Abstract: Background : Approximately 45% of new ALL cases occur in adults ≥ 20 years of age (Howlader et al. SEER Cancer Statistics. 2015), and approximately 50% of adult patients relapse with poor subsequent outcomes (Oriol et al. Haematologica. 2010; Basson et al. JCO. 2011). Promising early efficacy and manageable safety were previously reported with anti-CD19 CAR T cells (KTE-C19) in adult patients with R/R ALL (Shah et al. ASCO 2017. #3024). Here we report updated results of the ZUMA-3 trial. Methods : Adult patients (≥ 18 years of age) with R/R ALL (Philadelphia+ eligible), & gt; 5% bone marrow (BM) lymphoblasts; Eastern Cooperative Oncology Group performance status (ECOG) 0-1; and adequate renal, hepatic, and cardiac function were eligible. Patients with active graft-versus-host disease or clinically significant infection were not eligible. Patients received a target dose of 1 × 106 CAR T cells/kg or 2 × 106 CAR T cells/kg after lymphodepletion with 25 mg/m2/day fludarabine for 3 days and 900 mg/m2/day cyclophosphamide given on the last day. The primary endpoint of phase 1 was incidence of dose-limiting toxicities (DLTs). Key secondary endpoints included incidence of adverse events (AEs), incidence of minimal residual disease-negative (MRD-) responses, duration of remission (DOR), relapse-free survival (RFS), and overall survival (OS). Exploratory endpoints included levels of anti-CD19 CAR T cells in blood and levels of cytokines in serum. Results : As of the data cut-off date (DCO; April 26, 2017), 22 patients have been enrolled, and 16 patients received KTE-C19 on study. Four patients had not received treatment by the DCO, 1 patient did not receive KTE-C19 due to an AE after conditioning, and 1 patient received KTE-C19 under compassionate use. All 16 patients who received KTE-C19 prior to the DCO were included in the safety analysis, and all patients who had the opportunity to be followed for 8 weeks prior to the DCO were included in the efficacy analysis (n = 11). Of the 16 patients dosed with KTE-C19, 63% were male, 56% had ECOG 1, and 50% had received ≥ 2 previous lines of treatment, including 3 patients with prior blinatumomab. Nineteen percent of patients had undergone prior allogeneic stem cell transplant, 31% had R/R to ≥ second-line therapy, 31% had primary refractory disease, and 19% experienced first relapse within 12 months of first remission. Most patients (81%) had baseline BM blasts ≥ 60%. Six patients received the 2 × 106 cells/kg dose and 10 received the 1 × 106 cells/kg dose. No DLTs were observed. One patient experienced a grade 5 event of cytokine release syndrome (CRS) at the 2 × 106 cells/kg dose, and no other KTE-C19-related grade 5 AEs were observed. In the 16 patients who received KTE-C19, all of whom were followed for at least 4 weeks, the most common grade ≥ 3 AEs were hypotension (56%), anemia (50%), pyrexia (50%), and decreased platelet counts (44%). Grade ≥ 3 CRS and neurologic events (NE) were reported in 25% and 63% of patients, respectively. Tocilizumab (toci) or steroids were given for AE management in 94% and 75% of patients, respectively. In the 11 patients eligible for the efficacy analysis, objective response rate was 82%, including 8 (73%) patients with a complete remission (CR or CR with partial hematopoietic recovery), and 1 (9%) with blast-free BM. All remissions were MRD- as determined by flow cytometry. All 5 (100%) of the other patients who were too early for inclusion in the efficacy analysis had MRD- bone marrow with varying degrees of count recovery at the time of the DCO. Median follow-up was 6.8 months; 4 patients relapsed 63 - 168 days after treatment with KTE-C19. Efficacy was comparable between patients who recieved KTE-C19 doses of 1 × 106 and 2 × 106 CAR T cells/kg. Data from additional patients, including those treated with a lower dose of 0.5 × 106 CAR T cells/kg, as well as updated safety, efficacy, biomarker, and product characteristic analyses across dosing groups will be presented. Conclusions : In this ongoing phase 1 study, KTE-C19 has shown promising efficacy in adult patients with R/R ALL. The safety profile was generally manageable and additional approaches to improve the benefit:risk profile are being explored. ZUMA-3 continues to enroll additional patients at the 0.5 × 106 CAR T cells/kg dose level. Disclosures Wierda: AbbVie: Consultancy, Honoraria, Research Funding; Karyopharm: Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria; Juno: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Kite: Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; Emergent: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding; The University of Texas MD Anderson Cancer Center: Employment; Acerta: Research Funding. Oluwole: Kite Pharma: Membership on an entity's Board of Directors or advisory committees. Schiller: Kite Pharma: Research Funding. Topp: Regeneron: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding; Celgene: Other: Travel; Macrogenics: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Research Funding. Kersten: Kite Pharma: Honoraria; Novartis: Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Millenium/Takeda: Honoraria, Research Funding; Mundipharma: Honoraria; Gilead Sciences: Honoraria; BMS: Honoraria; MSD: Honoraria; Amgen: Honoraria. Mojadidi: Kite Pharma: Employment, Equity Ownership. Xue: Kite Pharma: Employment, Equity Ownership. Mardiros: Kite Pharma: Employment, Equity Ownership. Jiang: Kite Pharma: Employment, Equity Ownership. Shen: Kite Pharma: Employment, Equity Ownership. Aycock: Kite Pharma: Employment, Equity Ownership. Stout: Kite Pharma: Employment, Equity Ownership. Wiezorek: Kite Pharma: Employment, Equity Ownership. Jain: Kite Pharma: Employment, Equity Ownership.

