Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has been highly effective in treating pediatric B-cell acute lymphoblastic leukemia (B-ALL), reporting complete responses one month after infusion in close to 90% of patients. However, these responses are not durable in all patients, and up to 50% will relapse with CD19+ or CD19- disease within two years of infusion. Relapse of CD19+ disease is likely due to loss of functional CAR T-cells. There is interest in developing strategies to enhance persistence of CAR T-cells, but the exact mechanisms that drive persistence in a patient are poorly understood. The initial quality of T-cells isolated, the design of the CAR, and conditions during ex vivo manufacturing can all impact T-cell differentiation and exhaustion. As T-cells lack endogenous nutrient stores, they are dependent on their local milieu to fuel their proliferation, cytotoxicity and persistence. We previously demonstrated how the unique metabolic fate of CD19/41BBz CAR T-cells supports their longevity following adoptive transfer. Still, how T-cells respond to the metabolic environment during manufacturing and what impact this has on long-term functional persistence in patients is poorly understood. In two recent clinical trials performed at the Children's Hospital of Philadelphia evaluating a murine (NCT04276870) and humanized (NCT03792633) CD19/41BBz CAR for pediatric patients with B-ALL, we observed that two modifications made to the manufacturing process appeared to have impacted long-term functional persistence of CAR T-cells: base media formulation and cytokine supplementation. This led to four manufacturing conditions across these trials: X-VIVO IL-2, X-VIVO IL-7/IL-15, OpTmizer IL-2, and OpTmizer IL-7/IL-15. Initial clinical response and expansion of CAR T-cells appear similar across manufacturing conditions when compared to historical X-VIVO IL-2 manufacturing conditions used in prior murine (NCT02906371) and humanized (NCT02374333) pediatric CD19/41BBz CAR T-cell studies; however, cells produced in X-VIVO IL-2 demonstrate a longer duration of functional persistence in patients as defined by B-cell aplasia. Additionally, there were significantly increased numbers of CAR T-cells in the X-VIVO IL-2 group, measured by qPCR, in peripheral blood of patients 3-months post-infusion compared to other manufacturing groups. Switching manufacturing back to original X-VIVO IL-2 condition rescues the loss of CAR T-cells at month 3 to pre-manufacturing change levels. Bulk and single-cell RNA-sequencing reveals that media formulation, but not cytokine stimulation, drives major transcriptional differences in patient CAR T-cells both pre-infusion and at peak expansion post-infusion, including differences in master transcriptional regulators. T-cells grown in X-VIVO demonstrate an increased effector/effector memory signature, increased cell volume at end of manufacturing harvest, upregulation of cholesterol biosynthesis pathway genes, differential granzyme profile, and increased CD8+ T-cells. Healthy donor CAR T-cells grown in X-VIVO demonstrate superior expansion and tumor control in a xenograft model of leukemia compared to donor-matched OpTmizer-grown cells, culminating in increased survival. In a targeted metabolomics screen, we identified several differences in metabolite abundance across formulations. We note that X-VIVO was enriched with metabolites which lie at the intersection of TCA cycle activity, one carbon metabolism, as well as arginine, ornithine, and inosine metabolism, several of which have been implicated in T-cell stemness and mitochondrial function. Based on this data, we propose that metabolite bioavailability during manufacturing of CD19/41BBz CAR T-cells has a large impact on the phenotype, transcriptional signature, and overall in vivo functional competence of infused cells, which can determine long-term durability of pediatric B-ALL patient responses.

