Developing Multispecific T cell Engaging Therapy for Mixed Phenotype Acute Leukemia

Mixed-phenotype acute leukemia (MPAL) is a high-risk leukemia subtype presenting with both myeloid (e.g. CD33) and lymphoid (e.g. CD19) surface antigens. Cure rates in children and adolescents remain lower than for most other acute lymphoblastic leukemia (ALL) subtypes treated in pediatrics, with overall survival ranging from 75-80%. Outcomes for adults are significantly worse, with overall survival ranging from 20-50% for patients over 40 years of age. Uniform approaches to therapies have been challenging to implement, in large part due to the absence of clinical trials available for these patients. Physicians variably implement therapeutic strategies for ALL or acute myeloid leukemia (AML), frequently alternating between the two approaches. The most accepted treatments employed in adults or children with a poor response to initial therapy include hematopoietic stem cell transplant (HCT) after inducing remission with either ALL or AML therapy. The fact that B-Myeloid MPAL cells frequently express both CD19 and CD33 while no non-neoplastic human cells express both targets creates an opportunity to selectively kill MPAL cells with minimal toxicity. We hypothesize that an MTE engineered to require CD19 AND CD33 expression will selectively kill MPAL cells compared to an MTE that requires CD19 OR CD33 expression. We propose to achieve this by engineering an MTE that binds both CD19 and CD33 on MPAL cells and CD3 on T cells and by tuning the affinity of the CD19 and CD33 binders so that the MTE binds poorly to single positive (SP) normal cells that express either CD19 or CD33 but binds well to double positive (DP) cancer cells that express both. For this we take advantage of both affinity and avidity, the latter of which occurs only when the CD19/CD33 MTE interacts with both CD19 and CD33 on MPAL cells. Such therapies could be used to induce deep remissions, improve outcomes when integrated with chemotherapy, and/or serve as a bridge to HCT. Bispecific T cell engaging therapies such as blinatumomab, recognize CD19, is standard of care for treatment of relapsed childhood ALL, and is being incorporated into upfront regimens for adult and childhood ALL. However, patients treated with blinatumomab require months of intravenous immunoglobulin (IVIG) replacement therapy due to the consequential depletion of healthy CD19 positive B cells, which share CD19 as a critical surface antigen with most B-cell ALL. We will show preliminary data that demonstrate our initial in vitro success in targeting CD19/CD33/CD3 antigens as well as improved specificity by generating lower affinity variants of known CD19 binders. In this proposal, we will further engineer and generate MTEs using additional affinity variants of CD19 and CD33 binders to enhance specificity, and also evaluate the toxicity of these MTEs on normal B-cells. We will further test our MTEs in pre- clinical mouse trials to assess the efficacy of MTEs targeting these common antigens expressed on MPAL cells. The future goal of this work is to advance these data into human clinical trials. Project Number: 1R21CA296748-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Mignon Loh (+1 co-PI) | Institution: SEATTLE CHILDREN'S HOSPITAL, SEATTLE, WA | Award Amount: $434,361 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZCA1 SRB-F (M2)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11212310