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Synthesis and Immunological Application of HIV and Tumor-Associated Carbohydrate Antigens

Synthesis and Immunological Application of HIV and Tumor-Associated Carbohydrate Antigens. Michael Giuliano Gellman Group December 6th, 2007. poliovirus. http://www.math.sunysb.edu. Lipid A - E. Coli. Boons, G.-J. et al. J. Am. Chem. Soc . 2007 , 129 , 5200. Antigens and Vaccines.

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Synthesis and Immunological Application of HIV and Tumor-Associated Carbohydrate Antigens

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  1. Synthesis and Immunological Application of HIV and Tumor-Associated Carbohydrate Antigens Michael Giuliano Gellman Group December 6th, 2007

  2. poliovirus http://www.math.sunysb.edu Lipid A - E. Coli Boons, G.-J. et al. J. Am. Chem. Soc. 2007, 129, 5200. Antigens and Vaccines • immunogen - agent capable of eliciting an immune system response • antigen - contraction of “antibody generating” • lipids • proteins/peptides • carbohydrates Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2000, 39, 836.

  3. Antigen IgG IgM Immune Response to Foreign Antigens • Adaptive Immune Response • Cell - mediated • Helper T Cells • Cytotoxic T Cells • Generation of specific antibodies and long-term immunity • Innate Immune Response • takes place in bloodstream • Macrophages • B Cells • Antibodies (non-specific) • An effective vaccine illicits both of these responses

  4. Types of Vaccines ** • ** First officially licensed carbohydrate conjugate vaccines (95-100% clinical efficacy): • ProHIBIT (1987) http://phil.cdc.gov

  5. Scaffold E E Carrier E Conjugate Vaccines Adjuvant: an organic or inorganic immunostimulant administered with a vaccine Epitope: The antigen itself Scaffold: - must not itself be immunogenic - biomimetic antigen presentation Carrier: - contains peptide sequence that stimulates cell-mediated immune response - capable of presenting a high epitope density (i.e. multivalent presentation)

  6. Efforts Toward an HIV-1 Vaccine

  7. Antibody Evasion by HIV-1 • Dense coating of high-mannose oligosaccharides excludes the vast majority of recognizable protein components of gp120 • Facilitates association with mannose-binding receptors on antigen-presenting dendritic cells. http://www3.niaid.nih.gov

  8. Fab • Typical IgG antigbody; 2 antigen binding sites Fc } • IgG 2G12 antibody; 4 antigen binding sites The 2G12 Antibody • Discovered in mid 1990’s - exhibits novel rearrangement of binding domains • Goal: Elicit an immune response with epitope specificity similar to 2G12 Calarese, D.A. et al. Science. 2003, 300, 2065.

  9. = Mana1-2Man linkage 2G12 Recognition of Oligomannose Early Scaffold Designs: Man9GlcNAc2 Calarese, D.A. et al. Science. 2003, 300, 2065. Wang, L.-X. et al. Org. Biomol. Chem.2004, 2, 483. Wang, L.-X. et al. Chem. Biol. 2004, 11, 127.

  10. An Early Semi-Synthetic Epitope Cluster Wang, L.-X. et al. Org. Biomol. Chem.2004, 2, 483. Wang, L.-X. et al. Org. Biomol. Chem. 2003, 1, 3507.

  11. An Early Semi-Synthetic Epitope Cluster • Competitive Binding (ELISA) Results vs. gp120: • Man9GlcNAc2Asn - IC50= 960 mm • Trivalent cluster - IC50= 21mm Wang, L.-X. et al. Org. Biomol. Chem.2004, 2, 483. Calarese, D.A. et al. Science. 2003, 300, 2065.

  12. The Importance of Epitope Design Wang, L.-X. et al. Org. Biomol. Chem.2004, 2, 483. Wang, L.-X. et al. Org. Biomol. Chem. 2003, 1, 3507. Wang, L.-X. et al. Chem Biol.2004, 11, 127.

