Thesis presented March 31, 2022

Abstract:
Screening of a VHH cDNA library raised from alpacas immunised with a lysate of T24 cells (human urine bladder cancer cell line over-expressing CD105), with soluble CD105 by phage display and phage ELISA resulted in 19 VHHs that bind to human CD105 protein, an endothelial co-receptor of TFG-β involved in the regulation of angiogenesis and tumour development. The selected anti-CD105 VHH cDNAs were amplified, sequenced and analysed with ExPASY, GeneDoc and BLAST tools. The nineteen sequences showed the typical structural features of a VHH and all of them were different. The VHH cDNAs were subcloned from the phagemid vector pHEN2 into pET22b(+) vector for expression and purification of recombinant VHH proteins by affinity chromatography on Ni-NTA agarose columns. Thirteen recombinant VHH proteins maintained the ability to bind CD105 as determined by ELISA. These anti-CD105 VHHs were microarrayed on biochips to evaluate their specificity and affinity to CD105 membrane-bound on cells by Surface Plasmon Resonance imaging (SPRi). SPRi data showed that the recombinant VHHs bind SC cells that overexpress CD105 on their surface but not or little THP-1 cells that do not, proving their specificity. The average of SPRi reflectivity variation among the 13 VHHs at 2 hours of assays was 4.59 with SC cells and 2.69 with the THP-1 cells, cell density average was 578 cells/area with SC cells and 367 cells/area with THP-1 cells and the average of the approximation to the apparent affinity constant was 110.19 x 10^-2 with SC cells and 106.26 x 10^-2 with THP-1 cells. The VHH were grouped according to their specificity and affinity approximation, six had high specificity and affinity approximation (3 of them even higher than the positive control), five were specific but had low affinity approximation and two had low specificity and affinity approximation. The most efficient (six top-ranked) anti-CD105 VHHs, that bind with high affinity and specificity to CD105 in solution (sCD105) and on cells (membrane-bound CD105), are proposed as CD105 detection tools for research both in studies that seek a better understanding of the CD105 function in vitro and in vivo and in application as nanoprobes for the diagnosis and therapeutics of diseases in which angiogenesis plays an important role.

Keywords:
VHH, CD105, SPRi, biochip, nano-probes

On-line thesis.