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A highly efficient murine IL-4 antagonist as a model for rational drug design
218
Immunotherapy of allergy
Materials and Methods: Costar RIA plates were coated with the monoclonal anti human IgE antibody BSW17 and used for biopanning of phage libraries displaying random peptides from 6 to 15 amino acids in length. Both pill and pVlll libraries were screened displaying either linear or disulfide bridge constrained peptides. Phage particles strongly binding to BSW17 were enriched and analyzed by DNA sequencing. Peptides containing amino acid sequences deduced from phage display (BSW17 mimotopes) were chemically synthesized, coupled to a protein carrier (KLH) and used to immunize rabbits. Crude rabbit sera as well as IgG fractions purified by affinity chromatography on a Sepharose-mimotape peptide column were tested for the presence of specific anti human IgE antibodies. Futthemore, anaphylactogenicity of the anti IgE immune response induced by mimotope immunization on human blood ceils was determined using a soluble leukotriene release assay (CAST-ELISA). Results: Two types of mimotopes with sequence and/or structural homology to different FIX domains were identified by phage libraty screening: one mimicking the C&3 (370-375) region postulated to be involved in receptor binding (I), the other mimicking the C&4 (51X-508) region speculated to be involved in target cell triggering (3). Chemically synthesized mimotope peptides coupled to immunogenic carder proteins were found to be recognized by BSWI 7 as were peptide conjugates containing the corresponding original Fc&fragments. Immunization of rabbits with these mimotope conjugates resulted in the generation of anti peptide antibodies which also isolype-specifically recognize human IgE. Like the model antibody BSW17, the mimotope-induced anti IgE antibodies are not anaphylactogenic on human blood cells. Concludon: Mimotope peptides from random peptide phage display library screening provide immunogens for active immunization against a specific target. The anti BSW17 mimobodies are basic structures for anti allergy vaccine development
[l] Jardieu, P.; Curr. Op. Immunol. 7: 779 (1995). [2] Rudolf et al.; J. Immunol. 157: 5646 (1996). [3] Stanworth et al.; Lancet 336: 1279 (lQQ0).
0.5.13.3
T cell response to PLA2 and PLA2 derived peptldes: Modulatlon bv conventional bee venom lmmunotheraby
F. Spertini ‘, Ft.Kammerer ‘, A. Keltner 2, N. Dufour I, Y. Chvatchko l, G. Corradin 2. ’ Division of /mmunok?gyand Allergy; Centre Hospitalier Universitaire Vaudoik, Laosanne, Switzerland, 2institute of Bkxhemishy University of Lausanne, Laosanne, Switzerland Introduction: Immunological mechanisms of desensitization are still incompletely understood. Safer methods of immunotherapy with reduced risks of anaphylaxis need to be developed. The aim of this study was to evaluate the effects of conventional venom immunotherapy (VIT) on PLA2 specific T cells and on T cell reactivity to short and long synthetic peptides mapping PLA2. Meterlals and Methods: Proliferation of a CD4+ enriched PBMC fraction and cytokine secretion by T cell lines from bee venom hypersensitive patients undergoing VIT in response to PLA2 and PLA2 synthetic peptides. Result!~ T cell proliferation in response to three 40 to 60 amino acid long synthetic peptides mapping the entire PLA2 with an overlap of 10 residues (1-59, 51-99, 90-134) steadily increased during the first 14 weeks of VIT corresponding to the antigen incremental dose period. These results contrast with low proliferation indices obtained with short 15 amino acid long peptides, and the inability to characterize the immunodominant region of the molecule with short peptides. The end of VIT (3 to 5 years) conversely corresponded to a marked decrease in T cell responsiveness to PLA:! or its long synthetic peptides whereas in parallel a shift in the pattern of cytokine secretion by T cell lines from a THOto a THI-type pattern was observed. Conclusion: After a transient increase in T cell proliferative response, late VIT was characterized by T cell hyporesponsiveness to allergen and a modulation of cytokine secretion from a THOto a T~j-type pattern. Long peptides mapping the whole PLA2, by their capacity to recruit multiple T cell epitopes, appear to be efficient T cell stimulators and may represent potential candidates for peptide immunotherapy.
