Metastatic Pancreatic Ductal Adenocarcinoma (PDAC) is a root cause of lethality in patients. The metastasis is promoted by abundant proteases in the PDAC microenvironment. Synthetic immuno-oncology opens new avenues towards overcoming limitations of standard immunotherapy agents, previously unsuccessful in PDAC. Anti-tumour cytokines such as IL-12 poorly infiltrate the stroma of solid tumours and trigger toxicity. Here, by exploiting the abundant proteases in PDACs, a targeted and tolerable masked Collagen-Binding Domain CBD-Il12 (mCBD-Il12) was produced by fusing the mask to the p35 subunit of Il-12 through a cleavable linker. This linker is cleaved by PDAC-specific proteases, leading to activation of mCBD-Il12 upon tumour infiltration.
Human PDAC samples were analysed for the expression of MMP2/9 and PLAU in established subtypes (classical and quasi-mesenchymal). mCBD-IL12 with a linker sensitive to such protease activity was therefore produced in HEK293F cells and purified using immobilised metal affinity and size exclusion chromatography. The ability of various murine tissues and their associated proteases to cleave the linker was verified through western blotting. Finally, the molecule was tested in a metastatic and mesenchymal pancreatic syngeneic mouse model.
The quasi-mesenchymal subtype was found to express MMP2/9/PLAU to a higher degree than the other Collisson/Sadanandam PDAC subtypes. In addition, we found the linker to be readily cleaved by murine pancreatic tumour lysate in contrast with non-cancerous pancreatic tissue in vitro. Interestingly, mCBD-Il12 was found to considerably reduce the number of liver metastases and ascites volume compared to wild-type Il-12 or controls in a murine PDAC model. The ascites colour varied from white in wild-type Il-12 to light red in mCBD-Il12, and dark red in controls. Analysis of the blood-based cytokines is pending.
MCBD-Il12 is a novel bioengineered immunotherapy molecule that seems to suppress liver metastases in murine PDAC models. Further studies are required to investigate the changes in the tumour microenvironment and immune infiltrates involved.
The authors.
Cancer Research UK - Convergence Science Centre.
A. Mansurov, J. Ishihara: Financial Interests, Institutional, Stocks/Shares: Arrow Immune Inc; Financial Interests, Institutional, Other, Inventor: Patent number: US62/878,574, 2019. All other authors have declared no conflicts of interest.