Background – Fibroblast activation protein α (FAP) is a post-proline protease and solid-tumor target which Avacta’s pre|CISION^® peptide moiety linker selectively uses as a differentiated means to target the tumor microenvironment (TME). The potential of the pre|CISION^® technology is highlighted by the promising clinical data from AVA6000, a pre|CISION^® enabled Doxorubicin peptide drug conjugate. FAP expression is tightly regulated in normal adult tissues, with low to undetectable levels in most healthy cells. However, it becomes highly upregulated in the TME where it is expressed by cancer-associated fibroblasts (CAFs). This selective expression pattern has been observed in over 90% of epithelial tumors, including breast, colorectal, pancreatic, lung, brain, and ovarian cancer.

Methods – We implemented a multi-faceted approach to further the understanding of FAP biology and the potential application for pre|CISION^® medicines across a broad range of tumors. We conducted a comprehensive analysis of FAP using real-world data from Tempus AI’s LENS database encompassing over 140,000 tumor samples across 30+ solid tumor types. We evaluated FAP pan-cancer RNA expression, spatial organization of FAP in the TME, as well as correlations with biomarkers. We further analyzed FAP expression pre- and post-treatment and performed in-depth analysis to evaluate FAP expression in tumor subtypes and primary or metastatic lesions. Single-cell RNA-sequencing (scRNA-seq) analysis provided insights into FAP-expressing cell populations as well as correlations between CAF abundance and FAP. FAP expression was evaluated at the protein level using IHC analysis across multiple tumor indications. Multi-immunofluorescence IHC (multi-IF) was used to evaluate the spatial localization of FAP relative to tumor cells and vasculature.

Results – Our findings reveal high FAP expression across a range of tumor indications and demonstrate a correlation with novel biomarkers of interest. IHC analysis confirms a direct relationship between FAP RNA and protein expression, while multi-IF imaging and concentric partitioning analysis for a range of solid tumor indications shows the spatial association of tumor cells, blood vessels and CAFs expressing FAP. ScRNA-seq data confirms high level FAP expression on fibroblasts in stromal-rich tumors. Finally, co-culture models were established with tumor cells and matched fibroblasts which represent tumors with differing levels of FAP enzyme activity. Avacta’s preCISION^® peptide-drug conjugates elicit bystander cell kill over a range of physiological FAP levels within these co-culture models.

Conclusions – Combined, these analyses emphasize the significance of FAP as an important stromal target for focused delivery of targeted oncology therapeutics, and guide further development of the preCISION^® pipeline.

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