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 we selected several CTLA-4-based variants capable of binding to human alphavbeta3 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.
Hufton
S E
SE
Target Quest B.V; Provisorium, Maastricht, The Netherlands.
van Neer
N
N
van den Beuken
T
T
Desmet
J
J
Sablon
E
E
Hoogenboom
H R
HR
D000069594:Abatacept; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D060908:CTLA-4 Antigen; D015195:Drug Design; D015723:Gene Library; D006801:Humans; D018796:Immunoconjugates; D008024:Ligands; D017510:Protein Folding; D019030:Receptors, Vitronectin / Q000737:chemistry; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D008024:Ligands; D019030:Receptors, Vitronectin; D000069594:Abatacept