Overcoming the breast tumor microenvironment by targeting MDSCs through CAR-T cell therapy.
Authors: Nalawade Saisha Abhay , Shafer Paul , Bajgain Pradip , McKenna Katie , Ali Arushana , Kelly Lauren , Joubert Jarrett , Watanabe Norihiro , Leen Ann , Parihar Robin , Vera Valdes Juan Fernando , Hoyos Velez Valentina 
Published: June 2021

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Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.