10.5.13.4
] A hlghly efflclent murlne IL-4 antagonist as a model for ratlonal drug deslgn
S.M. Grunewald, S. Kunzmann, 8. Schnarr, J. Ezemieks, W. Sebald, A. Duschl. Biozentrum, Physiotogische Chemie II, Am Hub/and, Wuerzburg, Germany Introduction: IL-4 is a major regulator in the immune system, which plays a central role in THYdominated diseases like type I allergies, helminthic infections and some autoimmune diseases. The inhibition of the IL-4 system should lead to central immunomodulation in these maladies, which might help to cure symptoms. To address this in mice, we developed a highly efficient murine IL-4 antagonistic protein (QY) on the basis of inhibition of receptor activation (1).
Tuesa’ay,24 June 1997 - Oral presentations
Material and Methods: The amino acids glutamine 116 and tymsine 119 of murine IL-4 were both substituted by aspartic acid residues using site directed mutagenesis. Mutated and wild type IL-4 proteins were produced in SF-9 insect cells following infection with recombinant baculoviruses. Proteins were purified and in vitro binding assays were performed with the recombinant extracellular domain of murine IL-4 receptor u using the BlAcore 2000 system (Pharmacia). Inhibition of IL-4 induced cell proliferation was determined by PHlTdR incorporation. CD23 expression on splenic B-cells was measured by a modified ELISA. After incubation of the cells with cytokines, protein tyrosine phosphorylation was determined by immunoprecipitation and immunobloting of cell lysates with specific antibodies. Results: The QY mutant binds with similar affinity to the murine lL-4R (I subunit as wild type 11-4,but is functionally inactive and completely inhibits IL-4 induced proliferation of lipopolysacchatide (LPS) stimulated splenic B cells, the T cell line CTLM and the pre-B cell line BAIF3 in a dose dependent manner. In contrast to wild type 11-4,the QY mutant shows no detectable upregulation of CD23 expression on LPS stimulated splenic B cells. The QY mutant also abolishes tyrosine phosphorylation of cellular proteins in IL-4 stimulated BAJFB cells. Conclusion: These results indicate that the QY mutant is a highly efficient antagonist for IL-4 in the mouse system. This antagonist is therefore a promising tool to study in vivo in mice, whether the inhibition of the IL-4 system is a suitable target e.g. for the development of anti allergic drugs. [I] Grunewald, SM., S. Kunzmann, B. Schnarr, J. Ezemieks, W. Sebald, and A. Duschl. 1997. A mutine IL-4 antagonistic mutant protein completely inhibits IL-4 induced cell proliferation, differentiation and signal transduction. J. Biol. Chem. 272: 1480-1483.
10.5.13.5
1 IgG complexes lnhlblt IgE production and antigen presentatlon
by human B cells
P. Baselmans, E.-M. PUlabauer, R. Bheekha Escura, J. H&k, M. Woisetschkiger, G.C. Mudde. SANLIOZ Research Institute, Vienna, Austria Introduction: Previously we reported that CD23 (Fc~Rll) expressing EBV B cells, can bind and use monomeric IgE for enhanced antigen presentation. This was in contrast to the low affinity FcyRll receptor (CD32), which was not able to bind either monomeric or complexed IgG sufficiently enough for antigen uptake and presentation. Here, we report that the same IgG complexes, which mediate antigen presentation through monocytic cells, inhibit B cell function, both at the level of antigen presentation and at the level of antibody production. Msterials and Methods: ChimeticNIP specificantibodiesof IgE, IgGl and lgG3 were used to study 1) antigen uptake and presentation, 2) their influence on IgE switch induction by normal human B cells after in vitro antigen specific cognate stimulation, with HLA matched Th2 cells and after non specific stimulation by aCD40 + 11-4.IgE and IgG antibody titers were measured by ELISA. Results: The addition of IgG containing antigen complexes to cultures of EBV-B cells, which presented antigen to specific T cell clones, either via antigen pinocytosis or IgE mediated antigen uptake caused inhibition of T cell proliferation. In addition, normal human tonsillar B cells, stimulated by Th2 cells in an antigen specific and cognate system, were completely blocked in their ability to produce IgE or IgG antibodies. Moreover, non specific stimulation of antibody production by B cells by aCD40 + IL-4 was also blocked by the addition of IgG antigen complexes. The negative regulation of IgG complexes was confirmed by using aCD32 antibodies. Conclusions: The data confirmthe observationthat IgE antibodiesenhance the immune response, by CD23 mediated antigen presentation of B cells, whereas IgG antibodies prevent antigen presentation and antibody production by B, while directing the immune response to antigen presenting cells which express CD64 and or CD32a.
