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 light chain V regions. Polymerase chain reaction products were cloned into the pDAB 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 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.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant GM-46400. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by a grant by the American Cancer Society and a NIH Cancer Center grant to the Wistar Institute.

¶

Supported by a National Research Development Award.

**

Supported by NIH Grant RO1 CA 31615.

§§

Supported by grants from the American Foundation for AIDS Research and NIH.

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The abbreviations used are: mAb, monoclonal antibodies; CDRs, complementarity determining regions; GM-CSF, granulocyte-macrophage colony-stimulating factor; PCR, polymerase chain reaction; IL, interleukin; cpm, counts/minute; IPTG, isopropyl--thio-galactopyranoside; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; rAb, recombinant antibody.

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VonFeldt, J. M., Monfardini, C., Fich, S., Rosenbaum, H., Kieber-Emmons, T., Williams, R. M., Kahn, S. A., Weiner, D. B., and Williams, W. V.(1995) Pept. Res., in press.

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