Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides

We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based prote in scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linke d neuropeptide. Upon expression as periplasmic-targeted proteins in Escheri chia coli, molecules with superior solubility characteristics to the wild-t ype V-domain were produced. These mutations in CTLA-4 ablated binding to it s natural ligands CD80 and CD86, whereas binding to a conformation-dependen t anti-CTLA-4 monoclonal antibody showed that the V-domain framework remain ed correctly folded. Secondly, to develop a system for library selection, w e displayed both wild-type and mutated CTLA-4 proteins on the surface of fd -bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on pha ge bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step pa nning enriched 5,000 to 2,600-fold respectively over wild-type phage, Bacte riophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifi cally bound somatostatin receptors on transfected CHO-K1 cells preincubated with 1 mu g/ml tunicamycin to remove receptor glycosylation. Binding was s pecific, as 1 mu M somatostatin successfully competed with CTLA-4R- Som3, C TLA-4R- Som3 also activated as well as binding preferentially to non-glycos ylated receptor subtype Sst4. The ability to substitute CDR-like loops with in CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. (C) 1999 Wiley-Liss, Inc.