Methods for generating genetically modified T cells

Abstract

The present invention provides a method for generating genetically modified T cells, comprising the steps of: a) providing a sample comprising T cells; b) preparing the sample by centrifugation; c) concentrating the T cells; d) activating the T cells using a modulating agent; e) genetically modifying the T cells by transduction with lentiviral vector particles; and f) removing the modulating agent, thereby generating a sample of genetically modified T cells, wherein the method is performed in 144 hours or less, less than 120 hours, less than 96 hours, less than 72 hours, less than 48 hours, or less than 24 hours. In one embodiment of the present invention, the enrichment of the T cells is performed by magnetic cell separation using magnetic particles directly or indirectly coupled to CD4- and / or CD8-specific antibodies or antigen-binding fragments thereof, and the magnetic particles can be removed from the cells after separation.

Description

[Technical field]

[0001] The present invention relates to the field of generating genetically engineered T cells, and in particular to generating genetically engineered T cells within a short period of time and with low concentrations of contaminants and / or unwanted cells in a target population. [Background technology]

[0002] Clinical manufacturing of genetically modified T cells is a complex process. Patients' peripheral blood mononuclear cells (PBMCs), which are often rich in T cells, are activated before genetic modification with viral or non-viral vectors. The modified T cells are then expanded to reach the cell numbers required for therapy, after which the cells are finally formulated and / or cryopreserved before reinfusion. The cell products must undergo a number of quality control assays and meet all shipping standards and Good Manufacturing Practice (GMP) guidelines. Thus, until now, adoptive cell transfer (ACT) in which genetically modified T cells are used has often been performed by researchers who have developed their own manufacturing processes for small-scale clinical trials by using makeshift devices and basic facilities. Meanwhile, automated processes in closed systems are also available (e.g., WO2015162211A1, WO2019046766A1). WO2019032929A1 discloses a method for genetically engineering T cells, in which a sample comprising T cells is incubated under stimulatory conditions and a nucleic acid is introduced into the stimulated T cells during at least a portion of the incubation.

[0003] There is a need in the art for improved methods, preferentially automated processes, for generating genetically modified T cells, e.g., to reduce toxicity of the generated T cells and / or to shorten process times, e.g., to allow improved administration to patients in need thereof. Summary of the Invention

[0004] Residual modulatory agents contaminating the drug product may be harmful when infused, as they may lead to undesired activation of T cells in vivo. This may lead to rapid release of inflammatory cytokines, causing severe cytokine release syndrome, fever, hypotension, organ failure, and even death. In addition, residual lentiviral vectors contaminating the drug product in soluble and / or cell-associated forms may be harmful when infused, as they may induce undesired immune responses, such as complement activation, antibody-dependent cell-mediated cytotoxicity, including adoptive immune responses against antigens delivered by the lentiviral vector and / or transduction of non-target cells in vivo. Transduction of non-target cells and subsequent expression of the transgene may induce undesired side effects, such as induction of undesired immune responses, carcinogenesis, altered viability, altered proliferation, altered physiological state, and altered native function.

[0005] Surprisingly, it has been found that the process for generating modified T cells disclosed herein can be shortened from the start of the process to less than 144 hours, less than 120 hours, less than 96 hours, less than 72 hours, less than 48 hours, or even less than 24 hours, during which time molecules, reagents potentially harmful to the patient are removed during the process and / or at the end of the process as clean-up and an additional safety layer, i.e. the sample containing T cells is prepared into a sample containing genetically modified T cells that can then be directly (re)infused to a patient in need thereof. The genetically modified T cells may be T cells expressing a chimeric antigen receptor, and the application may be for treating cancer in a patient.

[0006] It was surprising that there was no need to expand the genetically modified T cells in vitro to the cell numbers known to be required for effective treatment of patients, since further expansion of these genetically modified T cells to therapeutically effective amounts would occur in vivo. The expansion of the number (quantity) of genetically modified T cells in a sample generated as disclosed herein may be less than 10-fold, preferentially less than 5-fold, compared to the number (quantity) of T cells in the sample prepared at the beginning of the process. This is possible due to the high quality of the genetically modified T cell composition / sample generated by the method disclosed herein, i.e., low contamination with reagents, lentiviral vectors, and non-genetically modified T cell components.

[0007] The present invention has been successful in demonstrating that CAR T cells generated in vitro within a few days, e.g., 3 days (72 hours) or less, using the methods disclosed herein in the absence of an explicit expansion step, surprisingly promote robust anti-tumor activity in vitro and in vivo, demonstrating that in vivo, but not in vitro, expansion is essential for the generation of functional CAR T cells (see Example 10).

[0008] Although data is shown for a method carried out over 3 days, it is clear that in vivo effects will be observed in samples produced by this method in less than 72 hours (3 days), e.g., 48 hours or 24 hours, and the time period for the produced cells to cause the in vivo effect of killing cancerous cells will simply be delayed. Figure 9 provides data showing that production times can be further shortened, with only a reduction in gene transfer efficiency. [Brief description of the drawings]

