Recombinant Antibodies in Bioactive Peptide Design

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody. Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDABL vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones 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 revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.

Toward a Better Understanding of the Basis of the Molecular Mimicry of Polysaccharide Antigens by Peptides

“…11). This is in agreement with previous observations showing that aromatic amino acids are considered as ideal residues for mimicking glycan side chain structures (69,86) and that ␤-turn/extended structures may be accurate conformational mimics of helices (87,88). Yet, except for the number of residues involved, discriminating between the binding modes of the three peptides remains difficult.…”

Construction and Binding Kinetics of a Soluble Granulocyte-Macrophage Colony-stimulating Factor Receptor α-Chain-Fc Fusion Protein

“…The purified DNA was checked using two different pairs of restriction enzymes (BamHI/SalI and BamHI/ XbaI), and the expected size of the inserts was confirmed on a 2% analytical agarose gel (ϳ350 bp and ϳ4,750 bp for the BamHI/SalI digestion, and ϳ1,200 -1,300 bp, ϳ2,500 bp, and ϳ4,300 bp for the BamHI/XbaI digestion). The sequence of the insert was also confirmed by sequencing according to previously published protocols (20).…”

Rational Design of Granulocyte-Macrophage Colony-stimulating Factor Antagonist Peptides

“…Molecular modeling of the rAb 23.2 allowed the identification of complementarity determining regions (CDRs) as sites of structural mimicry of GM-CSF, focusing attention on the CDRI region mimicking residues on the B and C helices of GM-CSF. After synthesis and characterization of CDRI, CDRII, and CDRII peptides, the CDRI peptide exhibited specific GM-CSF receptor binding and antagonist bioactivity (28). Thus, these studies suggest that residues on the B and C helices of GM-CSF mediate binding to the low affinity receptor (GM-CSFR␣ alone).…”

“…This implicates these sites in intermolecular interactions with the GM-CSFR. We also have used a recombinant antibody (rAb) as a GM-CSF mimic (28). Molecular modeling of the rAb 23.2 allowed the identification of complementarity determining regions (CDRs) as sites of structural mimicry of GM-CSF, focusing attention on the CDRI region mimicking residues on the B and C helices of GM-CSF.…”

“…Here we describe the development of two cyclic peptide GM-CSF mimics (1785 and 1786) obtained from structural analysis of the GM-CSF region mimicked by rAb 23.2 (28). Cysteines were introduced in the peptide structures at the amino and carboxyl termini to allow cyclization.…”