Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering.
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 (KD). 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.
Sandström
K
K
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 90 Stockholm, Sweden.
Xu
Z
Z
Forsberg
G
G
Nygren
P-A
PA
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000276:immunology / Q000378:metabolism; D018122:B7-1 Antigen / Q000276:immunology / Q000378:metabolism*; D015374:Biosensing Techniques; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism*; D018968:CD58 Antigens / Q000378:metabolism; D016466:CHO Cells; D060908:CTLA-4 Antigen; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D015398:Signal Transduction / Q000502:physiology*; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D018106:CD28 Antigens; D018968:CD58 Antigens; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D008024:Ligands; D019151:Peptide Library; D013205:Staphylococcal Protein A