13;142(6):959-968. doi: 10.1039 / c6an02622b. PMID: 28232992).

[0070] In one example, Fast Blue Salt B can be used to strongly attach antibodies to polystyrene surfaces. As seen in Figure 8, binding of antibodies to polystyrene surfaces with Fast Blue Salt B allows for the ability to produce a product with a dramatic reduction in antibody binding to the final surface (from 55.8% to 5.6%) within one day. In this case, Fast Blue Salt B is utilized to strongly attach antibodies to surfaces in the manner described below. However, any other approach employing Fast Blue or related compounds to more strongly attach antibodies to surfaces can also be utilized. Notably, the essential element for the Fast Blue approach is the use of Fast Blue compounds, and numerous modifications of the incubation times, wash solutions, and temperatures described herein may yield similar results. Here, polystyrene tissue culture vessels were first incubated with 1 mM Fast Blue B Salt solution in sodium phosphate buffer, pH 6.8, at 40°C for 30 minutes. The vessels were then washed three times with sodium phosphate buffer and incubated with CD3 and CD28 antibodies (approximately 150 ng / cm² of CD3 and 300 ng / cm² of CD28) in sodium phosphate buffer for 16 hours at 4°C. The antibody solution was removed, and the surfaces were incubated with phosphate buffer containing 0.1% Tween 20 for 1 hour, followed by three washes with phosphate buffer. Monocyte-depleted PBMCs were then incubated in the vessels, utilizing a method identical to the rapid manufacturing workflow described herein, without the use of Fast Blue, and including less than one day (~17-20 hours) of cell culture before harvest. Furthermore, virus can be added simultaneously with the addition of cells or after a short delay (~1-6 hours).

[0071] In addition to the ability of Fast Blue Salt B to strongly attach antibodies to solid surfaces, it also maintains the ability of CD3 / CD28 antibodies to activate T cells. As can be seen in Figure 9, monocyte-depleted PBMCs stimulated with CD3 / CD28 antibodies previously bound to the surface of polystyrene plates using the Fast Blue Salt B method were highly activated, as measured by CD69 expression.

[0072] Furthermore, monocyte-depleted PBMCs activated using antibody-coated plates with the Fast Blue salt B method were also efficiently transduced with virus using co-incubation of cells with surface-bound antibody and subsequent exposure to virus. In this study, GFP lentivirus was added, the product removed from the plate, and washed 20 hours later. As a control, wells were coated with CD3 / CD28 antibodies using the standard method of passive antibody adsorption in PBS without Fast Blue salt B. After washing, the cells were cultured in a new container for 3 days, and GFP expression was detected by flow cytometry. The Fast Blue method was found to result in comparable viral transduction efficiencies (15.2% and 14.6% of GFP-positive cells were transduced with the Fast Blue method versus the Fast Blue-free control method, respectively).

[0073] Specific T cell activation reagents were found to function more efficiently when using the rapid, sub-day manufacturing workflow described herein. To compare specific T cell activation reagents, monocyte-depleted PBMCs (200,000 cells in 100 μl) were cultured in 96-well plates containing TexMACs containing IL-7 (10 ng / ml) and IL-15 (5 ng / ml). (登録商標) (Miltenyi Biotec) medium, 3% CTS (登録商標) Cells were seeded in immune cell serum substitute (ThermoFisher Scientific). GFP lentiviral vectors and the indicated activation reagents (using the manufacturer's suggested concentrations) were added, and the cells were cultured for 4 days. After 20 hours, cells were washed and the medium was replaced to remove free virus and free activation reagents. Cloudz (登録商標) For cells containing the reagent, lysis buffer was used to remove the activation reagent. GFP expression was measured by flow cytometry. As seen in Figure 1A, Cloudz (登録商標) TransAct human T cell activation reagent (登録商標) , and CTS CD3 / CD28 Dynabeads (登録商標)Compared to the conventional method, the rapid manufacturing workflow was more efficient and maximized transduction efficiency. It is noteworthy for many figures that the amount of viral vector employed was kept low to easily visualize differences between conditions, rather than using excessive amounts of virus to maximize transduction efficiency. In Figure 1, the ability of the rapid manufacturing workflow to lead to high transduction efficiency can be observed using high levels of lentiviral vector (however, the vector copy number per transduced cell is maintained below 5). As can also be seen in Figure 1B, the rapid manufacturing workflow also works with soluble CD3 and / or soluble CD3 and CD28 antibodies (Figure 1C). For example, the use of OKT3, a widely used CD3 antibody, is possible with this workflow (Figure 1C). When using a manufacturing process with monocyte-depleted PBMCs, the use of CD3 alone performs similarly in terms of transduction efficiency compared to supplementation with CD3 and CD28 antibodies. Of note, OKT3 CD3 antibody stimulation significantly improved transduction efficiency in this manufacturing workflow. (登録商標) The results showed that Transactin functioned more efficiently than surface-bound (e.g., plate or flask) CD3 antibodies, as well as soluble CD3 antibodies, when used in a rapid manufacturing workflow, resulted in similar activation of CD4 and CD8 T cells, as measured by CD69 expression at the time of product harvest (20 hours after initiation of culture). (登録商標) It was observed that treatment with soluble / surface-bound CD3 resulted in a decreased level of CD69 upregulation in CD4 T cells compared to the group treated with soluble / surface-bound CD3. This result indicates that either soluble CD3 or CD3 bound to culture vessels can be used efficiently for T cell activation using a fast workflow. Furthermore, these activation reagents were compatible with the TransAct (登録商標) It appears to be superior to other substances such as