Background & AimT cell fitness and differentiation status are critical factors that influence the manufacturing (MFG) of effective autologous cell therapy products, including chimeric antigen receptor (CAR) T cells. Our retrospective analysis from over 80 clinical trial subjects with heme malignancies revealed considerable variability in T cell composition, with younger “fit” naïve T cells (TN) and/or T memory stem cells (TSCM) in the starting material positively correlating with ex vivo proliferative capacity, in vivo expansion, persistence, and clinical response. Conversely, leukapheresis comprising of older T cells, like effector memory (TEM) and terminally differentiated effectors (TEFF) had an adverse impact. Here, we evaluated the potential of using highly fit precursors (TN/TSCM) for CAR T MFG to generate improved cell therapy doses.Methods, Results & ConclusionFit T cells were obtained via immunomagnetic depletion with anti-CD45RO microbeads from healthy donors and patients. MFG feasibility, along with product phenotype and function, were compared to conventional CAR T cells. Both CAR T cell products induced effective CAR-mediated anti-tumor responses in vitro and in vivo. Prominently, MFG from CD45RO depleted starting material generated CD4 and CD8 CAR T cells with a less differentiated phenotype (CD45RA, CD27, CCR7, CD95, CD28), increased polyfunctionality and increased stemness gene signatures (TCF7, LEF1). In contrast, standard MFG of donor-matched bulk T cell material generated CAR T products which were more differentiated (CD45RO+CD27) and exhibited gene signatures prone to dysfunction, exhaustion, and senescence (TBX21, MAF). Additionally, using an in vitro model of CAR T cell exhaustion, CD45RO-depleted products retained a more memory-like phenotype and were resistant to dysfunction as compared with conventional products.These results show that while both products are potent in primary challenges, the CD45RO depletion approach for CAR T MFG may generate a product with improved persistence and capacity to respond to repeated tumor challenges without succumbing to exhaustion. In summary, this study demonstrates feasibility of our precision MFG strategy to enrich TN/TSCM upfront to produce a younger, potent CAR T product that can mediate effective anti-tumor response, exhibit self-renewal capacity, respond to repeated antigen exposures, and orchestrate a robust and, importantly, durable response.

Akt1 and Akt2, isoforms of the serine threonine kinase Akt, are essential for T cell development. However, their role in peripheral T cell differentiation remains undefined. Using mice with germline deletions of either Akt1 or Akt2, we found that both isoforms are important for Th17 differentiation, although Akt2 loss had a greater impact than loss of Akt1. In contrast to defective IL-17 production, Akt2 -/- T cells exhibited enhanced IL-4 production in vitro under Th2 polarizing conditions. In vivo , Akt2 -/- mice displayed significantly diminished IL-17A and GM-CSF production following immunization with myelin oligodendrocyte glycoprotein (MOG). This dampened response was associated with further alterations in Th cell differentiation including decreased IFNγ production but preserved IL-4 production, and preferential expansion of regulatory T cells compared to non-regulatory CD4 T cells. Taken together, we identify Akt2 as an important signaling molecule in regulating peripheral CD4 T cell responses.

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has achieved tremendous success treating B cell malignancies; however, some patients fail to respond due to poor autologous T cell fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR T cell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR T cells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the T cell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of T cells from patients with leukemia that previously failed CAR T cell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient T cells, providing a strategy for increasing patient responses to CAR T cell therapy.