  13. Continued Structure-Activity Studies • For the 2G12 - gp120 interaction to be exploited there are 2 requirements to be met by synthetic chemists: • Efficient routes to Man9GlcNHAc2 and other oligomannose species • 2) Design of a scaffold that presents the given epitope in an orientation advantageous to binding by 2G12 extremely important for synthetic vaccine design

  14. One-Pot Oligomannose Assembly 1 2 3 Wong, C.-H. et al. Angew. Chem. Int. Ed. 2004, 43, 1000. Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.

  15. One-Pot Oligomannose Assembly 4 5 Wong, C.-H. et al. Angew. Chem. Int. Ed. 2004, 43, 1000. Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.

  16. Glycosyl Donor Activation Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313.

  17. Evaluation 1 2 3 4 5 Wong, C.-H. et al. Angew. Chem. Int. Ed. 2004, 43, 1000. Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724. Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372.

  18. Assembly of Branched Oligosaccharides 6 7 Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372. Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.

  19. Assembly of Branched Oligosaccharides 8 Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372. Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.

  20. Glycosyl Donor Activation Frasier-Reid, B. Tetrahedron Lett. 1990, 31, 4313.

  21. Evaluation 6 7 8 Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372. Wong, C.-H. et al. J. Am. Chem. Soc. 1999, 121, 724.

  22. Insights from Binding Studies • Tightest binding oligosaccharide was Man9GlcNAc2 • Scaffold-conjugated or surface-immobilized epitopes bound 2G12 tighter than soluble ones. • Co-crystal structures of branched oligosaccharides and 2G12 confirmed high specificity for Mana1-2Man linkages. Man9GlcNAc2 a b a) Mana1-2Man arm in major binding site b) Branches in secondary binding sites a Wilson, I. A.; Wong, C.-H. et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 13372. Calarese, D.A. et al. Science. 2003, 300, 2065.

  23. Fully Synthetic Man9GlcNHAc2 Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2004, 43, 2562. Danishefsky, S.J. et al. Tetrahedron Lett.. 2003, 44, 1791.

  24. Synthesis of the Chitobiose Trisaccharide Danishefsky, S.J. et al. Tetrahedron Lett.. 2003, 44, 1791. Seeberger, P.H.; Danishefsky, S.J. et al. Chem. Eur. J. 1997, 3, 1617.

  25. Synthesis of Man9GlcNHAc2 Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2004, 43, 2562.

  26. Sinay Radical Activation/Glycosylation Sinay, P. et al. Carbohydr. Res. 1992, 236, 73. Sinay, P. et al. Synlett. 1990, 572. Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2004, 43, 2562.

  27. Conjugate Vaccine Design Considerations Scaffold E E Carrier E • Three independently conducted binding studies and two crystallographic studies all arrived at the same conclusions: • Man9GlcNHAc2, in particular, the highly branched nonamannose portion, bound the tightest to 2G12 and likely in a multivalent fashion. • Carrier and scaffold must be designed such that: • antigens are presented similarly to their arrangement on the surface of HIV-1 • each has the appropriate immunogenicity

  28. Vaccine Candidates - Wang • Keyhole Limpet Hemocyanin (KLH) and tetanus toxoid used as carrier/cell-mediated immunostimulant. • Very long, hydrophobic linker regions. • Rabbits were innoculated with either peptide or KLH conjugates with adjuvant • Sera taken after four doses showed high IgG titer… • …mostly specific for the linkers! Wang, L.-X. et al. Bioconjugate Chem.. 2006, 17, 493.

  29. Vaccine Candidates - Danishefsky • Scaffold Assembly and Conjugation: Danishefsky, S.J. et al. J. Am. Chem. Soc. 2007, 129, 11042. Dumy, P. et al. Org. Lett. 2003, 5, 243. Bogusky, M. J,. et al. Biochemistry. 2003, 42, 3214.

  30. Vaccine Candidates - Danishefsky • Key features and evaluation: • Conjugation to a proven, superior immunostimulatory carrier • Short, biomimetically structured linker • OMPC conjugate showed stronger binding to 2G12 than any construct evaluated previously • In vivo immunological evaluations are currently underway. Danishefsky, S.J. et al. J. Am. Chem. Soc. 2007, 129, 11042. Wilson, C.B. Eur. J. Immunol.2001, 31, 2373.

