Abstract: Chimeric antigen receptor modified (CAR) T cells have revolutionized the treatment of blood cancers, though some patients still show a poor response in either CAR expansion, effector response, or persistence. 1 In this study, we determined the features of pre-infusion CAR-transduced T cells that generated optimally functional responses after infusion. Methods Using both the pre-infusion product and PBMCs isolated at weeks 1–4, 8, and 3-months post-infusion from 15 patients undergoing experimental anti-CD19 CAR T cell treatment for refractory or relapsed B-ALL, we generated a comprehensive single cell gene expression and T cell receptor (TCR) sequencing dataset on over 180,000 CAR T cells (figure 1). Results As expected, pre-infusion CAR T cells tend to highly express genes associated with proliferation, while post-infusion CARs show signs of either cytotoxic effector differentiation or dysfunctional terminal differentiation. Sequencing of the endogenous TCR, at the single cell level, allows us to track the trajectories of clonally and transcriptionally related cells (figure 2). Post-infusion cells with significant cytotoxic effector function share TCRs with a statistically defined subset of CARs in the pre-infusion sample (figure 3). Using a machine learning approach, we found that potent effector precursor CAR T cells have a specific transcriptional profile distinct from the other pre-infusion CAR T cells, including markers of early effector function such as increased EOMES, GNLY, GZMH, GZMK, KLRD1, and IFNγ. Formalizing this signature, we have developed a robust classifier that can predict with 82.8% accuracy whether a CAR T is likely to become a favorable effector based on its pre-infusion profile (figure 4). This prediction model can be used to evaluate the extent to which a patient‘s generated CAR product will be able to mount a robust response after encountering its target. Additionally, there are a number of genes, as a part of this signature, that are expressed on the cell surface and can be utilized as a method to differentiate the effector precursor pre-infusion CAR T cells from other pre-infusion CARs, including CD52, CD74, CD86, and LAG3, among others. Abstract 152 Figure 1 Clustering of 184, 791 CAR-transduced T cells based on gene expression Abstract 152 Figure 2 Alluvial plot depicting CAR T cell lineage tracing using the endogenous T cell receptor Abstract 152 Figure 3 Visualization of CAR T cell clusters with arrows indicating the shared TCRs between pre-infusion and post-infusion cells Abstract 152 Figure 4 Machine learning classifier of pre-infusion, early effector CAR T cell phenotype Conclusions Our findings suggest a therapeutic approach that enriches these cells prior to infusion resulting in superior per cell CAR effector activity. Reference Xu X, Huang S, Xiao X, Sun Q, Liang X, Chen S, et al. Challenges and Clinical Strategies of CAR T-cell Therapy for Acute Lymphoblastic Leukemia: Overview and Developments. Front Immunol 2020; 11 :569117. Ethics Approval This study was approved by St. Jude Children’s Research Hospital’s Institutional Review Board (IRB); IRB number Pro00007661. All patients consented to the use of materials for the research study.

Abstract: T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.

Abstract: The goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion in leukemia patients as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans. A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells during manufacturing and from peripheral blood mononuclear cells (PBMCs) of 15 patients enrolled on our institutional, autologous CD19-CAR T cell therapy study (NCT03573700). CAR T cell expansion and persistence were determined by measuring vector copy numbers in the PBMCs of treated patients. We had previously established novel exhaustion DNA methylation datasets that delineate between progenitor and fully exhausted T cells. These datasets served as a guide for stratifying our post-infusion CAR T cells along the exhaustion developmental trajectory. Our data show that CD19-CAR T cells lose repressive DNA methylation at effector loci (e.g. PRF1, TBET) while gaining methylation at genes associated with memory potential (e.g. LEF1, TCF7). We confirmed these epigenetic changes are coupled to endogenous human T cell effector and memory differentiation by cross-referencing our epigenetic data with publicly available transcriptional profiles for antigen-specific effector and long-lived memory CD8 T cells from individuals vaccinated for yellow fever. Furthermore, we show that CAR T cells were unable to mount an in vivo recall response after relapse of antigen-positive disease or recovery of endogenous B cells. These observations support the conclusion that CD19-CAR T cells acquire stable epigenetic exhaustion programs that limit their protective capacity. This work was supported by the National Institutes of Health (1R01AI114442 to BY and LRP to CCZ), the National Comprehensive Cancer Network Young Investigator Award (to CZ), Alex’s Lemonade Stand Foundation Young Investigator Grant (to CZ), Stand Up to Cancer- SU2C (to BY), the American Lebanese Syrian Associated Charities (ALSAC) to BY, and Assisi foundation to BY.