Background: Efforts to augment the efficacy of cell therapies include development of 4th generation chimeric antigen receptor (CAR) T-cells delivering a transgenic activator protein to the tumor target. Interleukin-18 (IL-18) is a pro-inflammatory cytokine shown to enhance CAR T-cell proliferative potency and antitumor activity in pre-clinical models (Hu et al, Cell Reports 2017). Methods: We are conducting a first-in-human trial using huCART19-IL18, a 4th generation autologous CAR T-cell product transduced by lentiviral vector to co-express humanized anti-CD19 CAR and IL-18 in patients (pts) with relapsed/refractory B-cell non-Hodgkin lymphomas (NHL) or chronic lymphocytic leukemia (CLL) (NCT04684563). We are using a Bayesian optimal interval dose titration design exploring doses between 3 and 300 million huCART19-IL18 cells per pt. The ex vivo culture time for manufacturing is reduced to 3 days to further improve T-cell activity/persistence and to shorten the time from apheresis to treatment. For this ongoing phase 1 trial, pts must be ≥ 18 years old, have CD19+ relapsed/refractory B-cell NHL or CLL, and have had at least 2 lines of therapy including failure of prior CAR T-cell therapy (if indicated by FDA label). The primary objective is to define the recommended phase 2 dose and evaluate the safety of huCART19-IL18; secondary objectives are feasibility, efficacy, and characterization of pharmacokinetics. Following apheresis, optional bridging therapy is permitted. HuCART19-IL18 cells are administered as a single IV infusion 2-5 days after lymphodepleting chemotherapy (LD). Pts with clinical benefit are eligible to receive retreatment. Dose-limiting toxicity (DLT) observation period is 28 days after infusion. Responses are assessed using Lugano criteria for NHL and revised iwCLL criteria for CLL at months (M) 3, 6, 9, and 12. Results: As of July 14, 2022, 9 pts have enrolled, and 8 pts have been infused and are evaluable for safety (DLBCL 3, MCL 2, THRBCL 1, HGBL 1, FL 1). At enrollment, median age was 65 years (56-75), 75% were males, median ECOG PS was 1 (0-1), the median number of prior therapies was 6.5 (range 4-13), 7/8 (88%) pts relapsed after prior CAR-T (3 post axi-cel, 3 post tisa-cel, 1 post brex-cel). The best responses to prior CAR T-cell therapy were CR in 3, PD in 3, PR in 1. Seven pts (88%) received systemic bridging therapy including 5 (63%) who also had radiation. Pts receiving LD were treated with bendamustine (90 mg/m2 x 2 days). With staggered enrollment to allow for DLT evaluation, the median time from apheresis to infusion was 47 days (26-82). The first pt was infused with DL1A (3x106 cells) without LD. Subsequently, 2 were infused with DL1B (3x106 cells), 1 with DL2 (7x106), and 2 with DL3 (3x107), all after LD. Manufacturing for 2 products did not meet the target dose but exceeded minimum infusible dose and pts were treated: 1 with DL2 (7x106) and 1 with dose between DL2 and DL3 (2.8x107). Cytokine release syndrome (CRS) occurred in 4 (50%) pts: Grade (G)1 in 2, G2 in 1, G3 in 1 with median onset at 7.5 days (2-8) and median duration of 5.5 days (5-11); 2 pts required anti-cytokine therapy. Neurotoxicity occurred in 2 (25%) pts: 1 on day 20 lasting for 2 days (G1) and 1 on day 8 for 6 days (G2). Other non-hematologic G3 or higher adverse events at least possibly related to huCART19-IL18 included infections in 2 (25%), hypotension in 2 (25%), and AST elevation in 1 (12.5%) in the setting of CRS. There have been no study-related deaths. Of 7 pts who are evaluable for response (DLBCL 3, THRBCL 1, MCL 2, FL 1), the ORR at M3 is 100% (CR 57%, PR 43%). Of the 3 pts with PR at M3, 1 pt was re-treated with huCART19-IL18 at M4 (achieved CR at M3 after re-treatment and remains in CR), 1 pt was taken off study in PR to pursue alternative therapy, and 1 pt progressed at M5 with CD19-negative disease and is receiving alternative therapy. None of the 4 pts who achieved CR at M3 have progressed to date including FL pt refractory to axi-cel who is in sustained CR over 12 M after huCART19-IL18 at DL1B (Figure 1A). All pts are alive at median follow-up of 8 M (1.9-14.1). Figure 1B shows huCART19-IL18 cell expansion and persistence for each pt. Conclusions: In this first-in-human study, huCART19-IL18 shows a manageable toxicity profile and encouraging early efficacy across all dose levels in heavily pretreated pts with CD19+ NHL including those who did not respond to prior 2nd generation CAR T-cell products. Enrollment continues at DL3. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The field of cancer immunotherapy has led to successful treatments such as monoclonal antibodies (MAb), inhibitory receptor (IR) blockade, and adoptive cell transfer (ACT). ACT modified to express chimeric antigen receptors (CARs) can redirect T cells to tumor antigens and has shown remarkable clinical efficacy in patients with relapse and refractory leukemia and lymphoma. 