[0009] [Figure 1]Schematic diagram for generating genetically modified T cells in a short period of time. A sample containing T cells is prepared, such as human whole blood, leukapheresis, buffy coat, PBMC, expanded or isolated T cells. Optionally, the sample contains serum containing a substance that inhibits genetic modification by lentiviral vectors. To allow efficient transduction, the serum is removed by washing. In addition, the T cells are polyclonally activated with a regulator that binds CD3 and CD28, and then genetically modified using lentiviral vectors. As a cleanup, the regulator is removed to obtain purified genetically modified T cells. [Diagram 2] Schematic diagram of the removal of the modulatory activating agent. T cells are polyclonally activated with a modulatory reagent comprising an antibody or antigen-binding fragment thereof specific for CD3 and an antibody or antigen-binding fragment thereof specific for CD28. Both antibodies or their fragments are directly or indirectly coupled to a biodegradable linker. The modulatory activating reagent can be removed by washing or by adding an enzyme that specifically degrades the linker, thereby releasing the CD3- and CD28-specific antibody or fragment thereof. In addition, the activating reagent can be removed by chemical cleavage of the CD3- and CD28-specific antibody or antigen-binding fragment thereof. The removal of the modulatory agent or fragment thereof from the cells can be performed by one or several washing steps. [Diagram 3]Schematic diagram of the removal of magnetic enrichment reagents. CD4+ and / or CD8+ T cells are separated by magnetic cell separation, such as MACS, where magnetic particles are directly or indirectly contacted with T cells coupled with CD4- and / or CD8-specific antibodies or antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof are coupled to the magnetic particles via biodegradable linkers. The coupled magnetic particles can be removed by washing or by adding an enzyme that specifically degrades the linker, thereby releasing the CD4- and / or CD28-specific antibodies or fragments from the magnetic particles. Additionally, the magnetic particles can be removed by chemical cleavage. Removal of the magnetic particles or fragments thereof can be performed by one or several washing steps. [Figure 4] Schematic diagram of the removal of reagents for indirect magnetic labeling of T cells. T cells can be indirectly labeled by contacting magnetic particles with T cells coupled to CD4 and / or CD8 specific antibodies or antigen-binding fragments thereof via a biotinylated biodegradable linker, the magnetic particles being coupled to biotin specific antibodies or antigen-binding fragments thereof. The magnetic particles can be released from the T cells by adding an enzyme that specifically digests the biodegradable linker and / or by adding biotin (as a competitor). Additionally, the indirectly coupled magnetic particles can be removed by washing and / or chemical cleavage. Removal of the cleaved agents or magnetic particles can be performed by one or several washing steps. [Diagram 5]Removal of activation reagent by washing. Enriched T cells were polyclonally stimulated with T Cell TransAct™ (Miltenyi Biotec), a regulatory reagent comprising an antibody or antigen-binding fragment specific for CD3 and an antibody or antigen-binding fragment specific for CD28, directly coupled to a biodegradable linker. T cells 20 hours after stimulation with the regulatory activation reagent were washed, and the presence of bound biodegradable linker was measured by flow cytometry at several time points after stimulation. The stimulating reagent was efficiently removed by washing, as detected by a decrease in biodegradable linker levels over time. [Figure 6] Removal of the regulator by washing and enzyme activity. Enriched T cells were polyclonally stimulated with T Cell TransAct™ (Miltenyi Biotec), a regulator reagent comprising an antibody or antigen-binding fragment specific to CD3 and an antibody or antigen-binding fragment specific to CD28, directly or indirectly coupled to a biodegradable linker. 20 hours after stimulation, the T cells bound to the regulator activation reagent were washed, and the presence of the biodegradable linker was measured by flow cytometry 24 hours later. 26 hours after stimulation, an enzyme specific to the biodegradable linker was added, and the presence of the biodegradable linker was measured over time at several time points after stimulation. The stimulatory reagent was efficiently removed by washing and adding an enzyme specific to the biodegradable linker. [Figure 7] The enzyme specific for the biodegradable linker is non-toxic. Enriched T cells were polyclonally stimulated with T Cell TransActTM (Miltenyi Biotec), a regulatory reagent that contains an antibody or antigen-binding fragment specific for CD3 and an antibody or antigen-binding fragment specific for CD28, directly or indirectly coupled to a biodegradable linker. The enzyme was added to the T cells 26 hours after stimulation, and the viability was measured 24 hours later by flow cytometry with PI staining. The enzyme specific for the biodegradable linker is not harmful to the enriched and activated T cells, since comparable viability was detectable with or without the enzyme specific for the biodegradable linker. [Figure 8]Efficient removal of non-cellular components by cumulative washing. The efficiency of cumulative washing and removal of non-cellular components was calculated based on two different washing regimes, either 2.6-fold dilution after each individual washing step or 5-fold dilution after each individual washing step. The calculated cumulative dilution efficiency was normalized to undiluted (i.e., 100%). In the case of 2.6-fold serial dilution, the ratio of non-cellular components is below 0.001% after 11 consecutive washing steps. In the case of 5-fold serial dilution, the ratio of non-cellular components is below 0.001% after 7 consecutive washing steps. [Figure 9] Setting up a process for genetically engineering T cells in 3 days. T cells transduced on day 0 and incubated with dextranase, an enzyme specific for biodegradable linkers, on day 1 showed the lowest transduction efficiency levels, indicating insufficient T cell stimulation. This was confirmed by the analysis of T cells stimulated for longer periods by later addition on days 2 or 3, where higher transduction efficiency levels were detectable compared to T cells incubated with the enzyme on day 0. Better transduction efficiency, closer to the conventional protocol, was observed for stimulated T cells transduced on day 1 and incubated with dextranase on days 2 or 3. [Figure 10]Efficient removal of regulators in the CliniMACS® Prodigy system of engineered T cells generated within 3 days. Leukapheresis samples of healthy donors with up to 1e9 CD4 / CD8 cells were automatically processed in the CliniMACS® Prodigy system to generate CAR T cells within 3 days. 4e8 T cells were polyclonally stimulated with regulator MACS® GMP T Cell TransAct™ (Miltenyi Biotec) and genetically modified with VSV-G pseudotyped lentiviral vectors. On day 2, 10 ml of a solution containing dextranase was automatically added to specifically degrade the biodegradable linker, releasing antibodies or fragments specific for CD3 and CD28 and neutralizing the activity of the regulator. As a control, a production run on the CliniMACS® Prodigy system was performed under the same conditions and with the same donor material, but without the addition of enzymes specific for the biodegradable linker. Figure 10A: For both T cell engineering runs on the CliniMACS® Prodigy system, the presence of the biodegradable linker was assessed by flow cytometry on formulated cells stained with an antibody specific for the biodegradable linker. Figure 10B: Compared to the CliniMACS® Prodigy run without added enzyme, the biodegradable linker was efficiently removed in the CliniMACS® Prodigy system, as only a small percentage of linker-positive cells were detectable. In addition, the mean intensity level (MFI) of the biodegradable linker for all viable cells was at background levels when enzyme was added. [Figure 11]T cell stimulation levels in the CliniMACS® Prodigy system upon enzymatic removal of the activation reagent on day 2. The effect of removal of the regulator on the stimulation levels was assessed by flow cytometry staining for CD25 and CD69, both reported to be reliable T cell activation markers (CD25: REA570; CD69: REA824; MiltenyiBiotec). Unstimulated T cells from the same donor derived from small scale culture served as control, and T cells retrieved from the CliniMACS® Prodigy system were analyzed with or without enzymatic treatment. Compared to unstimulated control cells, significantly higher mean intensity levels of both activation markers were detected in T cell samples treated or not with dextranase. This confirmed that stimulation up to day 2 was already sufficient for the upregulation of both activation markers. This also shows that regulators can be removed already on day 2 or even before, without affecting stimulation. [Figure 12] Proliferation of stimulated T cells in the CliniMACS® Prodigy system for genetically engineering T cells within 3 days. No T cell expansion was detectable on day 3 in three independent production runs, suggesting that T cells were fully activated but T cell proliferation had not yet begun (see also FIG. 12). Consequently, the production protocol for genetically modifying T cells within 3 days is too short to support T cell expansion in vitro. [Figure 13] Assessment of CAR expression kinetics on a small scale. Figure 13A: After 5 days, transduction efficiency reached a plateau level of 18-22%, confirming stable transgene delivery and transgene expression. Figure 13B: At 2 days post-transduction, 16% of T cells were already CAR positive, but no distinct CAR-positive population was yet detectable. At later time points, a distinct CAR-expressing population was detected by flow cytometry. [Figure 14]Assessment of CAR expression kinetics in large-scale production on the CliniMACS® Prodigy system. In contrast to the small-scale experiments (see FIG. 13), CAR expression in the CliniMACS® Prodigy system did not reach plateau levels at early time points. Two days after transduction, 19% of T cells were CAR positive. Transduction efficiency increased to 75% at later time points, indicating that CAR was not yet fully expressed two days after transduction. [Figure 15] Optimization of CAR T cell manufacturing parameters on the CliniMACS® Prodigy system. Isolated stimulator T cells were genetically modified with 2.5 ml of VSV-G pseudotyped CD20 / CD19 tandem CAR-encoding lentiviral vector for 1e8 T cells (see FIG. 15: condition I) and in parallel with the same lentiviral vector volume for 4e8 T cells (see FIG. 15: condition II). Condition II also supports culture at higher cell density by increasing the volume and by performing an initial shaking step. On day 2, the same volume of dextranase was applied to both T cell manufacturing conditions. On day 3, manufactured T cells were washed and harvested multiple times and total T cell numbers were determined by cell counting. Washed and harvested cell samples of both CAR T cell manufacturing conditions were cultured in 24-well in an incubator for an additional 8 days to allow reliable assessment of transduction efficiency when steady-state levels of CAR expression are typically observed. Figure 15A: The transduction efficiency of condition II was 32%, whereas that of condition I was only 20%. Importantly, a higher LV dose per cell (MOI) was applied in condition I. Figure 15B: Not only was a higher transduction efficiency determined in condition II, but four times more (i.e., 4e8) T cells were transduced. This resulted in an almost seven-fold increase in the yield of CAR-transduced T cells in condition II compared to condition I. [Figure 16]Cytokine expression levels of CAR T cells generated within 3 days. Stimulated, CD20-CAR transduced, dextranase-treated T cells produced within 3 days in the CliniMACS® Prodigy system were co-cultured with CD20, GFP-expressing Raji cells at various effector-to-target ratios (E:T), and after 24 hours, the presence of inflammatory cytokines such as interferon gamma (IFN-g), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2 was evaluated using the MACSPlex cytokine kit assay (Miltenyi Biotec). In CD20 CAR-transduced T cells generated within 3 days, IFN-g, GM-CSF, and IL-2 levels were detectable in an E:T-dependent manner at high levels, even above the quantification level. In contrast, no cytokines were detectable in unstimulated T cells and stimulated T cells that remained untransduced. This confirms the tumor antigen-specific response of CAR-transduced T cells produced within 3 days. [Figure 17]Cytotoxic activity of CAR T cells generated within 3 days. CAR T cells and Raji-GFP cells manufactured within 3 days were co-cultured for an additional 2 days and 50% of the cells were analyzed by flow cytometry to quantify the number of residual tumor cells and, consequently, the cytolytic ability of the CAR T cells (round 1; left). Another 20,000 Raji-GFP tumor cells were added to the remaining 50% of the co-culture to evaluate the potency of the CAR T cells in a second successive round of co-culture. In the second successive round of co-culture, additional tumor cells were added to evaluate the cytotoxic activity under conditions intended to be difficult for the CAR T cells (round 2: right). After 72 hours, flow cytometry was performed to quantify the number of residual tumor cells in the second round of co-culture. At high E:T ratios (i.e., 1.25:1), nearly 100% of the Raji cells were lysed in the first and also in the second round. In contrast, only 50% and 40% of residual target cells were detectable in the untransduced controls. At an E:T ratio of 0.425:1, functionality was comparable to that at 1.25:1, but at a lower overall level. In the presence of CAR-transduced T cells, 60% of tumor cells were lysed in the first and second rounds of co-culture. In contrast, in the presence of untransduced CAR T cells, only 40% of tumor cells were lysed in the first round, and no killing was detectable in the second round. When comparing untransduced T cells with CAR-transduced T cells, no specific killing was detectable in the first round at an E:T ratio of 0.15:1. In summary, the functionality of CAR-transduced T cells produced within 3 days was confirmed in vitro, since fewer tumor cells were detectably present after two successive rounds of co-culture, compared to untransduced controls. [Figure 18]In vivo functionality of CAR T cells generated within 3 days. The in vivo functionality of CAR-transduced T cells generated within 3 days was confirmed in 6-8 week old NOD scid gamma (NSG) (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) mice. All experiments were performed in compliance with the Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and in compliance with the regulations of the German Animal Protection Act. In brief, leukapheresis samples from healthy donors were automatically processed on the CliniMACS® Prodigy system and CAR T cells were generated within 3 days (see Figure 18, top). On day 0, the bag containing the leukapheresis sample was sterilely connected to the CliniMACS Prodigy® tubing set 520 by welding. The cells were automatically washed and labeled with CD4 and CD8 CliniMACS reagents to enrich for T cells. 2e8 T cells were transferred to the culture chamber in IL-7 / IL-15-containing medium and polyclonally stimulated using MACS® GMP T Cell TransAct™ (Miltenyi Biotec) in a culture volume of 200 ml. On day 1, the isolated stimulated T cells were genetically modified with a VSV-G pseudotyped lentiviral vector to induce the expression of CD22 / CD19 tandem-CAR. A bag containing 10 ml of lentiviral vector was sterilely connected to the tubing set and automatically transferred to the chamber containing the T cells. On day 2, 10 ml of a solution containing dextranase was sterilely connected to the tubing set and automatically added to the chamber containing the T cells to specifically degrade the linker. This releases antibodies or fragments thereof specific for CD3 and CD28, inhibiting the activity of the modulator.After multiple washes, the cell product was analyzed by flow cytometry to determine transduction efficiency, viability, and cell composition at each step (see FIG. 19). A total of 3e6 or 6e6 T cells of the CAR-transduced groups per mouse were injected on the day of harvest (see FIG. 18, bottom). Tumors were established by intravenous inoculation of 5e5 firefly luciferase-expressing Raji cells before day 4 (see FIG. 17). Seven mice were treated per group. Two additional groups were established as negative controls. One group received tumor cells but no T cells (n=7; tumor only). One group received tumor cells and 3e6 untransduced T cells from the same donor cultured in parallel on a smaller scale (n=7). Tumor growth as well as antitumor responses were frequently monitored using an In vivo Imaging System (IVIS Lumina III). For this purpose, 100 μl of XenoLight Rediject D-Luciferin Ultra was injected i.p., after which mice were anesthetized using an isoflurane XGI-8 anesthesia system. Measurements were performed 6 min after substrate injection. At the end of the experiment, spleen, bone marrow, and blood were prepared and analyzed by flow cytometry to determine the frequency of tumor cells and T-cell subsets. [Figure 19] Cellular composition. The quality of the cell product was determined by flow cytometry by staining for CD45h, CD3, CD4, CD8, CD16 / CD56, 7-AAD, CD19, and CD14 in samples taken before enrichment, after enrichment, and after collection. The cellular composition after formulation was 67% CD4 T cells, 18% CD8 T cells, and 7% NKT cells. The frequency of NK cells, eosinophils, neutrophils, B cells, or monocytes was at background levels. This confirmed the T cell purity after enrichment. [Figure 20]Representative in vivo imaging data for selected groups. Tumor burden as well as antitumor activity of CAR T cells were frequently monitored by in vivo imaging. For the cohort containing mice that received 3e6 viable T cells, all mice, transduced and non-transduced, are shown. For the tumor-only group, 3 representative mice out of 7 are shown. Tumor burden rapidly increased in mice from cohorts that received only non-transduced T cells or tumor cells. Mice from both control groups had to be sacrificed 14 days after T cell injection because tumor burden reached a critical level. In contrast, mice that received CAR-transduced T cells showed a deceleration of increase at early time points, 3 and 7 days after T cell injection, in a dose-dependent manner compared to the control group. The level of tumor burden in the CAR-transduced T cell group peaked at 7 days after T cell injection, followed by a steady reduction in tumor burden to the level measured at the beginning of the experiment. [Figure 21] In vivo imaging data for all groups. Mean tumor burden measured over time as p / s + / - SEM is shown for all mice in all groups. Data for the 6E6 CAR-transduced T cell group (n=7) is included. Mice treated with 6E6 T cells showed a more rapid anti-tumor response than the 3E6 group. At day 14 post-T cell injection, tumor burden was substantially reduced to comparable low levels for both T cell doses. Control groups (i.e., tumor only and untransduced T cells) were unable to control tumor growth and mediate potent anti-tumor activity. [Figure 22]T cell abundance in bone marrow. Human T cell abundance in bone marrow of three randomly selected mice was quantified by flow cytometry by staining for CD45h, CD4, CD8, CD20, CD22, 7-AAD, and CD19 CAR detection (all Miltenyi Biotec). Numbers of each mouse are shown. For the control group, analysis was performed on bone marrow sampled on day 14. For the 3e6 CAR-transduced T cell group, three randomly selected mice were analyzed on day 18. As expected, no T cells were found in the tumor-only group. In the untransduced cohort, up to 20% of T cells were detectable. In contrast, the frequency of human T cells was highest in the cohort including mice infused with CAR-transduced T cells, up to 75%, indicating that CAR T cells homed to this microenvironment and expanded in vivo. [Figure 23] Tumor and T cell abundance in bone marrow. Human intracellular compartments were investigated in more detail to determine the frequency of human Raji tumor cells and human T cells. The frequency of all human cells was therefore set to 100%. Numbers for each mouse are shown. As expected, in the tumor-only cohort, no human T cells were found, only Raji cells. The bone marrow is a favorable microenvironment for Raji tumor cells. In contrast, for the CAR-transduced T cell population, only a small percentage of Raji cells was detectable in this organ. This is consistent with the presence of approximately 50% human CD4 T cells and approximately 50% human CD8 T cells in these representative mouse organs. 20-60% of the human cells were Raji cells in the untransduced T cell population, with a ratio of CD4 T cells to CD8 T cells of 2:1-3:1. [Figure 24]Abundance of T cell subsets in spleens. T cell abundance in spleens of three randomly selected mice was quantified by flow cytometry by staining for CD45h, CD4, CD8, CD20, CD22, 7-AAD, and CD19 CAR detection (all Miltenyi Biotec). Numbers of each mouse are shown. For the control group, analysis was performed on spleens sampled on day 14. For the 3e6 CAR-transduced T-cell group, three randomly selected mice were analyzed on day 18. As expected, no T cells were found in the tumor-only group. In the untransduced cohort, up to 10% of T cells were detectable. In contrast, the frequency of human T cells was highest in the cohorts including mice infused with CAR-transduced T cells, up to 40%. [Diagram 25] Abundance of T cell subsets in blood. Abundance of T cells circulating in the blood of three randomly selected mice was quantified by flow cytometry by staining for CD45h, CD4, CD8, CD20, CD22, 7-AAD, and CD19 CAR detection (all Miltenyi Biotec). Numbers for each mouse are shown. For the control group, analysis was performed on spleens sampled on day 14. For the 3e6 CAR-transduced T cell group, three randomly selected mice were analyzed on day 18. No T cells were found in the tumor-only group. Only a small percentage of T cells was found in the cohort containing mice with untransduced T cells. In contrast, the frequency of human T cells circulating in the blood was highest in the cohort containing mice infused with CAR-transduced T cells, up to 25%. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] One aspect of the invention is a method for generating genetically modified T cells, comprising: a) providing a sample comprising T cells; b) preparing the sample by centrifugation; c) enriching the T cells of step b (enriching the T cells from the prepared sample); d) activating the enriched T cells using modulatory agents; e) genetically modifying the activated T cells by transduction with lentiviral vector particles; f) Removing the regulator Including, A sample of genetically modified T cells is then generated, Methods are provided, wherein the method is performed in 144 hours or less, less than 120 hours, less than 96 hours, less than 72 hours, less than 48 hours, or less than 24 hours.

[0011] To date, the most common side effect after CAR T cell infusion is the onset of immune activation, known as cytokine release syndrome (CRS). This is a systemic inflammatory response caused by cytokines released immediately after infusion by the infused CAR T cells upon recognition of potentially high-load tumor cells expressing the CAR antigen. This toxicity can be at least partially reduced by manufacturing CAR T cells within a short period of time, since not all CAR T cells express the CAR at this early time point, and the CAR expression level increases steadily but slowly thereafter (see Example 10).

[0012] For example, the combination of removing the modulating agents and / or magnetic particles used in the T cell enrichment disclosed herein and performing the methods disclosed herein in 144 hours or less, 120 hours or less, 96 hours or less, 72 hours (3 days) or less, 48 ​​hours or less, or 24 hours or less, allows for successful application in in vivo treatment of patients suffering from, for example, cancer, where the number of T cells in the generated sample of the method is less than 10-fold or less than 5-fold compared to the number of T cells in the prepared sample.

[0013] A prepared sample (or sample preparation) comprising T cells may be prepared from a subject, such as a human (the sample comprising T cells provided by the subject). The prepared sample may be human whole blood, a subject's leukapheresis, buffy coat, PBMCs, expanded or isolated T cells.

[0014] Preparation of the sample may result in volume reduction, rebuffering, serum removal, red blood cell reduction, platelet removal, and / or washing.

[0015] Alternatively, the method may begin with step a of providing a sample comprising T cells.

[0016] Alternatively, the method may start with step b, in which a sample containing T cells is prepared by centrifugation. This alternative step b may be followed by steps c to f.

[0017] In said method, the sample in step a) comprises human serum, and said serum is removed by step b).

[0018] The human serum may contain a component that reduces the transduction efficiency of lentiviral vector particles into cells. The component of human serum that may reduce transduction efficiency may be a component of the subject's complement system or may be a neutralizing antibody (see, e.g., DePolo et al., 2000, Molecular Therapy 2:218-222).

[0019] Removal of human serum may be performed by washing (washing steps) achieved by said centrifugation. The washing steps may be performed by a series of medium / buffer exchanges (at least two exchanges), whereby human serum and / or its components are removed from the T cells.

