Affinity-optimized bispecific CAR-T cells targeting HLA-G and CD70 for the treatment of clear cell renal cell carcinoma

Background Therapeutic options for clear cell renal cell carcinoma (ccRCC) are limited. Recent clinical trials showed the efficacy of chimeric antigen receptor (CAR)-T cell therapy in treating ccRCC. However, heterogeneous expression of targeting antigen drives immune escape and impairs therapeutic efficacy. Bispecific CARs co-targeting two tumor-associated antigens may overcome this limitation.Methods Human leucocyte antigen-G (HLA-G) and CD70 expression in normal and cancer tissues were analyzed using public RNA sequencing databases and immunohistochemistry of patient samples. An HLA-G CAR was engineered using its natural receptor human inhibitory receptors Ig-like transcript 4 (ILT4). The binding affinity of ILT4 to HLA-G was improved through structure-based rational design. A CD70 nanobody sequence was then incorporated into the HLA-G CAR to generate an HLA-G/CD70 bispecific CAR, together with a CD70-targeting short hairpin RNA (shRNA) to prevent CAR-T cell fratricide. A panel of linkers between the two antigen-binding domains was compared with optimize the CAR signal, and lead candidates were evaluated in vitro and in vivo using heterogeneous tumor models.Results HLA-G and CD70 were identified as ideal bispecific CAR-T targets to overcome the heterogeneity of ccRCC. An ILT4 mutant (Mut-5) with enhanced binding affinity to HLA-G was obtained. The HLA-G CAR derived from this high-affinity ILT4 mutant elicited enhanced antigen-specific cytotoxicity. An HLA-G/CD70 bispecific CAR was subsequently developed by connecting the affinity-optimized ILT4 mutant and a CD70 nanobody via an optimized (G4S)2 linker. To avoid fratricide of the bispecific CAR-T cells mediated by the expression of CD70, we incorporated a CD70 shRNA into the vector. The bispecific CAR showed specific cytotoxicity against both antigens, respectively, and mediated high antitumor activity toward double-positive tumor cells. Bispecific CAR-T cells effectively suppressed heterogeneous tumor growth in vivo and displayed less exhaustion compared with single-target CAR-T cells.Conclusions We established an affinity-optimized HLA-G/CD70 bispecific CAR as a promising strategy to overcome antigen heterogeneity-mediated resistance and immune escape in ccRCC.