90% of pediatric patients with acute lymphoblastic leukemia (ALL) respond to CD19 CAR T cell therapy (CAR T19), although only 26-35% of patients with chronic lymphocytic leukemia (CLL) show complete responses (CR). It is unclear why responses are less frequent in CLL compared to ALL. The heavy pre-treatment received by CLL patients likely contributes to lower response rates with CAR T cell therapy, and disease progression is worsened due to profound T cell defects characterized by elevated expression of IRs such as PD-1 and CTLA-4. Fraietta et. al. assessed CLL patient apheresis samples and CART19 products to show that CR was associated with elevated levels of CD27+PD1-CD8+ T memory cells whereas the non-responders (NR) showed an exhausted phenotype with high levels of multiple IRs. We studied 14 patients with advanced, heavily pretreated CLL who received at least one dose of CART19. Patients with CRs exhibited high in vivo expansion and persistence of infused CAR T cells, as opposed to NR’s. Importantly, at peak levels of in vivo CAR expansion, NR’s had elevated levels of CTLA-4 expression which correlated with poor CLL patient responses to CART19 therapy. In summary, these data suggest that eliminating CTLA-4 mediated T cell inhibition can be clinically beneficial. Thus, we tested the hypothesis that disruption of CTLA4 would improve CAR T cell efficacy in CLL based on numerous observations. Our data demonstrate that knockout (KO) of CTLA-4 in T cells from healthy donors using CRISPR Cas9 technology leads to maintenance of surface CAR expression and higher tumor clearance in a chronic re-stimulation model using CART19 cells against NALM6 tumor cells. In xenograft models of ALL, KO of CTLA-4 increases the anti-tumor efficacy of CART19 cells. We then performed CTLA4 disruption in T cells banked from CLL patients that did not respond to CAR T cell therapy to determine whether dysfunctional CAR T infusion products can be invigorated by CTLA-4 KO. In both chronic re-stimulation model and xenograft models of NALM6, CTLA-4 KO CD19 CAR T cells from CLL patients maintained surface CAR expression, exhibited enhanced tumor clearance, and higher survival rates relative to subject-matched unedited CAR T cells. Thus, suggesting that CTLA4 disrupted CAR T cell products may enhance the success rate of CAR T cell therapy for CLL patients. This technology can be feasibly expanded to other tumor indications to increase the overall efficacy of CAR T cells. These IND-enabling studies will support the translation of this therapy to the clinic. Citation Format: Sangya Agarwal, Angela Aznar Gomez, Tong Da, Shunichiro Kuramitsu, Weimin Kong, Pranali Ravikumar, Mercy Gohil, Megan M. Davis, Joseph A. Fraietta, Gabriela Plesa, David L. Porter, Regina M. Young, Carl H. June. Disruption of cell-intrinsic checkpoint regulator CTLA-4 in CD19 directed CAR T cells provides clinical efficacy in CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5571.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA < 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Background & Aim NY-ESO-1 is a cancer testis antigen with ectopic expression on multiple myeloma (MM), melanoma and sarcoma but restricted expression on normal tissues, rendering it a good candidate target for cancer immunotherapy. We recently reported a phase 1 pilot trial of multiplex CRISPR/Cas9 genome-edited NY-ESO-1 TCR cells (NYCE T cells; NCT03399448) for patients (pts) with advanced MM, synovial sarcoma and myxoid/round cell liposarcoma (MRCL) demonstrated safety and feasibility as well as persistence of T cells. Here, we characterize the phenotype and function of the ex vivo manufactured NYCE T cells from the 3 pts that were infused in the trial. Methods, Results & Conclusion The 3 infusion products (2 MM – pt #35 and Pt #7, and 1 MRCL – pt #39) were used to compare expanded T cells with either CRISPR/Cas9 editing without TCR transduction or with Mock-editing and TCR transduction from the same pt. We examined the phenotypic markers of T cell differentiation and checkpoint inhibitors by multicolor flow cytometry and assessed in vitro anti-tumor efficacy via a luciferase-based killing assay. We found heterogeneity in the composition of T cell subsets from all 3 infusion products with various CD4 and CD8 ratios, ranging from 0.12-1.1. Transduction efficiencies (determined by Vbeta8.1+ staining) varied between 1-11% and different levels of knockout efficiency in TRAC, TRBC and PDCD1 genes were observed by digital PCR. Three NYCE T cell infusion products displayed a terminally differentiated T cell phenotype similar to CRISPR/Cas9 edited counterpart. In contrast to Mock-edited T cells from the same pt, NYCE T cells exhibited lower levels of differing combinations of exhaustion-associated markers (PD1, TIM3, LAG3, EOMES and CTLA4) in both CD4+ and CD8+ T cell compartments. All 3 infusion products elicited antigen-specific killing of NY-ESO-1 expressing target cells. We demonstrate that NYCE T cells are heterogeneous in composition. Furthermore, we showed that CRISPR/Cas9 editing does not affect T cell differentiation and anti-tumor efficacy, but does reduce levels of exhaustion-associated markers expressed by the final products. This work provides an in-depth characterization of the heterogeneous product composition and function of the first multiplex CRISPR/Cas9-edited T cells tested in humans.

