Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands

We have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands, To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3, CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ, Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display me selected several CTLA-4-based variants capable of binding to human alpha v beta 3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.