Design and Cytotoxicity Evaluation of a Cancer-targeting Immunotoxin Based on a Camelid Nanobody-PE Fusion Protein

Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy. This study aimed to engineer a recombinant chimeric fusion protein by combining a nanobody-targetin...

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Veröffentlicht in:Iranian journal of immunology Jg. 21; H. 4; S. 302
Hauptverfasser: Khoshbakht, Mona, Forghanifard, Mohammad Mahdi, Aghamollaei, Hossein, Amani, Jafar
Format: Journal Article
Sprache:Englisch
Veröffentlicht: Iran Shiraz Institute for Cancer Research 31.12.2024
Schlagworte:
ADP Ribose Transferases - genetics
ADP Ribose Transferases - immunology
Animals
Annexin V
Apoptosis
Apoptosis - drug effects
Bacterial Toxins - immunology
Breast Neoplasms - immunology
Breast Neoplasms - therapy
Camelus
Cancer
Cell Line, Tumor
Cell proliferation
Cytotoxicity
Drug resistance
Endoplasmic Reticulum Chaperone BiP
Enzyme-linked immunosorbent assay
Exotoxins
Exotoxins - immunology
Exotoxins - pharmacology
Female
Flow cytometry
Fusion protein
Heat-Shock Proteins - genetics
Heat-Shock Proteins - immunology
HEK293 Cells
Humans
Immunotoxins
Immunotoxins - pharmacology
Internalization
MCF-7 Cells
Nanobodies
Neoplasms - immunology
Neoplasms - therapy
Proteins
Pseudomonas aeruginosa Exotoxin A
Recombinant Fusion Proteins - pharmacology
Single-Domain Antibodies - immunology
Single-Domain Antibodies - pharmacology
Tumor cell lines
Virulence Factors - immunology
Western blotting
GRP78
Nanobody
Recombinant Protein
Exotoxin
Breast Cancer
ISSN:1735-1383, 1735-367X, 1735-367X
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Zusammenfassung:Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy. This study aimed to engineer a recombinant chimeric fusion protein by combining a nanobody-targeting domain with an exotoxin effector domain. The chimeric protein was designed to bind surface-expressed GRP78 on cancer cells, facilitating internalization and inducing apoptosis to inhibit proliferation and survival. Using a flexible linker, we designed two constructs linking VHH nanobody domains to Pseudomonas exotoxin (PE) domains II, III, and Ib. These constructs were then optimized for expression in E. coli BL21 (DE3) using the pET28a vector. Following the expression of the recombinant proteins, we purified them and tested their binding capability, cytotoxicity, and ability to induce apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7, as well as in control cell lines HEK-293 and MDA-MB-468. The binding affinity was measured using a cell-based ELISA, internalization was assessed through Western blotting, cytotoxicity was evaluated by an MTT assay, and apoptosis was determined using flow cytometry with an Annexin V kit. The immunotoxin specifically bound to cancer cells expressing csGRP78. The results of the cytotoxicity test showed that the cytotoxic effect of two constructs, I and II, depended on concentration and time. With an increase in both components, the effect of recombinant proteins also increased. Both constructs were able to penetrate and induce apoptosis in csGRP78+ cells. These immunotoxin structures showed therapeutic potential against GRP78-expressing cancers, making them suitable candidates for targeted therapy pending in vivo studies.
Bibliographie:ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:1735-1383
1735-367X
1735-367X
DOI:10.22034/iji.2024.104052.2878