Growth of solid tumors is dependent on successful angiogenesis, in which the key factor is the vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). VEGF, following binding to a specific receptor (KDR, a.k.a. VEGFR2) undergoes cellular internalization. Endothelial cells of tumor vessels, as opposed to those lining up normal blood vesels, are characterized by an increased expression of VEGFR2 receptors.

The aim of this study was to investigate a novel two-domain protein, the cognitive part of which allows directing the chimeric protein to endothelial cells lining up tumor blood vessels whereas the other, effector domain would entail destruction of targeted cells.

We constructed a synthetic gene coding for a chimeric protein ABRaA-VEGF₁₂₁ which combines abrin A chain (ABRaA - a plant toxin that inactivates ribosomes [Wang et al., 2004]) linked to human VEGF₁₂₁ via a short aminoacid spacer (G₄S). ABRaA-VEGF₁₂₁ expression was carried out in E. coli BL21(DE3) strain. The protein was isolated from insoluble fraction as inclusion bodies, then it was solubilized and purified (>95%). Additionally, LPS was removed from ABRaA-VEGF₁₂₁ preparations (<0.0025 EU/ 1μg protein). The obtained protein migrates, under non-reducing conditions, as a ~84kDa homodimer and a ~42kDa monomer; it shows immunoreactivity towards anti-hVEGF₁₂₁ monoclonal antobodies.

ABRaA-VEGF₁₂₁ shows strong cytotoxicity towards PAE/KDR cells overexpressing KDR receptor (LC₅₀ = 0.067 µg/ml), and induces in them apoptotic death, as opposed to PAE cells overexpressing VEGFR1 receptor (PAE/hFlt-1) or wild PAE cell line (LC₅₀ ≈ 27.3 µg/ml).

ABRaA-VEGF₁₂₁ inhibits protein biosynthesis in a cell-free protein translation system. Preincubation of ABRaA-VEGF121 with anti-hVEGF₁₂₁ monoclonal antobodies eliminated cytotoxicity associated with this protein. This indicate that both domains are biologically active.

In vivo studies of anticancer properties of ABRaA-VEGF₁₂₁ demonstrated inhibition of tumor growth, as observed in the B16(F10) murine melanoma tumor model. Histochemical analysis demonstrated selective destruction of tumor vessels mediated by ABRaA-VEGF₁₂₁.

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