[0020] In the method, the T cells are prepared and enriched in less than 2 hours, preferentially in less than 1 hour.

[0021] In said method, said T cells are activated (stimulated) with said modulating agent for less than 72 hours, preferentially less than 48 hours, more preferentially less than 24 hours, i.e., addition of said modulating agent and removal of said modulating agent occur within said time.

[0022] In said method, transduction of said activated T cells is initiated 2 days after stimulation of said T cells with a modulating agent, preferentially 1 day after said stimulation, more preferentially simultaneously with said stimulation.

[0023] In the method, the regulatory activating agent may be removed (step f) in less than 2 hours, preferentially less than 1 hour, more preferentially less than 30 minutes after adding the regulatory agent to the T cells (step d).

[0024] In said method, said genetic modification of said T cells by transduction with lentiviral vector particles (step e) may be performed in less than 2 days, preferentially in less than 1 day, more preferentially in less than 12 hours.

[0025] In said method, the T cells of the prepared sample may be enriched for CD4 positive and / or CD8 positive T cells by using CD4 and / or CD8 as positive selection markers, prior to said genetic modification of the T cells, and / or the T cells of the prepared sample may be depleted of cancer cells contaminating the sample comprising T cells by using a tumor associated antigen (TAA) as a negative selection marker. The TAAs can be selected from one or more of the following markers, e.g., CD19, CD20, CD22, CD30, CD33, CD70, IgK, IL-1Rap, Lewis-Y, NKG2D ligand, ROR1, CAIX, CD133, CEA, c-MET, EGFR, EGFRvIII, EpCam, EphA2, ErbB2 / Her2, FAP, FR-a, GD2, GPC3, IL-13Ra2, L1-CAM, mesothelin, MUC1, PD-L1, PSCA, PSMA, VEGFR-2, BCMA, CD123, and CD16V.

[0026] The enrichment of said CD4+ and / or CD8+ T cells and / or depletion of cancer cells from the prepared sample can be performed by a separation step. The separation can be performed by flow cytometry methods such as FACSorting (fluorescence activated cell sorting), magnetic cell separation such as MACS, or microchip-based cell sorting such as MACSQuant® Tyto®. The use of a magnetic cell separation step is preferred.

[0027] In the method, the enrichment of CD4 and / or CD8 positive T cells comprises: i) contacting T cells with magnetic particles that are directly or indirectly coupled to an antibody or antigen-binding fragment thereof specific for CD4 and / or CD8, wherein the magnetic particles and the antibody or antigen-binding fragment thereof coupled thereto can be removed; ii) separating CD4 and / or CD8 T cells in a magnetic field; iii) removing said magnetic particles from the enriched T cells after separation. This is carried out by a magnetic cell separation step comprising:

[0028] In the method, the enrichment of CD4 and / or CD8 positive T cells comprises: i) contacting T cells with magnetic particles that are directly or indirectly coupled to an antibody or antigen-binding fragment thereof specific for CD4 and / or CD8, wherein the magnetic particles and the antibody or antigen-binding fragment thereof coupled thereto may be removed by washing; ii) separating CD4 and / or CD8 T cells in a magnetic field; iii) removing the magnetic particles from the enriched T cells after separation by washing. This is carried out by a magnetic cell separation step comprising:

[0029] In the method, the enrichment of CD4 and / or CD8 positive T cells comprises: i) contacting T cells with magnetic particles that are directly or indirectly coupled to an antibody or antigen-binding fragment thereof specific for CD4 and / or CD8, wherein the magnetic particles and the antibody or antigen-binding fragment thereof coupled thereto can be chemically and / or enzymatically cleaved; ii) separating CD4 and / or CD8 T cells in a magnetic field; iii) removing said magnetic particles from the enriched T cells after the separation step by chemical and / or enzymatic cleavage of said magnetic particles and said antibodies or antigen-binding fragments thereof coupled thereto. This is carried out by a magnetic cell separation step comprising:

[0030] The removal of the magnetic particles from enriched T cells after a separation step by chemical and / or enzymatic disruption may be performed within the magnetic field or may be performed after removal of the magnetic field.

[0031] Methods and systems for removing magnetic particles from cells that are directly or indirectly bound to said cells are well known in the art.

[0032] Some exemplary methods and systems for reversibly labeling cells with magnetic particles that result in detachment of the magnetic particles from the cells are listed herein.

[0033] One strategy exploits the specific competition of non-covalent interactions. US20080255004 discloses a method of reversibly binding to a solid support, such as a magnetic particle, using an antibody that recognizes a target moiety, conjugated to a modified biotin, such as desthiobiotin, and modified streptavidin or avidin, which are bound to the solid support. The binding interaction of the modified binding partner is weaker compared to the strong and specific binding between biotin and streptavidin, and dissociation is promoted in the presence of such a competitor. EP2725359B1 describes a reversible magnetic cell separation system based on the non-covalent interaction between a ligand-PEO-biotin-conjugate that recognizes a target moiety and an anti-biotin-antibody with a magnetic particle, which can be released by adding a competitor molecule, biotin, streptavidin, or an auxiliary reagent.

[0034] In the method, the enrichment of CD4 and / or CD8 positive T cells comprises: i) contacting T cells with magnetic particles indirectly coupled via a linker to an antibody or antigen-binding fragment thereof specific for CD4 and / or CD8, wherein the magnetic particles and the antibody or antigen-binding fragment thereof coupled thereto can be removed by adding a competitor that competes with the binding between the linker and the antibody or antigen-binding fragment thereof; ii) separating CD4 and / or CD8 T cells in a magnetic field; iii) removing said magnetic particles from the enriched T cells after the separation step by adding a competitor. This is carried out by a magnetic cell separation step comprising:

[0035] In said method, said competitor is biotin, streptavidin, or an auxiliary reagent.

[0036] Besides these competitive release mechanisms, mechanical agitation, chemically cleavable or enzymatically degradable linkers are mentioned for label removal. WO 96 / 31776 describes a method for releasing magnetic particles from target cells after separation by enzymatically cleaving a part of the particle coating or a part present in the linking group between the coating and the antigen recognition moiety. One example is the application of magnetic particles coated with dextran and / or linked to the antigen recognition moiety via dextran. Subsequent cleavage of the isolated target cells from the magnetic particles is initiated by the addition of dextranase, a dextran degrading enzyme. Related methods in EP 3037821 disclose detection and separation of target moieties, for example by fluorescent signals, using conjugates with enzymatically degradable spacers.

[0037] Recently, there has been an increasing interest in the technique of using antigen recognition moieties, whose binding to target moieties is characterized by low affinity constants.To ensure specific and stable labeling by such low affinity antigen recognition moieties, the structure of label conjugate must include the multimerization of antigen recognition moieties, which provides high binding strength.Low affinity antigen recognition moieties can dissociate from target moieties when multimerization is disrupted, providing the opportunity to release detection moieties and antigen recognition moieties from target moieties under the best conditions.

[0038] This reversible multimeric staining was first described in US Patent No. 7,776,562 and also in US Patent No. 8,298,782, where the multimerization is established by non-covalent interactions. An exemplary low affinity peptide / MHC monomer with StreptagII is multimerized with streptactin, and the multimerization can be released by adding the competitor molecule biotin.

[0039] This method is outlined in U.S. Patent No. 9,023,604 with respect to the characterization of receptor binding reagents corresponding to antigen recognition moieties to allow for reversible labeling.-4 sec -1 A receptor binding reagent characterized by a size of 100 or larger is multimerized with a multimerization reagent having at least two binding sites Z that reversibly non-covalently interact with a binding partner C to provide a complex with high avidity for a target antigen. A detectable label is attached to the multivalent binding complex. Reversibility of multimerization is initiated upon disruption of the bond between the binding partner C and the binding site Z of the multimerization reagent. For example, in the case of Fab-StreptagII / streptactin multimers, multimerization can be released by the competitor biotin.

[0040] EP 0819250 B1 provides a method for releasing magnetic particles bound to cell surfaces via affinity reagents, such as antibodies or antigen-binding fragments thereof, in which the magnetic particles are released by the action of a glycosidase specific for a glycosidic linkage present in (a) the coating of the particles and (b) at least one of the linking groups between the coating and the affinity reagent.

[0041] In the specification of European Patent No. 3336546 A1, a) Basic formula (I): A n -PB m -C q -X o (I) At least one conjugate having the formula: In the formula, A: antigen recognition moiety P: Enzymatically degradable spacer B: First binding portion C. Second binding moiety X: Detection part n, m, q, and o are integers from 1 to 100; preparing a conjugate, wherein B and C are non-covalently bonded to each other, and A and B are covalently bonded to P; b) labeling the target moiety recognized by the antigen recognition moiety A with at least one conjugate; c) detecting the labeled target moiety with a detection moiety X; d) B m and C. q By disrupting the non-covalent bond between q -X o a step of cutting the e) Enzymatic cleavage of the spacer P removes the binding moiety B from the labeled targeting moiety. m Step of cutting discloses a method for detecting a target moiety in a sample of a biological specimen.

[0042] The method of EP 3336546 A1 can be used not only to detect target moieties, i.e. target cells expressing such target moieties, but also to isolate target cells from a sample of biological specimen. The isolation procedure relies on the detection of the target moiety. For example, the detection of the target moiety by fluorescence can be used to trigger an appropriate separation process, such as that carried out in a FACS or TYTO separation system. In the method of EP 3336546 A1, the well-known magnetic cell separation process can also be used as a detection and separation process, where the magnetic particles are detected by a magnetic field.

[0043] In a preferred embodiment of the invention, the magnetic particles directly coupled to CD4 and / or CD8 specific antibodies or antigen-binding fragments thereof are coupled via a biodegradable linker, which is degraded by adding an enzyme that (specifically) digests the biodegradable linker. The biodegradable linker may be or may contain a polysaccharide, and the enzyme that specifically digests the glycosidic linkage is a hydrolase.

[0044] The biodegradable linker may be or may comprise dextran, and the enzyme that (specifically) digests dextran may be dextranase.

[0045] In another preferred embodiment of the invention, said magnetic particles indirectly coupled to antibodies specific for CD4 and / or CD8 or antigen-binding fragments thereof, such as Fab, comprise two components: i) said Fab specific for CD4 and / or CD8, coupled to a linker, such as dextran, or to a tag, such as PEO-biotin; ii) coupled via a magnetic particle that is coupled to an antibody or antigen-binding fragment thereof specific for said tag, e.g. biotin, where after combining components i and ii and after contacting T cells with said indirectly coupled magnetic particles, the magnetic particles can be disrupted (removed) by adding a competitor that competes with said tag, e.g. biotin (as a competitor).

[0046] In another preferred embodiment of the invention, said magnetic particles indirectly coupled to antibodies specific for CD4 and / or CD8 or antigen-binding fragments thereof, such as Fab, comprise two components: i) a biodegradable linker, such as dextran, coupled to a tag, such as PEO-biotin; ii) coupled via a magnetic particle that is coupled to an antibody or antigen-binding fragment thereof specific for said tag, e.g. biotin, and after combining components i and ii and contacting T cells with said indirectly coupled magnetic particles, the magnetic particles can be detached from said T cells by adding an enzyme that specifically digests the biodegradable linker, such as dextranase, and / or by adding a competitor that competes with said tag, e.g. biotin (as a competitor).

[0047] In said method, said competitor is biotin, streptavidin, or an auxiliary reagent.

[0048] The principle of this embodiment of the invention is illustrated in FIG. 4 with respect to the release / decoupling principle.

[0049] In the method, the modulating agent comprises a CD3-specific antibody or antigen-binding fragment thereof and / or a CD28-specific antibody or antigen-binding fragment thereof coupled directly or indirectly via a linker, and the CD3- and CD28-specific antibodies or antigen-binding fragments thereof can be removed.

[0050] In the method, removal of modulating agent from the cells may further be performed by one or more washing steps.

[0051] In the method, the CD3- and CD28-specific antibodies or antigen-binding fragments thereof are included, and the CD3- and CD28-specific antibodies or antigen-binding fragments thereof may be chemically and / or enzymatically cleaved, and the modulator is removed by chemical and / or enzymatic cleavage of the CD3- and CD28-specific antibodies or antigen-binding fragments thereof. Removal of the cleaved modulator from the cells may be further performed by one or more washing steps.

[0052] The above described methods and systems for removing magnetic particles from cells that are directly or indirectly coupled to said cells may also be suitable, may be transferred and / or may be applied to the removal of said modulators comprising an antibody or antigen-binding fragment thereof specific for CD3 and an antibody or antigen-binding fragment thereof specific for CD28 that are directly or indirectly coupled via a linker.

