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

• Published in: Proteins, structure, function, and bioinformatics Vol. 36; no. 2; pp. 217 - 227
• Main Authors: Nuttall, Stewart D., Rousch, Mat J. M., Irving, Robert A., Hufton, Simon E., Hoogenboom, Hennie R., Hudson, Peter J.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01.08.1999
• Subjects: Abatacept; Amino Acid Sequence; Animals; Antigens, CD - metabolism; Antigens, Differentiation - biosynthesis; Antigens, Differentiation - chemistry; Antigens, Differentiation - genetics; Antigens, Differentiation - metabolism; B7-1 Antigen - metabolism; B7-2 Antigen; bacteriophage; Bacteriophages - genetics; Bacteriophages - metabolism; Binding, Competitive; Capsid - biosynthesis; Capsid - genetics; CHO Cells; Cloning, Molecular - methods; Cricetinae; CTLA-4; CTLA-4 Antigen; display; Glycosylation; Humans; Immunoconjugates; Membrane Glycoproteins - metabolism; Membrane Proteins; Models, Molecular; Molecular Sequence Data; peptide; Peptide Library; Peptides - metabolism; Protein Binding; Protein Engineering; Protein Folding; receptor; Receptors, Somatostatin - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Solubility; somatostatin; Somatostatin - biosynthesis; Somatostatin - chemistry; Somatostatin - genetics; Somatostatin - metabolism
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/(SICI)1097-0134(19990801)36:2<217::AID-PROT7>3.0.CO;2-X

We have designed and engineered the human cytotoxic T‐lymphocyte associated protein‐4 (CTLA‐4) variable (V‐like) domain to produce a human‐based protein 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‐linked neuropeptide. Upon expression as periplasmic‐targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild‐type V‐domain were produced. These mutations in CTLA‐4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation‐dependent anti‐CTLA‐4 monoclonal antibody showed that the V‐domain framework remained correctly folded. Secondly, to develop a system for library selection, we 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 phage bound specifically to immobilized CD80‐Ig and CD86‐Ig and in one‐step panning enriched 5,000 to 2,600‐fold respectively over wild‐type phage. Bacteriophage displaying CTLA‐4 with somatostatin in CDR3 (CTLA‐4R‐Som3) specifically bound somatostatin receptors on transfected CHO‐K1 cells pre‐incubated with 1μg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 μM somatostatin successfully competed with CTLA‐4R‐Som3. CTLA‐4R‐Som3 also activated as well as binding preferentially to non‐glycosylated receptor subtype Sst4. The ability to substitute CDR‐like loops within CTLA‐4 will enable design and construction of more complex libraries of single V‐like domain binding molecules. Proteins 1999;36:217–227. © 1999 Wiley‐Liss, Inc.