Developing and Characterizing the Anti-Tumor Efficacy of a Tri-Specific CAR T Cell Targeting CD19, CD20 and CD22

Chimeric antigen receptor (CAR) T cell therapy targeting proteins such as CD19 and BCMA has achieved considerable success in treating B cell cancers with several multicenter clinical trials reporting an overall response rate greater than 90%. However, relapse occurs in up to 50% of patients receiving treatment and remains a major limitation in part due to antigen downmodulation. To circumvent this, we have developed and generated CAR T cells using a bicistronic CAR vector (“DuoCAR”) simultaneously targeting 3 antigens: CD19, CD20, and CD22 and compared them to CD19-Targeted CAR (“CAR19”) T cells using in vitro and in vivo readouts. Purified CD3+ T cells were activated, transduced with either DuoCAR- or CAR19-encoding lentivirus, and expanded in rhIL2. Overall, DuoCAR T cells showed superior ex vivo expansion compared to regular CAR19 T cells. In our in vitro studies, DuoCAR T cells showed robust upregulation of T cell activation markers, such as CD25, Granzyme B, and HLA-DR, and potent target cell killing when co-cultured with the B cell lymphoma cell line, Raji, or CRISPR-derived Raji CD19 knockout (KO) cells while CAR19 T cells were capable of doing so only with parental Raji cells. Furthermore, when cultured with Raji CD19, CD20, or CD22 KO cell lines, DuoCAR T cells produced high levels of IFNg, IL2, and TNF. In vivo, we observed that DuoCAR T cells were capable of eradicating both Raji and Raji CD19 KO cells in tumor-bearing NSG mice within a week and displayed upregulation of T cell activation markers along with increased levels of serum cytokines. Furthermore, DuoCAR T cells displayed strong engraftment and persistence in both the bone marrow and spleen of treated mice several weeks after tumor clearance suggesting that they can potentially provide long-term immunity against relapse. Overall, our results demonstrate the feasibility of engineering tri-specific CAR T cells and their strong capabilities in eradicating both wild-type and antigen-loss tumor cells in vitro and in vivo, raising the likelihood of superior anti-tumor efficacy in B cell lymphoma treatment despite potential antigen downmodulation.