In vitrostudies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy

Abstract

The sequence asparagine-glycine arginine (NGR), flanked by Cysteine (Cys) residues so as to form a disulfide-bridge (CNGRC), has previously been found to target and bind specifically to aminopeptidase N (APN), which is highly expressed on the surface of tumor cells. The goal of this study was to develop and evaluate the potential of fusion proteins carrying the CNGRC sequence linked to the enzyme carboxypeptidase G2 (CPG2) for targeted cancer therapy. We refer to this strategy as ligand-directed enzyme prodrug therapy (LDEPT). We constructed two forms of the CNGRC-CPG2 fusions, containing one or two copies of the cyclic NGR motif and designated CNGRC-CPG2 (X-CPG2) and CNGRC-CPG2-CNGRC (X-CPG2-X), respectively. In vitro binding assays of the purified constructs showed that both X-CPG2 and X-CPG2-X bound with high affinity to cancer cells expressing high levels of APN, compared to their binding to cells expressing low levels of APN. Further in vitro studies of the constructs to assess the therapeutic potential of LDEPT were carried out using cells expressing high and low levels of APN. Using methotrexate, it was demonstrated that cancer cell survival was significantly higher in the presence of the fusion proteins, due to the hydrolysis of this cytotoxic drug by CPG2. Conversely, when the prodrug ZD2767P was used, cancer cell killing was higher in the presence of the fused CPG2 constructs than in their absence, which is consistent with CPG2-mediated release of the cytotoxic drug from the prodrug. Furthermore, the doubly-fused CPG2 construct (X-CPG2-X) was significantly more effective than the singly-fused construct (X-CPG2).

Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

“…The single and double CPG2–CNGRC fusion proteins produced showed “biobetter” features in binding specificity, stability and in vitro cytotoxicity. 18 Several PEGylation strategies have been established to improve the therapeutic potency of proteins/drugs through enhancing their stability and lowering immunotoxicity. 21 To enhance the therapeutic efficacy and to reduce the protein immunogenicity of the produced fusion proteins here we conjugated the previously generated CNGRC–CPG2 fusion proteins with PEG polymer and studied the effect of PEGylation on the anti-cancer targeting effectiveness.…”

“… 21 Two different studies found that conjugation of CPG2 either with the fusion peptide (CNGRC) or PEG polymer formed fusion proteins and PEGylated CPG2, respectively with higher serum stability and lower ex vivo immunotoxicity compared with the nonfused or non-PEGylated CPG2. 10 , 18 In this study, we produced new PEGylated fused CPG2 proteins which tested for their stability and immunotoxicity. Our data showed that the serum stability of the PEGylated CPG2 fusion proteins was not changed in relation to the non-PEGylated ones ( Figure 4 ).…”

“…As in our previous study, 18 we used 2 cell lines as models for high (HT1080) and low (A549) APN-expressing cell lines. In a 96 well plate 25 000 cells/well were seeded, and the following day the cells were fixed with 4% paraformaldehyde.…”

“…13 Several studies have exploited such cancer marker binding peptides to design a tumor targeted drug and/or enzyme and to overcome the low specificity of anticancer therapy. [14][15][16][17] Our recent study presented a developed fusion protein using the APN-binding peptide (CNGRC) conjugated to the therapeutic enzyme CPG2, 18 where the resulting constructs; N-terminal single and N-, C-termini double fusion proteins found to provide new therapeutic fusion proteins that can be used in the liganddirected enzyme prodrug therapy (LDEPT). The fusion proteins produced showed notably enhanced enzyme kinetics of CPG2 and protein stability, and lowered ex vivo immunotoxicity compared to those of the nonfused CPG2 (wild type (WT)).…”

Tumor-Targeting Peptides: The Functional Screen of Glioblastoma Homing Peptides to the Target Protein FABP3 (MDGI)

“…A previous study showed that adding an extra cysteine residue to a cyclic nonapeptide, iRGD (CRGDK/RGPD/EC), improved the tumor penetrating function [33]. In addition, one cysteine residue may interact with cysteine(s) in other peptides to form peptide complexes [34]. It is also possible that the single cysteine residue in the original CooP sequence interacts with the cysteine residue (Cys 125 ) on the FABP3 sequence to stabilize the complex.…”