404. Development of Combinational Cellular Immunotherapy to Treat Pediatric Acute Myeloid Leukemia Patients

Abstract

Hematopoietic stem cell transplantation is the standard of care for pediatric acute myeloid leukemia (AML) patients, but relapse still occurs in approximately 40% of AML patients. Hence there is an unmet need to improve disease control with fewer adverse acute and late effects. We therefore aim to develop ‘HLA-matched’ modified cellular therapies based on a platform of cord blood transplantation (CBT) to treat pediatric AML patients. In the majority of AML patients the tumor oncogene Wilms tumor 1 (WT1) is overexpressed and can serve as a target for tumor specific therapy. In addition to our previously developed cord blood-derived dendritic cell vaccine (CB-DC) we are currently developing gene modified T lymphocytes from the same CB-unit to specifically target WT1 expressing AML to support and increase the anti-leukemic effects of CBT. Lentiviral vectors were developed to express recombinant T-cell receptors (TCR) specifically recognizing HLA-A2+ restricted WT1 peptide. TCR expression was improved by codon-optimization of the cDNA sequence including cysteines in the alpha and beta chain constant domains. These WT1-specific TCR α/β-chains were incorporated in a single lentiviral vector linked by a ‘self-cleaving’ T2A peptide to improve efficiency. The order of the alpha and beta TCR chains, and inclusion of a furin cleavage site in the expression cassette did not enhance TCR expression in Jurkat and primary T cells derived from either peripheral blood or cord blood. Primary T cells were stimulated with T2 cells and CB-DCs pulsed with the specific peptide, which resulted in T cell activation as measured by CD69 and CD137 upregulation, as well as IFNgamma production. To reduce the risk of off-target antigen recognition of gene-modified T cells, and to further improve efficacy, CRISPR/Cas9 can be used to eliminate endogenous TCR expression. Cas9 protein and guide RNA (gRNA) were delivered to GFP positive reporter Jurkat cells using plasmid and mRNA electroporation (EP), integrating and non-integrating lentiviral vectors and adenoviral vectors with the highest efficiency of knockout being achieved by EP. Two gRNAs targeting the constant domains of the alpha chain and three targeting those of the beta chain resulted in similar knockout efficiencies in Jurkat cells (approximately 25%). Of note, combining the gRNAs did not result in higher knockout efficiencies. Gene-editing at the molecular level was confirmed by next generation sequencing. We envision that addition of TCR-engineered T cells (introduction of tumor-specific TCR and knockdown of endogenous TCR), derived from the same cord blood as used for the transplantation, will more efficiently and with improved safety prevent early relapse after CBT by targeting WT1 AML.