[0074] It has generally been thought in the art that sequential T cell activation followed by viral transduction is necessary to maximize viral transduction efficiency. However, at least using our particular manufacturing workflow, the results here demonstrate that simultaneous T cell activation and viral transduction result in transduction efficiencies essentially equivalent to sequential methods. For example (see Figure 2A), monocyte-depleted PBMCs were transfected with Cloudz (登録商標) Activate T cells using a T cell activation reagent and manufacture Cloudz using the manufacturing process described in the section on detailed examples of rapid manufacturing workflows. (登録商標) The cells were transduced with a lentiviral CD19 chimeric antigen receptor (CAR) vector either simultaneously with or 24 hours after the addition of CAR. Additionally, similar experiments were performed with a CD19 CAR vector and a BCMA CAR vector (see Figure 2B). As can be seen in Figures 2A and 2B, using the described manufacturing workflow, there is no significant difference in the level of CAR expression when using simultaneous or sequential viral transduction.

[0075] While in the majority of cases, no significant differences in transduction efficiency were observed when cell activation and viral transduction were performed simultaneously, in certain cases a small advantage was observed for a short (up to 3 hours) post-activation / pre-transduction period, which still allowed for product production in less than a day. For example, in the product production shown in the figure, a short pre-incubation step of 1 hour or more resulted in a small advantage of ~10% higher transduction efficiency levels. There was no statistically significant advantage to incubation for longer than 1 hour.

[0076] Both traditional and previously reported high-throughput T cell manufacturing approaches use culture conditions that almost exclusively employ cytokines such as IL-2, IL-15, and / or IL-7. To evaluate the optimal cytokines for use in a high-throughput manufacturing workflow, the manufacturing process described in the Detailed Example of a High-Throughput Manufacturing Workflow section was performed using IL-2 (300 μg / ml), IL-15 (5 ng / ml), IL-7 (10 ng / ml), a combination of IL-15 and IL-7, or the complete absence of exogenous cytokines (Figure 4). When comparing CD19 CAR expression in T cells 72 hours after transduction between the different cytokine groups, no difference in transduction efficiency was observed. Surprisingly, there was no difference in transduction efficiency even in the complete absence of cytokines. Therefore, to further reduce costs and prevent stimulatory effects on T cells that may lead to undesired differentiation / activation, the manufacturing process can be further simplified to be performed in the absence of any exogenous cytokines. As seen in Figure 1B, high-speed T cell production was also nearly equally efficient in directing GFP transduction of T cells when comparing productions performed in the presence or absence of cytokines.

[0077] Another important characteristic of the manufacturing process is the incubation period. Because one goal of the manufacturing workflow is to limit that period to maintain the proportion of naive T cells, we compared manufacturing processes utilizing incubation periods of 6 and 17 hours. At the end of both of these time points, Cloudz (登録商標) The reagents were removed using lysis buffer, the cells were washed to remove virus, suspended in fresh medium without virus and activation reagent, and cultured for a total of 72 hours to measure CAR expression. As seen in Figure 4, a 6-hour culture period with lentiviral vector and T cell activation reagent is insufficient to result in significant CD19 CAR expression as measured by flow cytometry on day 3. Intermediate periods of more than a few hours and less than approximately 15 hours are considered unfavorable due to limitations on what manufacturing can be performed during a typical run time.

[0078] If the manufacturing platform can utilize PBMCs or monocyte-depleted PBMCs as the starting material and the product is cultured for less than one day, the final product will consist of additional mononuclear cells in addition to T cells. To assess the product's composition, three manufacturing runs were performed using the methods described in the Detailed Example of a Rapid Manufacturing Workflow section. As can be seen in Table 2, T cells comprised a large proportion of the product, but the product also contained smaller numbers of B cells, NK cells, and monocytes. Because the preferred method for the workflow involves producing a cryopreserved product, this testing was performed after the product was thawed. Because granulocytes are a minor component of the starting apheresis product and are also highly sensitive to freeze / thawing, virtually no granulocytes were detected in the product.

[0079] Table 2. Composition of thawed UF-KURE19 product (17-hour production) from three manufacturing runs [Table 2]

[0080] Product composition was determined using flow cytometry with CD3, CD4, CD8, CD19, CD56, and CD14 antibodies, and 7-AAD to assess survival.

