Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering
2003 (English)In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 16, no 9, 691-697 p.Article in journal (Refereed) Published
CD28 is one of the key molecules for co-stimulatory signalling in T cells. Here, novel ligands (affibodies) showing selective binding to human CD28 (hCD28) have been selected by phage display technology from a protein library constructed through combinatorial mutagenesis of a 58-residue three-helix bundle domain derived from staphylococcal protein A. Analysis of selected affibodies showed a marked sequence homology and biosensor analyses showed that all investigated affibodies bound to hCD28 with micromolar affinities (K-D). No cross-reactivity towards the related protein human CTLA-4 could be observed. This lack of cross-reactivity to hCTLA-4 suggests that the recognition site on hCD28 for the affibodies resides outside the conserved MYPPPYY motif. The apparent binding affinity for hCD28 could be improved through fusion to an Fc fragment fusion partner, resulting in a divalent presentation of the affibody ligand. For the majority of selected anti-CD28 affibodies, in co-culture experiments involving Jurkat T-cells and CHO cell lines transfected to express human CD80 (hCD80) or LFA-3 (hLFA-3) on the cell surface, respectively, pre-incubation of Jurkat cells with affibodies resulted in inhibition of IL-2 production when they were co-cultured with CHO (hCD80(+)) cells, but not with CHO (hLFA-3(+)) cells. For one affibody variant denoted Z(CD28:5) a clear concentration-dependent inhibition was seen, indicating that this affibody binds hCD28 and specifically interferes in the interaction between hCD28 and hCD80.
Place, publisher, year, edition, pages
2003. Vol. 16, no 9, 691-697 p.
affibody, CD28, CD80, combinatorial, co-stimulation, CTLA-4, Fc-fusion, inhibition, LFA-3, ligands, phage display, selection, bacterial receptor domain, monoclonal-antibody, escherichia-coli, fusion proteins, t-cells, binding, libraries, construction, cd28, rejection
IdentifiersURN: urn:nbn:se:kth:diva-22911DOI: 10.1093/protein/gzg086ISI: 000186148300008OAI: oai:DiVA.org:kth-22911DiVA: diva2:341609
QC 201005252010-08-102010-08-10Bibliographically approved