Fully human antibody V 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 domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) V and mesothelin (MSLN) V sequences and redirect T cell with V based-CAR. The antitumor activity and mode of action of PSMA V and MSLN V were evaluated in vitro and in vivo compared with the traditional scFv-based CARs.

Results: Human V domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. V 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 domains can prevent tumor escape in tumor with heterogeneous antigen expression.

Conclusions: Fully human antibody V 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 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.