[0081] Since significant expression of proteins such as CARs after lentiviral transduction does not occur within 17-20 hours in T cells, to assess the in vitro activity of transduced CD19 CAR-T products, we produced CD19 CAR-T cells from monocyte-depleted PBMCs using the manufacturing workflow described in the section "Detailed Example of Rapid Manufacturing Workflow." After 20 hours of activation / transduction, cells were washed free of virus and then transduced with CloudZ. (登録商標)The T cell activation reagent was removed. The cells were then cultured for a total of 3 days, followed by evaluation of cytotoxic activity against target RAJI human lymphoma cells and measurement of CAR surface expression. As seen in Table 3, the rapidly produced CAR-T cells were able to efficiently lyse RAJI tumor cells.

[0082] Table 3. Results of in vitro cytotoxicity test of rapidly produced CD19 CAR-T cells after 3 days of culture [Table 3]

[0083] As described in the section on detailed examples of rapid manufacturing workflows, monocyte-depleted PBMCs were transduced with a CD19 CAR lentiviral expression vector to transduce T cells into CloudZ. (登録商標) The cells were activated with a T cell activation reagent. Free virus was removed, the T cell activation reagent was dissolved after 20 hours, and cell culture was continued for 3 days. The cytotoxic activity of CD19 CAR-T cells against RAJI tumor cells was assessed by flow cytometry, measuring the loss of calcein AM dye from tumor cells after 4 hours of co-culture with CAR-T cells. CD19 CAR expression was measured by flow cytometry using an FMC63-specific antibody (AcroBiosystems).

[0084] Because the CD19 CAR protein is not significantly expressed on the surface of T cells when harvested after 17-20 hours of culture using standard flow cytometry, we also assessed T cell activity in a mouse model to demonstrate the efficacy of the product. This method allows for full expression of the CAR in vivo, allowing T cells to subsequently acquire their cytotoxic activity against human CD19-expressing cells. To demonstrate the improved efficacy of our rapid-manufacturing CAR-T products, this in vivo study utilized cryopreserved cells produced in 17 hours, starting with monocyte-depleted PBMCs and transduced with a CD19 CAR lentiviral vector, following the workflow described in the "Detailed Example of the Rapid Manufacturing Workflow" section. This product expressing the CD19 CAR was designated UF-KURE19 cells. Cells produced using the same workflow but maintained in culture for 6 days instead of 17 hours were designated Kure19. The cells were tested in a circulating mouse model of human lymphoma, involving intravenous injection of human RAJI tumor cells into immunodeficient mice (NSG), followed by a single intravenous injection of the CAR-T product 7 days after tumor cell injection. Traditionally, ~5 million CAR T cells have been used in this model to demonstrate significant efficacy, but lower doses were utilized due to the anticipated increased efficacy of the rapid-manufacturing product. For the UF-Kure19 product, doses of 2 million and 4 million CAR-positive T cells were utilized in the UF-KURE19 cohort, while the 6-day manufactured product had 2 million CAR-positive T cells. As can be seen in Figure 5, UF-KURE19 cells demonstrate significant efficacy at both low and high dose levels. In contrast, the 6-day culture product demonstrates reduced tumor development compared to vehicle-treated mice, but efficacy is dramatically reduced when compared to the UF-KURE19 product. Similar to the mouse studies employing Kure19, the UF-KURE19-injected group tolerated the treatment well (as monitored by weight change, food intake, appearance, and behavior) and showed no obvious signs of toxicity.

[0085] CD19 CAR-T cells have been shown to persist for months and even years (as measured by transgene detection). For example, the Tisa-cel product has been shown to persist for at least two years in some patients and to have favorable clinical outcomes (10). Human T cell proliferation in the blood of NSG mice bearing RAJI lymphoma tumors was measured by flow cytometry. Table 4 shows the average number of human T cells detected per microliter of mouse peripheral blood at the indicated time points after injection of RAJI tumor cells (0.5 million i.v.) into female NSG mice (measurements were performed in n = 3–5 mice per time point). Note that the blood samples for these measurements were derived from the mouse efficacy study described above. As shown in Table 4, the efficacy of UF-KURE19 cells was significantly higher than that of KURE19 CAR-T cells produced at 6 days, correlating with human T cell proliferation.

[0086] Table 4. T cells detected by flow cytometry per microliter of mouse blood [Table 4] ND = Not Measured

