Inhibition of TGF- β- receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer .

Background Immunotherapy with chimeric antigen receptor (CAR)-engineered T-cells is effective in some hematologic tumors. In solid tumors, however, sustained antitumor responses after CAR T-cell therapy remain to be demonstrated both in the pre-clinical and clinical setting. A perceived barrier to the efficacy of CAR T-cell therapy in solid tumors is the hostile tumor microenvironment where immunosuppressive soluble factors like transforming growth factor (TGF)-beta are thought to inhibit the cellular immune response. Here, we analyzed whether CAR T-cells specific for the receptor tyrosine kinase-like orphan receptor 1 (ROR1) antigen, that is frequently expressed in triple-negative breast cancer (TNBC), are susceptible to inhibition by TGF-beta and evaluated TGF-beta-receptor signaling blockade as a way of neutralizing the inhibitory effect of this cytokine. Methods CD8(+) and CD4(+) ROR1-CAR T-cells were prepared from healthy donors and their antitumor function analyzed using the TNBC cell line MDA-MB-231 in vitro and in a microphysiologic 3D tumor model. Analyses were performed in co-culture assays of ROR1-CAR T-cells and MDA-MB-231 cells with addition of exogenous TGF-beta. Results The data show that exposure to TGF-beta engages TGF-beta-receptor signaling in CD8(+) and CD4(+) ROR1-CAR T-cells as evidenced by phosphorylation of small mothers against decapentaplegic homolog 2. In the presence of TGF-beta, the cytolytic activity, cytokine production and proliferation of ROR1-CAR T-cells in co-culture with MDA-MB-231 TNBC cells were markedly impaired, and the viability of ROR1-CAR T-cells reduced. Blockade of TGF-beta-receptor signaling with the specific kinase inhibitor SD-208 was able to protect CD8(+) and CD4(+) ROR1-CAR T-cells from the inhibitory effect of TGF-beta, and sustained their antitumor function in vitro and in the microphysiologic 3D tumor model. Combination treatment with SD-208 also led to increased viability and lower expression of PD-1 on ROR1-CAR T-cells at the end of the antitumor response. Conclusion We demonstrate the TGF-beta suppresses the antitumor function of ROR1-CAR T-cells against TNBC in preclinical models. Our study supports the continued preclinical development and the clinical evaluation of combination treatments that shield CAR T-cells from TGF-beta, as exemplified by the TGF-beta-receptor kinase inhibitor SD-208 in this study.