Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 101-102

REPORTE CORTO/SHORT REPORT

Presented in the Congress Biotecnologia Habana 94. La Habana, Cuba, Nov. 28 - Dec. 3, 1994

MOLECULES MODULATING IMMUNE/INFLAMMATORY RESPONSES, DESIGN AND DEVELOPMENT OF POTENTIAL THERAPEUTICS

Manuel J. Arana1, Nelson Santiago1, Glay Chinea1, Belkis Torres1, Tirso Pons1, Maribel Guerra1, Eduardo Lopez2, M. Callejo3, Hilda E. Garay1, Osvaldo Reyes1.

1Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162. 2Center for Pharmaceutical Chemistry, and 3Finlay Institute, La Habana, Cuba.

Code Number: BA95042
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The role of different bacteria-derived agents and cytokines on the onset and development of immune and inflammatory responses has been broadly studied. Gram-negative bacteria LPS binding to inflammatory cell membranes is related to the activation of a cytokine cascade constituting a central pathogenic mechanism on sepsis, and also related with activation of HIV replication in monocytes and acute graft-vs-host diseases (1). Therefore the design of anti-LPS molecules as drugs may be useful in the profilaxis and treatment of LPS-mediated diseases. At the same time, based on some natural proteins that function as cytokine antagonists maintaining basic structural features of receptor ligands, some groups have engineered cytokines to obtain both partial agonists or antagonists. Studies based on structure- activity analysis of IL/2/IL-2R interactions, indicating that binding and activation events can be separated or modulated to elicit a subset of IL-2 responses excluding other (2), open the possibility to engineer IL-2 proteins with potential use as immunosupressing drugs (antagonist) or improved immunotherapeuticals (partial agonist). We briefly described herein preliminary results in using different approaches to obtain molecules modulating immune/inflammatory responses: (a) LPS neutralizing peptides, considering a charged cluster along the sequence of different LPS binding proteins, (b) IL-2 antagonists and partial agonists, using the phague displaying technology to expose hIL-2 and to select desired analogues obtained by random mutagenesis.

EXPERIMENTAL PROCEDURES

Peptides were synthesised using the tea bag method (3) and purified by RF-HPLC. Peptides were derived from: bovine BPI (KIRGKWKARKNFIK), human LPB (RVQRWKVRKSFFK), rabit CP18 (RKRLRKFRNK-IKEKLKKIGQK) and anti-LPS factor from L. poliphemus (CHYRIKPTFRRLKWKYKGKFWC). A polymyxin B (PMB) LPS- binding peptide(CKKLFKCKTK) and a HPV/E7 peptide (DMVDTGFGAMNFADLQPNK SDVPIDI) were also used. PBMC were cultured in the presence of P. aeruginose LPS 20 ng/mL and equimolar concentrations of the different peptides (1.25 and 100 nM). TNF concentrations of 18-24 h PBMC cultures were determined using L929 target cells as described (4). All DNA manipulations were performed essentially as previously described (5). The biological activity of human IL-2 was tested using the IL-2 dependent murine T lymphocyte cell line CTLL-2 (6).

RESULTS AND DISCUSSION

Peptides corresponding to particular highly positive charge clusters of the sequence of different LPS-binding proteins were all able to inhibit in a dose-depending manner the LPS induction of TNF production by human peripheral blood mononuclear cells (figure 1). These peptide sequence seem to correspond to the LPS binding sites of the proteins as previously proposed for BPI, LBP and LALF (7), similarly to positive charge oligopeptides based on the structure of PMB that were already shown to bind LPS and reduced mortality in animals (8). This result will permit us the design of stabilized peptides or hybrid proteins with potential therapeutic properties in neutralizing endotoxin. In the purpose to obtain IL-2 analogues from a collection of random IL-2 mutants we have first cloned the hIL-2A125 fused to the pelB signal peptide from E. carotova in a phagemid vector. The phage pIII/hIL-2 hybrid protein was assembled on otherwise wild-type phage capsids after been targeted to the periplasm of E. coli. The hIL-2 hybrid protein was exposed on phages maintain biological activity as tested in a CTLL-2 cell proliferation assay. Using mutD5 mutagenesis we will obtain a phage-exposed IL- 2 mutant collection from which virions will be initially picked on IL-2R subunit expressing cell lines and further selected in proper biological assays according to desired functional features.

Fig. 1.-Effect of peptides on LPS induction of TNF production in PBMC

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2. HEATON, K.M. et al. (1993). Cell. Immunol. 151: 590

3. HOOEHTEN, R. et al (1986). Biotechniques 6: 522

4. FLICK, D.A. and G. E. GIFFORD (1984). J. Immunol. Methods. 68: 167

5. SAMBROOK, J. et al. (1989). Molecular Clonning: A Laboratory Manual. 2nd ed.. Cold Spring Harbor Laboratory Press. N.Y.

6. GILLIS, S. et al. (1978). J. Immunol. 120: 2027.

7. HOESS, A. et al. (1993). EMBO J. 12: 3351

8. RUSTICI, A. et al. (1993). Science 259: 361.

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud

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