[0087] As mentioned above, the use of cytokines in the rapid manufacturing workflow was not necessary to achieve significant transduction efficiency. Therefore, the impact of cytokine use on the memory / differentiation state of T cells at harvest (20 hours) was assessed by flow cytometry. Furthermore, this study employed different cytokines or the absence of cytokines in the manufacturing process, as shown in Figure 10, to better evaluate the impact of cytokines on the product. Finally, this study also compared the use of monocyte-depleted PBMCs versus isolated T cells as starting material. The manufacturing workflow described in the Detailed Example of Rapid Manufacturing Workflow section was utilized, except that purified T cells were used as starting material for a subset of samples. RosetteSep (登録商標)T cells were purified from peripheral blood using a Human T cell Enrichment Kit (StemCell Technologies). As seen in Table 5, when comparing products produced starting from isolated T cells with those produced starting from monocyte-depleted PBMCs, it was observed that the CD8+ T cell components of the products were not identical. In particular, when starting from PBMCs, there were 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 they are thought to increase potentially beneficial cytotoxic activity. The levels of highly beneficial naive T cells, known to correlate with CAR-T efficacy, were similar in the CD8+ T cell compartment whether starting from PBMCs or isolated T cells. However, the percentage of beneficial central memory T cells in PBMCs was higher compared to isolated T cells, and the levels of more differentiated, undesirable effector memory cells were lower. In the CD4+ T cell compartment, the percentage of highly beneficial naive T cells was elevated when starting from PBMCs compared to isolated T cells. Therefore, based on the phenotypic analysis of the product, a workflow starting with PBMCs provides a more favorable workflow that may be predicted to result in improved clinical outcomes and superior in vivo persistence of CAR-T cells. Furthermore, this study also investigated the use of different cytokines (IL2, IL7, IL-15) or the absence of cytokines during manufacturing. As seen in Table 5, when starting with PBMCs, there were no significant differences in T cell memory / differentiation phenotypes across all conditions over the 20-hour workflow. Surprisingly, when starting with monocyte-depleted PBMCs, cytokines were further suggested to be a non-essential component of this manufacturing workflow. Interestingly, when starting with isolated T cells, a decrease in naive T cells was observed when cytokines were not employed, suggesting that the use of cytokines may be more important for manufacturing when using isolated T cells as the starting material. PBMCs may provide an endogenous source of cytokines.

[0088] Table 5. Phenotype of T cells produced using the fast workflow [Table 5] TIFF2026501322000006.tif155169

[0089] T cells were manufactured using the rapid workflow described in the Detailed Example of Rapid Manufacturing Workflow section, or using an identical workflow except using isolated T cells as the starting cell material. After 20 hours, cells were assessed for T cell phenotype by flow cytometry.

[0090] Because in vitro studies indicated that cytokines did not appear to be necessary to efficiently transduce T cells or maintain the desired memory / differentiated T cell phenotype when using a rapid manufacturing process starting with PBMCs, we performed mouse in vivo efficacy studies to further confirm these results. Starting with monocyte-depleted PBMCs, CD19 CAR-T cells were manufactured using a rapid manufacturing workflow with either IL-7 (10 ng / ml) and IL-15 (5 ng / ml) or no cytokines during culture. The same human lymphoma tumor model (RAJI) as described above in NSG mice was employed. In this case, 7 days after tumor cell injection, 1.2 × 10 CAR-T cells per mouse were injected. 6 CD19 CAR-positive T cells were injected into the mice. As can be seen in Figure 6, the CAR-T product manufactured in the complete absence of exogenous cytokines efficiently controlled tumor development in a manner similar to that of the product manufactured with cytokines. All vehicle control mice died of disease progression before imaging was performed on day 45. Of note, the low dose of 1.2 × 10 6 CD19 CAR-positive T cells were highly effective, as seen in Figure 6A, further demonstrating the efficacy of a fast-track CAR-T product. This study also clearly demonstrates that exogenous cytokines are not required to generate a fast-track (<1 day) CAR-T product starting from PBMCs.

[0091] In addition to similar efficacy, circulating human T cells were not reduced in mice when the cytokine-free product was utilized. Human T cells in mouse blood from the experiment shown in Figure 6, 41 days after tumor cell injection, were quantified using a human-specific CD3 antibody and flow cytometry analysis. Cytokine-free CAR-T group: 4495 human T cells per microliter of mouse blood. IL7 / IL15 CAR-T group: 1618 human T cells per microliter of mouse blood.

[0092] A detailed example of a rapid manufacturing workflow In this workflow, selected representative modifications are shown in parentheses. In addition to this relatively manual workflow shown below, the entire workflow can be performed in a fully closed and / or semi-automated or automated manner. For example, cell washing, harvesting, etc. can be performed using automated equipment. Manufacturing process starting with monocyte-depleted PBMCs to produce CAR-T products in less than one day A. Obtaining Starting Cells (Day -1, or 0) Autologous peripheral blood mononuclear cells were obtained from patients using leukapheresis (or peripheral blood draw). The cell product was processed immediately and stored overnight. (The cell product can also be cryopreserved and processed at a later date, if desired.) B. Processing of Apheresis Samples (Day 0) The apheresis sample was washed by centrifugation to remove plasma and reduce the number of contaminating platelets. (If peripheral blood samples were used instead of apheresis samples, PBMCs were isolated using methods such as Ficoll-based isolation.) The apheresis sample was then diluted with medium such as TexMACS (Miltenyi Biotec) supplemented with CTS immune cell serum substitute (ThermoFisher Scientific) and incubated in a tissue culture flask or cell culture bag at 37°C (±2°C) in a culture incubator to allow monocytes to adhere at a concentration of up to 5 million cells per ml. After at least 2 hours of monocyte adherence to the flask or bag, nonadherent cells were transferred to a new tissue culture flask or bag. The volume was adjusted to 2 million cells per ml with medium containing IL-7 (10 ng / ml) and IL-15 (5 ng / ml). (The medium can be prepared without the addition of any exogenous cytokines, or with the addition of IL-7 alone, IL-15 alone, or other cytokines.) Activation of CT cells (day 0) Cloudz (登録商標) CD3 / CD28 T cell activation reagent (R&D Systems / Biotechne) can be added to the flask containing the monocyte-depleted, processed apheresis product. (Soluble CD3 antibody, soluble CD3 / CD28 antibody, surface-bound CD3 antibody with or without CD28 soluble antibody, Immunocult (登録商標) (Stemcell Technologies), or any other T cell activation reagent can be used.) D. Cell transduction (day 0) Immediately after addition of the T cell activation reagent, 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 incubated at 37°C (±2°C) in a 5% (±0.5%) CO incubator for 17-20 hours. E. Cell Harvesting (Day 1) Cloudz (登録商標)If cells were activated using a 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 a freezing bag or vial.

