Production and Conjugation of Truncated Recombinant Diphtheria Toxin to VEGFR-2 Specific Nanobody and Evaluation of its Cytotoxic Effect on PC-3 Cell Line.
Cation exchange chromatography
Immunotoxin
Immunotoxin therapy
MTT
Prostate cancer
Targeted therapy
Journal
Molecular biotechnology
ISSN: 1559-0305
Titre abrégé: Mol Biotechnol
Pays: Switzerland
ID NLM: 9423533
Informations de publication
Date de publication:
Nov 2022
Nov 2022
Historique:
received:
21
11
2021
accepted:
25
03
2022
pubmed:
29
4
2022
medline:
30
9
2022
entrez:
28
4
2022
Statut:
ppublish
Résumé
Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay. Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 μg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells.
Identifiants
pubmed: 35478310
doi: 10.1007/s12033-022-00485-1
pii: 10.1007/s12033-022-00485-1
doi:
Substances chimiques
Antineoplastic Agents
0
Cyclohexanes
0
Diphtheria Toxin
0
Immunotoxins
0
Single-Domain Antibodies
0
Sodium Dodecyl Sulfate
368GB5141J
Nickel
7OV03QG267
KDR protein, human
EC 2.7.10.1
Vascular Endothelial Growth Factor Receptor-2
EC 2.7.10.1
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1218-1226Subventions
Organisme : Iran University of Medical Sciences
ID : 33778
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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