P437: effective immunotherapy of pediatric t(8;21) acute myeloid leukemia by repurposing cd3xcd19 targeting bispecific antibodies

Background: t(8;21) acute myeloid leukemia (AML) is one of the most frequent subtypes of leukemia in pediatric patients with overall favorable outcome. Yet, the backbone of treatment mainly relies on intensive chemotherapy with 15% relapse rate. To further improve treatment outcomes, targeted/immune-based therapies are urgently needed. However, identification of novel immunotherapy targets exclusively expressed on AML cells has been challenging. Aims: The t(8;21) AML subtype has been shown to express varying levels of CD19 and this led us to question whether CD19 targeting immunotherapies approved for acute lymphoblastic leukemia (ALL) and mixed phenotype acute leukemia (MPAL) can be repurposed in CD19+ pediatric AML as well. Methods: We first assessed the incidence and outcome of CD19 positivity in pediatric AML patients by performing a retrospective medical records study on cytogenetic, flow cytometry and outcome data. Pediatric patients in the Dutch NOPHO-DBH AML-2012 cohort, diagnosed between January 2012 and October 2022 were included. Next, to determine if CD19+ AML cells could be killed by T-cells engaged through blinatumomab, a CD3xCD19 bispecific antibody, we used an ex-vivo co-culture system. Our system comprised t(8;21) patient-derived xenograft (PDX) cells, grown on mesenchymal stem cell (MSC) feeders in an in-house optimized cell culture medium supporting the survival and proliferation of AML cells, and varying ratios of CD3+ T-cells from healthy donors. Activation markers in T-cells and killing efficiency in AML population were determined in presence and absence of blinatumomab by flow cytometry. Results: In total, 167 AML patients were analyzed. Of these, 18 (10.8%) were positive for CD19 based on diagnostic flow cytometry data. In CD19+ patients, 11 carried t(8;21), one patient had t(1;11), one had t(9;11), two had t(16;21), and for 3 patients the cytogenetic data were unknown. The median age of the CD19+ patients was 10.5 years, and 77.8% were male. All CD19+ patients achieved complete remission (CR) within two induction courses of chemotherapy. Five patients had a relapse (27.8%), and one patient died (5.6%). Four of these patients were CD19+ in their relapse (80%). The 5-year event-free (EFS) and overall survival (OS) were 65.0% and 85.7%. Next, we assessed the response of t(8;21) patient-derived cells towards CD19 bispecific antibodies. The t-distributed stochastic neighbor embedding (tSNSE) plots showed homogenous CD19 expression in both CD34+ (immature) and CD11b+ and/or CD15+ (more differentiated) subpopulations, suggesting that both immature and more mature AML cells could be irradicated by blinatumomab activated T-cells. Accordingly, in co-culture of T-cells (Effectors) with AML (Target) cells at a ratio of 1:1 and 1:10 E:T, 85% and 50% killing of AML cells were observed respectively in the presence of blinatumomab after 48 hours, while in the absence of blinatumomab, the killing% was negligible. T-cell activation markers CD137, CD69, and CD25 increased substantially following co-culture of AML cells and T-cells with blinatumomab, suggesting that the bispecific antibody-mediated T-cell activation results in the observed AML cell killing. Summary/Conclusion: CD19 positivity can be exploited as an immunotherapy target in t(8;21) pediatric AML. Our data elucidates the efficacy of blinatumomab in eradicating AML cells, thus supporting clinical evaluation of its safety and efficacy in this pediatric AML subtype. Keywords: Immune therapy, Acute myeloid leukemia, AML1-ETO, Pediatric