[0093] Description of methods that enable release testing of vector copy number and replication-competent virus in high-speed genetically modified T cell products: A major obstacle in the area of ​​high-speed T cell manufacturing workflows for viral vectors is performing the necessary product release testing, and therefore, the method described below was developed. Current product release testing requirements for retroviral- or lentiviral-transduced cell therapy products include assessing replication-competent lentivirus and vector copy number. Both of these assays have significant false-positive results when using traditional qPCR-based assays, even when free plasmid contamination is present at low levels. Residual free plasmid from 293 cell transfection is present in viral vectors. At early points in manufacturing after viral transduction, residual vector-derived plasmid remains, making it nearly impossible to eliminate false-positive results using traditional testing methods. This false-positive reactivity is a major obstacle, limiting the ability of high-speed manufactured products to be used in patients.

[0094] Because the DNA of interest for both viral integration and replicating vectors is integrated viral DNA, the manufacturing workflow included herein, combined with a method for removing free residual plasmids, allows PCR or other molecular testing for both vector copy number and replicating lentivirus to be performed without false positive reactions. Notably, free plasmid DNA is significantly smaller than genomic DNA. A centrifugation-based method is used to remove small DNA while preserving large genomic DNA. Using this method, false positive reactions from plasmid DNA do not occur, overcoming a major obstacle in high-speed CAR-T manufacturing while still being able to measure genomically integrated DNA.

[0095] To remove free plasmid DNA from CAR-T products cultured for a short period (e.g., 0–3 days), centrifugation can be used to remove small DNA fragments (e.g., less than 10 kb) while preserving genomic DNA (e.g., using a solution such as a salt / polymer solution that preferentially precipitates high-molecular-weight DNA). For example, the PacBio Short Read Eliminator Kit (PacBio; Menlo Park, CA) can be used, enabling this separation in a single centrifugation step (circulomics.com / store / Short-Read-Eliminator-Kit-p131401036). In addition to this commercially available reagent, an alternative method is to use 4% PVP 360,000, 1.2 M KCL, and 20 mM Tris-HCl pH 8 in place of the commercially available SRE buffer in the protocol described below, which has previously been shown to efficiently remove small fragments (e.g., less than 10 kb from total DNA) (11). The purified DNA can then be used directly in assay tests (e.g., qPCR assays). Although this kit was not designed for this specific application, it works well and provides a simple, cost-effective, and fast approach.

[0096] One example of how this method can be employed is to first isolate total DNA using any commercially available total DNA isolation kit capable of isolating human genomic DNA (e.g., DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). The method described below, or a similar approach, can then be used to remove low-fragment DNA. 1. Using a wide-bore pipette tip, add Buffer SRE to the starting genomic DNA sample and mix well by pipetting. If you are not using PacBio or other commercially available reagents, you can add 4% PVP 360,000, 1.2M KCL, 20mM Tris-HCl pH 8 to all DNA samples in a 1:1 volume ratio. 2. The tube was centrifuged at 10,000 x g for 30 minutes at room temperature. 3. The supernatant was removed with a pipette. 4. 70% ethanol was added to the tube. 5. Centrifuge at 10,000 x g for approximately 2 minutes at room temperature. 6. The supernatant was removed and the wash with 70% ethanol was repeated. 7. Buffer EB (or 10 mM Tris-HCl pH 8 if not using the PacBio kit) was added to the tube and incubated at 50°C for 10-30 minutes, followed by resuspending the DNA pellet.

[0097] The use of the low-fragment removal workflow in combination with the rapid manufacturing workflow demonstrated an efficient process, allowing release testing of genetically modified T cell products at early time points in culture (e.g., from 17 hours to 3 days). For example, CAR-T products manufactured using the 17-20 hour workflow were harvested at 20 hours, with an alternative aliquot harvested at 72 hours. Total DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen), and low-fragment removal DNA was prepared using the Short Read Eliminator Kit (PacBio). The low-fragment removal DNA, as well as the total DNA samples, were tested for replication-competent lentivirus using a real-time PCR assay against VSV-G, and for vector integration using real-time PCR assays against GAG and the housekeeping gene PTBP2. The primers and fluorescent probes used for the PCR reactions can be seen in Table 6.

