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

Abstract

Objectives: Aminopeptidase N (APN) is an enzyme highly expressed in metastatic cancers and could be used in targeted cancer therapy. Our previous work showed the successful construction of CNGRC-carboxypeptidase G2 (CPG2) and CNGRC-CPG2-CNGRC fusion proteins. Our conjugates and prodrugs were effective in targeting high APN-expressing cancer cells. In the present study, we aim to produce long-acting fusion proteins to overcome 2 of the main drawbacks of antibody-directed enzyme prodrug therapy. Methods: N-terminal and N-, C-terminal fusion CPG2, CNGRC-CPG2, and CNGRC-CPG2-CNGRC, respectively, were PEGylated using polyethylene glycol (PEG) maleimide (40K). We examined the effect of PEGylation on the therapeutic efficacy of the new products. The resulting PEGylated fusion proteins were tested for their stability, ex vivo immunotoxicity, binding capacity to their target on high HT1080, and low A549 APN-expressing cells. The catalytic activity of the resulting PEGylated fusion CPG2 proteins was investigated. Pro-drug "ZD2767P" cytotoxic effect in association with PEG CPG2-CNGRC fusion proteins on cancer cells was studied. Results: Our work demonstrated that the properties of the PEGylated single-fused proteins were significantly improved over that of un-PEGylated fused CPG2, and its kinetic activity and APN-binding affinity were not negatively affected by the PEGylation. Significantly, The PEGylated single-fused CPG2 had lower immunogenicity than the un-PEGylated CPG2. Our results, however, were different in the case of the PEGylated double-fused CPG2. Although its stability in human serum under physiological conditions was not significantly affected, the kinetic activity and its binding affinity to their cellular marker (APN) were substantially reduced. When the study was performed with high and low APN-expressing cancer cell lines, using the prodrug ZD2767p, the PEGylated fusion CPG2 demonstrated cancer cell killing effects. Conclusion: We have successfully produced PEGylated-CNGRC-CPG2, which is bioactive and with lower immunogenicity in ligand-directed enzyme prodrug therapy for cancer treatment.

Keywords: PEGylated CPG2 conjugates; aminopeptidase N; immunogenicity; ligand-directed enzyme prodrug therapy; long-acting drugs; targeted cancer therapy.

Figure 1.

Identification of PEGylated CPG2 proteins. The resulting PEG–CPG2 fusion proteins (WT “PEG-CPG2”, single “PEG X-CPG2” and double “PEG X-CPG2-X” fusion proteins) were separated on SDS-PAGE and stained with Coomassie brilliant blue. Un-PEGylated proteins (WT, X-CPG2, and X-CPG2-X) in lanes 1, 3, and 5, and PEGylated proteins are shown in lanes 2, 4, and 6, respectively. Abbreviations: PEG, polyethylene glycol; CPG2, carboxypeptidase G2.

Figure 2.

Circular dichroism spectra of CPG2 fusion proteins before and after PEGylation. (A) The combined far UV spectra of CPG2 “WT”, single “X-CPG2” and double “X-CPG2-X” fusion proteins. (B) The combined far UV spectra of PEG CPG2, PEG X-CPG2, and PEG X-CPG2-X fusion proteins. (C) Table presents the results of CDNN deconvulation analysis of the PEGylated and non-PEGylated CPG2 fusion proteins CD spectra, showing their secondary structure composition. Darker shades indicate an increase in the percentage composition. Abbreviations: UV, ultraviolet; PEG, polyethylene glycol; CPG2, carboxypeptidase G2; CD, circular dichroism.

Figure 3.

Enzyme kinetics of the PEGylated and non-PEGylated CPG2 fusion proteins. (A) The catalytic activity of CPG2 presented in the graph of MTX hydrolysis rate by CPG2. (B) The enzyme kinetics parameters (K_m, V_max, and K_cat) of the PEGylated and non-PEGylated CPG2 fusion proteins, Graph pad PRISM 6 software was used to calculate the parameters. (C) Michaelis–Menten plots of the CPG2 (in fusion proteins) enzymatic action on MTX hydrolysis. Abbreviations: MTX, methotrexate; PEG, polyethylene glycol; CPG2, carboxypeptidase G2.

Figure 4.

PEGylated CPG2 fusion proteins stability in human serum. Serum samples from healthy donors were incubated at 37 °C with 0.1 µL/µg of the purified PEGylated and non-PEGylated CPG2 fusion proteins. The enzymatic (MTX hydrolytic) activity of CPG2 in each sample was tested every 48 h, and the percentage of remaining catalytic activity was plotted following normalization to a 100% activity at 0 time point. Abbreviations: MTX, methotrexate; PEG, polyethylene glycol; CPG2, carboxypeptidase G2.

Figure 5.

Ex vivo T-cell proliferation assay. Study of the immunotoxicity of PEGylated and non-PEGylated CPG2 fusion proteins. PBMCs from normal healthy donors were co-incubated with the CPG2 fusion proteins “10 µg/mL”, in addition to the vehicle (PBS) as negative control and LPS as positive control. After 48 h cells incubated with the resulting CCK-8 solution for 4 h, and the resulting absorbance at 450 nm plotted for each donor (D). Abbreviation: LPS: lipolysaccharide; MTX, methotrexate; PEG, polyethylene glycol; CPG2, carboxypeptidase G2.

Figure 6.

In vitro binding strength of PEGylated and non-PEGylated CPG2 fusion proteins to the APN-expressing cancer cell lines. Low “A549” and high “HT1080” APN expressing cancer cell lines were cultured in 96-well plate with either PBS (as negative control) or increasing concentrations of the purified PEGylated and non-PEGylated CPG2 fusion proteins (0.063, 0.127, 0.192, and 0.255 µM). After 1 hour, cells were washed twice and ELISA assay using HRP-tagged anti-His6 antibody was utilized to explore the binding capacities of the PEGylated fusion proteins. Abbreviations: APN, Aminopeptidase N; PEG, polyethylene glycol; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay

Figure 7.

The effect of MTX on cell viability following treatment with PEGylated and non-PEGylated fusion proteins. High “HT1080, HepG2 and MDA-MB468” and low “A549, MDA-MB231 and FaDu” APN-expressing cancer cell lines were incubated with the purified PEGylated fusion proteins, followed by treatment with MTX along with PBS “ negative control” and MTX only “positive control for 48 h. The MTT assay was employed to obtain the percentage of cell viability. The resulting survival results are normalized to the negative control (taken as 100% survival). Abbreviations: MTX, methotrexate; APN, aminopeptidase N; PEG, polyethylene glycol.

Figure 8.

The cytotoxic effect of the prodrug ZD2767P in combination with PEGylated and non-PEGylated fusion proteins. Cancer cell lines expressing high and low APN levels were treated with the prodrug ZD2767P following incubation with the purified PEGylated fusion proteins. Cells were treated with PBS as negative control, and then the cell viability level was assessed using MTT assay. The resulting survival results are normalized to the negative control as 100% survival. Abbreviations: APN, aminopeptidase N; PEG, polyethylene glycol.