Patent EA-004228-B1 (EA)
Title: Immune response modulator alpha-2 macroglobulin complex
Inventor: PIZZO SALVATORE V
IPC: A61K47/48
Filed: 1999-04-01
Abstract: 1. A stable complex comprising at least one intact biomolecule with a nucleophilic group and activated alpha2 macroglobulin having an intact bait region, wherein each of said intact biomolecule with a nucleophilic group is bound to said alpha2 macroglobulin via a bond, consisting of said nucleophilic group covalently bound to gamma-glutamyl group of said cleaved thiol ester of said alpha2-macroglobulin. 2. The stable complex of claim 1 wherein said biomolecule is selected from the group consisting of peptides, proteins, carbohydrates, cytokines, growth factors, hormones, enzymes, toxins, anti-sense RNA, drugs, oligonucleotides, lipids, DNA, antigens, immunogens, allergens, and combinations thereof. 3. The stable complex of claim 2 wherein said biomolecule is selected from the group consisting of KQIINMWQEVGKAMYACTRPNYNKRKRIHIGPGRAFYTTK (SEQ ID NO:1); KQIINMWQEVGKAMYACTRPNNNTRKSIRIQRGPGRAFVTI (SEQ ID NO:2); CTTPAQGNSMFPSCCCTKPTDGNC (SEQ ID NO:3); and TRILTIPQSLDSCTKPTDGNC (SEQ ID NO:4). 4. The stable complex of claim 1 wherein said biomolecule has a molecular weight of from about 0.5 kilodaltons to about 100 kilodaltons. 5. An immunogen comprising a stable complex of claim 1, wherein said at least one intact biomolecule with a nucleophilic group is an antigenic molecule having at least one epitope. 6. The stable complex of claim 2, wherein said antigen is selected from the group consisting of consisting of HIV antigens, hepatitis virus antigens, peptides thereof, fragments thereof, hybrid peptides thereof, chimeric peptides thereof, and hybrid synthetic peptides thereof. 7. The stable complex of claim 6, wherein said antigen or HIV antigen fragments comprise gp120, gp160 or a fragment thereof. 8. The stable complex of claim 6, wherein said hepatitis virus antigens or fragments thereof comprise HbsAg or a fragment thereof. 9. A stable complex comprising at least one intact biomolecule with a nucleophilic group and activated alpha2 macroglobulin having an intact bait region, wherein each of said intact biomolecule with a nucleophilic group is bound to said alpha2 macroglobulin via a binding consisting of said nucleophilic group covalently bound to gamma-glutamyl group of said cleaved thiol ester of said alpha2-macroglobulin, wherein said stable complex prepared by the sequential steps of activating alpha2-macroglobulin by incubation with a nucleophilic compound to form nucleophile-activated alpha2-macroglobulin, removing excess said nucleophilic compounds, and incubating said nucleophile-activated alpha2-macroglobulin with said biomolecule, whereby said stable complex is formed. 10. The stable complex of claim 9, wherein said biomolecule is selected from the group consisting of peptides, proteins, carbohydrates, cytokines, growth factors, hormones, enzymes, toxins, anti-sense RNA, drugs, oligonucleotides, lipids, DNA, antigens, immunogens, allergens, and combinations thereof. 11. The stable complex of claim 10, wherein said antigen is selected from the group consisting of consisting of HIV antigens, hepatitis virus antigens, peptides thereof, fragments thereof, hybrid peptides thereof, chimeric peptides thereof, and hybrid synthetic peptides thereof. 12. The stable complex of claim 11, wherein said antigen or HIV antigen fragments comprise gp120, gp160 or a fragment thereof. 13. The stable complex of claim 11, wherein said hepatitis virus antigens or fragments thereof comprise HbsAg or a fragment thereof. 14. The stable complex of claim 9 wherein said biomolecule is selected from the group consisting of KQIINMWQEVGKAMYACTRPNYNKRKRIHIGPGRAFYTTK (SEQ ID NO:1); KQIINMWQEVGKAMYACTRPNNNTRKSIRIQRGPGRAFVTI (SEQ ID NO:2); CTTPAQGNSMFPSCCCTKPTDGNC (SEQ ID NO:3); and TRILTIPQSLDSCTKPTDGNC (SEQ ID NO:4). 15. The stable complex of claim 9 wherein the molecular weight of said biomolecule is from about 0.5 kilodaltons to about 100 kilodaltons. 16. An immunogen comprising a stable complex of claim 9, wherein said at least one intact biomolecule with a nucleophilic group is an antigenic molecule having at least one epitope. 17. The stable complex of claim 9 wherein said nucleophilic compound has the formula RNH2, wherein R is selected from the group consisting of hydrogen and an alkyl group of 1 to 6 carbon atoms. 18. The stable complex of claim 17 wherein said nucleophilic compound is selected from the group consisting of ammonia, methylamine, ethylamine, and combinations thereof. 19. The stable complex of claim 9 wherein said incubating of said nucleophile activated alpha2-macroglobulin with said biomolecule is carried out at a temperature ranging from about 35 degree C to about 55 degree C. 20. The stable complex of claim 9 wherein said incubation step is carried out at a temperature ranging from about 37 degree C to about 50 degree C, and a period of time ranging from about 1 hour to about 24 hours. 