CAR-T cells based on a TCR mimic nanobody targeting HPV16 E6 exhibit antitumor activity against cervical cancer

The E6 and E7 oncoproteins of human papillomavirus (HPV) are considered promising targets for HPV-related cancers. In this study, we evaluated novel T cell receptor mimic (TCRm) nanobodies targeting the E6_29-38 peptide complexed with human leukocyte antigen (HLA)-A∗02:01 in the chimeric antigen receptor (CAR) format. We isolated two dromedary camel nanobodies, F5 and G9, through phage display screening. F5 bound more efficiently to the complex expressed on cells, including peptide-pulsed T2, overexpressed 293E6, and cervical cancer lines CaSki and SS4050, compared to G9. CAR-T cells based on the F5 nanobody specifically killed target cells, including 293E6, CaSki, and SS4050 <i>in vitro</i>, through activation of nuclear factor of activated T cells (NFAT) and nuclear factor κB (NF-κB) signaling. Importantly, F5 CAR-T cells inhibited the growth of CaSki and SS4050 tumor xenografts in mice. These findings demonstrate that HPV-16+ cervical cancer can be targeted by F5 nanobody-based CAR-T cells, offering a valuable alternative strategy for treating HPV-16+ malignancies.