The present thesis describes the development of a novel-binding molecule based on VH dimers. The objective was to profit from the good qualifications of that domain to bind antigens. The very large diversity of this domain in terms of sequence variations, as well as the higher number of residue contact interactions that generally forms with the antigen makes VH domains an ideal candidate to improve binding properties. The combination of two VHs coupled in a similar way to the VLNH would create new, non-natural antigen binding surfaces with yet undefined properties. These binders could have higher affinities than classical VL/VH, considering the number of amino acids contacts. In addition, the use of only VHs allows the possibility to form homodimers, which would provide symmetric binding surfaces that could recognisie antigen molecules with high symmetry, such as dsDNA. The aim of the present thesis was to study whether VH domains could dimerise. We found useful to know if they could be expressed by mammalian cells, bacteria and be displayed on phages. In addition, we wanted to find out if they could bind specifically an antigen. In the present thesis we demonstrate that VHs are efficiently expressed in mammalian cells as dimers showing a specific binding activity. These dimers are oriented similarly to VL/VH couples and are relatively stable even if they are not covalently linked. They can also be expressed in bacteria and displayed on phages keeping their binding specificities, thus, making them a biotechnological tool with high potentiality. Using the phage display technology we selected specific hetero- and homo-dimeric binders showing that also symmetrical surfaces are possible to be formed. In addition, we present data that strongly suggest the involvement of both domains in the antigen recognition. Other aspects related to the requirements of scVHD (single chain VH dimer) formation and purification such linkers and expression system were also investigated.
|Autori:||Sepulveda Toepfer, Jorge|
|Titolo:||VH dimers (VHDs): a novel binding molecule|
|Relatore/i esterni:||Burrone, Oscar|
|Data di pubblicazione:||11-lug-2001|
|Appare nelle tipologie:||8.1 PhD thesis|