[0098] Table 6. PCR primers and probes used for GAG, VSV-G, and PTPBP2 qPCR assays. [Table 6]

[0099] PCR products were quantified using a standard probe consisting of linearized plasmids designed to express single copies of GAG, VSV-G, and PTBP2. As seen in Figure 7A, qPCR reactions performed with low-fragment-removed DNA did not detect VSV-G and were therefore negative for replication-competent lentivirus, as expected for two different products produced at 20 h. On the other hand, when total genomic DNA was utilized without low-fragment removal, both samples gave false-positive qPCR results for VSV-G, likely due to contamination with low-fragment-free plasmid. We performed further assays to evaluate whether alternative approaches, such as cell washing or culturing for 3 days (rather than 20 h), could overcome the false-positive reactivity in testing for replication-competent lentivirus. Specifically, we harvested lentivirally transduced CD19 CAR-T products at 20 and 72 h and thoroughly washed the cells before genomic DNA isolation. Specifically, we used cells (5 × 10 6 The cells were washed with 50 mL of PBS 15 times for the 72-hour samples and 5 times for the 20-hour samples. In all cases (20- and 72-hour samples of both products), there was a false-positive reactivity when using the VSV-G qPCR assay. Therefore, increasing the incubation period to 3 days and thoroughly washing the cells was not sufficient to reduce this false-positive reactivity.

[0100] Of note in testing replication-competent lentiviruses is that no positive tests have been reported to the FDA for clinical products in the past decade, supporting the conclusion that the consistently observed positive reactions in 17-20 hour and 72 hour rapid product manufacturing are due to false positive reactions (fda.gov / media / 113790 / download). Furthermore, when the incubation period was extended to 8 days, the VSV-G qPCR assay became negative, suggesting that free plasmid DNA had been lost by this time point and that true replication-competent virus was not present.

[0101] Similar to testing for replication-competent lentivirus, another important release test for virally transduced cell therapy products is vector integration, often measured by qPCR for the GAG ​​gene. The presence of free plasmid contamination in viral vectors can result in false-positive results. To assess the vector multiplicity of infection (MOI) and baseline complete vector integration, we first performed GAG / PTPB2 qPCR to determine the vector copy number of CD19 CAR lentiviral-transduced T cells maintained in culture for 8 days. As seen in Figure 7B, an MOI of 10:1 resulted in a peak vector copy number per transduced T cell (i.e., CD19 CAR expression) of 1.5. Next, we used a rapid manufacturing workflow, except that we harvested cells at 20 hours, day 3, and day 7, to determine the vector copy number per transduced cell when the MOI of the CD19 CAR vector was 10:1. Additionally, we utilized an SRE kit (PacBio) to remove low-fragment DNA. As can be seen in Figure 7C, the results of vector integration were similar across all time points. Of note, surface expression of the CD19 CAR was fully expressed by around 72 hours but very low at 20 hours.

[0102] Finally, gel electrophoresis of total DNA samples prepared from two 20-hour rapid T cell productions before and after low-fragment removal demonstrates that, as expected, genomic DNA is preserved and therefore only undesired low-molecular-weight fragments are removed (Figure 7D). (登録商標) Quantification of DNA concentration using a spectrophotometer (ThermoFisher Scientific) did not reveal any measurable change in DNA concentration before or after low-fragment removal, indicating that only trace amounts of DNA were removed.

[0103] As will be appreciated, the CAR-T cell manufacturing workflow described herein provides significant improvements over the prior art. Specifically, but not by way of limitation, 1. Manufacturing is primarily performed using unfractionated cells derived from apheresis products or apheresis cells in which monocytes have been removed by adherence to a solid surface. Furthermore, peripheral blood mononuclear cells (PBMCs) or monocyte-depleted PBMCs isolated directly from peripheral blood can be utilized. Therefore, purified T cells, which have been considered crucial for high-speed CAR-T workflows involving viral transduction, are not required. To our knowledge, this is the first one-day or less protocol to employ PBMCs, rather than isolated T cells, as the starting material for high-speed genetically modified T cell manufacturing using viral transduction. 2. Cell activation can be achieved using a variety of methods. Magnetic beads require several days to shed cells, and therefore methods employing such beads delay product recovery and / or result in significantly reduced product yield due to loss of bead-bound cells. Furthermore, we have unexpectedly discovered that the use of certain non-magnetic bead-based T cell activation reagents improves transduction efficiency. The activation approach described herein provides improved transduction efficiency while avoiding the increased cost and manufacturing complexity associated with the use of magnetic beads required by prior art methods. 3. The manufacturing process described herein is the first rapid CAR-T manufacturing workflow described to be able to run efficiently in the absence of exogenous cytokines. The addition of cytokines during manufacturing significantly increases costs and can also result in alterations of T cells. Because a major advantage of a rapid manufacturing workflow is to maintain the starting population of T cells (e.g., naive T cells) as close as possible to the original cells obtained from the patient, the absence of exogenous cytokines is considered a major advantage. 4. The described use of a completely closed system for producing T cell products using bags allows for the provision of a convenient kit-based product, where virus / media / activation reagents, etc. are provided in the bag and subsequently mixed with the apheresis product. Using this method, the product can be manufactured outside of a clean room, facilitating widespread use and manufacturing of the product, reducing costs and increasing accessibility. 5. The methods described herein overcome a major obstacle in the field of required product release testing. Current release testing requirements for retroviral- or lentiviral-transduced cell therapy products include assessing replicative lentivirus and vector copy number. When using traditional qPCR-based assays, both of these assays produce significant false-positive results, even when free plasmid contamination is at low levels. Residual free plasmids from virus-producing cells, such as 293 cells, are present in viral vectors. At early manufacturing points after viral transduction, residual plasmids from the vector remain, making it nearly impossible to eliminate false-positive results using traditional testing methods. This false-positive reactivity is a major obstacle for other groups, limiting the ability of rapidly manufactured products to be used in patients. Because the DNA of interest for both virally integrated and replication-competent vectors is the integrated viral DNA, the manufacturing workflow encompassed herein, combined with a method to remove free, residual plasmid DNA, allows for false-positive-free qPCR reactions for both vector copy number and replication-competent lentivirus. Notably, free plasmid DNA is significantly smaller than genomic DNA. A centrifugation-based method is used to remove small DNA while preserving large genomic DNA, overcoming a major obstacle to rapid CAR-T manufacturing by eliminating false-positive reactions from plasmid DNA and allowing measurement of DNA integrated into the genome.