[0053] In the method, the removal of the modulator of the CD3 and / or CD28 specific antibody or antigen-binding fragment thereof comprises: a) a competitive reaction comprising the step of adding a competitor that competes with the tag, e.g. biotin (as a competitor), wherein said modulating agent comprises an antibody or an antigen-binding fragment thereof, such as a Fab, specific for CD3 and / or CD28, indirectly coupled via two components, said two components being: i) an antibody or antigen-binding fragment thereof, such as a Fab, specific for CD3 and / or CD28, coupled to said tag, such as PEO-biotin, or an antibody or antigen-binding fragment thereof, such as a Fab, coupled via a linker, such as a dextran, which is coupled to said tag, such as PEO-biotin, and ii) a competitive reaction, which may be an antibody or an antigen-binding fragment thereof, such as a Fab, specific for a tag, e.g., biotin, and said components i) and ii) are combined and contacted with a cell; and / or b) enzymatic cleavage comprising the step of adding an enzyme that biodegrades the linker, with the proviso that the linker is a biodegradable linker (i.e., an indirect or direct linkage of two antibodies or antigen-binding fragments thereof via a linker). This is carried out by:

[0054] In another preferred embodiment of the invention, the regulator comprises two components: i) an antibody or antigen-binding fragment thereof, such as a Fab, specific for CD3 and / or CD28, coupled to a tag, such as PEO-biotin, or an antibody or antigen-binding fragment thereof, such as a Fab, specific for CD3 and / or CD28, coupled via a linker, such as a dextran, which is coupled to a tag, such as PEO-biotin; ii) an antibody or an antigen-binding fragment thereof, such as a Fab, specific for a tag, e.g., biotin; and an antibody or antigen-binding fragment thereof, such as a Fab, specific for CD3 and / or CD28, indirectly coupled via After combining components I and ii, and contacting the combined components with T cells, the combined components can be disrupted (removed) by adding a competitor that competes with the tag, e.g., biotin (as a competitor).

[0055] The biodegradable linker may be or may include a polysaccharide, and the enzyme that specifically digests the glycosidic linkage may be a hydrolase.

[0056] The biodegradable linker may be or may include dextran, and the enzyme that specifically digests dextran may be dextranase.

[0057] In a preferred embodiment of the invention, the modulating agent comprises an antibody or antigen-binding fragment thereof specific for CD3 and / or an antibody or antigen-binding fragment thereof specific for CD28, directly coupled via a biodegradable linker, which is degraded by adding an enzyme that specifically digests the biodegradable linker. The biodegradable linker may be or may include a polysaccharide, and the enzyme that specifically digests glycosidic linkages is a hydrolase.

[0058] The biodegradable linker may be or may include dextran, and the enzyme that specifically digests dextran may be dextranase.

[0059] The method, wherein following genetic modification of T cells by transduction with lentiviral vector particles, residual lentiviral vector particles are removed.

[0060] Removal of said residual lentiviral vector particles may be performed prior to, subsequent to or after removal of said modulatory agent and / or said removal of said magnetic particles.

[0061] The removal of said residual lentiviral vector particles may be performed by washing, which results in a 10-fold or less, preferably a 100-fold reduction in residual vector particles in the sample comprising the genetically modified T cells.

[0062] A washing step may be performed by a series of medium / buffer exchanges (at least two exchanges) to remove the residual lentiviral vector particles from the sample containing the genetically modified T cells. Exchanges may be performed by separation of cells and medium / buffer by centrifugation, sedimentation, adhesion, or filtration, followed by medium / buffer exchange.

[0063] A 10-fold or less, preferably 100-fold reduction in residual vector particles in a sample containing genetically modified T cells by washing can be achieved, for example, by: i) Separating the cells and medium / buffer ii) Removal of 90%, preferably 99%, of the medium / buffer volume. iii) Add fresh medium / buffer to bring to original volume iv) Resuspending the cells in medium / buffer This can be achieved by:

[0064] The washing steps may be performed in a sequential manner that can result in a cumulative reduction of lentiviral vectors (i.e., two washing steps with a 10-fold reduction per step results in a 100-fold cumulative reduction).

[0065] The removal of the residual lentiviral vector particles can be carried out by incubation with a substance that inactivates the lentiviral vector particles and / or reduces their stability, which can be washed away after said incubation, which is not carried out for longer than 3 hours, preferentially not for longer than 1 hour.

[0066] Such substances that inactivate lentiviral vector particles and / or reduce their stability may be, for example, heparin, antiretroviral agents, complement factors of human blood, neutralizing antibodies contained in human blood, or weakly basic buffers.

[0067] The antiretroviral agent may be an inhibitor of a viral enzyme, such as, for example, zidovudine (AZT) or raltegravir.

[0068] Complement factors and / or neutralizing antibodies contained in blood, eg, human blood, can be isolated by methods well known in the art.

[0069] The weakly basic buffer may have a pH value of about 7-9 that is sufficiently mild so as not to harm the T cells of the sample. Such buffers are described, for example, in Holic et al. (Hum Gene Ther Clin Dev. 2014 Sept;25(3):178-85).

[0070] In the methods, depleted human serum as disclosed herein, or substances isolated therefrom, such as complement factors and / or neutralizing antibodies that inhibit productive transduction of lentiviral vector particles into T cells, can be added to the genetically modified T cells, thereby removing and / or neutralizing residual lentiviral vector particles.

[0071] The methods disclosed herein are preferentially automated methods carried out in a closed system.

[0072] The methods disclosed herein can be carried out entirely as automated processes, preferentially in closed systems under GMP conditions.

[0073] Such a closed system allows operation under GMP or GMP-like conditions ("sterile") and results in clinically applicable cell compositions. Exemplarily, CliniMACS Prodigy® (Miltenyi Biotec GmbH, Germany) is used herein as a closed system. This system is disclosed in WO 2009 / 072003. However, it is not intended to limit the use of the method of the present invention to CliniMACS® Prodigy.

[0074] The CliniMACS Prodigy® system is designed to automate and standardize the complete cell product manufacturing process. It combines CliniMACS® separation technology (Miltenyi Biotec GmbH, Germany) with a wide range of sensor-controlled cell processing functions. Salient features of the device include: Single-use CentriCult for standardized cell processing and culture TM Chamber Cell enrichment and depletion capabilities alone or in combination with CliniMACS® Reagent (Miltenyi Biotec GmbH) Cell culture and expansion capabilities with temperature and CO2 gas exchange controlled Final product formulation in pre-defined media and volumes Ability to program the device using Flexible Programming Suite (FPS) and GAMP5 compatible programming languages ​​to customize cell processing Custom tube sets for various applications

[0075] The centrifugation chamber and the culture chamber may be the same. The centrifugation chamber and the culture chamber may be used in various conditions. For example, in the case of separation or transduction, a high rotation speed (i.e., a large g-force) may be applied, while the culture step may be performed, for example, with slow rotation or even in idling. In another variant of the present invention, the chamber changes rotation direction in a vibrational manner, which causes the chamber to shake and the cells to be maintained in suspension. Thus, in the process of the present invention, the T cell stimulation step, the genetic modification step, and / or the culture step may be performed under stationary or shaking conditions in the centrifugation or culture chamber.

[0076] In said method, the number of T cells in the generated sample may be less than 10-fold, preferentially less than 5-fold, compared to the number of T cells in said provided sample.

[0077] In the method, the T cells generated undergo fewer than four cell divisions, preferentially fewer than three.

[0078] Further expansion of such genetically modified T cells to therapeutically effective amounts will occur in vivo, eliminating the need to expand genetically modified T cells in vitro to cell numbers known to be required for effective treatment of patients (see, e.g., Ghassemi et al., 2018, Cancer Immunol Res 6:1100-1109). This is made possible by the high quality of the genetically modified T cell compositions / samples generated by the methods disclosed herein, i.e., low contamination with ungenetically modified T cell components and toxic substances.

[0079] The omission of in vitro expansion / propagation of genetically modified T cells to larger cell numbers allows for a reduction in the time required to prepare a clinically applicable composition comprising the modified T cells.

[0080] The genetically modified T cells may be genetically modified to express a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any accessory molecule on their cell surface.

[0081] For final formulation, the genetically modified T cells may be washed by centrifugation and the culture medium replaced with a buffer appropriate for subsequent use, such as infusion of the resulting cell composition into a patient.

[0082] If desired, the genetically modified T cells can be separated from the unmodified T cells, for example, again using magnetic separation techniques.

[0083] In one aspect, the invention provides a cell composition obtainable by the methods disclosed herein.

[0084] In one embodiment of the invention, the cell composition is a pharmaceutical cell composition, optionally comprising a pharmaceutical carrier.

[0085] The methods of the invention may include any of the embodiments of the invention and / or steps described herein in any order and / or combination that results in a functional method for generating genetically modified T cells as disclosed herein.

[0086] In addition to the above applications and embodiments of the present invention, further embodiments of the present invention are described below, but are not intended to be limited to such embodiments.

[0087] Embodiment In a preferred embodiment of the invention, T cells are genetically modified to express chimeric antigen receptors in an automated process in a closed system, for example by using the CliniMACS® Prodigy (Miltenyi Biotec GmbH).

[0088] For example, a sample containing T cells from a human suffering from cancer can be prepared. Human serum from the prepared sample containing T cells can be washed away by a centrifugation step.

[0089] CD4+ and / or CD8+ T cells can be enriched in a magnetic separation step using anti-CD4 and / or anti-CD8 antibodies or antigen-binding fragments thereof coupled to magnetic particles via dextran. After the CD4+ and / or CD8+ T cells are separated from a sample containing T cells in a magnetic field, the magnetic particles are removed from the enriched cells by adding dextranase, which disrupts the binding of the antibodies or their fragments to the magnetic particles by cleaving the dextran chains.

[0090] The enriched CD4+ and / or CD8+ T cells can be activated for 24 hours using as modulating agents an antibody or antigen-binding fragment thereof specific for CD3 and an antibody or antigen-binding fragment thereof specific for CD28, coupled via a linker comprising dextran.

[0091] Lentiviral vector particles containing a nucleic acid encoding a CAR can be added to a sample containing activated CD4+ and / or CD8+ T cells. Transduction can be performed for 24 hours during or after stimulation.

[0092] After transduction of the lentiviral particles into CD4+ and / or CD8+ T cells, the regulator is washed off or removed by adding dextranase, which disrupts the binding of the antibodies or their fragments to each other by cleaving the dextran chains. The residual lentiviral vector particles in the sample containing the genetically modified T cells are reduced by 10-fold or less, preferentially 100-fold or less, by repeated washing. As a result, a pure sample containing the genetically modified T cells is achieved in 144 hours or less, 120 hours or less, 96 hours or less, 72 hours or less, 48 ​​hours or less, or 24 hours or less, and the expansion of the genetically modified T cells in the generated sample is less than 10-fold, preferentially less than 5-fold, compared to the amount of T cells in the original prepared sample containing T cells. The sample or composition containing the genetically modified T cells can be applied to the human, and the genetically modified T cells can express a CAR that recognizes a TAA in the human.

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

[0094] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the method or composition, but may include unspecified elements, whether essential or not.

[0095] As used herein, the terms "modulator," "activator," and "stimulator" may be used interchangeably.

[0096] The modulating agent may be selected from the group consisting of agonist antibodies or antigen-binding fragments thereof, cytokines, recombinant costimulatory molecules, and small drug inhibitors. The modulating agent is an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof coupled to beads or nanostructures. The modulating agent may be a nanomatrix, the nanomatrix comprising a) a matrix of mobile polymer chains, and b) an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof attached to said matrix of mobile polymer chains, the nanomatrix being 1-500 nm in size. The anti-CD3 antibody and the anti-CD28 antibody or fragments thereof may be attached to the same or separate matrix of mobile polymer chains. When the anti-CD3 antibody and the anti-CD28 antibody or fragments thereof are attached to separate matrix of mobile polymer chains, fine tuning of the nanomatrix for stimulating T cells is possible. The nanomatrix may be biodegradable. The nanomatrix may be collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan, or extracellular matrix composition. The polysaccharide may include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan, guar gum, or alginate. The selection of the degradative enzyme agent will depend on the glycosidic linkage. If the polymeric coating is a polysaccharide, the polysaccharide will be selected to have glycosidic linkages that are not normally found in mammalian cells. For example, hydrolases that recognize specific glycosidic structures can be used as enzymes, such as dextranase, which cleaves α(1->6) linkages; cellulase, which cleaves β(1->4) linkages; amylose, which cleaves amylases; pectin, which cleaves pectinase; chitin, which cleaves chitinase.

[0097] In addition, the small nanomatrices disclosed, for example, in WO 2014 / 048920 A1, are capable of being sterile filtered, which is an important feature for activating T cells under conditions complying with strict GMP standards, i.e. in a closed system.

[0098] The term "depletion" as used herein refers to a process of negative selection that separates desired cells from undesirable cells, here typically cancer cells, that are labeled with an antibody or antigen-binding fragment thereof that is coupled to a solid phase such as a particle, fluorophore, or hapten.

[0099] The term "particles" as used herein refers to solid phases such as colloidal particles, microspheres, nanoparticles, or beads. Methods for producing such particles are well known in the art. The particles may be magnetic particles. The particles may be in solution or suspension, or may be lyophilized prior to use in the present invention. The lyophilized particles are then reconstituted in a convenient buffer before contacting with the sample to be treated according to the present invention.

[0100] The term "magnetic" in "magnetic particles" as used herein refers to all subtypes of magnetic particles, particularly ferromagnetic, superparamagnetic, and paramagnetic particles, that can be prepared by methods well known to those skilled in the art. "Ferromagnetic" materials are strongly influenced by magnetic fields and are capable of retaining their magnetic properties even when the magnetic field is removed. "Paramagnetic" materials have only weak magnetic susceptibility and quickly lose their weak magnetism when the magnetic field is removed. "Superparamagnetic" materials are highly susceptible to magnetism, i.e., they become strongly magnetic when placed in a magnetic field, but lose their magnetism as quickly as paramagnetic materials.

[0101] The link between the antibody (or its antigen-binding fragment) and the particle can be covalent or non-covalent. Covalent linkage can be, for example, linkage with the carboxyl group of a polystyrene bead, or linkage with the NH2 or SH2 group of a modified bead. Non-covalent linkage is, for example, by biotin-avidin, or fluorophore-coupled particles linked to anti-fluorophore antibodies. Methods for coupling antibodies to particles, fluorophores, haptens such as biotin, or larger surfaces such as culture dishes are well known to those skilled in the art.

