A clinical-grade automated platform for the manufacturing of CAR-γδ T cells for immunotherapy.

Gamma delta (γδ) T cells are promising candidates for adoptive immunotherapy due to their unique biological and safety profiles. In particular, ex vivo expanded Vγ9Vδ2 T cells engineered with chimeric antigen receptors (CARs) have gained considerable interest, as they combine potent innate-like antitumor activity with a low risk of graft-versus-host disease and cytokine release syndrome, making them ideal for off-the-shelf cancer immunotherapies. However, as they represent only 1-5% of peripheral T cells, effective activation and expansion protocols are essential to generate sufficient numbers for clinical use. In this study, we describe an automated and highly efficient process for the expansion of Vγ9Vδ2 T cells and the manufacturing of CAR γδ T cells using the CliniMACS Prodigy closed system. This platform integrates cell separation, activation, genetic modification and expansion ensuring reproducibility and compliance with GMP standards. Starting with an alpha-beta (αβ) T cell and B cell magnetic depletion, we achieved a 4.59-log αβT cell depletion and a 3.97-log B cell depletion. By carefully optimizing activation, transduction and expansion, we were able to achieve up to 374-fold increase in Vγ9Vδ2 T cells, resulting in an average number of 6.64 x 109 (4.76×109 - 8.98×109) cells after 14 days of culture using the TCT-LS process. Using a GMP-grade lentiviral vector pseudotyped with the envelope protein from baboon endogenous virus (BaEV) encoding a CD123 chimeric antigen receptor (CAR) at a multiplicity of infection (MOI) of 0.12, we achieved a mean γδ T cell transduction efficiency of 57.4%. Flow cytometry analysis of the final cell product showed an average composition of 89.83% γδ T cells, 9.82% NK cells, and 0.012% residual αβ T cells. The low average vector copy number (VCN) of 1.4 underscores the safety and efficiency of the transduction protocol. Functional assays against a leukemic cell line confirmed robust anti-tumor activity of CAR-γδ T cells produced using this platform, demonstrating the feasibility of large-scale manufacturing and highlighting their potential for future clinical immunotherapy applications.