Investigating the intracellular interactions of clec14a and the characterisation of monoclonal antibodies targeting clec14a

Lodhia, Puja (2016). Investigating the intracellular interactions of clec14a and the characterisation of monoclonal antibodies targeting clec14a. University of Birmingham. Ph.D.

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Abstract

CLEC14A is a tumour endothelial marker known to regulate sprouting angiogenesis. While the extracellular interactions of CLEC14A have previously been studied, the intracellular interactions of CLEC14A are unknown. Fascin was identified as a binding partner for the cytoplasmic tail of CLEC14A using a yeast two hybrid screen. Interaction of CLEC14A with fascin was confirmed by proximity ligation and co-localisation was observed in HUVEC filopodia. This data indicated that interaction of CLEC14A and fascin may be important for filopodia formation during sprouting angiogenesis. Binding studies with domain deletion mutants of fascin revealed the CLEC14A binding site to be located within a highly conserved region of the β-trefoil 3 domain between amino acids 323 and 384. In addition, phosphorylation of S274 was found to regulate this interaction.
Five monoclonal antibodies against CLEC14A had the potential to be developed into anti-angiogenic cancer therapeutics. The functional properties of these antibodies were explored in \(in\) \(vitro\) assays. Clones 1 and 3 were found to inhibit cell migration while clone 4 disrupted tubule formation. Clones 3 and 4 were developed into antibody drug conjugates (ADCs). These ADCs demonstrated potent cytotoxicity localised to the tumour endothelium \(in\) \(vivo\). These results indicate that targeting CLEC14A could be an effective strategy to disrupt the tumour vasculature.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):