[0102] In principle any sorting technique can be used for enrichment, isolation or selection. This includes, for example, affinity chromatography or any other antibody-dependent separation technique known in the art. Any ligand-dependent separation technique known in the art can be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cells. A particularly powerful sorting technique is magnetic cell sorting. Methods for magnetically separating cells are commercially available, for example, from Invitrogen, Stem cell Technologies, Cellpro, Seattle, or Advanced Magnetics, Boston. For example, monoclonal antibodies can be directly coupled to magnetic polystyrene particles, such as Dynal M450 or similar magnetic particles, and used, for example, for cell separation. Dynabead technology is not column-based; instead, these magnetic beads with attached cells enjoy liquid-phase dynamics in the sample tube, and the cells are isolated by placing the tube on a magnetic rack. However, in a preferred embodiment for enriching CD4+ and / or CD8+ T cells from a sample containing T cells according to the invention, the monoclonal antibody or antigen-binding fragment thereof is used in conjunction with colloidal superparamagnetic microparticles having an organic coating, e.g. with polysaccharides (Magnetic Activated Cell Sorting (MACS) technology (Miltenyi Biotec, Bergisch Gladbach, Germany)). Such particles (nanobeads or microbeads) may be directly conjugated with the monoclonal antibody or may be used in combination with anti-immunoglobulin, avidin or anti-hapten specific microbeads.

[0103] MACS technology allows for cell separation by incubating cells with magnetic nanoparticles coated with antibodies against a specific surface antigen. This causes cells expressing that antigen to attach to the magnetic nanoparticles. The cell solution is then transferred to a column placed in a strong magnetic field. During this step, cells that attach to the nanoparticles (which express the antigen) remain in the column, while other cells (which do not express the antigen) pass through. In this way, cells can be separated as positive or negative for a particular antigen / marker.

[0104] In the case of positive selection, after the column is removed from the magnetic field, cells expressing the antigen of interest that are attached to the magnetic column are washed into a separate container.

[0105] In the case of negative selection, the antibodies used are directed against surface antigens known to be present on cells other than the cells of interest. After the cell / magnetic nanoparticle solution is run through the column, cells expressing these antigens will bind to the column and the cells of interest will be included in the flow-through fraction, which is then collected. These cells are "untouched" since they are not labeled by the antibodies coupled to the nanoparticles.

[0106] This procedure can be carried out using direct or indirect magnetic labeling. In the case of direct labeling, a specific antibody is directly coupled to the magnetic particles. Indirect labeling is a convenient alternative when direct magnetic labeling is not possible or desirable. This labeling strategy can use a primary antibody against any cell surface marker, a specific monoclonal or polyclonal antibody, or a combination of primary antibodies. The primary antibody can be unconjugated, biotinylated, or fluorophore-conjugated. Magnetic labeling is then achieved using anti-immunoglobulin microbeads, anti-biotin microbeads, or anti-fluorophore microbeads.

[0107] The term "disruption" as used herein in the context of disrupting a magnetic particle or modulating agent for activation means: By cleaning only and / or by adding a competitor followed by washing, and / or by chemical disruption, i.e. by adding substances (non-proteinaceous chemical compounds) that break the covalent bonds, and / or by enzymatic cleavage followed by washing, and / or By inputting energy to break the covalent bond (physical scission) Refers to removal.

[0108] The term "competition reaction" in the context of separation, as used herein, refers to a magnetic particle or modulating agent for activation that comprises two non-covalently linked components, one component that binds to the cell via an antibody or antigen-binding fragment specific for CD3, CD28, CD4, and / or CD28, and comprises a tag and a second component that binds to the tag, such that the binding to the tag can be abolished by the addition of a competitor.

[0109] A competitor may compete with and / or displace one of the components, the magnetic particle, the modulator, or the antibody or antigen-binding fragment thereof, for example because of a higher affinity for the corresponding component or because the concentration of the competitor molecule is higher compared to the concentration of the magnetic particle or modulator that is indirectly coupled to an antibody or antigen-binding fragment thereof specific for CD3, CD28, CD4, and / or CD8.

[0110] The term "enzymatic cleavage", as used herein in the context of cleaving a magnetic particle or a modulating agent, refers to an antibody or antigen-binding fragment thereof specific for CD3, CD28, CD4, or CD8 that is directly or indirectly linked via a biodegradable linker, which can be specifically biodegraded, digested, or cleaved by the activity of the enzyme, thereby splitting the magnetic particle or modulating agent into at least two separate molecules. Thus, a single released antibody or antigen-binding fragment thereof, such as a Fab, specific for CD3 or CD28, does not further affect the activation of the T cell to which it is bound. In addition, if the antibody or antigen-binding fragment thereof, such as the Fab, has a low affinity and / or a high k(off) rate, the antibody or antigen-binding fragment thereof, such as the Fab, will be removed from the cell to which it is bound.

[0111] The term "chemical cleavage", as used herein in the context of cleaving a magnetic particle or a modulating agent, refers to an antibody or antigen-binding fragment thereof specific for CD3, CD28, CD4, or CD8 that is directly or indirectly linked via a chemically degradable linker, which can be specifically degraded or cleaved by adding a non-proteinaceous chemical that breaks the covalent bond under physiological conditions, thereby splitting said magnetic particle or modulating agent into at least two separate molecules. Examples of reactions suitable for chemical cleavage under physiological conditions may be reduction, such as reduction of disulfide bonds with a reducing agent or reduction of diazo bonds with dithionite, or oxidation, such as cleavage of glycol residues with periodate.

[0112] Thus, a single released antibody or its antigen-binding fragment, such as a Fab, specific for CD3 or CD28 has no further effect on the activation of the T cell to which it is bound. In addition, if the antibody or its antigen-binding fragment, such as the Fab, has a low affinity and / or a high k(off) rate, the antibody or its antigen-binding fragment, such as the Fab, will be removed from the cell to which it is bound.

[0113] The term "physical cleavage", as used herein in the context of cleaving a magnetic particle or a modulating agent, refers to an antibody or antigen-binding fragment thereof specific for CD3, CD28, CD4 or CD8, which is directly or indirectly linked via said physically cleavable linker, being capable of being specifically decomposed or cleaved by an energy input that breaks the covalent bond under physiological conditions, thereby splitting said magnetic particle or modulating agent into at least two separate molecules. An example of a reaction suitable for physical cleavage under physiological conditions may be a photoreaction, such as photocleavage of a photosensitive linker with UV or visible light, as exemplified by cleavage of an ortho-nitrobenzyl derivative with near-UV light (300-365 nm). Thus, a released single antibody or antigen-binding fragment thereof, such as a Fab, specific for CD3 or CD28, does not further affect the activation of the T cell to which it is bound. In addition, if the antibody or antigen-binding fragment thereof, such as the Fab, has a low affinity and / or a high k (off) rate, the antibody or antigen-binding fragment thereof, such as the Fab, will be removed from the cell to which it is bound.

[0114] The term "marker" as used herein refers to a cellular antigen that is specifically expressed by a certain cell type. Preferentially, the marker is a cell surface marker, and can perform enrichment, isolation, and / or detection of live cells. The marker can be a positive selection marker, such as CD4, CD8, and / or CD62L, or a negative selection marker (e.g., depletion of cells expressing CD14, CD16, CD19, CD25, CD56).

[0115] The term "expression", as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven within a cell by its promoter.

[0116] The term "antigen-binding molecule" as used herein refers to any molecule that preferably binds to or is specific for a desired target molecule of a cell, i.e., an antigen. The term "antigen-binding molecule" includes, for example, an antibody or an antigen-binding fragment thereof. The term "antibody" as used herein refers to a polyclonal or monoclonal antibody that can be generated by methods well known to those skilled in the art. The antibody may be of any species, e.g., mouse, rat, sheep, human. If non-human antigen-binding fragments are to be used for therapeutic purposes, they can be humanized by any method known in the art. The antibody may also be a modified antibody (e.g., oligomer, reduced, oxidized, and labeled antibody).

[0117] The term "antibody" includes both intact molecules and antigen-binding fragments such as Fab, Fab', F(ab')2, Fv, and single chain antibodies. In addition, the term "antigen-binding fragment" includes any molecule other than an antibody or antibody fragment that preferentially binds to a desired target molecule of a cell. Suitable molecules include, but are not limited to, oligonucleotides known as aptamers that bind to a desired target molecule, carbohydrates, lectins, or any other antigen-binding protein (e.g., receptor-ligand interactions). The coupling of the antibody to the particle or nanostructure can be covalent or non-covalent. Covalent coupling can be, for example, to the carboxyl groups of polystyrene beads, or to the NH2 or SH2 groups of modified beads. Non-covalent coupling can be, for example, by biotin-avidin, or fluorophore-coupled particles that are linked to anti-fluorophore antibodies.

[0118] The term "specifically binds" or "specific" with respect to an antigen-binding molecule, such as an antibody or fragment thereof, refers to an antigen-binding molecule (in the case of an antibody or fragment thereof to an antigen-binding domain) that recognizes and binds to a particular antigen in a sample, such as CD4, but does not substantially recognize or bind other antigens in said sample. An antigen-binding domain of an antibody or fragment thereof that specifically binds to an antigen from one species may also bind to that antigen in another species. This cross-species reactivity does not violate the definition of "specific" as used herein. An antigen-binding domain of an antibody or fragment thereof that specifically binds to an antigen, such as the CD4 antigen, may substantially bind to various variants (allelic variants, splice variants, isoforms, etc.) of said antigen. This cross-reactivity does not violate the definition that the antigen-binding domain is specific for an antigen, such as CD4.

[0119] The term "genetically modified T cells" or "genetically engineered T cells" can be used interchangeably and thus refer to containing and / or expressing a foreign gene or nucleic acid sequence that modifies the genotype or phenotype of the cell or its progeny. In particular, these terms refer to the fact that cells can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins, such as CARs, that are not naturally expressed in such cells. Genetic modification of cells includes, but is not limited to, transfection, electroporation, nucleofection, transduction using retroviral vectors, lentiviral vectors, non-integrating retro or lentiviral vectors, transposons, designer nucleases including zinc finger nucleases, TALEN, or CRISPR / Cas.

[0120] The genetically modified T cells obtained by the methods disclosed herein can be used for subsequent steps, such as research, diagnostic, pharmacological or clinical applications known to those skilled in the art.

[0121] The genetically modified T cells can also be used as a pharmaceutical composition in therapy, for example cell therapy, or in the prevention of disease. The pharmaceutical composition can be implanted into an animal or human, preferentially into a human patient. The pharmaceutical composition can be used to treat and / or prevent disease in a mammal, particularly a human, where treatment and / or prevention possibly involves administering a pharmacologic effective amount of the pharmaceutical composition to the mammal. The pharmaceutical composition of the present disclosure can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0122] The term "therapeutically effective amount" means an amount that provides a therapeutic benefit to a patient.

[0123] The genetically modified T cell composition obtained by the method of the present invention can be administered either alone or as a pharmaceutical composition in combination with a diluent and / or other components such as cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may comprise the genetically modified T cells of the present disclosure in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline and phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0124] The term "activation," as used herein, refers to inducing a physiological change with a cell that increases the function, proliferation, and / or differentiation of a target cell.

[0125] The term "transduction" refers to the transfer of genetic material from a viral agent, such as a lentiviral vector particle, into a eukaryotic cell, such as a T cell.

[0126] Tumor-associated antigen (TAA), as used herein, refers to an antigenic substance produced in tumor cells. Tumor-associated antigens are tumor or cancer markers that are useful for identifying tumor / cancer cells in diagnostic tests and are potential candidates for use in cancer therapy. Preferentially, TAA may be expressed on the cell surface of tumor / cancer cells.

[0127] The term "removal of a modulatory agent," as used herein, refers to physically removing the modulatory agent from the T cell and / or inactivating the modulatory agent to the point where it no longer has an effect on the activity of the T cell.

[0128] Lentivirus is a genus of Retroviridae that causes chronic and fatal diseases characterized by long latency periods in humans and other mammalian species. The best known lentivirus is the human immunodeficiency virus HIV, which can efficiently infect non-dividing cells, so lentivirus-derived retroviral vectors are one of the most efficient methods of gene delivery.

[0129] To generate retroviral vectors, such as lentiviral vectors, the gag / pol and env proteins required for assembly of vector particles are provided in trans by a packaging cell line, e.g., HEK293T. This is usually accomplished by transfecting the packaging cell line with one or more plasmids containing the gag / pol and env genes.

[0130] The term "removal of residual lentiviral vector particles," as used herein, refers to physically removing residual lentiviral vector particles from the T cells and / or inactivating the residual lentiviral vector particles to the extent that they are no longer effective to genetically modify the T cells.

[0131] The term "residual lentiviral vector particles," as used herein, refers to the portion of lentiviral vector particles in a sample that contains T cells that have not transduced T cells.

[0132] The term "the method is performed in 144 hours or less, 120 hours or less, 96 hours or less, 72 hours or less, 48 ​​hours or less, or 24 hours or less" means that the duration of the process disclosed herein from the initiation of the process, i.e., from providing a sample containing T cells to a sample containing genetically modified T cells that can then be directly (re)infused to a patient in need thereof, does not take longer than the respective time frame.

[0133] In blood, serum is the plasma that is neither blood cells (serum does not contain white blood cells - leukocytes, or red blood cells - erythrocytes) nor clotting factors and does not contain fibrinogen. Serum contains all proteins that are not used in blood clotting, as well as all electrolytes, antibodies, antigens, hormones, and any exogenous substances. Human serum is serum derived from humans.

