Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-01-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/3/625 |
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| author | Mohammed S. Aldughaim Fatimah Alsaffar Michael D. Barker |
| author_facet | Mohammed S. Aldughaim Fatimah Alsaffar Michael D. Barker |
| author_sort | Mohammed S. Aldughaim |
| collection | DOAJ |
| description | 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 <i>Escherichia coli</i> 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. |
| first_indexed | 2024-03-09T03:41:53Z |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T03:41:53Z |
| publishDate | 2021-01-01 |
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| series | Molecules |
| spelling | doaj.art-d61fc340f4644a22b9433b336669b82c2023-12-03T14:40:14ZengMDPI AGMolecules1420-30492021-01-0126362510.3390/molecules26030625Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour SiteMohammed S. Aldughaim0Fatimah Alsaffar1Michael D. Barker2Research Centre, King Fahad Medical City, P.O. Box 59046, Riyadh 11525, Saudi ArabiaDepartment of Oncology and Metabolism, Medical School, University of Sheffield, Beech Hill Rd, Sheffield S10 2RX, UKDepartment of Oncology and Metabolism, Medical School, University of Sheffield, Beech Hill Rd, Sheffield S10 2RX, UKBroad-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 <i>Escherichia coli</i> 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.https://www.mdpi.com/1420-3049/26/3/625angiogenesiscarboxypeptidase G2TIMP3VEGFR2 |
| spellingShingle | Mohammed S. Aldughaim Fatimah Alsaffar Michael D. Barker Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site Molecules angiogenesis carboxypeptidase G2 TIMP3 VEGFR2 |
| title | Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site |
| title_full | Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site |
| title_fullStr | Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site |
| title_full_unstemmed | Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site |
| title_short | Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site |
| title_sort | coupling of a novel timp3 peptide to carboxypeptidase g2 for pro drug activation at the tumour site |
| topic | angiogenesis carboxypeptidase G2 TIMP3 VEGFR2 |
| url | https://www.mdpi.com/1420-3049/26/3/625 |
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