Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor

doi:10.1016/j.molimm.2011.11.013

Molecular Immunology

Volume 50, Issues 1–2, February 2012, Pages 35-41

https://doi.org/10.1016/j.molimm.2011.11.013 Get rights and content

Abstract

Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics.

Highlights

► VEGFR2 is an important tumor-associated receptor on the endothelial cells. ► Blockade of the VEGFR2with nanobody lead to the inhibition of neovascularization. ► VEGFR2-specific nanobody can recognize antigen on the cell surface by FACS. ► VEGFR2 nanobody potently inhibited endothelial tube formation.

Introduction

Angiogenesis plays an important role in the growth, invasion and metastasis of cancer. Blockade of angiogenesis is an attractive approach for the treatment of this disease (Folkman, 2007). Recent research has focused on the development of antibodies (Abs) and small molecules that target the tumor-associated endothelial cells (Youssoufian et al., 2007, Zhang et al., 2009). One of the important tumor-associated receptors on the endothelial cells is vascular endothelial growth factor receptor-2 (VEGFR2, fetal liver kinase-1, FLK1, or kinase-insert domain receptor, KDR). VEGFR2 belongs to the human VEGF receptor 1–3 family, which has a strong tyrosine kinase activity and transduces the Vascular Endothelial Growth Factor (VEGF) signals in endothelial cells, producing the downstream signaling that leads to cell proliferation, tube formation, the inhibition of apoptosis, and eventually tumor progression (Olsson et al., 2006). Because the interaction of VEGF with its receptors seems to be essential for tumor angiogenesis, blockade of the VEGF receptor signaling may lead to the inhibition of neovascularization and tumor metastasis.

Serum of camelidae contains an important fraction of functional antibodies, called heavy-chain antibodies that are naturally devoid of light chains. Camelid heavy-chain antibodies, therefore, recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody™ (Nb) (Muyldermans et al., 2009, Rahbarizadeh et al., 2011). Unique hydrophilic amino acids within the framework-2 region of the VHH make that Nanobodies act as autonomous single-domain antigen-binding Nanobodies. In addition, the hypervariable regions [i.e. complementary determining regions (CDRs)] of Nanobodies are on average longer than those of conventional antibodies, most probably to compensate for the absence of the antigen-binding regions of the light chain (Bond et al., 2003). Nanobodies have many inherent, advantageous properties, such as low molecular mass (15 kDa), a strict monomeric behavior, low immunogenicity, high affinity, high solubility and stability and high yield expression of recombinant VHH in bacteria or yeasts (Buelens et al., 2010). These characteristics make VHHs useful next-generation reagents in immunoassays and for therapeutic applications (Goldman et al., 2006, Saerens et al., 2008). As with other recombinant antibody fragments, VHH fragments isolated from hyper-immunized or naïve libraries are highly specific based on the recognition of unique epitopes on target antigens. Antigen-specific Nanobodies have been reported for a wide-range of targets ranging in size from immunogenic proteins as part of cells, parasites or viruses, to individual enzymes or toxins and to low-molecular weight haptens (Doyle et al., 2008, Hmila et al., 2010, Lafaye et al., 2009, Roovers et al., 2007, Thys et al., 2010).

In this paper, we present the first examples of VEGFR2-specific Nanobodies and demonstrate the ability of one of these Nanobodies, named 3VGR19, to bind VEGFR2 on the cell surface. Furthermore, we show that Nanobody 3VGR19 inhibits capillary tube formation in vitro.

Section snippets

Cell lines and proteins

HEK293, 293KDR and HUVECs were used in this study. 293KDR is a stably transfected cell line expressing about 2.5 × 10⁶ VEGFR2 per cell (Backer and Backer, 2001). HEK293 and 293KDR were grown in DMEM medium supplemented with 10% FBS. HUVECs were grown in M199 medium supplemented with 10% FBS and Low Serum Growth Supplement (Invitrogen). Cultures were maintained on plastic flask and incubated at 37 °C in 5% CO₂.

Recombinant extracellular domain of VEGFR2 was purchased from R&D system.

Cell based-immunization and serum response

Two young male

Immunization and serum respond

To raise an immune response against VEGF receptor type 2, we immunized camels with a stably transfected cell line expressing high levels of VEGFR2. Blood was collected before each injection, and the immune response was monitored by ELISA using serial dilutions of the camel sera. Specific antibody titers raised rapidly after the second injection as evaluated on methanol fixed 293KDR cells (Fig. 1, panel A) and on recombinant VEGFR2 (Fig. 1, panel B). These data demonstrated the successful

Discussion

Angiogenesis plays an important role in the growth, invasion and metastasis of solid tumors. Not surprisingly, anti-angiogenesis treatment has proven to be an effective treatment for solid tumors (Folkman, 1972). Anti-angiogenesis treatments are based on restricting neovascularization through blocking the interaction between vascular growth factors and vascular endothelial cells in tumor tissues, thereby inducing necrosis and preventing tumor growth and metastasis. The most important vascular

Acknowledgments

The authors wish to express their deep gratitude to all who provided support during the course of this research, especially Jan Van Gompel in CMIM and Parviz Rohi in ASRI. We thank the cellular and molecular interaction department-VUB for providing fellowship for M.B. to come to Belgium. This project was financially supported by the Pasteur Institute of Iran.

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Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor

Abstract

Highlights

Introduction

Section snippets

Cell lines and proteins

Cell based-immunization and serum response

Immunization and serum respond

Discussion

Acknowledgments

Mol. Immunol.

FEBS Lett.

J. Immunol. Methods

J. Mol. Biol.

J. Thromb. Haemost.

Biochem. Biophys. Res. Commun.

Mol. Immunol.

Cell. Signal.

Exp. Mol. Pathol.

Mol. Immunol.

Dev. Comp. Immunol.

J. Invest. Dermatol.

Biochem. Biophys. Res. Commun.

J. Biotechnol.

Vet. Immunol. Immunopathol.

J. Control. Release

Biochem. Biophys. Res. Commun.

Curr. Opin. Pharmacol.

Antiviral Res.

Biochem. Biophys. Res. Commun.

Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins

Bioconjug. Chem.

Development of second-generation VEGFR tyrosine kinase inhibitors: current status

Curr. Oncol. Rep.

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae

Antimicrob. Agents Chemother.