[0134] As used herein, the term "subject" refers to an animal. Preferentially, the subject is a mammal, such as a mouse, a rat, a cow, a pig, a goat, a chicken, a dog, a monkey, or a human. More preferentially, the individual is a human. The subject may be a subject (patient) suffering from a disease, such as cancer, but the subject may also be a healthy subject.

[0135] The term "closed system" as used herein refers to any closed system that reduces the risk of cell culture contamination during the carrying out of culture processes, such as the introduction of new substances by transduction, and during the carrying out of cell culture steps, such as proliferation, differentiation, activation, and / or cell separation. Such a system allows operation under GMP or GMP-like conditions ("sterile") and results in clinically applicable cell compositions. Exemplarily, CliniMACS Prodigy® (Miltenyi Biotec GmbH, Germany) is used herein as a closed system. This system is disclosed in WO 2009 / 072003. However, it is not intended to limit the use of the method of the present invention to CliniMACS Prodigy®.

[0136] The process of the present invention can be carried out in a closed system (closed cell sample processing system) including a cylinder, a pump, a valve, a magnetic cell separation column, and a centrifugation chamber including a base plate and a cover plate, connected by a tubing set. A blood sample or other source including T cells can be transferred to and from the tubing set by sterile docking or sterile welding. A suitable system is disclosed in WO2009 / 072003.

[0137] The closed system may include multiple tubing sets (TS) where cells are transferred between the TS by sterile docking or sterile welding.

[0138] The different modules of the process can be carried out in different functionally closed TSs and the product of one module (cells) produced in one tube set can be transferred by sterile means to another tube set, for example T cells can be magnetically enriched in a first tube set (TS) TS100 by Miltenyi Biotec GmbH and the positive fraction containing the enriched T cells is welded out of the TS100 and welded into a second tube set TS730 by Miltenyi Biotec GmbH for further activation, modification, culture and washing.

[0139] The term "automated method" or "automated process" as used herein refers to any process that is automated by the use of a device and / or a computer and computer software. An automated method (process) requires less human intervention and less human time. In some cases, the method of the present invention is automated if at least one step of the method is performed without any human assistance or intervention. Preferentially, the method of the present invention is automated if all steps of the method disclosed herein are performed without any human assistance or intervention other than connecting fresh reagents to the system. Preferentially, the automated process is performed in a closed system such as the CliniMACS Prodigy® disclosed herein.

[0140] The closed system may include: a) a sample processing unit including an input port and an output port coupled to a rotating vessel (or centrifugation chamber) having at least one sample chamber, the sample processing unit configured to provide a first processing step to the sample or to apply centrifugal force to the sample to rotate the vessel such that the sample is deposited in the chamber to separate at least a first and a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, the sample separation unit including a separation column holder, a pump, and a plurality of valves configured to at least partially control fluid flow through a separation column disposed in the fluid circuit and holder, the separation column configured to separate labeled and unlabeled components of the sample flowing through the column.

[0141] The rotating vessel can also be used as a temperature-controlled cell incubation and culture chamber (CentriCult Unit = CCU). This chamber can be filled with a defined gas mixture provided by an attached gas mixing unit (e.g., using pressurized air / N2 / CO2 or N2 / CO2 / O2).

[0142] All agents may be connected to the closed system before the process begins, including all buffers, solutions, culture media, and nutritional supplements, MicroBeads used for washing, transfer, suspension, culture, cell harvesting, or immunomagnetic cell sorting in the closed system, or such agents may be welded or connected by sterile means at any point during the process.

[0143] The cell sample containing the T cells can be prepared in a transfer bag or other suitable container that can be connected to a closed system by sterile means.

[0144] The term "preparing a (cell) sample comprising T cells" refers to the preparation of a cell sample, preferentially a human cell sample of blood origin. Usually, the cell sample may consist of blood cells derived from a donor or a patient. Such blood products may be in the form of whole blood, buffy coat, leukapheresis, PBMCs or any clinical sampling of blood products. Such blood products may be fresh or frozen in origin.

[0145] The term "washing" refers, for example, to the replacement of the medium or buffer in which the cells are maintained. The replacement of the supernatant may be partial (e.g., removing 50% of the medium and adding 50% fresh medium). This is often done for dilution or nutrient purposes. Or the replacement of the supernatant may be total. To obtain a more radical replacement of the original medium in which the cells are maintained, several washing steps may be combined. The washing steps often include pelleting the cells by centrifugal force and removing the supernatant. In the method of the invention, the cells may be pelleted, for example, by spinning the chamber at 300×g, and the supernatant may be removed during the rotation of the chamber. The medium may be added during the rotation or during the steady state.

[0146] Generally, the wash or washing step may be performed once or by a series of medium / buffer exchanges (at least two exchanges, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 exchanges) to remove substances intended to be removed from the T cells, such as human serum and / or components thereof, magnetic particles, or residual lentiviral vector particles. The exchanges can be performed by separation of cells and medium / buffer by centrifugation, sedimentation, adhesion, or filtration, followed by exchange of medium / buffer.

[0147] In general, a CAR may comprise an extracellular domain (extracellular portion) that includes an antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide. In some embodiments of the invention, the antigen-binding domain of the CAR binds to a tag or hapten that is coupled to a polypeptide (a "haptenylated" or "tagged" polypeptide), which can bind to a disease-associated antigen, such as a tumor-associated antigen (TAA), which may be expressed on the surface of a cancer cell.

[0148] Such CARs may also be referred to as "anti-tag" CARs or "adapter CARs" or "universal CARs", as disclosed, for example, in US Pat. No. 9,233,125 B2.

[0149] The hapten or tag may be directly or indirectly coupled to a polypeptide (tagged polypeptide), which is capable of binding to said disease-associated antigen expressed on the target (cell) surface.

[0150] "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a particular organelle (such as the endoplasmic reticulum) and / or to the cell surface.

[0151] Generally, an "antigen-binding domain" refers to a region of a CAR that specifically binds to an antigen, such as a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). The CAR of the present invention may comprise one or more antigen-binding domains (e.g., tandem CARs). Generally, the targeting region of a CAR is extracellular. The antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a diabody. Any molecule that specifically binds to a given antigen, such as an affibody or a ligand-binding domain derived from a naturally occurring receptor, can be used as an antigen-binding domain. In many cases, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of an immunoglobulin heavy and light chain are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S)3-linker".

[0152] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as that for which the CAR is to be used.For example, when it is intended to be used for human therapy, it may be beneficial for the antigen-binding domain of the CAR to comprise human antibody or humanized antibody or its antigen-binding fragment.Human antibody or humanized antibody or its antigen-binding fragment can be produced by various methods well known in the art.

[0153] "Spacer" or "hinge" as used herein refers to a hydrophilic region between the antigen-binding domain and the transmembrane domain. The CAR of the present invention may include an extracellular spacer domain, but may also omit such a spacer. Spacers can include, for example, Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences, or combinations thereof. A well-known example of a spacer is the CD8 alpha hinge.

[0154] The transmembrane domain of the CAR may be derived from any desired natural or synthetic source of such a domain. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived from, for example, CD8 alpha or CD28. If the important signaling and antigen recognition modules (domains) are present in two (or even more) polypeptides, the CAR may have two (or more) transmembrane domains. The division of the important signaling and antigen recognition modules allows small molecule-dependent, titratable, reversible control of CAR cell expression by the small molecule-dependent heterodimerization domain of each polypeptide of the CAR (e.g., WO2014127261A1).

[0155] The cytoplasmic signaling domain (or intracellular signaling domain) of a CAR is involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to a specialized function of a cell, for example in T cells, the effector function may be cytolytic activity or helper activity, including secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein that is sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0156] Well-known examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signaling following antigen receptor engagement.

[0157] In general, T cell activation may be mediated by two different classes of cytoplasmic signaling sequences. The first initiates antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domains), and the second provides secondary or costimulatory signals in an antigen-independent manner (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0158] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs).

[0159] Examples of ITAM-containing primary cytoplasmic signaling domains commonly used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. The most prominent are sequences derived from CD3 zeta.

[0160] The cytoplasmic domain of the CAR may be designed to contain a CD3ζ signaling domain by itself, or may be combined with any other desired cytoplasmic domain. The cytoplasmic domain of the CAR may contain a CD3ζ chain portion and a costimulatory signaling region (domain). The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for efficient lymphocyte response to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0161] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked to each other randomly or in a specified order, with or without a linker. A short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the linkage. A well-known linker is the glycine-serine doublet.

[0162] As one example, the cytoplasmic domain may comprise the signaling domain of CD3ζ and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3ζ and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3ζ, the signaling domain of CD28, and the signaling domain of CD137.

[0163] Also, as described above, any of the extracellular portions or transmembrane or cytoplasmic domains of the CAR may contain heterodimerization domains to separate the important signaling and antigen recognition modules of the CAR.

[0164] The CAR may be further modified to include one or more operable elements at the level of the nucleic acid encoding the CAR to eliminate CAR-expressing immune cells by a suicide switch. The suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In one embodiment, the nucleic acid expressing and encoding the CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0165] In some embodiments, the endodomain may contain a primary cytoplasmic signaling domain or a costimulatory region, but not both. In such embodiments, an immune effector cell comprising a CAR of the present disclosure is activated only if another CAR comprising the deleted domain also binds to its corresponding antigen.

[0166] In some embodiments of the invention, the CAR may be a "SUPRA" (split, universal, and programmable) CAR, where the "zipCAR" domain can link an intracellular costimulatory domain and an extracellular leucine zipper (WO 2017 / 091546). This zipper can be targeted, for example, by a complementary zipper fused to the scFv region, making the SUPRA CAR T cells tumor specific. This approach will be particularly useful for generating universal CAR T cells against a variety of tumors. Adapter molecules can be designed to be tumor specific, providing the option to change the specificity after adoptive transfer. This is important in the context of selective pressure and antigen avoidance.

[0167] The CAR that can be expressed in the genetically modified T cells obtained by the methods disclosed herein can be designed to include any portion or part of the above-referenced domains described herein in any order and / or combination that results in a functional CAR, i.e., a CAR that mediates an immune effector response of an immune effector cell expressing the CAR disclosed herein. EXAMPLES

[0168] Short-term manual generation of engineered T cells Samples containing T cells were prepared from the buffy coat and PBMCs were isolated. Blood products were diluted in a 1:2 or 1:3 ratio with CliniMACS® buffer and 30 mL was layered on a 15 mL cushion of Pancoll human. Tubes were centrifuged at 450 × g for 30 min at room temperature with medium brake. After centrifugation, cells at the interface were carefully aspirated and washed three times with 50 mL of CliniMACS® buffer to remove platelets and residual Pancoll. T cells were isolated using CD4 and CD8 specific microbeads (Miltenyi Biotec) according to the manufacturer's instructions. T cells were cultured at 1 × 10 in TexMACS medium containing human AB serum (10% (vol / vol) GemCell), IL-7 (10 ng / mL), and IL-15 (5 ng / mL).6 T cells were seeded into 24-well plates with 2 mL of T cell suspension per well at a concentration of 1000 cells / mL. To activate T cells, T Cell TransAct TM After 24 hours of culture in an incubator at 37°C and 5-10% CO2, a lentiviral vector encoding a therapeutic CAR is added at an MOI of 2. 24 hours after transduction, T Cell TransAct TM The enzyme dextranase, which specifically degrades the biodegradable linker present in the microbeads, was added at 1:100 for 1 hour at 37°C to release both reagents from the T cells. TM Non-cellular components, such as cellular components and degraded components of the microbeads, are separated from the transduced T cells by centrifugation at 450×g for 10 min. The supernatant is removed and fresh medium is added to the same volume. The washing procedure is repeated three times to reduce impurities. The transduced T cells are analyzed by flow cytometry to determine transduction efficiency and to perform functional assays, such as a killing assay in which they are co-cultured with tumor target cells expressing the CAR antigen. EXAMPLES

[0169] Rapid and automated generation of genetically modified T cells A sample of T cells derived from the donor's leukapheresis is prepared in a bag. The bag is connected with a sterile weld to the tubing set installed in the CliniMACS Prodigy® device. CliniMACS buffer, CliniMACS CD4 and CD8 reagents (Miltenyi Biotec GmbH) and activation reagents are also connected to the same tubing set. The enrichment step, which takes between 30 min and 2 h in total, is started as part of a fully automated process. In detail, the tubing set is automatically primed with buffer and then the leukapheresis product is transferred to the chamber of the tubing set, where it is washed three times with CliniMACS buffer to remove serum and platelets. The cells are magnetically labeled with CliniMACS CD4 and CD8 reagents and captured on a column placed in a magnetic field. The labeled cells captured on the column are rinsed several times and eluted into a target cell fraction bag. A portion of the enriched cells is transferred to the CentriCult via a sterile weld connection. TM The cells are transferred to a chamber and formulated in MACS GMP TexMACS medium supplemented with IL-7 / IL-15 (all from Miltenyi Biotec GmbH). As part of the automated process, an activation step is initiated and the activation reagent MACS GMP TransAct is automatically added to the culture. Within 24 hours after enrichment, the bag containing the lentiviral vector is sterile welded to a tubing set and the lentiviral vector suspension is transferred to the CentriCult TM The cells are transferred to a chamber. After 24-48 hours, the activation reagent and magnetic particles are degraded by adding dextranase specific for the biodegradable linker present in the activation reagent and magnetic particles. After 1 hour, non-cellular components such as residual lentiviral vectors, degradative enzymes, and degraded components of the activation reagent and CliniMACS CD4 and CD8 reagents are removed by washing. The genetically modified T cells are automatically formulated in a solution suitable for human infusion. EXAMPLES

[0170] Administration of CAR T cells with additional cleanup steps CAR T cell therapy is provided, for example, to treat pediatric and adult patients with relapsed or refractory CD19-positive B-cell malignancies. The clinical method for preparing engineered T cells is based on Example 2, in which patient cells (derived from BM, blood, or leukapheresis) are connected to the CliniMACS Prodigy® device, rapidly processed (i.e., preferably in less than 24 hours), and reinfused to the patient. The duration of the process can be adjusted to match the timing of the required patient preparation regimen (e.g., chemotherapy treatment for lymphodepletion) and to meet medical needs and clinical applicability (e.g., clinical protocols, patient health status, reactivity of the doctors, hospitalization). The advantages of the described invention are that it allows for rapid treatment and patient care, as well as "bedside" preparation of drug products. In the present invention, a solution for rapid cell preparation is described in which potentially harmful substances such as viral vectors and activation reagents are removed before infusion. For example, residual activation reagents contaminating drug products can be harmful when infused because they can lead to in vivo T cell activation, which can lead to rapid release of proinflammatory cytokines, causing severe cytokine release syndrome, fever, hypotension, organ failure, and even death.

