Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site.

Mohammed S Aldughaim,Michael D Barker,Fatimah Alsaffar

doi:10.3390/molecules26030625

Abstract

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

Highlights

Broad-spectrum cytotoxic drugs, such as nitrogen mustards, have been the mainstay of cancer therapy for many years
In antibody directed enzyme prodrug therapy (ADEPT), enzymes that convert prodrugs to active drugs are first targeted to the cancer site by a tumour-specific monoclonal antibody
Solubility testing was carried out to determine whether the carboxypeptidase G2 (CPG2) was expressed predominantly in the cytosol or in inclusion bodies

Summary

Introduction

Broad-spectrum cytotoxic drugs, such as nitrogen mustards, have been the mainstay of cancer therapy for many years. Such drugs target cancer cells but all proliferating cells, resulting in severe side-effects, which limits dosage and potential efficacy in the long-term [1]. In ADEPT, enzymes that convert prodrugs to active drugs are first targeted to the cancer site by a tumour-specific monoclonal antibody. After clearance of the enzyme from normal tissue, the prodrug is administered to be activated at the cancer site [5]. ADEPT is advantageous due to the accumulation of the enzyme–antibody conjugate within the tumour vasculature after clearance from normal tissues

Methods

Results

Discussion

Conclusion