Abstract: 3009 Background: Engineered T-cell therapy has shown promise in B-cell malignancies and melanoma, but clinical investigation in epithelial cancers has been limited. Methods: We conducted a phase I/II clinical trial of T cells genetically engineered to express a T-cell receptor that targets an HLA-A*02:01-restricted epitope of E6 (E6 TCR T Cells) for patients with metastatic HPV-16+ carcinoma. The cell dose was escalated in cohorts of single patients (1 x 10 9 , 1 x 10 10 , and 1-2 x 10 11 cells). Patients received a nonmyeloablative conditioning regimen of cyclophosphamide and fludarabine, a single infusion of E6 TCR T Cells, and systemic high-dose aldesleukin. Results: Twelve patients were treated, 9 at the highest cell dose, plus one retreatment. The cancer types were 6 cervical, 4 anal, 1 oropharyngeal, and 1 vaginal. No dose-limiting toxicity, autoimmune adverse events, or cytokine storm were observed. Two patients with anal cancer treated at the highest cell dose experienced partial tumor responses lasting 6 and 3 months after treatment. The patient with a 6-month response had complete regression of one tumor and partial regression of two tumors that were resected upon progression; she has no evidence of disease 22 months after treatment. T-cell receptor gene transfer efficiency was 45 and 51% in the responding patients, and 47-76% (median 61%) in the non-responding patients. Responding patients showed robust levels of E6 TCR T cell memory (30 and 46% of circulating T cells 1-month after treatment). Non-responding patients showed wide-ranging levels of E6 TCR T cell memory (range 4-53%, median 29%). Expression of programmed cell death protein 1 (PD-1) by circulating E6 TCR T Cells 1-month after treatment was low in all patients ( 〈 5%). The patient with a 6-month response had 7% E6 TCR T Cells in a resected tumor 10 months after treatment, 25% of which expressed PD-1. A patient with no response had no detectable E6 TCR T Cells in a resected tumor 3 months after treatment. Conclusions: E6 TCR T-cell therapy was safe at doses up to 2 x 10 11 cells. Regression of metastatic HPV+ carcinoma occurred in two patients following treatment, suggesting that TCR T-cell therapy can mediate epithelial cancer regression. Clinical trial information: NCT02280811.

