Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells

Abstract

Background Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. Methods CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function. Results The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%. Conclusions Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Spermidine Promotes Nb CAR-T Mediated Cytotoxicity to Lymphoma Cells Through Elevating Proliferation and Memory

“…Our previous findings proved that nanobody based CAR-T cells possessed a highly efficient cytotoxicity to tumor cells, more notably, Bispecific Nb CAR-T cells (target CD19 and CD20 simultaneously) was more effective than single target. 60 Therefore, we further explore the killing efficiency of Nb CAR-T cells combined with spermidine. As mentioned, 5μM of spermidine treating brought remarkable cytotoxicity to CD19 CAR-T cells than 0.5μM, thus same dose was utilized to Nb CAR-T cells.…”

“…Subsequently, previously constructed Nb CAR-T cells were used to explore the effects of spermidine. 60 Previous results have shown that nanobody-based CAR-T cells can effectively activation (an activation marker) and proliferation when stimulated by Daudi cells, resulting in lysis of these tumor cells. It was worth mentioning that we found the positive rate had a significant impact on CAR-T cells anti-tumor activity.…”

Antibody variable region engineering for improving cancer immunotherapy

“…With the first US FDA‐approved nanobody monotherapy in 2019, considerably more effort has been invested in the feasibility of nanobody‐based CAR‐T therapy. Bispecific nanobody anti‐CD19+CD20 CAR‐T cells were constructed and killed B cell lines in vitro with high specificity and enhanced proliferation [ 168 ].…”

Emerging applications of nanobodies in cancer therapy

“…Multiple nanoCAR-T cell products are under preclinical investigation for diverse target antigens on both hematological malignancies as well as solid tumors (Table 5). Antigens that have been targeted with nanoCARs are: CD38 (1G3) , CD19 (Wang et al, 2021), CD20 (De Munter et al, 2018;Wang et al, 2021) andCD33 (De Munter et al, 2020) in hematological malignancies, and mucin-1 (AR32) (Bakhtiari et al, 2009;Iri-Sofla et al, 2011;Khaleghi et al, 2012), VEGFR2 (3VGR19) (Hajari and prostate-specific membrane antigen (PSMA) (NBP) (Hassani et al, 2019) in solid tumors. Targeting the tumor microenvironment (TME) instead of TAAs might provide a more generally applicable CAR therapy.…”