[0171] In addition, residual lentiviral vector contaminating the drug product in soluble and / or cell-associated form may be harmful when infused, as it induces undesirable immune responses such as complement activation, antibody-dependent cell-mediated cytotoxicity, including adoptive immune responses against antigens delivered by the lentiviral vector and / or transduction of non-target cells in vivo. Transduction of non-target cells and subsequent expression of the transgene may induce undesirable side effects such as induction of undesirable immune responses, carcinogenesis, altered viability, altered proliferation, altered physiological state, and altered native function. EXAMPLES

[0172] Setting up a process to genetically engineer T cells in 3 days T cells from two healthy donors were enriched for naïve T cells with a human pan T cell isolation kit (Miltenyi Biotec) and on day 0 were transfected with T Cell TransAct™, a regulatory reagent containing an antibody or antigen-binding fragment specific for CD3 and an antibody or antigen-binding fragment specific for CD28, directly or indirectly coupled to a biodegradable linker. TM (Miltenyi Biotec). On the same day or day 1, stimulated T cells were transduced with VSV-G pseudotyped GFP-encoding LVs at an MOI of 2 in 24-well plates at 1e6 cells / ml in IL-7 / IL-15-containing TexMACS medium. Dextranase, which specifically digests the biodegradable linker, was added on days 1, 2, or 3, and transduction efficiency was assessed by flow cytometry on day 10. T cells stimulated on day 0 and transduced on day 1, but without dextranase, served as a control for the conventional protocol to genetically engineer T cells. As illustrated in Figure 9, T cells transduced on day 0 and incubated with the enzyme specific for the biodegradable linker showed the lowest transduction efficiency level, indicating insufficient T cell stimulation. This was confirmed by the later analysis of stimulated T cells added on days 2 or 3, where higher levels of transduction efficiency were detectable compared to T cells incubated with the enzyme on day 0. Higher transduction efficiencies, close to those of the conventional protocol, were observed in stimulated T cells transduced on day 1 and incubated with dextranase on days 2 or 3. EXAMPLES

[0173] Removal of regulators and analysis of T cell activation levels in the CliniMACS® Prodigy system for genetic T cell engineering within 3 days Leukapheresis samples from healthy donors with up to 1e9 CD4 / CD8 cells were automatically processed on the CliniMACS® Prodigy system to generate CAR T cells within 3 days. On day 0, the bag containing the leukapheresis sample was connected to a CliniMACS Prodigy® tubing set 520 by sterilization with welding. The cells were automatically washed and labeled with CD4 and CD8 CliniMACS reagents to enrich for T cells. 4e8 T cells were transferred to the centrifuge and culture chamber in IL-7 / IL-15 containing medium and incubated in a culture volume of 200 ml with the regulator MACS® GMP T Cell TransAct™. TM(Miltenyi Biotec). On day 1, isolated activated T cells were genetically modified with a VSV-G pseudotyped lentiviral vector at an MOI of 3 to induce the expression of a CD20 / CD19-specific tandem CAR. A bag containing 10 ml of lentiviral vector was sterilely connected to the tubing set and automatically transferred to the chamber containing the T cells. On day 2, 10 ml of a solution containing an enzyme specific for the biodegradable linker was sterilely connected to the tubing set and automatically added to the chamber containing the T cells to specifically degrade the linker. This resulted in the release of antibodies or their fragments specific for CD3 and CD28 and the inhibition of the activity of the modulator. As a control, a CliniMACS® Prodigy system run was performed under the same conditions and with the same donor material but without the addition of the enzyme specific for the biodegradable linker. After multiple washes, a cell product was obtained that was suitable for therapeutic applications. For both T cell engineering runs on the CliniMACS® Prodigy system, the presence of the biodegradable linker was assessed by flow cytometry on the formulated cells by staining with an antibody specific for the biodegradable linker. As illustrated in Figures 10A and 10B, the biodegradable linker was efficiently removed in the CliniMACS® Prodigy system, as only a small percentage of linker-positive cells were detectable, compared to the CliniMACS® Prodigy run without added enzyme. In addition, the mean intensity level (MFI) of the biodegradable linker for all viable cells was at background levels when enzyme was added (see Figure 10B). In contrast, there was a high mean intensity level (MFI) in the CliniMACS® Prodigy run without added enzyme.

[0174] The effect of removing the regulators on stimulation was evaluated by flow cytometry staining for CD25 and CD69. Both CD25 and CD69 have been described as reliable T cell activation markers (CD25: clone REA570, and CD69: REA824 (both Miltenyi Biotec): CD69 is a more early activation marker than CD25). Unstimulated T cells from the same donor derived from small scale culture served as control. T cells recovered from the CliniMACS® Prodigy system with or without enzymatic treatment were analyzed. Highly significant mean intensity levels of both activation markers were detected in both T cell engineering conditions compared to unstimulated control cells (see Figure 11). This confirmed that stimulation by day 2 was already sufficient to induce the upregulation of both activation markers. This also shows that regulators can be removed already on day 2 without affecting stimulation. EXAMPLES

[0175] Assessment of the expansion potential of stimulated T cells on the CliniMACS® Prodigy system for genetically engineering T cells within 3 days Multiple manufacturing runs with stimulated T cells were performed as described in Example 5, but with different starting T cell numbers ranging from 1e8 to 4e8, in the presence of dextranase added on day 2. Input T cell numbers on day 0 were compared with output T cell numbers obtained on day 3. T cell expansion proliferation was not detectable on day 3, suggesting that T cells were sufficiently stimulated but proliferation had not yet begun (see also FIG. 12). Consequently, the manufacturing protocol for genetically modifying T cells within 3 days is too short to support T cell expansion in vitro. The data also suggest that the yield of recoverable CAR T cells is efficiently increased by increasing the starting cell number. EXAMPLES

[0176] Assessment of CAR expression kinetics on small scale and CliniMACS® Prodigy systems CAR expression kinetics is particularly important for the success of CAR T cell therapy when applying short-term manufacturing processes. Infused CAR T cells expressing therapeutic CAR molecules do not remain fully non-functional because tumor antigens cannot be recognized. During this time, tumor progression may continue in the patient, making it more difficult for CAR T cells to respond to higher tumor burdens. To date, the most common side effect after infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). This is a systemic inflammatory response caused by cytokines released shortly after infusion by infused CAR T cells upon recognition of potentially high-load tumor cells expressing CAR antigens. Since not all CAR T cells express CARs at this early time point and at high CAR expression levels, this toxicity can be at least partially reduced by manufacturing CAR T cells within a short period of time.

[0177] In a small study, CD4 / CD8 enriched T cells from two healthy donors were used in a T Cell TransAct study. TM (Miltenyi Biotec). On day 1, 1e6 cells / ml of stimulated T cells were transduced with CAR-encoded LVs at an MOI of 9 in 24-wells, and the kinetics of CAR expression was determined by flow cytometry by day 13 as the ratio of transduced cells (i.e., transduction efficiency) using a CAR detection reagent (e.g., CD19 CAR antibody, anti-human, 130-115-965, MiltenyiBiotec) containing the CAR antigen peptide directly or indirectly coupled to PE. As illustrated in Figure 13, 2 days after transduction, 16% of T cells were CAR positive, but no distinct population expressing CAR was yet detectable. By day 5, the transduction efficiency level reached a plateau level of 18-22% and the CAR-expressing population was distinct.

[0178] In a large-scale study on the CliniMACS® Prodigy system, 2e8 CD4, CD8 enriched T cells were cultured in the culture chamber on day 0 using the MACS® GMP T Cell TransActTM (Miltenyi Biotec) in 100 ml of IL-7 / IL-15-containing medium. On day 1, isolated stimulated T cells were genetically modified with a VSV-G pseudotyped CD19 CAR-encoding lentiviral vector at an MOI of 62.5 by sterilely connecting the LV-containing bag to the tubing set. On day 2, a 10 ml solution containing dextranase specific to the biodegradable linker of the regulator was sterilely connected and automatically added to the chamber containing the T cells. On day 3, a sample of the cell suspension was analyzed by flow cytometry after staining with a CAR detection reagent (e.g., CD19 CAR antibody, anti-human, 130-115-965, Miltenyi Biotec) containing a CAR antigen peptide directly or indirectly coupled to PE. As illustrated in FIG. 14, 2 days after transduction, 19% of the T cells were CAR positive. The culture process in the CliniMACS® Prodigy was extended to allow analysis at later time points. The transduction efficiency increased to 75% on day 10, indicating that CAR was still not fully expressed 2 days after transduction. EXAMPLES

[0179] Optimization of CAR T cell manufacturing parameters on the CliniMACS® Prodigy system The manufacturing process of CAR T cells is a complex process that depends on multiple parameters and has a high degree of donor variability. Optimizing gene transfer efficiency and T cell culture offers the possibility to reduce the amount of lentiviral vector required and obtain a higher number of (CAR) T cells. In two separate T cell engineering runs, 1e8 or 4e8 CD4,CD8 enriched T cells were transfected with the MACS GMP T Cell TransAct™ on the CliniMACS Prodigy system. TM(Miltenyi Biotec) on day 0 in IL-2-containing medium. On day 1, isolated stimulated T cells were genetically modified with 2.5 ml of VSV-G pseudotyped CD20 / CD19 tandem CAR-encoding lentiviral vector (see FIG. 15: condition I) for 1e8 T cells and in parallel with the same lentiviral vector volume for 4e8 T cells. For the 4E8 CAR T cell production run, the process active matrix was further modified to allow culture at higher cell density by increasing the volume and performing an initial shaking step immediately after adding the lentiviral vector volume (see FIG. 15: condition II). On day 2, for both T cell production runs, the same volume of dextranase was applied by sterile connection and automated addition to the culture chamber. On day 3, the produced T cells were washed multiple times, harvested, and total T cell numbers were determined by cell counting. Washed and harvested cell samples of both CAR T cell manufacturing runs were cultured for an additional 8 days in 24-wells in an incubator, allowing reliable assessment of transduction efficiency at later time points with CAR detection reagents containing CAR antigen peptides directly or indirectly coupled to PE (e.g., CD19 CAR antibody, anti-human, 130-115-965, MiltenyiBiotec). As illustrated in Figure 15A, the transduction efficiency of condition II was 32%, whereas the transduction efficiency of condition I was only 20%, despite the application of a higher LV dose per cell (MOI) in condition I. This indicates that the parameters of condition II favor a higher frequency of CAR T cells, highlighting the potential for CAR T cell manufacturing protocol optimization. Not only was a higher transduction efficiency determined in condition II, but 4e8 T cells were transduced. This resulted in an almost 7-fold increase in the total number of CAR-transduced T cells in condition II compared to condition I. EXAMPLES

[0180] In vivo function of CAR T cells generated within 3 days The function of CAR T cells is typically assessed in vitro when co-cultured with tumor cells expressing the CAR antigen. Within a short period of time and upon contact with the CAR antigen, CAR T cells release inflammatory cytokines such as interferon-gamma (IFN-g), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2. In addition, granzyme B and perforin B are released, reducing the number of viable tumor cells. These functional assays were performed to characterize the functionality of CAR T cells produced within 3 days. T cells from two healthy donors were enriched for naïve with a human pan T cell isolation kit (Miltenyi Biotec) and on day 0 were transfected with T Cell TransAct, a modulating reagent comprising an antibody or antigen-binding fragment specific for CD3 and an antibody or antigen-binding fragment specific for CD28, directly or indirectly coupled to a biodegradable linker. TM(Miltenyi Biotec). On day 1, stimulated T cells at 1e6 cells / ml in IL-7 / IL-15-containing TexMACS medium were transduced with VSV-G pseudotyped CD20 CAR-encoding LVs at an MOI of 10 in 24-well plates. Dextranase was added on day 2, and transduced T cells were washed and harvested on day 3, and co-cultures were set up at various effector-to-target (E:T) ratios. Transduction efficiency was 70% on day 3, as measured by flow cytometry using CAR-specific detection reagents. A total of 50,000, 17,000, 6,000, or 2,000 T cells were added in triplicate to 40,000 CD20-expressing Raji cells in RPMI / 10% FCS / L-glutamine medium in 96-well flat-bottom plates. Transgenic Raji cells expressing GFP (Raji-GFP) were used to allow identification and quantification of tumor cells in co-culture by flow cytometry and to determine the cytotoxic activity of the manufactured CAR T cells. As a control, co-cultures with Raji-GFP cells were established in triplicate in parallel with unstimulated and untransduced T cells. In addition, tumor cells were co-cultured with stimulated but untransduced T cells. In this way, potentially non-specific cytotoxic activity is easily detected. 24 hours after setting up the co-culture, 100 μl of supernatant was removed from each well and cytokine expression levels were evaluated by flow cytometry using the MACSPlex cytokine kit assay (Miltenyi Biotec). In CD20 CAR-transduced T cells generated within 3 days, IFN-g, GM-CSF, and IL-2 levels were detectable in an E:T-dependent manner at high levels, even above the quantification level (see Figure 16). In contrast, no cytokines were detectable in unstimulated T cells and in stimulated T cells that remained untransduced, confirming the specific antitumor response of CAR-transduced T cells produced within 3 days.