Abstract: Major limiting factors for cell therapy in solid tumors include clonal heterogeneity and the related lack of universally expressed tumor-specific antigens. As the only truly polyclonal cell product in advanced development, tumor infiltrating lymphocytes (TILs) offer the broadest diversity of tumor reactivity but can be limited by suboptimal effector function in situ. Here we present a novel platform designed to leverage the diverse TCR repertoire of TILs while amplifying their anti-tumor activity via a synthetic costimulatory antigen receptor (CoStAR). Methods A CoStAR molecule encoding an extracellular folate receptor alpha (FOLR1)-targeting single-chain fragment variable (scFv) and intracellular CD28 and CD40 signaling sequences was transduced into peripherally harvested T cells (healthy donor) and primary ovarian cancer TILs. Coculture experiments were performed with engineered cell lines and autologous tumor digests expressing varying levels TCR-stimulus and/or FOLR1. Cytolytic activity, activation markers, proliferation and cytokine secretion were measured. Results Anti-FOLR1 CoStAR T cells activated with TCR stimulation and FOLR1 displayed increased activation markers (eg, CD137, 〉 50% increase), improved cytolytic activity, cytokine production (eg, IL-2, 〉 15-fold) and ~7-fold increased proliferation when compared to either unmodified cells or when stimulated via TCR alone. Importantly, no T cell effector function, as measured by cytolytic activity, cytokine secretion, upregulation of activation markers or proliferation, was observed when CoStAR T cells were cocultured with targets expressing only FOLR1, underscoring the costimulation-only mechanism of action and reliance on native TCR repertoire for tumor recognition. Furthermore, increased activity was also observed when primary ovarian cancer anti-FOLR1 CoStAR TILs were cocultured with autologous tumor. Conclusions CoStAR is a novel platform that leverages synthetic biology and T-cell-intrinsic circuits to create a product with markedly increased functional activity including cytotoxicity, proliferation, and cytokine expression while retaining broad, patient-specific neoantigen recognition to limit both antigen escape and off-tumor toxicity. Instil plans to initiate its first-in-human clinical trial with ITIL-306, an investigational anti-FOLR1 CoStAR TIL product in 1H 2022. Additional scFv targets are being evaluated for clinical application across a broad range of solid tumor histologies.

Abstract: Introduction: Site-specific gene correction of the point mutation causing sickle cell disease (SCD) in hematopoietic stem cells (HSCs) constitutes a precise strategy to generate a life-long source of gene-corrected erythrocytes that do not sickle. However, low efficiency of homology-directed repair (HDR) in primitive reconstituting HSCs is currently a limit to the use of therapeutic genome editing for treatment of severe genetic blood disorders. To identify the mechanism(s) that underlie decreased HDR efficacy in primitive HSCs relative to that in more mature progenitor populations, we assessed: efficiency of gene delivery and expression after electroporation of in vitro transcribed mRNA; functional ZFN-mediated endonuclease activity; cell cycle status; gene expression of key HDR genes; and cytotoxic responses; in the following immunophenotypically-defined human cell populations: HSCs (CD34+/CD38-/CD90+CD45RA-); multipotent progenitors (MPPs) (CD34+/CD38-/CD45RA-/CD90-); and progenitor cells (CD34+/CD38+). Methods: CD34+ cells were enriched from human G-CSF-mobilized peripheral blood and cultured for 1-3 days prior to electroporation of in vitro transcribed mRNA encoding GFP or a pair of zinc finger nucleases (ZFN). The ZFNs, designed to target the sickle mutation in exon 1 of the human beta-globin gene, were co-delivered with one of the homologous donor templates containing the corrective base (A/T): an integrase-deficient lentiviral vector (IDLV) or a 101bp single-stranded oligodeoxynucleotide (oligo). Percentages of alleles containing insertions/deletions (indels) and/or HDR-mediated gene correction were analyzed by high throughput sequencing (HTS). Acute cytotoxicity was determined by flow cytometry, identifying viable cells as 7AAD/AnnexinV neg. cells. To assess HDR-mediated gene correction in vivo after three months, gene-edited cells were transplanted ( 〉 1E6 viable CD34+ cells/mouse, I.V.) one day after electroporation into irradiated (250cGy) NOD/SCID/IL2R gamma-/- (NSG) mice. Results: In HSCs, MPPs and progenitor populations, no differences were observed in delivery and expression from electroporated GFP mRNA [%GFP(+) and MFI]. To assess the activity of ZFN mRNA in the stem and progenitor populations, ZFNs were delivered to CD34+ cells through electroporation of in vitrotranscribed mRNA. The CD34+ cells were then FACS-sorted into the respective populations and HTS was