Targeting and Elimination of T Cell Subpopulations by Chimeric Antigen Receptor-modified T Cells

Abstract

Chimeric antigen receptors (CARs) demonstrated a high potential for the elimination of tumor cells in cancer immunotherapy. The first successful application of CAR-expressing T cells aimed at the elimination of B cells in hematological malignancies. However, despite being a breakthrough, which highlighted the potential of the CAR T cell technology, its application in targeting T cells currently has limitations. On the one hand, it is the limited choice of target molecules that are presented on malignant cells, but not on healthy cells. On the other hand, the design of the signaling components included into CAR constructs needs to be ‘fine-tuned’ to provide higher efficiency and persistence of the CAR-modified T cells. Adapting the CAR T cell platform to treat T cell malignancies poses challenges in terms of the targeting approach. Redirecting the CAR-expressing T cells requires a careful selection of the target antigen, because unlike for B cells, the elimination of the entire T cell population is not a feasible strategy. Therefore, for selective elimination of defined subpopulations, I explored the possibility to redirect CAR-expressing cells against TCR Vβ clonotypes. As a model and proof of principle, I used TCR Vβ 5.3+ subpopulation. To this end, I generated an anti-Vβ 5.3 CAR in a dual chain format (anti-Vβ 5.3 dcCAR). In this study, I could show that the dcCAR T cells selectively eliminate TCR Vβ 5.3+ T cell line in vitro. Moreover, co-culture experiments with isolated primary human T cells showed specific recognition of the target subpopulation. Taken together, these findings demonstrate the potential of this targeting approach to specifically eliminate defined T cells subpopulations. Since the cytoplasmic domains in currently used CAR molecules have an impact on the functionality and efficiency of the CAR-expressing T cells, the effect of the immunoglobulin tail tyrosine (ITT) motif in the anti-Vβ 5.3 dcCAR was assessed. For this purpose, the CD28-derived ITT motif included in most conventional CAR backbones was exchanged with the ITT motif from the lymphoid and myeloid co-receptor DAP10. The modified costimulatory domain of the anti-Vβ 5.3 dcCAR showed increased signaling capacity in comparison to the conventional CD28 ITT-containing domain, but similar cytotoxic efficiency exerted by both dcCAR-expressing Bw58 cells. The findings of this study provide a groundwork for further investigation of CAR molecules intended to target selectively defined TCR Vβ-specific subpopulations, by utilizing other Vβ-specific monoclonal antibodies as variable domains of CARs. Moreover, the improved signaling potential as a result of the ITT exchange in the CARs, provides a basis for expanding the current knowledge about the potential role of additional signaling elements in CAR activity-modulating co-receptors. Altogether, these findings could contribute to the improvement of the CAR T cell platform in the future.