[0181] Co-cultured T cells and Raji-GFP cells were co-cultured for an additional 2 days and 50% of the cells were analyzed by flow cytometry to quantify the number of residual tumor cells and, consequently, the potency of the CAR T cells (Round 1; left). Another 20,000 Raji-GFP tumor cells were added to the remaining 50% of the co-culture to evaluate the potency of the CAR T cells in the presence of more tumor cells, similar to a challenging condition for the CAR T cells. After another 72 hours, flow cytometry was performed to quantify the number of residual tumor cells in the second round of co-culture (Round 2: right). At a high E:T ratio of 1.25:1, nearly 100% of the Raji cells were killed in the first and second round of co-culture (see Figure 17). In contrast, only 50% and 40% of the target cells were detectable in the untransduced controls of the first and second co-cultures. At an E:T ratio of 0.425:1, a similar pattern of functionality was detectable as at 1.25:1, but at a lower overall level. In the presence of CAR-transduced T cells, 60% of tumor cells were lysed in the first and second rounds of co-culture. In contrast to untransduced controls, 40% of tumor cells were lysed in the first round and no killing was measured in the second round. At an E:T ratio of 0.15:1, the frequency of T cells was too low to induce cytotoxic activity against Raji-GFP tumor cells. In summary, the functionality of CAR T cells generated within 3 days was confirmed by in vitro assays showing cytokine release and specific killing of CAR-transduced T cells. EXAMPLES

[0182] In vivo function of CAR T cells generated within 3 days The in vivo functionality of CAR-transduced T cells generated within 3 days was assessed using 6- to 8-week-old NOD scid gamma (NSG) (NOD.Cg-Prkdc scid Il2rg tm1Wjl All experiments were performed in accordance with "Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes" and in compliance with the regulations of the German Animal Protection Act.

[0183] Briefly, leukapheresis samples from healthy donors were automatically processed on the CliniMACS® Prodigy system and CAR T cells were generated within 3 days (see Figure 18, top). On day 0, the bag containing the leukapheresis sample was sterilely connected to a CliniMACS Prodigy® tubing set 520 by welding. The cells were automatically washed and labeled with CD4 and CD8 CliniMACS reagents to enrich for T cells. 2e8 T cells were transferred to the culture chamber in IL-7 / IL-15-containing medium and cultured in a 200 ml culture volume using the MACS® GMP T Cell TransAct™. TM(Miltenyi Biotec). On day 1, isolated activated T cells were genetically modified with a VSV-G pseudotyped lentiviral vector to induce expression of a CD22 / CD19 tandem-CAR. A bag containing 10 ml of lentiviral vector was sterilely connected to the tubing set and automatically transferred to the chamber containing the T cells. On day 2, a 10 ml solution containing dextranase was sterilely connected to the tubing set and automatically added to the chamber containing the T cells to specifically degrade the linker. This releases antibodies or their fragments specific for CD3 and CD28 and inhibits the activity of the modulator. After multiple washes, the cell products were analyzed by flow cytometry to determine the transduction efficiency, viability, and cell composition at each step (see Figure 19). Cell composition was determined by staining for CD45h, CD3, CD4, CD8, CD16 / CD56, 7-AAD, CD19, and CD14. After formulation, 67% CD4 T cells, 18% CD8 T cells, and 7% NKT cells were detected. The frequency of NK cells, eosinophils, neutrophils, B cells, or monocytes was at the lowest level of detection. Transduction efficiency was analyzed by flow cytometry using a CAR detection reagent (e.g., CD19 CAR antibody, anti-human, 130-115-965, Miltenyi Biotec) containing the CAR antigen peptide directly or indirectly coupled to PE. The transduction efficiency on the day of harvest was 21%. When analyzed after an additional 8 days of small-scale long-term culture, when stable CAR expression levels were achieved, the transduction efficiency increased to 73%. Raji tumors were established by intravenous inoculation of 5e5 firefly luciferase-expressing Raji cells 4 days before harvesting the engineered T cells (see Figure 17). 3e6 or 6e6 total T cells from the CAR transduced groups were injected into 7 mice per group on the day of harvest (see FIG. 18, bottom). Two additional groups were established as negative controls: one group received 5e5 tumor cells but no T cells (n=7; tumor only); one group received 5e5 tumor cells and 3e6 untransduced T cells from the same donor cultured in parallel on a smaller scale (n=7).Tumor growth as well as antitumor responses were monitored frequently using an In vivo Imaging System (IVIS Lumina III). For this purpose, 100 μl of XenoLight Rediject D-Luciferin Ultra was injected ip, after which mice were anesthetized using an isoflurane XGI-8 anesthesia system. Measurements were performed 6 min after substrate injection.

[0184] All mice in the groups that received 3e6 untransduced T cells and 3e6 transduced T cells are shown (see FIG. 20). For the group that did not receive T cells (i.e., tumor only), 3 representative mice out of 7 mice are shown. Tumor burden rapidly increased in all mice that received untransduced T cells or no T cells. The increase in tumor burden over time is comparable in both control groups. Mice in both control groups had to be sacrificed 14 days after T cell injection before reaching the critical tumor burden level. In contrast, mice in the group that received CAR-transduced T cells manufactured within 3 days showed a deceleration of increase at early time points in a dose-dependent manner 3 and 7 days after T cell injection compared to the control group. The level of tumor burden in the CAR-transduced T cell group peaked at 7 days after T cell injection. Tumor progression was completely reversed, as detected by a steady and uniform reduction in tumor burden in all mice to the level initially measured at the beginning of the experiment. Representative in vivo imaging data is shown for all mice in the group receiving 3e6 CAR-transduced T cells and the same dose of untransduced T cells. For the tumor-only group, a representative mouse is shown.

[0185] In Figure 21, tumor burden is depicted as the mean and SEM of all groups, including the group that received the highest dose of 6E6 CAR-transduced T cells per mouse per group (n=7). As expected, the 6e6 group had the fastest anti-tumor response, but tumor burden at the end of the experiment was comparable to the 3e6 CAR T cell group. This data confirms that CAR T cells generated without any expansion within 3 days mediate a strong anti-tumor response. This result was confirmed by flow cytometry data to quantify human tumor cells and human T cell subsets in spleen, bone marrow, and blood. Three randomly selected mice from the "tumor only" and "3E6 untransduced T cell" control groups were sacrificed on day 14, and the abundance of human cells, Raji cells, and T cell subsets in these organs was quantified by staining for CD45h, CD4, CD8, CD20, CD22, 7-AAD, and CD19 CAR detection (all MiltenyiBiotec). Three mice out of seven from the CAR-transduced T cell group were randomly selected and sacrificed on day 18 for similar analysis. As expected, no T cells were found in the tumor-only group (see FIG. 22). In the untransduced cohort, only up to 20% of T cells were detectable. In contrast, the frequency of human T cells was highest in the cohort containing mice infused with CAR-transduced T cells, up to 75%. Thus, T cells are more abundant in the cohort containing transduced T cells than in the cohort with untransduced T cells. This indicates that the CAR T cells are capable of expanding and persisting. This is consistent with the data shown in FIG. 21, which indicates that the CAR-transduced T cells are capable of controlling tumors, whereas the abundance of untransduced T cells was relatively low and they were unable to control tumor growth.

[0186] FIG. 23 further shows that the effect of the untransduced group is that the tumor is still present, in contrast to the transduced group, which shows the disappearance of tumor cells and a greater CD8 expansion.

[0187] The cellular composition of the human subsets was investigated in more detail by determining the frequency of cell subsets with only human cells (see Figure 23). Again, no human T cells were found in the tumor-only group. In the untransduced T cell group, 20-60% of the human cells were residual Raji cells, with CD4 to CD8 ratios ranging from 2:1 to 3:1. In the CAR-transduced T cell group, approximately 50% human CD4 T cells and 50% human CD8 T cells were found, with residual Raji cells in the CAR-transduced group only detectable at near background levels. This data suggests that the CD8 T cell subset was specifically expanded in vivo in the cohort containing mice with CAR-transduced T cells.

[0188] When the spleen was analyzed, no human T cells were found in this lymphoid organ in the tumor-only cohort (see Figure 24). In the cohort containing untransduced T cells, the T cell frequency was determined to be up to 10%. In contrast, the CAR-transduced T cell population had a much higher frequency of human T cells, up to 40% human T cells, as measured by in vivo imaging data. This confirmed T cell expansion and antitumor activity.

[0189] In conclusion, the in vivo data confirmed the in vitro functional data and showed that untransduced T cells were unable to control the proliferation of Raji cells. In contrast, the highest percentage of human T cells was found in the bone marrow, which is the favorable microenvironment for Raji engraftment and expansion. This indicates that CAR T cells expanded for 3 days are also able to home to such a microenvironment and promote antitumor activity.

[0190] Thus, CAR T cells generated within 3 days surprisingly promoted robust antitumor activity in vitro and in vivo even in the absence of an explicit expansion step, demonstrating that in vivo, but not in vitro, expansion is essential for the generation of functional CAR T cells.

Claims

1. 1. A method for generating genetically modified T cells, comprising: a) providing a sample comprising T cells; b) preparing the sample by centrifugation; c) enriching the T cells of step b; d) activating the enriched T cells using modulatory agents; wherein the modulating agent comprises an antibody or antigen-binding fragment thereof specific for CD3 and an antibody or antigen-binding fragment thereof specific for CD28, which are directly or indirectly coupled to a bead or nanostructure via a chemically cleavable or enzymatically degradable linker, and the CD3 and CD28 specific antibodies or antigen-binding fragments thereof can be chemically and / or enzymatically cleaved, and the modulating agent is removed by chemical and / or enzymatic cleavage of the CD3 and CD28 specific antibodies or antigen-binding fragments thereof. e) genetically modifying the activated T cells by transduction with lentiviral vector particles; wherein after genetic modification of T cells by transduction with said lentiviral vector particles, residual lentiviral vector particles are removed, said removal of residual lentiviral vector particles being performed by washing, said washing resulting in at least a 10-fold reduction in residual vector particles in a sample comprising genetically modified T cells. f) removing said modifier Including, Thereby, a sample of genetically modified T cells is generated; wherein the number of T cells in the generated sample is less than 10-fold compared to the number of T cells in the provided sample; the method is carried out in 144 hours or less; The method, wherein the genetically modified T cells do not need to be expanded in vitro prior to administration.

2. 2. The method of claim 1, wherein the sample in step a) comprises human serum, and the serum is removed in step b).

3. 3. The method according to claim 1 or 2, wherein in step c) the T cells are enriched for CD4 and / or CD8 positive T cells by using CD4 and / or CD8 as positive selection markers and / or depleted of cancer cells by using tumor associated antigens (TAA) as negative selection markers.

4. The enrichment of CD4 and / or CD8 positive T cells comprises: i) contacting the T cells with magnetic particles that are directly or indirectly coupled to an antibody or antigen-binding fragment thereof specific for CD4 and / or CD8, wherein the magnetic particles and the antibody or antigen-binding fragment thereof coupled thereto may be removed; ii) separating the CD4 and / or CD8 T cells in a magnetic field; iii) Removing the magnetic particles from the enriched T cells after separation. The method of claim 3, wherein the method is carried out by a magnetic cell separation step comprising:

5. The enrichment of CD4 and / or CD8 positive T cells comprises: i) contacting the T cells with magnetic particles that are directly or indirectly coupled to an antibody or antigen-binding fragment thereof specific for CD4 and / or CD8, wherein the magnetic particles and the antibody or antigen-binding fragment thereof coupled thereto can be cleaved chemically and / or enzymatically; ii) separating the CD4 and / or CD8 T cells in a magnetic field; iii) removing said magnetic particles from the enriched T cells after said separation step by chemical and / or enzymatic cleavage of said magnetic particles and said antibodies or antigen-binding fragments thereof coupled thereto.

5. The method of claim 4, wherein the method is carried out by a magnetic cell separation step comprising:

6. 6. The method of any one of claims 1 to 5, wherein the modulating agent comprises an antibody or antigen-binding fragment thereof specific for CD3 and / or an antibody or antigen-binding fragment thereof specific for CD28, which are directly coupled via a biodegradable linker, the biodegradable linker being degraded by adding an enzyme that specifically digests the glycosidic linkage of the biodegradable linker.

7. The method of claim 6 , wherein the biodegradable linker is or comprises a polysaccharide and the enzyme that specifically digests the glycosidic linkage is a hydrolase.

8. 8. The method of any one of claims 1 to 7, wherein the washing results in at least a 100-fold reduction in residual vector particles in the sample comprising the genetically modified T cells.

9. The method according to any one of claims 1 to 8, wherein the removal of residual lentiviral vector particles is carried out by incubation with a substance that inactivates lentiviral vector particles and / or reduces their stability.

10. 10. The method of any one of claims 1 to 9, wherein the depleted human serum of claim 2, or a substance isolated therefrom that inhibits productive transduction of lentiviral vector particles into T cells, is added to the genetically modified T cells, thereby removing and / or neutralizing residual lentiviral vector particles.

11. The method according to any one of claims 1 to 10, which is an automated method carried out in a closed system.

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