used to determine the percentage of alleles containing indels; the frequencies of indels were equivalent among the populations indicating equivalent ZFN mRNA activity. To evaluate the efficacy of site-specific HDR in HSCs and progenitor cells, ZFN mRNA was co-delivered with either an IDLV or an oligodeoxynucleotide donor template to modify the single base-pair involved in SCD. We observed lower percentage of HDR-mediated gene modification in the HSC population compared to progenitors with all donor templates. Due to the cell cycle phase restriction of HDR, we pre-stimulated CD34+ cells for 1-3 days prior to electroporation of ZFN mRNA and the oligo donor, and analyzed the cell cycle phases at the time of electroporation, and the frequencies of HDR and NHEJ produced by HTS. Only a small percentage of the immunophenotypic HSCs were in S/G2 phase after 24 hours of pre-stimulation; no HDR modification was observed in these cells. After 2-3 days of pre-stimulation, the HDR levels increased as the percentage of HSCs in S/G2 phase reached 20%. Importantly, assessment of relative cytotoxicity of the genome editing procedure (electroporation of ZFN mRNA and oligo donor) revealed a heightened sensitivity of HSCs/MPPs compared to progenitors, resulting in ~80% cell death in HSC vs. ~30% in progenitors under the conditions we are using. Transient expression of BCL-2 mRNA, co-electroporated with the genome editing reagents, improved HSC survival and significantly increased the numbers of HDR gene-corrected HSCs both in vitro and in vivo. Conclusions : These data indicate an elevated sensitivity to cytotoxicity from the gene editing process for HSCs compared to the mature progenitor cells under our conditions, which may explain the lower levels of gene modification seen using in vivo compared to in vitro assays. Transient overexpression of BCL-2 mRNA preserves HSC survival after HDR-based gene editing, increasing the frequency of gene-corrected HSCs. Disclosures Bjurström: UCLA: Patents & Royalties: 2016-290. Holmes:Sangamo BioSciences Inc: Employment.

Abstract: 2535 Background: ITIL-306 is an autologous tumor-infiltrating lymphocyte (TIL) therapy that integrates T-cell receptor (TCR)-specific antigen recognition (Signal 1) with robust costimulation via the novel CoStAR transgene upon engagement with FOLR1 (Signal 2; Sukumaran, et al. JITC. 2021;9:198). Here, we assessed IL-2 independent effector function by anti-FOLR1 CoStAR T cells in vitro and evaluated activity in a novel, more representative murine solid tumor model. Methods: For in vitro studies, healthy donor T cells were manufactured to express anti-FOLR1 CoStAR or left nontransduced (NTD). Product cells were stimulated with a single (Day [D]0; ±IL-2) or serial (D0, 7, 14, 21; no IL-2) addition of target cells expressing membrane-anchored OKT3 (Signal 1) and FOLR1 (Signal 2). T-cell activation and proliferation were measured. For in vivo studies, the carcinoembryonic antigen (CEA)-positive (Signal 1) H508 cell line was engineered to express FOLR1 (Signal 2) and injected into 6 mice/group (D–21). NTD or healthy donor T cells expressing HLA-A*02-restricted anti-CEA TCR only, anti-FOLR1 CoStAR only, or dual TCR+CoStAR were given intravenously (D0), and compared with PBS control. Tumor growth, survival, and T-cell expansion were assessed in 2 donors to D96. Results: After single and serial stimulation, only anti-FOLR1 CoStAR T cells showed sustained proliferation without exogenous IL-2. PD-1 positivity was low ( 〈 10%) on anti-FOLR1 T cells up to D24. In vivo, T-cell persistence on D14 was increased in dual TCR+CoStAR vs anti-CEA TCR only mice ( P 〈 .001) and all other groups ( P 〈 .01-.001). Tumor growth up to D58 was significantly lower in dual TCR+CoStAR ( 〈 .5 cm 3 ) vs all other groups (each 〉 2 cm 3 ; P 〈 .0001). Survival was significantly longer in dual TCR+CoStAR ( P 〈 .01) vs all other groups; only dual TCR+CoStAR mice were alive at D58, with 5/6 alive at D96. The second donor showed similar tumor control irrespective of exogenous IL-2 in responder mice, with 〉 50% alive at D96, and robust T-cell expansion in dual TCR+CoStAR mice. Conclusions: When combined with TCR-specific binding, CoStAR significantly enhanced T-cell proliferation, persistence, and antitumor activity in vivo vs TCR alone, resulting in tumor control and prolonged survival. Effects were not observed with CoStAR alone, underscoring that signaling through CoStAR alone does not induce T-cell effector function. The sustained proliferation of anti-FOLR1 CoStAR T cells without exogenous IL-2 support in vitro and in vivo supports a clinical TIL regimen free of high-dose IL-2. These results suggest that CoStAR may improve the clinical performance and benefit/risk profile of TILs, whereby fewer toxicities are expected with the removal of post-TIL IL-2 support. This will be explored in an upcoming first-in-human clinical study with ITIL-306.