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Development of a Recombinant Monospecific Anti-PLGF Bivalent Nanobody and Evaluation of it in Angiogenesis Modulation

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Abstract

During the past two decades, tumor therapy based on monoclonal antibody has been found as a confident therapeutic approach in solid tumors and hematologic malignancies. Nanobodies are the smallest fragment of an antigen-binding domain in heavy chain-only antibody originated from the Camelidae family. Accordingly, they are being recently developed rapidly as diagnostic and therapeutic agents. In this regard, targeting of angiogenic factors like Placenta growth factor (PLGF) via nanobodies show a high effectiveness. In the current study, we developed a recombinant anti-PLGF bivalent nanobody based on the affinity enhancement mutant form of anti-PLGF nanobody to suppress the angiogenesis progression. Thereafter, the bivalent nanobody (bi-Nb) was cloned and then expressed into a bacterial system. Afterward, the purity was authorized using western blot assay and the affinity was assessed using ELISA. In this regard, proliferation, 3D capillary tube formation, and migration assays were employed as functional assays. The obtained data were analyzed using t-test and P < 0.05 was considered as statistically significant. The results indicate that the bivalent nanobody could inhibit proliferation, mobility, and formation of endothelial cell capillary-like structure. Moreover, the EC50 was estimated for endothelial cell’s proliferation and capillary tube’s formation to be about 100 ng/ml and 65 ng/ml, respectively. Migration of MCF-7 was inhibited as about 69%, rather than the control. Accumulation of data have shown that targeting of angiogenic factors like VEGF via monoclonal antibodies or nanobodies can be useful in the suppression of tumor progression. Also, the inhibition of PLGF with monoclonal antibody indicated that it is significant in angiogenesis suppression. However, due to intrinsic properties of nanobodies, they are suggested to be used. Since the small size is rapidly removed through liver or kidney system, so it is important to use bivalent or polymeric forms for extending the half-life. Our findings indicated that the inhibition of PLGF can prevent growth and proliferation of endothelial cells and tumor cells through the bivalent nanobody. So, it is suggested as a novel therapeutic agent for angiogenesis suppression.

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Abbreviations

AMD:

Age-related macular degeneration

Ang:

Angiopoietin

bi-Nb:

Bivalent nanobody

bi-Nb-PLGF-pHEN6c:

Anti-PLGF bivalent nanobody-pHEN6c

CML:

Chronic myeloid leukemia

DMEM:

Dulbecco’s modified eagle medium

EGF:

Epidermal growth factor

ELISA:

Enzyme-linked immunosorbent assay

HGF:

Hepatocyte growth factor

HIF-1α:

Hypoxia-inducible factor-1 alpha

HRP-conjugated antibody:

Horse radish peroxidase-conjugated antibody

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PDGF:

Platelet-derived growth factor

PI3K:

Phosphatidylinositol-3 kinase

PLGF:

Placenta growth factor

PTM:

Post-translational modification

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

VEGF:

Vascular endothelial growth factor

VEGFR:

Vascular endothelial growth factor receptor

VHH:

Variable domain of the heavy chain of a heavy chain antibody

VWF:

Von Willebrand factor

MCF-7:

Michigan Cancer Foundation-7(a breast cancer cell line)

MTT:

3, 4, 5-Dimethylthiazol-2-yl-2–5-diphenyltetrazolium bromide

Ni–NTA:

Nickel ion-nitrilotriacetic acid

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

IgA:

Immunoglobulin A

IgG:

Immunoglobulin G

IPTG:

Isopropyl β-D-1 thiogalactopyranoside

TB medium:

Terrific broth medium

TMB:

3, 3′, 5, 5′-Tetramethyl benzidine

TNF-α:

Tumor necrosis factor alpha

HUVEC:

Human umbilical vein endothelial cell

DAB:

Diaminobenzidine

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Acknowledgements

This project was financially supported by North Khorasan University of Medical Sciences .and National Institute for medical research development (Nimad).

Funding

This project was financially supported by North Khorasan University of Medical Sciences, Bojnurd, Iran.

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Author notes

  1. Abolfazl Nikooharf and Roghaye Arezumand have equal first author role.

Authors and Affiliations

  1. Department of Medical Biotechnology and Molecular Science, North Khorasan University of Medical Science, Bojnurd, Iran

    Abolfazl Nikooharf, Roghaye Arezumand, Amir Hossein Khoshi & Hassan Namdar Ahmadabad

  2. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

    Kamran Mansouri

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  1. Abolfazl Nikooharf
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  2. Roghaye Arezumand
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  3. Kamran Mansouri
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  4. Amir Hossein Khoshi
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  5. Hassan Namdar Ahmadabad
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Contributions

RA: management the research group and analysis the result and writing the manuscript. AN: perform the assays and initial analysis and writing the manuscript. KM: help to performing the angiogenesis assays. AHK: writing the draft of manuscript. HNAA: writing the draft of manuscript.

Corresponding author

Correspondence to Roghaye Arezumand.

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Nikooharf, A., Arezumand, R., Mansouri, K. et al. Development of a Recombinant Monospecific Anti-PLGF Bivalent Nanobody and Evaluation of it in Angiogenesis Modulation. Mol Biotechnol 62, 580–588 (2020). https://doi.org/10.1007/s12033-020-00275-7

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