University of Twente Student Theses


Targeting integrins for self-stimulating cells with BMP-7 by using bispecific VHHs

Grobbink, A.M. (2021) Targeting integrins for self-stimulating cells with BMP-7 by using bispecific VHHs.

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Abstract:Osteoarthritis (OA) is one of the most common chronic health conditions and causes major disability worldwide. There is an urgent need to develop a minimally invasive, disease modifying treatment for the regeneration of cartilage. To date, BMP-7 has proven to be one of the most effective growth factors in enhancing the anabolic activity of chondrocytes. In this proof of principle study, bispecific VHHs were used to target Integrin β1 (ITGB1) and BMP-7 simultaneously to enable controlled BMP-7 delivery in vitro as a possible therapy for OA. First, the binding affinity of the two bispecific VHH (G7-FSH-14F8 and G7-WHR-3C9) was characterized with surface plasmon resonance imaging (SPRi). To localize and confirm binding and dual binding, immunofluorescence assays were performed. Pathway activation after BMP-7 stimulation in presence or absence of VHHs was assessed with luciferase, qPCR, and western blot assays. SPRi showed a high binding affinity of both bispecific VHHs for ITGB1 and BMP-7. Immunofluorescent assays showed binding of FSH-14F8 and Bi14F8 towards ITGB1 located at the focal adhesions and resulted in reduced binding of cells, whereas WHR-3C9 and Bi3C9 showed less impact on cell stretching and binding towards ITGB1 showed a diffuse signal over the entire cell surface. Furthermore, luciferase and qPCR assays resulted in higher expression levels after stimulation with Bi3C9, compared to Bi14F8. In conclusion, it was shown that both VHHs can be exploited for long term BMP-7 delivery. This study suggests that Bi3C9 showed preferable characteristics and should be used in future in vitro experiments. To target chondrocytes in vivo, bispecific VHHs targeting BMP-7 and a tissue-specific protein need to be developed. This thesis has clearly shown that bispecific VHHs can be used for targeted delivery of BMP-7 in vitro and shows great promise for disease-modifying treatments for the regeneration of cartilage.
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Programme:Biomedical Engineering MSc (66226)
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