[0104] Unless otherwise defined, 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.

[0105] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referenced throughout this disclosure are hereby incorporated by reference in their entirety, unless otherwise stated.

[0106] When a URL or other identifier or address is referenced, it is understood that such identifiers may change and certain information on the Internet may disappear or move, but that equivalent information may be obtained by searching the Internet, and that reference indicates that such information is available and publicly disseminated.

[0107] As used herein, abbreviations for any protecting groups, amino acids, and other compounds follow their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726-1732), unless otherwise indicated.

[0108] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter disclosed herein, representative methods, devices, and materials are described herein.

[0109] In certain instances, the nucleotides and polypeptides disclosed herein are available from GENBANK. (登録商標) and SWISSPROT. Information, including sequences, and other information regarding such nucleotides and polypeptides, contained in such publicly available databases is expressly incorporated herein by reference. Unless otherwise indicated and only where clear, references to such publicly available databases are to the most current versions of the databases at the time of filing this application.

[0110] Unless otherwise indicated, all numerical values ​​expressing properties such as quantities of ingredients and reaction conditions used in the specification and claims are understood to be modified in all instances by the term "about." Thus, 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 to be obtained by the subject matter disclosed herein.

[0111] Whenever any of the phrases "for example," "such as," "including," and the like are used herein, they are understood to be followed by the phrase "and without limitation," unless expressly stated otherwise. Similarly, "an example," "exemplary," and the like are understood to be non-limiting. The term "substantially" permits deviations from its description to the extent that they do not adversely affect the intended purpose. A stated term is understood to be modified by the term "substantially," even if the word "substantially" is not explicitly recited. Thus, for example, the phrase "wherein the lever extends vertically" means "wherein the lever extends substantially vertically," unless a precisely vertical orientation is necessary for the lever to perform its function.

[0112] The terms "comprising," "including," "having," and "involving" (and similarly, "comprises," "includes," "has," and "involves"), and their equivalents, are used interchangeably and have the same meaning. Specifically, each term is defined consistently with the general definition of "comprising" under U.S. patent law and, therefore, is to be construed as an open term meaning "including at least the following," and is not to be construed as excluding additional features, limitations, embodiments, etc. Thus, for example, "a process involving steps a, b, and c" means a process including at least steps a, b, and c. Whenever the word "a" or "an" is used, it is to be understood as "one or more," unless such interpretation is undue to the context. The terms "comprise," "have," "include," and "contain" (and their variants) are open-ended linking verbs that, when used in the claims, allow for the addition of other elements.

[0113] The use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims or the specification, means one or more, unless the context dictates otherwise.

[0114] As used herein, the term "about," when referring to a numerical value, or mass, weight, time, volume, concentration, or percentage, is meant to encompass variations from the specified amount of ±20% in some embodiments, ±10%, ±5%, ±1%, ±0.5%, ±0.1%, ±0.01%, and ±0.001%, where these variations are appropriate to perform the disclosed methods. The use of the term "or" in the claims is used to mean "and / or" when not explicitly referring to only alternatives or when the alternatives are not mutually exclusive.

[0115] As used herein, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. A number of numerical values ​​are disclosed herein, and it is understood that such numerical values ​​are also disclosed herein as "about" that numerical value in addition to the numerical value itself. For example, if the numerical value "10" is disclosed, "about 10" is also disclosed. Such numerical values ​​between two particular numerical values ​​are also understood to be disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0116] As used herein, "optional" or "optionally" means that a substantially described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur. For example, an optionally variant portion means that the portion may or may not be variant.

[0117] It will be understood that various details of the subject matter disclosed herein can be changed without departing from the scope of the subject matter disclosed herein. Also, the foregoing description is for purposes of illustration only, and not limitation. Obvious modifications and variations are possible in light of the above teachings. All such modifications and variations are within the scope of the appended claims when interpreted in accordance with the breadth and breadth to which they are fairly, legally, and equitably entitled.

