Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A.
Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.
Chen
Ting
T
Dyax Corp, 300 Technology Square, Cambridge, MA 02139, USA.
Hogan
Shannon
S
Conley
Greg
G
Pazmany
Csaba
C
Wu
Qi-Long
QL
McNeil
Gary L
GL
Bassill
Gary
G
Macintyre
Andrew N
AN
Martik
Diana
D
Nixon
Andrew E
AE
Sexton
Daniel J
DJ
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology* / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011266:Pregnancy-Associated Plasma Protein-A / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D013379:Substrate Specificity
D000906:Antibodies; D019151:Peptide Library; D011266:Pregnancy-Associated Plasma Protein-A