21. The stable complex of claim 20 wherein the temperature and time ranges of said incubation are selected from a temperature of about 37 degree C for about 24 hours, and a temperature of about 50 degree C from about 1 to about 5 hours. 22. A method for the preparation of a covalent complex between at least one intact biomolecule and alpha2-macroglobulin having an intact bait region, wherein said intact biomolecule with a nucleophilic group is bound to said alpha2 macroglobulin via a bond, consisting of said nucleophilic group covalently bound to gamma-glutamyl group of said cleaved thiol ester of said alpha2-macroglobulin, comprising the steps of: i) activating said alpha2-macroglobulin by incubation with a nucleophilic compound to form nucleophile-activated alpha2-macroglobulin; ii) removing excess said nucleophilic compound: and iii) incubating said nucleophile-activated alpha2-macroglobulin with said biomolecule for a period of time sufficient to form said complex. 23. The method of claim 22 wherein said nucleophilic compound has the formula RNH2, wherein R is selected from the group consisting of hydrogen and an alkyl group of 1 to 6 carbon atoms. 24. The method of claim 23 wherein said nucleophilic compound is selected from the group consisting of ammonia, methylamine, ethylamine, and combinations thereof. 25. The method of claim 22 wherein said incubating of said nucleophile activated alpha2-macroglobulin with said biomolecule is carried out at a temperature ranging from about 35 degree C to about 55 degree C. 26. The method of claim 22 wherein said incubation step is carried out at a temperature ranging from about 37 degree C to about 50 degree C, and a period of time ranging from about 1 hour to about 24 hours. 27. The method of claim 26 wherein the temperature and time ranges of said incubation are selected from a temperature of about 37 degree C for about 24 hours, and a temperature of about 50 degree C from about 1 to about 5 hours. 28. The method of claim 22, wherein said biomolecule is selected from the group consisting of peptides, proteins, carbohydrates, cytokines, growth factors, hormones, enzymes, toxins, anti-sense RNA, drugs, oligonucleotides, lipids, DNA, antigens, immunogens, allergens, and combinations thereof. 29. The method of claim 28, wherein said antigen is selected from the group consisting of consisting of HIV antigens, hepatitis virus antigens, peptides thereof, fragments thereof, hybrid peptides thereof, chimeric peptides thereof, and hybrid synthetic peptides thereof. 30. The method of claim 29, wherein said antigen or HIV antigen fragments comprise gp120, gp160 or a fragment thereof. 31. The method of claim 29, wherein said hepatitis virus antigens or fragments thereof comprise HbsAg or a fragment thereof. 32. The method of claim 28 wherein said biomolecule is selected from the group consisting of: KQIINMWQEVGKAMYACTRPNYNKRKRIHIGPGRAFYTTK (SEQ ID NO:1); KQIINMWQEVGKAMYACTRPNNNTRKSIRIQRGPGRAFVTI (SEQ ID NO:2); CTTPAQGNSMFPSCCCTKPTDGNC (SEQ ID NO:3); and TRILTIPQSLDSCTKPTDGNC (SEQ ID NO:4). 33. The method of claim 22 wherein the molecular weight of said biomolecule is from about 0.5 kilodaltons to about 100 kilodaltons. 34. The method of claim 22 wherein said method is carried out in the absence of a proteolytic enzyme capable to cleave said biomolecule and said bait region. 35. An immunogen comprising a biomolecule with a nucleophilic group in a complex with alpha2-macroglobulin having an intact bait region, said biomolecule having at least one epitope, wherein said alpha2-macroglobulin is capable of binding a receptor for alpha2-macroglobulin, said complex comprising at least one intact biomolecule and activated alpha2-macroglobulin with an intact bait region, wherein each of said intact biomolecule with a nucleophilic group is bound to the activated alpha2-macroglobulin via a binding consisting of said nucleophilic group covalently bound to a gamma-glutamyl group of said cleaved thiol ester of said alpha2-macroglobulin. 36. The immunogen of claim 35 wherein said biomolecule is selected from the group consisting of peptides, proteins, carbohydrates, cytokines, growth factors, hormones, enzymes, toxins, anti-sense RNA, drugs, oligonucleotides, lipids, DNA, antigens, immunogens, allergens, and combinations thereof. 37. The immunogen of claim 36 wherein said antigen is selected from the group consisting of consisting of HIV antigens, hepatitis virus antigens, peptides thereof, fragments thereof, hybrid peptides thereof, chimeric peptides thereof, and hybrid synthetic peptides thereof. 38. The immunogen of claim 37, wherein said antigen or HIV antigen fragments comprise gp120, gp160 or a fragment thereof. 39. The immunogen of claim 37, wherein said hepatitis virus antigens or fragments thereof comprise HbsAg or a fragment thereof. 40. The immunogen of claim 35, wherein said biomolecule is selected from the group consisting of: KQIINMWQEVGKAMYACTRPNYNKRKRIHIGPGRAFYTTK (SEQ ID NO:1); KQIINMWQEVGKAMYACTRPNNNTRKSIRIQRGPGRAFVTI (SEQ ID NO:2); CTTPAQGNSMFPSCCCTKPTDGNC (SEQ ID NO:3); and TRILTIPQSLDSCTKPT