Background CAR-T cell therapy has revolutionized the treatment of B-cell malignancies. However, the development of CAR-T therapies for T-cell malignancies has been slower, partially due to technical difficulties in T-cell antigen targeting CAR-T production such as fratricide and contamination of malignant T cells.¹ Although allogeneic αβ CAR-T cells from healthy donors provide blast-free treatment options, complications like GvHD and host rejection can arise. In this context, utilizing healthy donor-derived γδ T cells, free of the risk of GVHD and tumor contamination, presents a promising option for allogeneic CAR-T cell therapy.² In this study, we introduce allogeneic anti-CD5 γδ CAR T cells for treating T-cell malignancies.

Methods We employed bio-panning techniques to screen novel fully human single-chain variable fragments (scFvs) capable of recognizing the CD5 antigen. Next, we genetically modified γδ T cells obtained from a healthy donor by introducing functional CAR constructs, which incorporated CD5-specific scFv-fused 4–1BB/CD3z sequences and assessed thefunctionality and phenotypic features of anti-CD5 γδ CAR T cells through in vitro functional assays. We also evaluated the preclinical potency of these CAR T cells using immunodeficient mice models engrafted with CD5⁺ T cell lymphoma cell lines.

Results Several fully human scFvs that target CD5 were successfully identified and two scFv clones (A2 and C7) were utilized in generating anti-CD5 γδ CAR-T cells (γδCAR5) and tested. A2 γδCAR5 exhibited higher cell expansion and CAR-positive portion compared to C7 γδCAR5, although A2 γδCAR5 displayed higher exhaustion phenotypes. A2 γδCAR5 demonstrated robust cytokine production and cytotoxic activity in vitro. Furthermore, A2 γδCAR5 exhibited superior anti-tumor activity in a CD5⁺ T cell lymphoma xenograft model (HUT-78) compared to C7 γδCAR5. Interestingly, in the context of αβ T cells, C7 αβCAR5 showed higher cell expansion and greater anti-tumor effect than A7 αβCAR5, implying that the exhaustion of A2 CAR5 is more severe in the context of αβ T cells, compared to γδ T cells.

Conclusions Our successful generation and evaluation of anti-CD5 γδ CAR T cells provide the foundation for developing allogeneic CAR T cell therapy as a promising treatment of T cell malignancies.

References

1. Safarzadeh Kozani, Pouya, Pooria Safarzadeh Kozani, and Fatemeh Rahbarizadeh. ‘CAR-T cell therapy in T-cell malignancies: Is success a low-hanging fruit?.’ Stem cell research & therapy 2021;12(1):1–17.

2. Saura-Esteller, José, et al. ‘Gamma delta T-cell based cancer immunotherapy: past-present-future.’ Frontiers in Immunology 2022:2649.