Fully human antibody V(H) domains to generate mono and bispecific CAR to target solid tumors

BACKGROUND: Chimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs. METHODS: We used Humabody V(H) domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) V(H) and mesothelin (MSLN) V(H) sequences and redirect T cell with V(H) based-CAR. The antitumor activity and mode of action of PSMA V(H) and MSLN V(H) were evaluated in vitro and in vivo compared with the traditional scFv-based CARs. RESULTS: Human V(H) domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. V(H) modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody V(H) domains can prevent tumor escape in tumor with heterogeneous antigen expression. CONCLUSIONS: Fully human antibody V(H) domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, V(H) domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.