Background: Allogeneic CAR T cell therapy addresses logistical/manufacturing challenges inherent in autologous (auto) CAR T therapy. ALLO-501A, which uses Cellectis technologies, is an allogeneic anti-CD19 CAR T cell product whose a) disrupted TCRα gene may reduce GvHD risk, and b) edited CD52 gene may permit use of ALLO-647 (a humanized anti-CD52 mAb) to selectively deplete host T cells. Methods: The ongoing ALPHA2 study is a single-arm, open-label, 2 phase study of ALLO-501A in non-HLA matched patients (pts) with R/R LBCL and ≥2 prior lines of therapy. Prior auto CD19 CAR T therapy is allowed if tumors remain CD19⁺. Following lymphodepletion (LD) with ALLO-647 (60 mg or 90 mg), fludarabine 30 mg/m²/d x 3d (Flu), and cyclophosphamide 300 mg/m²/d x 3d (Cy), escalating doses of ALLO-501A (40 [DL1] or 120 [DL2] x 10⁶ viable CAR T cells) were administered. Retreatment was allowed for PD or SD with suboptimal CAR T expansion. Pts who had ≥SD at D28 could receive a second dose in a consolidation cohort. Phase 1 assessed safety/tolerability and cell kinetics of escalating doses of ALLO-501A following LD. Results: By 1/15/21, 11/11 enrolled pts received ALLO-647 (60 mg: n=6; 90 mg: n=5). Mean duration from enrollment to start of therapy was 6 days. After LD, 1 and 9 pt(s) were treated with ALLO-501A at DL1 and DL2, respectively; 1 pt developed CNS lymphoma and was not treated. Of 10 pts treated, 1 pt received retreatment and 4 pts were enrolled in the consolidation cohort. Pts had a median age of 60 years; 8 were ≥ stage III at diagnosis, 5 had IPI scores ≥3, and 3 had baseline LDH > 2x ULN. Median number of prior therapies was 3 (range 2 – 7); 3 pts had received auto CD19 CAR T cell therapy. 4/8 evaluable pts had rapidly PD at study entry. Median FU was 1.7 months. No dose modifications were required and no pt experienced DLTs. The most common AEs were anemia, leukopenia, neutropenia and thrombocytopenia (73%); and lymphopenia (64%). No GvHD or ICANS were reported. CRS was seen in 2 (18%) pts, both Grade < 3. Infusion-related reactions, all grade <3, were observed in 4 (36%) pts. D28 response data are available for 8 pts: 1 died of PD before D28; 4 additional pts had PD, including 2 who progressed 2 and 3 mos. after auto CAR T; 1 had SD; and 2 (both DL2) had CR. Of those in CR, 1 had peak ALLO-501A expansion at D14, persistence until D42, and ongoing CR at 4 mo; 1, with a 4-mo response to prior auto CAR T, had peak expansion at D28 and remains in ongoing CR at D56 after ALLO-501A with pending persistence. Conclusions: This dose escalation cohort contained heavily pretreated, actively progressing pts, some of whom had failed auto CAR T. Preliminary data suggest an acceptable safety profile following ALLO-501A and ALLO-647 and early signs of efficacy in LBCL. Enrollment into the consolidation cohort is ongoing; updated clinical/biomarker data of resistance and clinical activity will be presented. Clinical trial information: NCT04416984