Background & Aim Data from a recent clinical trial at the University of Pennsylvania (UPenn) demonstrate the safety of NYCE T cells, which are autologous, patient-derived T cells transduced with lentivirus to express NY-ESO-1 T cell receptor and multiplex-edited with CRISPR/Cas9 at three distinct genes. This pilot study was the first in the United States (US) to administer CRISPR-edited cells to a patient. The preclinical approach and data in support of this trial was reviewed by the National Institutes of Health Recombinant DNA Advisory Committee (NIH RAC) in 2016, followed by the acceptance of an Investigational New Drug (IND) application by the US Food and Drug Administration. The aim of this abstract is to introduce the experience of an academic institution in the development of a preclinical program to support submission of an IND application for a cell therapy product with CRISPR-mediated gene editing. Non-clinical assays, data, and FDA feedback on IND submissions for CRISPR-edited gene products at the Center for Cellular Immunotherapies at UPenn will be presented. Methods, Results & Conclusion The preclinical program for the US IND application for UPenn's NYCE T cell therapy product paired conventional T cell phenotyping assays and novel genome editing evaluations. A comprehensive IND application for NYCE T cells was packaged out of inter-disciplinary collaborations to evaluate safety and potency of CRISPR-edited, antigen-directed T cells as well as fulfill NIH RAC and FDA requests. Results of these analyses will be presented. Execution of the NYCE T cell IND led to preclinical strategies toward applying CRISPR-editing to improve upon other T cell therapies, such as chimeric antigen receptor (CAR) T cells. As such, UPenn is developing an "off-the-shelf" CAR T cell product with healthy donor T cells utilizing CRISPR-mediated knock-down of genes encoding endogenous allo-reactive proteins. The parallels between the NYCE and the off-the-shelf CAR T cell non-clinical development pathways will be discussed. Data from a recent clinical trial at the University of Pennsylvania (UPenn) demonstrate the safety of NYCE T cells, which are autologous, patient-derived T cells transduced with lentivirus to express NY-ESO-1 T cell receptor and multiplex-edited with CRISPR/Cas9 at three distinct genes. This pilot study was the first in the United States (US) to administer CRISPR-edited cells to a patient. The preclinical approach and data in support of this trial was reviewed by the National Institutes of Health Recombinant DNA Advisory Committee (NIH RAC) in 2016, followed by the acceptance of an Investigational New Drug (IND) application by the US Food and Drug Administration. The aim of this abstract is to introduce the experience of an academic institution in the development of a preclinical program to support submission of an IND application for a cell therapy product with CRISPR-mediated gene editing. Non-clinical assays, data, and FDA feedback on IND submissions for CRISPR-edited gene products at the Center for Cellular Immunotherapies at UPenn will be presented. The preclinical program for the US IND application for UPenn's NYCE T cell therapy product paired conventional T cell phenotyping assays and novel genome editing evaluations. A comprehensive IND application for NYCE T cells was packaged out of inter-disciplinary collaborations to evaluate safety and potency of CRISPR-edited, antigen-directed T cells as well as fulfill NIH RAC and FDA requests. Results of these analyses will be presented.