CRISPR/Cas9-based generation of CAR-expressing natural killer-like cells against acute myeloid leukemia

• Applicants: Professor Dr. Michael Hust; Professor Dr. Martin Sauer
• Subject Area: Hematology, Oncology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458675018

In general, low cost production methods of approved agents have to compensate for investments during early phases of drug development. This paradigm has been profoundly challenged by the appearance of clinically highly effective cellular biologicals such as chimeric antigen receptor- (CAR) engineered T cells for the treatment of B cell malignancies. Since the drug needs to be produced on demand on an individualized basis, production remains expensive resulting in very high costs for a single treatment. Thus, there is a burning economical and ethical need to develop “off-the-shelf” cell products that can be quickly released for a broad range of patients, independently of their HLA-type. We could very recently show and visibly publish that chimeric antigen receptor (CAR) expression during early phases of ex vivo generation of lymphoid progenitors suppressed BCL11B, leading to a downregulation of T cell–associated gene expression and acquisition of NK cell–like properties. This developmental shift, however, requires either a high tonic signaling activity by the CAR or continuous antigen-specific stimulation during differentiation.The central concept of this grant is to redirect lymphoid progenitor development for NK cell differentiation (as a potential source of an ubiquitously applicable cellular immunotherapeutic) by BCL11b disruption in early lymphoid progenitors that express CARs without tonic signaling activity. This would allow it’s use as a potential source of an ubiquitously applicable cellular immunotherapeutic across MHC barriers. Mechanisms of this differentiational shift are going to be explored. It is further hypothesized that anticipated limited in vivo persistence upon co-transplantation (HCT) into hematopoietic stem cell recipients will allow targeting of tumor-associated antigens that are shared with physiologic hematopoiesis. For proof of principle we intent to generate a murine CAR against murine CD123 in order to eradicate minimal residual disease as a source of potential relapse hypothesizing recovery of physiologic myelopoiesis (sharing CD123 expression) after disappearance of the CAR-bearing NK cell population. The grant application has the following objectives:Aim I: Generation of a suitable set of sgRNAs for selective disruption of the BCL11b gene locus, both, in murine and human precursor T cells.Aim II: Generation of BCL11b-disrupted murine and human CD123 CAR expressing lymphoid progenitors.Aim III: Comparative analysis of T- and NK cell reconstitution after co-transplantation of BCL11b-disrupted versus BCL11b naïve lymphoid progenitors expressing a CAR against murine CD123.Aim IV: Risk benefit analysis between anticipated potent anti-leukemia effects and hypothetically limited myelosuppression after co-transplantation of BCL11b-deficient CD123-expressing progenitors in vivo.

• DFG Programme: Research Grants