0.5.13.6
Intervention In house dust mite allergic responses wlth bacteria expresslng epltopes of allergens
R. Janssen ‘, C. Hetzel 2, S. Ely I, D. Young ‘, J. Lamb 2, J. Thole I. ’ Department of medical Micmbiollogy,Imperial College School of Medickm at St. Mary’s, Norfolk Place, London, UK, 2Depsrtment of Biology, Imperial College of Science, Technology and medicine, Prince Consort Road, London, UK Introduction: T-cellsplaya majorrole in regulation of the allergic response. The majority of allergen-specific CD4+ T-cells in atopic individuals produce Th2 -type cytokines such as 114,whereas those of non-atopic individuals produceThl-type cytokines like IFNy. Since mycobacteria and salmonellae have been shown to be potent inducers of Thl -type responses we are investigating whether immune responses to allergens can be modulated by recombinant bacteria expressing epitopes of allergens. Materials and Methods: A dominant CD4 epitope of the house dust mite allergen Der pl was expressed as part of the mycobacterial super oxide
Immunotherapy of allergy
Materials and Methods: Costar RIA plates were coated with the monoclonal anti human IgE antibody BSW17 and used for biopanning of phage libraries displaying random peptides from 6 to 15 amino acids in length. Both pill and pVlll libraries were screened displaying either linear or disulfide bridge constrained peptides. Phage particles strongly binding to BSW17 were enriched and analyzed by DNA sequencing. Peptides containing amino acid sequences deduced from phage display (BSW17 mimotopes) were chemically synthesized, coupled to a protein carrier (KLH) and used to immunize rabbits. Crude rabbit sera as well as IgG fractions purified by affinity chromatography on a Sepharose-mimotape peptide column were tested for the presence of specific anti human IgE antibodies. Futthemore, anaphylactogenicity of the anti IgE immune response induced by mimotope immunization on human blood ceils was determined using a soluble leukotriene release assay (CAST-ELISA). Results: Two types of mimotopes with sequence and/or structural homology to different FIX domains were identified by phage libraty screening: one mimicking the C&3 (370-375) region postulated to be involved in receptor binding (I), the other mimicking the C&4 (51X-508) region speculated to be involved in target cell triggering (3). Chemically synthesized mimotope peptides coupled to immunogenic carder proteins were found to be recognized by BSWI 7 as were peptide conjugates containing the corresponding original Fc&fragments. Immunization of rabbits with these mimotope conjugates resulted in the generation of anti peptide antibodies which also isolype-specifically recognize human IgE. Like the model antibody BSW17, the mimotope-induced anti IgE antibodies are not anaphylactogenic on human blood cells. Concludon: Mimotope peptides from random peptide phage display library screening provide immunogens for active immunization against a specific target. The anti BSW17 mimobodies are basic structures for anti allergy vaccine development
[l] Jardieu, P.; Curr. Op. Immunol. 7: 779 (1995). [2] Rudolf et al.; J. Immunol. 157: 5646 (1996). [3] Stanworth et al.; Lancet 336: 1279 (lQQ0).
