Adoptive cellular therapy for Ewing’s sarcoma
Tumor immunotherapy with chimeric antigen receptor (CAR)-modified T cells has shown substantial efficacy in early phase clinical trials. Targeting a single tumor associated antigen (TAA) could however result in creation of antigen escape variants. We have shown that simultaneous targeting of two TAAs with bispecific T-cell products can offset this antigen escape mechanism. To render T cells bispecific using a single molecule, we used systematic computational modeling to rationally design and construct a novel proof-of-principle bispecific CAR molecule that incorporates two scFv antigen-recognition domains, in tandem (TanCAR). Our data demonstrated that TanCAR-grafted T cells distinctly and specifically recognized and killed tumor cells with either target antigen and maintained their effector functionality despite downregulation of one target molecule, a characteristic that should circumvent antigen escape variants. Moreover, we saw synergistic functionality in the presence of both target molecules supporting the fact that expression of TanCARs can augment the activity of T cells. Using in silico modeling, we have created a novel TanCAR molecule consisting of HER2 (FRP5.scFv) and GD2 (14g2a.scFv)-recognition domains linked by a Gly-Ser linker. Our studies show that HER2/GD2 TanCAR T cells can recognize either antigen distinctly and both simultaneously. We hypothesize that targeting two distinct and validated TAAs on Ewing’s sarcoma cells will reduce the possibility of tumor escape that would eventually result in disease recurrence. Furthermore, this could improve T-cell activation, enhancing their persistence, survival and anti-tumor efficacy.
Aim I: Functional testing of HER2/GD2 bispecific TanCAR T cells in vitro. We will determine the anti-tumor efficacy, expansion and persistence of HER2/GD2 TanCAR (CD28.4-1BB.? co-stimulatory domain) T cells in comparison to HER2 or GD2 specific CAR T cells, in response to HER2/GD2 expressing Ewing’s sarcoma cells and target null controls using standard immunoassays in vitro.
Aim II: Functional testing of HER2/GD2 bispecific TanCAR T cells in vivo. We will study the expansion, persistence and anti-tumor efficacy of the TanCAR T cells in an orthotopic xenograft model of human metastatic Ewing’s sarcoma. We will also determine their ability to offset antigen escape, salvage tumor recurrence as well as study the expression pattern of TAAs before and after treatment.
Impact Statement: While cure rates for localized Ewing’s sarcoma has significantly improved, the outcome for patients with metastatic disease remains grimly unchanged over the last decade. Thus, there is a need for new more effective therapies that are preferably tumor targeted. Adoptive cell therapy with CAR T cells has the potential to fulfill this need. The high specificity of such T-cell therapies also makes them less toxic, reducing the organ toxicities and other long-term adverse effects endured by cancer survivors.