Background A barrier to successful adoptive cell therapy for solid tumors is target antigen heterogeneity and antigen escape. Activated T cells, including CAR T cells, have limited innate antigen capture/presentation capabilities.1 We engineered a novel TIM-4 CER that bears a toll/interleukin-1 (TIR) signaling domain from TLR2 designed to induce both direct cell-mediated cytotoxicity and initiate secondary immune responses, leading to improved solid tumor clearance and durability of response. CER-T cells offer potential pairing with multiple small molecule drugs to enhance the TIM-4 target ligand. We tested for antigen-presenting cell (APC)-like function, characterized transcriptional signatures, and quantified cytotoxic responses against EGFR-mutant NSCLC cells in combination with the epidermal growth factor inhibitor (EGFRi) osimertinib.
Methods CER-1236 contains a TIM-4 extracellular signaling domain fused with a TLR2-TIR domain, CD28 and CD3zeta. Using PS-coated beads labeled with a pH-sensitive dye (pHrodo), we quantified CER-1236 phagocytic uptake by FACs and fluorescent microscopy. Cytotoxic and proliferative responses against osimertinib-treated H1975 NSCLC cells was tested in vitro. We quantified APC activity by evaluating autologous HPV E7 TCR T cell activation and proliferation following CER-1236 co-culture with HLA-matched HPV-16 E7+ SCC152 tumor cells. To characterize transcriptional states, we performed bulk RNA-sequencing at rest and following activation. TLR2 inhibition was used to examine signaling pathway induction.
Results In combination with subtherapeutic osimertinib doses, CER-1236 eliminated 97% of targets by 72h at low effector:target ratios. EGFR inhibition drove target-dependent proliferation, production of TH1 cytokines (IFN-gamma, TNFalpha) and granzyme-B. Upon co-culture with PS-coated beads, 60% of CER-1236 T cells demonstrated increased bead uptake compared to untransduced or CER T cells expressing a TIM-4 PS-binding mutant (p<0.0001), APC assays demonstrated higher activation of E7-TCR T cells by CER-1236 than untransduced T cells, indicating occurrence of antigen uptake and presentation. Both cytokine production and antigen presentation were inhibited by a TLR2 inhibitor. Finally, transcriptional profiling identified a distinct signature related to APC-like function upon CER-1236 stimulation with enrichment of NF-kB, MAP kinase, TNFalpha and chemokine signaling.
Conclusions Novel TIM-4-containing CER T cells that include a TLR2 signaling domain synergize with osimertinib to eliminate EGFR-mutant NSCLC cells in vitro. Notably, CER T cells capture and present tumor cell antigen, and demonstrate APC-like transcriptional signatures. Antigen presentation, alongside inducible, target-specific cytotoxic function in single T cells, represent a potential advantage to initiate host immune responses against novel antigens for solid tumors.
Acknowledgements The authors would like to acknowledge Edson Oliveira, Phani Kukutla and the CERo team for helpful suggestions.
Lanzavecchia A, Roosnek E, Gregory T, Berman P, Abrignani S. T cells can present antigens such as HIV gp120 targeted to their own surface molecules. Nature. 1988; 334:530–532.
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