新型pre|CISION®技术化合物的表征与转化开发：在FAP裂解后向肿瘤微环境递送互补的双载荷

The proprietary pre|CISION® technology incorporates a peptidic substrate that is specifically cleaved by Fibroblast Activation Protein alpha (FAP). FAP is a post-proline protease that is overexpressed on the surface of cancer associated fibroblasts (CAFs) in many solid tumors, and facilitates delivery of pre|CISION® medicines specifically to the tumor microenvironment (TME). AVA6103 is a pre|CISION®-enabled exatecan candidate in clinical development. AVA6103 consists of exatecan, a potent Topoisomerase I (TOP1) inhibitor, covalently linked to a dipeptide containing a cleaving sequence (D-Ala-L-Pro), which is susceptible to hydrolysis by FAP but is resistant to hydrolysis by other mammalian proteases. The exquisite selectivity of the pre|CISION® substrate for FAP results in release of exatecan payload in the TME, greatly increasing the therapeutic window and hence reducing systemic exposure and associated toxicities. Drug combinations are commonly used in cancer therapy to improve outcomes and overcome resistance, therefore we engineered two complimentary payloads into a single pre|CISION® molecule. This may offer similar benefits whilst minimizing systemic toxicity. To create dual payload pre|CISION® Peptide-Drug Conjugates (PDCs), a series of compounds have been engineered with modifications in the capping-group and linker portions. Using structure-based drug design, clear structure-activity relationships have been established for affinity to, and susceptibility of linker cleavage by FAP and subsequent payload release. We now demonstrate how the use of the pre|CISION® technology platform has been widened to selectively control the release of multiple payloads from a single molecule via a FAP cleavable linker. This includes TOP1 inhibitors and DNA damage response inhibitors as the dual payloads. Selective payload release has been demonstrated in kinetic studies showing the ability to tune the rate of payload release and in cellular studies showing potent cytotoxicity and bystander activity in cell-based assays including tumor cell-fibroblast 3D co-culture models. Synergistic activity of two potentiating payloads and downstream pharmacodynamic markers for each payload have also been evaluated in cancer cell-lines with specific gene expression profiles. This has provided translational evidence for potential biomarkers and clinical indications. Delivery of payloads to mouse tumors in vivo has also been confirmed. We show the potential of the technology to deliver a therapeutically relevant combination of payloads specifically to the TME while reducing systemic dose-limiting toxicities. This broadens the utility of the pre|CISION® platform in the delivery of novel medicines.