PCT1:CO-STIM TCR T-cells to overcome the hostile tumor micro-environment and target triple-negative breast cancer.

Abstract

2590 Background: Adoptive T-cell therapy has demonstrated impressive efficacy in hematoligical cancers but the solid tumor micro-environment presents a unique challenge. However, recently TCR-T cell therapy has shown benefit in difficult-to-treat solid tumors and selection of specific tumor targets and control of the tumor micro-environment can unlock the broader potential of T cell therapy. Triple-negative breast cancer (TNBC) is a difficult-to-treat tumor as it lacks classical targets for hormone and antibody-based therapies. It harbors a highly immune-suppressive microenvironment and rarely responds to immune-checkpoint inhibitors. We sought to identify a novel target to make TNBC amenable for adoptive T-cell therapy with T-cell receptor (TCR)–engineered cells and applied a unique next-generation gene-engineering approach to make T-cells overcome the hostile microenvironment. Methods: (i) Discovery of TNBC-restricted target: We applied in silico analyses of >500 TNBC samples and >1,500 healthy tissues and validated findings with qRT-PCR and immune stainings of >300 TNBC samples as well as 40 healthy tissues. (ii) Discovery and selection of PCT1 TCR: We enriched ROPN1-specific TCRs from naïve repertoires and assessed specificity, sensitivity and performed preclinical safety studies. (iii) Development of TCR:CO-STIM technology to overcome immune suppression: we designed a panel of murine TCRs harboring different intracellular co-stimulatory domains, thereby providing additional stimulation to T cells aimed to overcome immune suppression in solid cancer. We tested their ability to extend anti-tumor durability in a murine melanoma model, performed comprehensive permutations to enable stable expression of fully human TCR:CO:STIM and applied it to multiple TCR specificities. Results: For TNBC, we identified that Ropporin (ROPN1), a protein expressed homogeneously in >90% of TNBC and persistent across disease stages but absent from healthy tissues, as an ideal target. We identified 13 clonal TCRs directed against 9 different ROPN1 epitopes. The lead TCR, termed PCT1 TCR , demonstrated high sensitivity and specificity towards ROPN1 + /HLA-A2 + cell lines and patient-derived organoids in 3D. Our TCR:CO-STIM technology significantly improved duration of response in a murine model and improved T cell fitness. Notably, when repeatedly challenged with ROPN1 + /HLA-A2 + TNBC cells, PCT1:CO-STIM , but not PCT1 TCR T-cells, could resist up-regulation of T-cell exhaustion markers and retained tumor-killing capacity for 3-10 extra rounds of stimulation. Importantly, PCT1:CO-STIM did show any signs of tonic signaling nor crossreactivity nor alloreactivity towards any major HLA-I allele. Conclusions: TCR:CO-STIM technology has shown enhanced activity of a selective and specific TCR targeted at ROPN1 and we are progressing PCT1:CO-STIM to the clinic for the treatment of TNBC.