Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering

Abstract

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.

Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications

“…The results revealed similar tumor uptake for both molecules. However, 68 Ga-ABY-002 had a significantly lower amount of radioactivity in blood, lung, spleen and the gastrointestinal tract as compared to 111 In-ABY-002, leading to higher T/B and tumor-to-organ ratios. Accordingly, the image contrast was slightly better on the 68 Ga-ABY-002 PET images then the 111 In-ABY-002 gamma camera images of the same mice obtained one day later after decay of the 68 Ga radioactivity.…”

“…Should the affinity of the primary leads not suffice, affinity maturation generally results in improved binders and may be achieved by either helix shuffling or sequence alignment combined with directed combinatorial mutagenesis [9,10]. The newly identified molecules with their altered binding surface generally keep PET 68 Ga, 76 Br, 64 Cu 18 F, 124 I [27] SPECT 111 In, 99m Tc, 125 I 57 Co [26] Bacterial toxin PE38 [50] Fc fusion RSV [84] Optical MRI Conjugated fluorescence [47,48] Fused fluorescent protein [90] …”

“…The gamma camera images showed high contrast and beside the tumor and the kidneys no other organ was visible. ABY-002 was also efficiently labeled with the positron emitter 68 Ga. Biodistribution and PET imaging studies performed in mice bearing SKOV-3 tumor xenografts showed specific tumor targeting [37]. The tumor uptake of 68 Ga-ABY-002 at 4 h post-injection (p.i.)…”

“…co-injection of 68 Ga-and 111 In-ABY-002 in mouse xenografts allowed site-by-site comparison of the same Affibody molecule with two different radiometals, one gamma emitter and one positron emitter. The results revealed similar tumor uptake for both molecules.…”

“…The first clinical investigation of 68 Ga-and 111 In-labeld ABY-002 in three patients with metastatic breast cancer showed that HER2-specific Affibody molecules have the potential to visualize HER2-expressing metastatic lesions. High contrast SPECT or PET images were obtained already 2-3 h after injection.…”