Investigating Proteolytic Cleavage of Drug-Linker Construct Targeting Plasmin with Anticancer Benzosuberene-Based Payload KGP18
Introduction
Pancreatic cancer is a highly aggressive malignant neoplasm that most often presents as advanced or metastatic disease. Despite recent advances in therapeutic treatment, pancreatic cancer is the third leading cause of cancer-related death in the U.S. with an estimated 12% 5-year survival. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 90% of all pancreatic cancers. In PDAC, proteases of the urokinase plasminogen activation system including plasmin are upregulated and strongly implicated in cancer metastasis, where they contribute to digestion of tissue barriers of the extracellular matrix (ECM) to promote tumor invasion. Plasmin is activated after cleavage of its precursor plasminogen by tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). As proof of principle, a synthetic drug-linker construct containing plasmin substrate properties was evaluated for its ability to release the incorporated cytotoxic anticancer payload KGP18 after plasmin cleavage. The payload has demonstrated potent vascular disruption and antimitotic activities.
Methods
The drug-linker construct was comprised of a plasmin-targeting tripeptide D-Ile-Phe-Lys linked to the maleimidocaproyl (MC) as a site for protein attachment, coupled with payload KGP18 with subnanomolar cytotoxicity via the self-immolative para-aminobenzyloxycarbonyl (PABC) spacer tethered to dimethylethylenediamine (DMED) carbonyl (MC-D-Ile-Phe-Lys-DMED-KGP18). Plasmin activity has preferential proteolytic cleavage at positively charged amino acid residues such as -Arg and-Lys and was assessed with the fluorogenic substrate D-Val-Leu-Lys-7-amino-4-methylcoumarin (D-VLK-AMC) with Tris buffered saline, pH 8, containing 0.1% Triton-X 100 at 37 ̊C. Substrate and released products were assessed by HPLC with 5-95% aqueous acetonitrile gradient containing 0.1% formic acid (drug-linker RT: 25.3 min and payload KGP18 RT: 29.4 min). Mechanism of action of the payload was investigated for its effects in the human cancer pancreatic cell line PANC-1 in a wound healing assay monitored with automated time lapse Biotek Lionheart microscopy.
Results
The stability of plasmin was confirmed and kinetic parameters (KM 308.2 ± 1.0 µM, Vmax 8.4 µM/sec) were determined at pH 8 with fluorogenic substrate D-VLK-AMC. The drug linker solubility was optimized for the cleavage reaction in Tris buffered saline pH 8, including 20% DMSO and 10% 1,2-propanediol. Plasmin cleavage of MC-D-Ile-Phe-Lys-DMED-KGP18 resulted in the release of payload as confirmed by HPLC. Subnanomolar cytotoxicity of KGP18 in PANC-1 cells (IC50 = 0.13 ±0.02 nM) was assessed in a sulforhodamine B (SRB) assay. The payload KGP18 inhibited PANC-1 cells migration and proliferation in a wound healing assay.
Conclusion
The drug linker MC-D-Ile-Phe-Lys-DMED-KGP18 was shown to be cleavable by plasmin and suitable for conjugation with phosphatidylserine (PS)-targeting antibodies and betabodies (BBs). BBs are uniquely designed to target PS, present at significant levels in the outer leaflet of plasma membranes of tumor cells and the tumor vasculature, whereas PS is largely confined to the inner leaflet in normal cells. The payload KGP18 showed subnanomolar cytotoxicity and potent cell migration and proliferation inhibition activities in PANC-1 cells.