PIN-bodies: a new class of antibody-like proteins with CD4 specificity derived from the protein inhibitor of neuronal nitric oxide synthase.
By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.
CNRS UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, Faculté de Pharmacie, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France. cedric.bes@cpbs.univ-montp1.fr
Cédric
C
Bès
Samuel
S
Troadec
Myriam
M
Chentouf
Hélène
H
Breton
Anne Dominique
AD
Lajoix
Frédéric
F
Heitz
René
R
Gross
Andreas
A
Plückthun
Thierry
T
Chardès
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D017951:Antigen Presentation / Q000187:drug effects; D015704:CD4 Antigens / Q000276:immunology*; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D013601:T-Lymphocytes / Q000187:drug effects
C110023:13B8-2 monoclonal antibody; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D010455:Peptides; D004398:Dyneins