| Summary: | 碩士 === 國立成功大學 === 生物化學暨分子生物學研究所 === 101 === Angiogenesis is the process that new blood vessels form from pre-existing vessels and provides the nutrients for tumor metastasis. Significant cross-talk exists between vascular endothelial growth factor receptor-2 (VEGFR2) and integrin αvβ3 that mediate angiogenesis. The interaction between integrin αvβ3 and VEGFR2 on endothelial cell is an important process during vascularization. The functional association between VEGFR2 and integrin αvβ3 is of reciprocal nature because each receptor is able to promote activation of its counterpart. This mutually beneficial relationship regulates a number of cellular activities involved in angiogenesis, including endothelial cell migration, survival, proliferation and tube formation. Therefore, the agents that inhibit both receptors would have important therapeutic potential. Here, we used the tenth module of fibronectin type III domain (10Fn3) as a molecular scaffold to develop dual-specific proteins that bound to VEGFR2 and integrin αvβ3. Integrin αvβ3-specific 10Fn3 mutant was expressed according to our previous study. VEGFR2-specific 10Fn3 mutants were expressed according to the results of mRNA display study. These dual specific mutants were created by linking VEGFR2-specific and αvβ3-specific 10Fn3 mutants using the (G4S1)2 or (G4S1)3 linkers. Two VEGFR-specific and four dual-specific proteins were successfully expressed in E. coli and purified to homogeneity. Cell adhesion inhibition assay showed that dual-specific proteins can retain their inhibitory affinities to integrin αvβ3 with the IC50 value of ~80 nM. In angiogenesis assay, dual-specific proteins inhibited not only HUVEC proliferation with an IC50 value of ~70 nM but also tube formation with an IC50 value of ~350 nM. In particular, dual-specific proteins exhibited synergistic effect on the inhibition of VEGF-mediated HUVEC migration with the IC50 value of ~130 nM. In contrast, integrin αvβ3-specific 10Fn3 mutant exhibited 10-fold less inhibitory activity with an IC50 value of ~2449 nM, and VEGFR2-specific 10Fn3 mutant only inhibits VEGF effect. The solubility of dual-specific proteins is ~ 0.3 mg/ml that is ~93-fold less than that of αvβ3-specific 10Fn3 mutant. The themosatbility of dual-specific proteins is ~55℃ that is 30℃ less than that of αvβ3-specific 10Fn3 mutant. These results suggest that the solubility and thermostability of dual-specific proteins should be improved. This study on molecular engineering approach is providing new and improved sources of clinically relevant dual VEGFR2- and αvβ3-specific drugs.
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