MAbTope: define and secure your epitope
What we need:
We are better than the other methods
Mapping the epitope of Golimumab on TNFα
Prediction - Docking
For the docking step, we need structures of the Ab and the target. TNFα forms a trimer that has been crystallized. On the other hand, we modeled the structure of the Golimumab. Here is shown the top-ranked pose among 108 generated by MAbTope. Four peptides were designed on the interface and used to proceed to experimental validation below.
Experimental validation 1 - Peptides validation
The purpose of the first round of validation was to determine which peptide(s) was (were) binding the Golimumab. Using first HTRF (Cisbio Bioassay’s system), we highlighted an energy transfer between P3 (which biotin was bound to an acceptor-fused streptavidin) and the Golimumab (detected by a cryptate-coupled anti-IgG). This observation was confirmed when spotting the peptides on a micro-array slide where P3 specifically bound the iFlur680-coupled Golimumab.
Experimental validation 2 - Alanine scanning
We proceeded to a second round of validation to identify the amino-acid residues within P3 which were actively implicated in the Golimumab interaction. We designed mutant variants of P3, replacing by Alanines the residues we suspected to be implicated. As shown in the graph, the mutants m1, m3 and m4 seemed to lose the interaction. On the contrary, m2 and m5 did not exhibited lower interaction with the Golimumab.
We identified here the sequence between aa169 and 183 of the TNFα as targeted but the Golimumab. Our mutation strategy further identified Y172, T174 and K175 as being the residues directly implicated in this interaction.