0.5.13.3
T cell response to PLA2 and PLA2 derived peptldes: Modulatlon bv conventional bee venom lmmunotheraby
F. Spertini ‘, Ft.Kammerer ‘, A. Keltner 2, N. Dufour I, Y. Chvatchko l, G. Corradin 2. ’ Division of /mmunok?gyand Allergy; Centre Hospitalier Universitaire Vaudoik, Laosanne, Switzerland, 2institute of Bkxhemishy University of Lausanne, Laosanne, Switzerland Introduction: Immunological mechanisms of desensitization are still incompletely understood. Safer methods of immunotherapy with reduced risks of anaphylaxis need to be developed. The aim of this study was to evaluate the effects of conventional venom immunotherapy (VIT) on PLA2 specific T cells and on T cell reactivity to short and long synthetic peptides mapping PLA2. Meterlals and Methods: Proliferation of a CD4+ enriched PBMC fraction and cytokine secretion by T cell lines from bee venom hypersensitive patients undergoing VIT in response to PLA2 and PLA2 synthetic peptides. Result!~ T cell proliferation in response to three 40 to 60 amino acid long synthetic peptides mapping the entire PLA2 with an overlap of 10 residues (1-59, 51-99, 90-134) steadily increased during the first 14 weeks of VIT corresponding to the antigen incremental dose period. These results contrast with low proliferation indices obtained with short 15 amino acid long peptides, and the inability to characterize the immunodominant region of the molecule with short peptides. The end of VIT (3 to 5 years) conversely corresponded to a marked decrease in T cell responsiveness to PLA:! or its long synthetic peptides whereas in parallel a shift in the pattern of cytokine secretion by T cell lines from a THOto a THI-type pattern was observed. Conclusion: After a transient increase in T cell proliferative response, late VIT was characterized by T cell hyporesponsiveness to allergen and a modulation of cytokine secretion from a THOto a T~j-type pattern. Long peptides mapping the whole PLA2, by their capacity to recruit multiple T cell epitopes, appear to be efficient T cell stimulators and may represent potential candidates for peptide immunotherapy.
10.5.13.4
] A hlghly efflclent murlne IL-4 antagonist as a model for ratlonal drug deslgn
S.M. Grunewald, S. Kunzmann, 8. Schnarr, J. Ezemieks, W. Sebald, A. Duschl. Biozentrum, Physiotogische Chemie II, Am Hub/and, Wuerzburg, Germany Introduction: IL-4 is a major regulator in the immune system, which plays a central role in THYdominated diseases like type I allergies, helminthic infections and some autoimmune diseases. The inhibition of the IL-4 system should lead to central immunomodulation in these maladies, which might help to cure symptoms. To address this in mice, we developed a highly efficient murine IL-4 antagonistic protein (QY) on the basis of inhibition of receptor activation (1).
Tuesa’ay,24 June 1997 - Oral presentations
Material and Methods: The amino acids glutamine 116 and tymsine 119 of murine IL-4 were both substituted by aspartic acid residues using site directed mutagenesis. Mutated and wild type IL-4 proteins were produced in SF-9 insect cells following infection with recombinant baculoviruses. Proteins were purified and in vitro binding assays were performed with the recombinant extracellular domain of murine IL-4 receptor u using the BlAcore 2000 system (Pharmacia). Inhibition of IL-4 induced cell proliferation was determined by PHlTdR incorporation. CD23 expression on splenic B-cells was measured by a modified ELISA. After incubation of the cells with cytokines, protein tyrosine phosphorylation was determined by immunoprecipitation and immunobloting of cell lysates with specific antibodies. Results: The QY mutant binds with similar affinity to the murine lL-4R (I subunit as wild type 11-4,but is functionally inactive and completely inhibits IL-4 induced proliferation of lipopolysacchatide (LPS) stimulated splenic B cells, the T cell line CTLM and the pre-B cell line BAIF3 in a dose dependent manner. In contrast to wild type 11-4,the QY mutant shows no detectable upregulation of CD23 expression on LPS stimulated splenic B cells. The QY mutant also abolishes tyrosine phosphorylation of cellular proteins in IL-4 stimulated BAJFB cells. Conclusion: These results indicate that the QY mutant is a highly efficient antagonist for IL-4 in the mouse system. This antagonist is therefore a promising tool to study in vivo in mice, whether the inhibition of the IL-4 system is a suitable target e.g. for the development of anti allergic drugs. [I] Grunewald, SM., S. Kunzmann, B. Schnarr, J. Ezemieks, W. Sebald, and A. Duschl. 1997. A mutine IL-4 antagonistic mutant protein completely inhibits IL-4 induced cell proliferation, differentiation and signal transduction. J. Biol. Chem. 272: 1480-1483.