[0118] References:

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[0120] 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.

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[0122] 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.

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Claims

1. A rapid method for producing a population of genetically modified T cells, Obtain a population of mixed mononuclear cells; Activating T cell populations contained within mixed mononuclear cell populations; and Exposing mixed mononuclear cells containing an activated T cell population to at least one viral vector adapted to transduce at least one T cell population contained within the mixed mononuclear cell population with an exogenous nucleotide, following a post-activation period of up to three hours; A method that includes this.

2. The method according to claim 1, wherein the period after activation is approximately 1 hour to approximately 3 hours, or the period after activation is approximately 1 hour.

3. The method according to claim 1, comprising recovering a mixed mononuclear cell population containing at least a population of genetically modified T cells up to 24 hours after exposure to at least one viral vector.

4. Activation is performed by exposing mixed mononuclear cells to an activator selected from one or more cytokines consisting of IL-2, IL-7, IL-15, IL-12, IL-18, and IL-21, and / or 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). The method according to claim 1, wherein activation is optionally performed by exposing mixed mononuclear cells to an activator selected from one or more of the group consisting of IL-7 and IL-15.

5. The method according to claim 1, further comprising the step of removing at least partially the monocyte population contained in the mononuclear cell population before the steps of activation and exposure to a viral vector.

6. The activation and exposure to the viral vector processes are carried out in the absence of exogenous cytokines. Optionally, the activation process is carried out by exposing a mixed mononuclear cell population to one or more of the following: a CD3 activator, a CD28 activator, a soluble or surface-bound CD3 antibody, or a soluble or surface-bound CD28 antibody. The method according to claim 5, wherein the activation step is optionally performed by exposing a mixed mononuclear cell population to a CD3 activator, or to one or both of a soluble or surface-bound CD3 antibody.

7. The method according to claim 1, wherein the steps of activation and exposure to a viral vector are carried out without prior T cell isolation or pre-activation steps.

8. Cryopreserving a mixed mononuclear cell population, including a population of genetically modified T cells; Obtain a mixed mononuclear cell population by apheresis or peripheral blood sampling; or The method according to claim 1, further comprising selecting a viral transduction vector from the group consisting of lentiviruses, retroviruses, and adenoviruses.

9. The method according to claim 1, further comprising the following steps following the step of exposure to a viral transduction vector: The steps include: isolating DNA from a transduced T cell population or its aliquots in order to provide an isolated DNA sample; and A step of separating plasmid DNA from the genomic DNA of a transduced T cell population by DNA size selection in order to provide an isolated genomic DNA sample in an isolated DNA sample or an aliquot thereof, thereby preventing contamination of the genomic DNA by plasmid DNA during subsequent analysis, wherein optionally the separation step is performed by fractional centrifugation.

10. Genetically modified T cells produced by the method described in any one of claims 1 to 9.

11. A kit for carrying out a rapid method for producing a genetically modified T cell population according to any one of claims 1 to 9, Bead-free T cell activator; A viral vector suitable for transducing a T cell population with foreign nucleotides; Suitable culture medium; A suitable cell washing solution; and Suitable containers for the sterile introduction of bead-free T cell activators, viral transduction vectors, culture media, and cell washing solutions. A kit that includes this.

12. The bead-free T cell activator is selected from one or more of the following groups: CD3 activator, CD28 activator, OX40 activator, CD2 activator, CD27 activator, ICAM-1 activator, LFA-1 (CD11a / CD18) activator, ICOS (CD278) activator, 4-1BB (CD137) activator, and CD3 antibody. The kit according to claim 11, further comprising optionally one or more cytokines selected from the group consisting of IL-2, IL-7, and IL-15.

13. The bead-free T cell activator is selected from one or more of the group consisting of one or more soluble or surface-bound CD3 antibodies and one or more soluble or surface-bound CD28 antibodies. The kit according to claim 12, wherein the bead-free T cell activator is optionally selected from one or more of the group consisting of one or more soluble or surface-bound CD3 antibodies.

14. Surface-bound CD3 antibody and / or surface-bound CD28 antibody are bound to the surface of the container. Optionally, surface-bound CD3 antibody and / or surface-bound CD28 antibody are bound to the surface of the container by a method other than passive adsorption. The kit according to claim 13, further optionally comprising a surface-bound CD3 antibody and / or a surface-bound CD28 antibody bound to the surface of the container by a method selected from the group consisting of covalent bonding, chemical crosslinking, glutardialdehyde coupling, treatment of the container with a bisdiazonium compound, and modification of the container surface by attachment of an antibody-binding protein.

15. Treatment with a bisdiazonium compound results in the binding of surface-bound CD3 antibody and / or surface-bound CD28 antibody. The kit according to claim 14, wherein optionally the bisdiazonium compound is Fast Blue Salt B.

16. The kit according to claim 11, wherein the viral vector is selected from the group consisting of lentiviruses, retroviruses, and adenoviruses.

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