Generating CAR γδ T Cells for Effective Targeting of B Lymphomas

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has attracted growing interest in the treatment of various malignancies and represents the most advanced technology to date. This approach involves the use of genetically engineered immune cells expressing a surface receptor, called CAR, that specifically detects tumor-associated antigens (TAAs). Here, we generate polyclonal CAR γδ T cells, as these cells do not require patient-donor compatibility and are ideal candidates due to their natural cytotoxicity. The cells are modified to express a B lymphoma-specific CAR, featuring one of the two designs: either 1) an anti-CD19 murine single-chain variable fragment (scFv), a CD28-hinge domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain; or 2) an anti-CD22 camelid single-domain antibody (sdAb), a CD8 hinge-domain, a 4-1BB costimulatory domain, and a CD3ζ activation domain. Using a recently developed packaging and producer line of 293SF-PacLV, we have produced and stocked the lentiviral vector (LV) for these CAR constructs. Lentiviral titration was performed on HEK 293A cells and validated by flow cytometry. γδ T cells were then transduced with the CAR lentivirus at different multiplicities of infection (MOIs). Analyses using flow cytometry showed a CAR transduction efficacy of over 30%, surpassing the clinical threshold required for therapeutic applications. Importantly, all γδ T cell subsets were efficiently transduced. This transduction rate represents a significant milestone in our research, and polyclonal CAR γδ T cells could be a promising off-the-shelf immunotherapy platform for treating B-cell malignancies.