Background
Adoptive T cell therapy (ATT) has shown efficacy in the treatment of hematologic malignancies. However, in solid cancer, response rates are currently unsatisfactorily low. One of the major challenges in the treatment of solid tumors is to overcome the poor lifespan and the loss of function of tumor-reactive T cells in the tumor microenvironment (TME). We aim to overcome these hurdles by engineering tumor-reactive T cells with chimeric costimulatory switch proteins (CSPs) which should prevent PD-1/L1-mediated inhibition and simultaneous turning it into activation of costimulatory pathways leading to functional enhancement and prolonged survival.
Materials and Methods
Two CSPs were generated combining the extracellular domain of PD-1 with an intracellular costimulatory domain of either CD28 or 4-1BB. T cells without CSP are controls. Primary human tumor-reactive CD8 T cells were engineered to express T cell receptors (TCRs) with HLA-A2 restricted recognition of either a tyrosinase or a renal cell carcinoma (RCC) peptide. CSP-engineered TCR-T cells and controls were in-depth characterized regarding cytokine release and cytotoxicity, phenotype, differentiation and metabolic state in vitro after co-culture (2D/3D-matrigel) in TME-adapted conditions (using melanoma or renal cell carcinoma (RCC) cell lines) and in vivo using an orthotopic human RCC mouse model. Blood, tumors and organs were analyzed for the presence and phenotype of T cells by flow cytometry.
Results
In vitro, CSP-engineered TCR-T cells showed enhanced cytokine release and cytotoxicity. CSPs enabled maintenance of function during repeated tumor challenge and nutrient-restricted conditions. Flow cytometry identified higher Ki67, IRF4 and BATF as well as markers of metabolic fitness in CSP-TCR-T cells compared to control T cells following co-culture with PD-L1 positive tumor cells. Induced changes were dependent on tumor cells expressing PD-L1 in the context of peptide/MHC ligands. In vivo, using an orthotopic implant of RCC tumor cells, ATT with TCR-T cells reduced tumor volume compared to the no T cell group, and only residual tumor cells were detected after histological examination in the CSP-TCR-T cell groups. Plasma IFN levels at day 3 after ATT were higher for CSP-TCR-T cells compared to control T cells.
Conclusions
We collected essential functional data using in vitro and in vivo models describing effects of PD-1-based CSPs on TCR-T cell phenotype and functional activity. We observed higher efficacy of PD-1-CSP-T cells in controlling orthotopic RCC xenografts, which is to our knowledge the first report of PD-1-CSP effects using non-affinity-enhanced TCR (isolated from human tumor-infiltrating T cells) and unmodified cancer cells with endogenous (not engineered) antigen/MHC and PD-L1. The results suggest that PD-1-CSPs can empower tumor antigen-reactive TCR-T cells for beneficial application in solid tumor therapy paving the way for in human clinical trials using TCR-T cells armored with CSPs.
Funded by Deutsche Krebshilfe, and ZIM/AppMic.
E. Noessner: None. C. Aman: None. A.J. Fischbeck: None. K. Junker: None. J. Linxweiler: None.