Investigating the Immunogenicity of CBFA2T3-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia for an Engineered T Cell Immunotherapy

The CBFA2T3-GLIS2 fusion oncogene is found in 18.4% of non-Down syndrome (DS) acute megakaryoblastic leukemia (AMKL) patients and is the oncogenic driver. These patients have the worst prognosis of acute myeloid leukemia patients (AML), having only a 14% overall survival, even with current treatments of chemotherapy and hematopoietic stem cell transplantation. These poor treatment outcomes highlight the need for novel therapies for AMKL. Recent novel therapies in other cancers engineer the immune system to target cancerous cells. These therapies include monoclonal antibodies, chimeric antigen receptor T-cell (CAR-T), and engineered T-cell receptor T-cell (TCR-T) therapies that function by binding to tumor-specific or tumor-associated antigens and stimulating a cytotoxic immune response. However, in the context of AMKL, there are currently no immunotherapy options. To explore potential AMKL targetable antigens, we screened the surfaceome of CBFA2T3-GLIS2+ patient cells that were expanded in a patient-derived xenograft (PDX) murine model. We eluted peptides from surface HLA:peptide complexes and utilized mass spectrometry for immunopeptidomics analysis. We identified 242 total peptides, 141 of which were not found in the human ligand atlas, indicating these peptides are leukemia-specific and not presented on normal cells. Of particular interest, we found a peptide from the BMP2 protein, which we have shown contributes to CBFA2T3-GLIS2 driven enhanced self-renewal, and a peptide from the DDX3X protein, which has been reported immunogenic in melanoma.

In an additional PDX murine model, mice engrafted with patient AMKL cells received healthy donor peripheral blood mononuclear cells (PBMCs) from a 6/6 HLA class I match individual. We observed variable reduction in leukemic burden and found those with the most significant reduction also showed a decrease in TCR clonotype diversity, suggesting the healthy T cells underwent clonal expansion directed against leukemia antigens. The clonally expanded TCRs were cloned into expression vectors and will be overexpressed in human CD8+ T cells to test functional response against artificial antigen-presenting cells (aAPCs) presenting the aforementioned peptides of interest through tandem minigene (TMG) assays and peptide stimulation assays. The identification of AMKL- directed effector CD8+ T cells and their corresponding immunogenic antigens will contribute towards the development of an immunotherapy for AMKL to improve patient outcomes.