Production of active long lasting CNGRC-CPG2 fusion protein using PEGylation to be used in Ligand Directed Cancer Therapy

Abstract

Aminopeptidase N (APN) is one of the important enzymes highly expressed in metastatic cancers, thus employed as a marker to target tumor cells. CPG2-CNGRC fusion protein is produced to target high APN expressing cancer cells, which with the prodrug results in high toxic effect. Since PEGylation of CPG2 has shown an improved favorable in vitro stability and immunotoxicity, we performed a site-directed PEGylation (thiol group directed) of the CPG2-CNGRC fusion protein and examined the effect of PEGylation on the resulting fusion protein's therapeutic efficacy. CPG2 kinetic activity was substantially enhanced following PEGylation of the single fusion protein (PEG CPG2-CNGRC). The binding affinity of the produced PEGylated fusion proteins to their cellular marker (APN) was notably reduced in case of the double fusion protein compared with non-PEGylated ones. Moreover, the cytotoxic effect of methotrexate and ZD2767P (prodrug) in association of the PEGylated fusion proteins was investigated and found that the cytotoxic effect of prodrug with PEGylated single fusion protein was improved significantly (low cell survival). Similar finding was found following MTX treatment where lower binding and kinetic activity of the PEGylated double fusion proteins resulted in higher MTX toxic effect (lower cell survival) in comparison with the non-PEGylated double fusion protein. Thus, although PEGylation is known for its usually favorable effect on the protein/drug pharmacodynamics, our results indicated that with our different fusion proteins (single and double fusion proteins) PEGylation improved their properties differintially.