Background With the progressive move of chimeric antigen receptor (CAR) T-cell therapy as a first-line treatment option for patients with B-cell malignancies, multiple efforts are being made to extend this therapy to other malignancies and broader patient populations. However, several limitations remain, including the impact of the tumor microenvironment and low persistence of the CAR T-cells post-infusion. Additional limitations are associated with the time-consuming and highly personalized manufacturing of autologous CAR T-cells. To overcome these challenges, further engineering of the CAR T-cells is needed. We present here short hairpin RNA (shRNA) interference as a mean to endow the CAR T-cells with novel attributes.
Methods We have selected an shRNA targeting CD3ζ which efficiently downregulates the T-cell receptor (TCR) complex cell surface expression, and have generated an allogeneic anti-BCMA CAR that co-expresses this shRNA to prevent the risk of graft-versus-host disease (GvHD). This construct (CYAD-211) was then evaluated in a Phase I clinical trial (NCT04613557) to validate the shRNA technology. In parallel, we have engineered a NKG2D-based CAR T-cell co-expressing an shRNA targeting MICA and MICB ligands, as a way to increase CAR T-cell persistence and prevent NKG2D mediated fratricide. This construct (CYAD-02) was also evaluated in a Phase I clinical trial (NCT04167696).
Results Insertion of the anti-CD3ζ shRNA into the CAR construct led to significant reduction in cell surface TCR expression and prevented TCR-mediated signaling. Preclinical data showed no evidence of GvHD when CYAD-211 was infused into NSG mice confirming efficient inhibition of the TCR activation, while the anti-tumor activity of the CAR was maintained. Clinical evaluation demonstrated that CYAD-211 was well tolerated in patients and the lack of observed GvHD despite engraftment provides proof of concept of the shRNA-driven downregulation of the TCR.
Addition of an shRNA targeting MICA/B along with NKG2D-based CAR led to a higher engraftment and enhanced anti-tumor activity of the CAR T-cells in preclinical studies. Clinical evaluation confirmed the good tolerability of CYAD-02 while showing an improved CAR T-cell persistence and activity when compared to NKG2D-based CAR without the shRNA.
Conclusions Our data provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without any signs of GvHD or can improve CAR T-cell characteristics while maintaining a good safety profile and efficacy. We are currently validating the technology to downregulate multiple-genes of interest simultaneously thereby providing a platform approach that could support the future of cell therapy.
Ethics Approval The clinical studies validating the technology obtained approval from Belgian and American competent authorities and Ethical Commitees. All that participants gave informed consent before taking part to the studies.
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