In vitro studies 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).

Keywords: antibody directed enzyme prodrug therapy; carboxypeptidase G2; glucarpidase; ligand directed enzyme prodrug therapy; targeted cancer therapy.

Figure 1. Schematic representation of the Ligand-Directed Enzyme Pro-Drug Therapy (LDEPT) strategy.

Tumor cells that express cancer specific receptors (such as aminopeptidase N “APN”) are bound via a specific ligand (such as CNGRC) that is attached via a linker to a therapeutic enzyme (such as CPG2) that can convert an inactive pro-drug (ZD2767P) to a cytotoxic compound at the site of tumor cells.

Figure 2

Structures of the prodrug compound (ZD2676P) used in this study and the cytotoxic compound produced following carbopeptidase G2 “CPG2”-mediated hydrolysis and removal of a glutamic acid moiety.

Figure 3. Cloning and production of CNGRC-CPG2 fusion proteins.

(A) Schematic representations of the structures of the constructed fusion proteins. (B) Left-hand panel: SDS-PAGE analysis of the purified fusion proteins, lanes A, B and C show purified WT, single and double fusion proteins respectively. Right-hand panel: western blotting with anti-CPG2 antibody.

Figure 4. Catalytic activity and kinetics of CPG2 and its fusion protein derivatives.

(A) E. coli BL21 (DE3) RIL cells expressing different CNGRC-CPG2 fusion proteins grown on folate supplemented agar plates. The yellow precipitates indicate CPG2 proteolytic activity. (+) and (-) are positive and negative controls respectively. P. CPG2: Pseudomonas putida CPG2 was used as a further positive control. (B) A graph showing the rate of methotrexate “MTX” hydrolysis by CNGRC-CPG2 proteins. Equal amounts of the proteins were used in these assays.

Figure 5. Far UV circular dichroism profiles of CPG2 and its fusion derivatives with the distribution of the β sheets and α-helix.

The figure shows combined molar ellipticity data for CPG2 (red line), the single fused construct (darker blue line) and the double fused construct (cyan line). ‘smooth 0’: CPG2, ‘smooth 1: X-CPG2, and ‘smooth 3′: X-CPG2-X. The obtained spectral data were corrected for the baseline buffer.

Figure 6

(A) Immunogenicity of CPG2 and its fusion protein derivatives. Human PBMCs from four donors were used as described in the Materials & Methods section. Cells treated with fusion proteins (X-CPG2 and X-CPG2-X) showed no significant differences in immunogenicity compared with the control groups. Student t-test used for the resulting values and relative comparison with the control non-treated cells “^* P < 0.05, ^** P < 0.001” (B) Stability of CPG2 and its fusion protein derivatives in human serum at 37°C. The catalytic activity of the proteins was assayed every 48 h over a period of 14 days.

Figure 7. The levels of APN expression in various cancer cell lines.

Left-hand panel: Different cancer cell lines were labeled with anti-CD13 antibody and the percentage of CD13 expression was determined as described in the Materials & Methods section. One way ANOVA with post hoc Tukey’s test for the resulting values presented with the following symbols indicating statisitical significance difference “p < 0.001” in comparison to the stated cell lines: * to MDA-MB231, # to MCF-7, $ to HepG2, & to T47D, % to SW620, b to MDA-MB 468, m to LoVo, £ to H661, ¥ to FaDu, § to SCC25 and ∆ to HT1080. Right-hand panels: fluorescence imaging of two low (MDA-MB 231 and A549) and two high (HT1080 and MDA-MB 468) APN expressing cell lines. An antibody specific for CD13 was detected using a secondary antibody labelled with Alexa fluor 488 (red) while nuclei were labelled with DAPI (blue).

Figure 8. In vitro binding assay of CPG2 and its fusion protein derivatives to cancer cells expressing low (A549) and high (HT1080) levels of APN.

Binding was measured as described in the Materials & Methods section.

Figure 9. Methotrexate detoxification by CPG2 and its fusion protein derivatives using cells expressing high- or low-levels of APN.

Cancer cell lines expressing low levels of APN (A549, MDA-MB231 and FaDu) or high levels of APN (HT1080, HepG2 and MDA-MB468) were treated with MTX. Cell viability was then measured as described in the Materials & Methods section. Two groups served as negative (non-MTX treated) and positive (treated with MTX only) controls. The results show the percentage of viable cells obtained following MTT assay. “student t-test ^* P < 0.001 relative to the non-treated control”.

Figure 10. Cell killing mediated by CPG2 and its fusion protein derivatives following addition of the prodrug ZD2767P.

(A) Cancer cell lines, expressing low or high levels of APN, were pre-incubated with CPG2 or its fusion protein derivatives, prior to addition of ZD2767P. Cell viability was then measured as described in the Materials & Methods section. “student t-test ^* P < 0.001 relative to the non-treated control”. (B) same as A but at different concentrations of the prodrug.

Figure 11. The use of a cytotoxic drug and a prodrug to demonstrate two uses of the LDEPT strategy.

In (A), accumulation of CPG2 on the surface of cancer cells leads to removal of methotrexate (MTX) thereby protecting cells from killing. In principle, this effect could be used to remove an excess of MTX. In (B) enzyme-mediated conversion of a prodrug to a cytotoxic compound leads to tumor killing.