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.
Bès
Cédric
C
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
Troadec
Samuel
S
Chentouf
Myriam
M
Breton
Hélène
H
Lajoix
Anne Dominique
AD
Heitz
Frédéric
F
Gross
René
R
Plückthun
Andreas
A
Chardès
Thierry
T
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