Compromised antigen binding and signaling interfere with bispecific CD19 and CD79a chimeric antigen receptor function

Abstract

Therapy with CD19 directed chimeric antigen receptor (CAR) T cells has transformed the treatment of advanced B-cell malignancies. However, loss of or low antigen expression can enable tumor escape and limit the duration of responses achieved with CAR-T cell therapy. Engineering bispecific CAR-T cells that target two tumor antigens could overcome antigen negative escape. We found that CD79a and b, which are heterodimeric components of the B cell receptor, were expressed on 84.3% of lymphoma cases by immunohistochemistry, and that 87.3% of CD79ab positive tumors coexpressed CD19. We generated three bispecific permutations: tandem, bicistronic and pooled products of CD79a-CD19 or CD79b-CD19 CAR-T cells and showed that bispecific CAR-T cells prevented the outgrowth of antigen negative cells in a CD19-loss lymphoma xenograft model. However, the tandem and bicistronic CAR-T cells were less effective than monospecific CD19 or CD79a CAR-T cells for the treatment of tumors that only expressed CD19 or CD79, respectively. When compared to monospecific CAR-T cells, T cells expressing a tandem CAR exhibited reduced binding of each target antigen and T cells expressing a bicistronic CAR vector exhibited reduced phosphorylation of downstream CAR signaling molecules. Our study showed that despite added specificity, tandem and bicistronic CAR-T cells exhibit different defects that impair recognition of tumor cells expressing a single antigen. Our data provide support for targeting multiple B cell antigens to improve efficacy and identify areas for improvement of bispecific receptor designs.