Banana Lectin Expressing CAR T Cells to Target Heterogenous Solid Tumors
Introduction
To overcome the heterogenous and dynamic expression of tumor antigen expression in solid tumors and the physical barrier of stroma, we developed a chimeric antigen receptor (CAR) using a lectin binder (H84T BanLec) which recognizes patterns of aberrant glycosylation (high mannose) preferentially expressed on tumor cells and surrounding stroma. Aberrant glycosylation patterns are a widely present hallmark of cancer, therefore incorporating H84T BanLec as part of a CAR on T cells engineered to target other tumor antigens should boost the efficacy of current therapies. Previously, we showed that H84T-CAR-expressing T cells effectively target and eliminate pancreatic tumors in preclinical models and we now demonstrate activity against non-small cell lung cancer (NSCLC) in both monolayer cultures and 3-dimensional spheroids composed of tumor cells, cancer stroma, and inhibitory monocytes. The tumor microenvironment (TME) of solid tumors also produces immunosuppressive factors, including TGFβ, that can inhibit T cell growth and effector function. We have shown T cells can be shielded from the inhibitory effects of TGFβ by expressing a dominant negative TGFβ type II receptor (DNR) and will now assess if the DNR will augment H84T CAR T cell activity in solid tumors.
Methods
We co-expressed the H84T BanLec CAR with a conventional scFv-derived CAR targeting HER.2 and compared the activity of dual-expressing H84T/HER.2 CAR-T cells against single-expressing CARs targeting NSCLC. We determined anti-tumor cytotoxicity by measuring residual GFP+ A549 NSCLC tumor cells in a sequential killing assay, using flow cytometry. We measured the activity of single and dual CAR T cells against lung stromal cells in both 2D and 3D spheroids and determined anti-tumor activity by loss of GFP intensity of tumor cells in multicellular spheroids using Incucyte Live Cell imaging. To test the inhibitory effect of TGFβ inhibition produced by the lung TME, we co-expressed DNR on H84T CAR T cells and measured both phsopho-SMAD2/3 expression in the presence of TGFβ by flow cytometry and the anti-tumor function of these DNR expressing H84T CAR-T cells. We analyzed the effects in both 2D and 3D tumor cultures, with and without cancer associated stroma.
Results
In sequential killing assays against tumor alone, we observed superior anti-tumor activity using dual expressing HER.2/H84T CAR T cells compared to T cells expressing either HER.2- or H84T-CAR. Similarly, in the presence of cancer-supportive stromal cells, HER.2/H84T dual CAR T cells have greater cytotoxicity against NSCLC than single CAR expressing T cells in 3D multicellular spheroids. Furthermore, expression of the DNR in H84T CAR T cells enhances their antitumor activity in the presence of TGFβ.
Conclusion
H84T CAR T cells have anti-tumor activity against NSCLC cancer cells and co-expressing H84T BanLec CAR with HER.2 CAR further enhances this effect in a tumor model that includes both cancer and supporting stromal cells. We are elucidating the mechanisms by which H84T CARs induce stromal disruption and enhance tumor infiltration in 3-dimensional and in vivo models. In addition, forced expression of a TGFβ DNR on H84T CAR-T cells further enhances their cytotoxicity, when the TME contains TGFβ and we are now investigating whether DNR expression on dual CAR T cells is similarly beneficial for a range of tumors.