10.5.13.5
1 IgG complexes lnhlblt IgE production and antigen presentatlon
by human B cells
P. Baselmans, E.-M. PUlabauer, R. Bheekha Escura, J. H&k, M. Woisetschkiger, G.C. Mudde. SANLIOZ Research Institute, Vienna, Austria Introduction: Previously we reported that CD23 (Fc~Rll) expressing EBV B cells, can bind and use monomeric IgE for enhanced antigen presentation. This was in contrast to the low affinity FcyRll receptor (CD32), which was not able to bind either monomeric or complexed IgG sufficiently enough for antigen uptake and presentation. Here, we report that the same IgG complexes, which mediate antigen presentation through monocytic cells, inhibit B cell function, both at the level of antigen presentation and at the level of antibody production. Msterials and Methods: ChimeticNIP specificantibodiesof IgE, IgGl and lgG3 were used to study 1) antigen uptake and presentation, 2) their influence on IgE switch induction by normal human B cells after in vitro antigen specific cognate stimulation, with HLA matched Th2 cells and after non specific stimulation by aCD40 + 11-4.IgE and IgG antibody titers were measured by ELISA. Results: The addition of IgG containing antigen complexes to cultures of EBV-B cells, which presented antigen to specific T cell clones, either via antigen pinocytosis or IgE mediated antigen uptake caused inhibition of T cell proliferation. In addition, normal human tonsillar B cells, stimulated by Th2 cells in an antigen specific and cognate system, were completely blocked in their ability to produce IgE or IgG antibodies. Moreover, non specific stimulation of antibody production by B cells by aCD40 + IL-4 was also blocked by the addition of IgG antigen complexes. The negative regulation of IgG complexes was confirmed by using aCD32 antibodies. Conclusions: The data confirmthe observationthat IgE antibodiesenhance the immune response, by CD23 mediated antigen presentation of B cells, whereas IgG antibodies prevent antigen presentation and antibody production by B, while directing the immune response to antigen presenting cells which express CD64 and or CD32a.
0.5.13.6
Intervention In house dust mite allergic responses wlth bacteria expresslng epltopes of allergens
R. Janssen ‘, C. Hetzel 2, S. Ely I, D. Young ‘, J. Lamb 2, J. Thole I. ’ Department of medical Micmbiollogy,Imperial College School of Medickm at St. Mary’s, Norfolk Place, London, UK, 2Depsrtment of Biology, Imperial College of Science, Technology and medicine, Prince Consort Road, London, UK Introduction: T-cellsplaya majorrole in regulation of the allergic response. The majority of allergen-specific CD4+ T-cells in atopic individuals produce Th2 -type cytokines such as 114,whereas those of non-atopic individuals produceThl-type cytokines like IFNy. Since mycobacteria and salmonellae have been shown to be potent inducers of Thl -type responses we are investigating whether immune responses to allergens can be modulated by recombinant bacteria expressing epitopes of allergens. Materials and Methods: A dominant CD4 epitope of the house dust mite allergen Der pl was expressed as part of the mycobacterial super oxide