Abstract: Early clinical studies of gene therapy for patients with X-linked Severe Combined Immunodeficiency (XSCID) only restored T cell immunity and carried a significant risk of iatrogenic leukemia. We developed a new gene therapy approach that utilizes a safety-modified lentiviral (LV) vector together with reduced exposure busulfan conditioning for newly diagnosed infants with XSCID (NCT01512888). Of the first enrolled 8 patients, 7 demonstrated robust reconstitution of T-, NK-, and B-cells with a median follow up of 16.4 months (range: 6.7 to 24.9 months; Mamcarz et al, N Engl J Med, 2019). Here we provide an update on our clinical study, which now includes 3 more patients (n=11 total), 8 months additional median follow-up (23.6 months; range: 1.5 to 33.9 months), more extensive analysis of T and B cell functional recovery, and detailed vector integration site studies. Overall, we successfully generated transduced autologous bone marrow (BM) CD34+ cells for all patients with a median vector copy number (VCN) of 0.45 VCN/cell (range: 0.16-1.13). Prior to the infusion of transduced CD34+ cells (median cell dose: 8.7 x106/kg; range: 4.5-19.0), patients received two daily doses of busulfan to target a cumulative area-under-the-curve (cAUC) of 22 mg*hr/L (achieved median: 22.3 mg*hr/L; range: 20.0-23.0). No severe adverse events, other than hematologic related to busulfan, were observed. All 11 patients had robust hematopoietic recovery within 3-4 weeks post cell infusion without blood product support. Nine patients, with a follow up of 〉 3 months, achieved normal for age T-cell and NK-cell numbers within 3-4 months post gene therapy. T-cells matured appropriately as assessed by normal receptor excision circles (TREC) levels and TCRvb repertoire analysis. In addition, phytohemagglutinin (PHA) stimulation assays demonstrated normal T-cell function. So far, 5 patients are off IVIG of whom 3 responded to vaccines. As previously reported, patient #1 demonstrated poor immune reconstitution. He received a 2nd infusion of transduced CD34+ cells without conditioning one year after his initial infusion, which resulted in functional T-cell immune reconstitution. Clinically, all patients with a follow up 〉 3 months recovered from pre-existing infections, are off protective isolation and prophylactic antimicrobials, and have normal growth in respect to height and weight. The median VCN at 12 months post gene therapy in seven patients, who have been followed for 〉 12 months, was 2.25 VCN/cell (range: 1.24-3.03) in T cells, 0.34 VCN/cell (range: 0.23-1.25) in B cells, 1.55 VCN/cell (range 1.27-3.39) in NK cells, and 0.08 VCN/cell (range: 0.03-0.76) in myeloid cells in peripheral blood, and 0.10 (range: 0.05-0.66) in CD34+ bone marrow cells, respectively. Detailed integration sites analysis for the first 7 patients, who received a single infusion of transduced CD34+ cells, revealed that the majority of sites were located in introns and intergenic regions throughout the human genome. The integration site pattern was highly consistent across patients with integration site clusters that had been previously described by us and others after LV transduction. In conclusion, LV gene therapy for XSCID using low dose busulfan conditioning and a novel LV vector is well tolerated and results in the development of a functional normal immune system without evidence of malignant transformation with a median follow up of almost 2 years. Thus, our approach may present a promising alternative to current therapies, which rely in part on high dose chemotherapy followed by allogeneic hematopoietic cell transplantation. Disclosures Mamcarz: American Lebanese Syrian Associated Charities: Research Funding; UpToDate: Honoraria; NHLBI: Research Funding; ASSISI Foundation of Memphis: Research Funding; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; California Institute of Regenerative Medicine: Research Funding. Zhou:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Lockey:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Boi:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Koon-Kiu:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Cross:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; NIH: Research Funding. Kang:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Ma:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Condori:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Dowdy:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Metais:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Langfitt:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Triplett:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Li:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Zhao:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Maron:Chimerix: Research Funding; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Astellas: Research Funding. Janssen:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Weiss:GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy. Youngblood:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Meagher:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Puck:Pfeizer: Other: spouse serves on Rare Disease Advisory Board; NIAID: Research Funding; Invitae: Other: spouse employment. Cowan:NIH NIAD: Research Funding; Leadiant: Consultancy; Rocket Pharma: Consultancy; bluebird bio: Consultancy; California Institute Of Regenerative Medicine: Research Funding; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria. Gottschalk:Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; TESSA Therapeutics: Other: Research Collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; EMD Serono: Honoraria; California Institute for Regenerative Medicine: Research Funding; Sanofi: Honoraria; NHLBI: Research Funding; Inmatics: Membership on an entity's Board of Directors or advisory committees; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; America Lebanese Syrian Associated Charities: Research Funding; ViraCyte: Consultancy; ASSISI fundation of Memphis: Research Funding.

Abstract: Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27−). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products. Significance: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007

Abstract: Current chimeric antigen receptor-modified (CAR) T cell therapy products are evaluated in bulk, without assessment of the possible heterogeneity in effector potential between cells. Conceivably, only a subset of the pre-infusion product differentiates into optimal effectors. We generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using both pre- and post-infusion CD19-CAR T cells from peripheral blood and bone marrow of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified potent effector post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. Effector precursor CAR T cells exhibited a unique transcriptional profile compared to other pre-infusion cells, and the number of effector precursor cells infused correlated with peak CAR T cell expansion. Additionally, we identified an unexpected cell surface phenotype (TIGIT+, CD62Llo, CD27−), conventionally associated with inhibiting effective T cell responses, that we used to successfully enrich for subsequent effector potential. Collectively, these results demonstrate that highly diverse effector potentials are present among cells in pre-infusion cell products, which can be exploited for diagnostic and therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the biological mechanisms underlying effector development in other CAR T cell therapy products. This work was supported by the National Institutes of Health (NIH)/National Cancer Institute grant P30CA021765, NIH grants U01AI150747 and R01AI136514 (PGT), the American Society of Transplantation and Cellular Therapy (AT), the American Society of Hematology (AT), the Key for a Cure Foundation (PGT), the Mark Foundation ASPIRE Award (PGT), and the American Lebanese Syrian Associated Charities (SG, PGT). Part of the laboratory studies were performed by the Center for Translational Immunology and Immunotherapy (CeTI2), which is supported by SJCRH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.