RECOMBINANT ANTIBODIES IN BIOACTIVE PEPTIDE DESIGN

Citation
C. Monfardini et al., RECOMBINANT ANTIBODIES IN BIOACTIVE PEPTIDE DESIGN, The Journal of biological chemistry, 270(12), 1995, pp. 6628-6638
Citations number
65
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
270
Issue
12
Year of publication
1995
Pages
6628 - 6638
Database
ISI
SICI code
0021-9258(1995)270:12<6628:RAIBPD>2.0.ZU;2-Q
Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific c ellular receptors which belong to a recently described supergene famil y, These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor i nteractions, One approach to dissecting out critical intermolecular in teractions is to develop analogs of specific interaction sites of pote ntial importance, Monoclonal antibodies have been employed for these p urposes in prior studies, Here we present application of recombinant a ntibody technology to the development of analogs of a site on GM-CSF b ound by a neutralizing anti-GM-CSF monoclonal antibody, Polyclonal ant isera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice, purified immunoglobulins were prepared and u sed to immunize syngeneic mice, Anti-anti-GM-CSF was developed which d emonstrated biological antagonist activity against GM-CSF-dependent ce llular proliferation, RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymeras e chain reaction performed with primers specific for murine kappa ligh t chain V regions, Polymerase chain reaction products were cloned into the pDAB(L) vector and an expression library developed, This was scre ened with anti GM-CSF neutralizing mAb 126.213, and several binding cl ones isolated, One clone (23.2) which inhibited 126.213 binding to GM CSF was sequenced revealing a murine kappa light chain of subgroup III , Comparison of the 23.2 sequence with the human GM-CSF sequence revea led only weak sequence similarity of specific complementarity determin ing regions (CDRs) with human GM-CSF, Structural analysis revealed pot ential mimicry of specific amino acids in the CDR I, CDR II and FR3 re gions of 23.2 with residues on the B and C helices of GM-CSF. A synthe tic peptide analog of the CDR I was bound by 126.213, specifically ant agonized GM-CSF binding to cells and blocked GM-CSF bioactivity, These studies indicate the feasibility of using recombinant antibody librar ies as sources of interaction site analogs.