Dual-Specificity CAR+ T Cells to Target B-Cell Malignancies and Opportunistic Fungal Infection

Lymphodepletion before adoptive cell transfer (ACT) can enhance anti-tumor responses by increasing access to homeostatic cytokines and by depressing the numbers of regulatory cells.  However, the lymphodepleted recipient is at risk for morbidity and mortality from pathogens. Indeed, opportunistic invasive fungal infection (IFI) by Aspergillus sp. results in a 60 to 85% in patients undergoing hematopoietic stem-cell transplantation. Antifungal agents such as polyenes, triazoles, and echinocandins can be rendered ineffective due to shifts in fungal epidemiology, emergence of drug resistant strains, and attendant toxicities in the recipients. Thus, other strategies are being developed that target fungi. Clinical-grade T cells genetically modified to express chimeric antigen receptors (CARs) have been successfully used to redirect specificity to target tumor-associated antigens. We and others have infused CD19-specific T cells in patients undergoing HSCT for investigational treatment of B-cell malignancies. This provides a platform to expand the role of adoptive immunotherapy to also target IFI. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-z (designated D-CAR) upon binding with carbohydrate cell wall in Aspergillus germlings. The D-CAR⁺ T cells exhibited specificity for b-1,3-gucan and inhibited hyphal growth of Aspergillus. The D-CAR was then expressed with our CD19-specific CAR (designated CD19RCD28) which is currently used in clinical trials. Our existing manufacturing platform was readily adapted to electro-transfer plasmids from the Sleeping Beauty (SB) system. Clinical-grade CD19-specific CAR⁺ T cells are produced after introducing two DNA plasmids coding for transposon (CD19RCD28) and transposase (SB11). The synchronous electro-transfer of a third SB-derived DNA plasmid coding for D-CAR resulted in CD19RCD28⁺D-CAR⁺ T cells. The resultant genetically modified T cells can target both CD19⁺ malignant B cells and Aspergillus. Generating CD19-specific T cells with multiple specificities will enable introduced fungal immunity to be long-lived and provides a foundation for engineering an immune response with that can target multiple antigens, such as to prevent the emergence of antigen-escape variants.