  31. Developing an Effective HIV-1 Vaccine • There is now proof of principle that a synthetic carbohydrate-based vaccine is feasible. • Lessons learned about carrier, scaffold, and linker strucutres: • dense epitope presentation • minimal exposure of linker and/or scaffold • choice of carrier/method of delivery Is there a logical next step?

  32. Multi-Component Anti-Tumor Vaccines:A Future Direction for HIV?

  33. Vaccines as Cancer Therapy • Correlation between tumor metastasis and re-occurrence and glycoprotein over-expression • Tumor cell surfaces are heavily coated with specific antigens and yet remain immunogenically silent • Goal: Break the immune system’s tolerance for tumor • carbohydrate antigens. Scaffold • Because tumor cell membranes often exhibit a diversity of antigens, a conjugate vaccine mirroring this may have the best chance for success. E1 E2 Carrier E3 Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2000, 39, 836.

  34. Tumor-Associated Carbohydrate Antigens Danishefsky, S.J. et al. Angew. Chem. Int. Ed. 2000, 39, 836.

  35. Glycoamino Acid Synthesis Yield: 93-99% d.r. > 20:1 Danishefsky, S.J. et al. J. Am. Chem. Soc. 2001, 123, 1890.

  36. Scaffold Assembly “Multigen” Danishefsky, S.J. et al. J. Am. Chem. Soc. 2001, 123, 1890.

  37. Evaluation of Multigen-KLH Conjugates • Five groups of mice were innoculated (three times), respectively, with multigen conjugate, each individual antigen conjugate, and a mixture of the individual conjugates of all three antigens (all with QS21 plant extract adjuvant). • Increased titers of both IgM (nonspecific) and IgG (specific) antibodies were observed for the construct itself and for each of the three antigens individually. . . A specific immune response for three distinct carbohydrate antigens. Danishefsky, S.J. et al. Proc. Natl. Acad. Sci. USA. 2002, 99, 13699. Danishefsky, S.J. et al. J. Am. Chem. Soc. 2001, 123, 1890.

  38. Assembly of a Three Component Vaccine Boons, G.-J. et al. Nature Chemical Biology. 2007, 3, 663. Boons, G.-J. et al. Angew. Chem. Int. Ed. 2005, 44, 5985.

  39. Evaluation of a Vesicle-based Vaccine • Seven groups of five mice were immunized four times with the vesicle construct shown at right or combinations of its individual components • IgG response to the vesicle construct was in vast excess compared to that of IgM: • memory-induced antibody production • robust T cell response • cytokine and complement activation via TLR-2 (adjuvant effects) Boons, G.-J. et al. Nature Chemical Biology. 2007, 3, 663. Boons, G.-J. et al. Angew. Chem. Int. Ed. 2005, 44, 5985.

  40. Conclusions • At the very least, proof of principle has been achieved in the development of a gp120 oriented HIV-1 vaccine. • Structural mimicry of the epitope itself and its environment are key. • Well-established single-antigen anti-cancer vaccines have been advanced to a new realm. • Multi-component vaccines are employing both multiple antigens and multiple kinds of immunogenic epitopes.

  41. Future Directions • The synergistic combinations of antigenic epitopes and immunogens in a structurally biomimetic fashion is likely applicable to HIV-1 vaccine development and may provide chemists and biologists with new tools to study and fight other infectious diseases. • The design of new methods of antigen/immunogen display and delivery may allow for the medical exploitation of new protein-carbohydrate, protein-protein, and cell-cell interactions.

  42. Acknowledgements Professor Samuel Gellman Josh Price Dr. W. Seth Horne Soo Hyuk Choi Li Guo Melissa Boersma Dr. Pil Seok Chae Johnathan Zhang Kim Peterson Will Pomerantz Jay Steinkruger Erik Hadley Emily Blamer Holly Haase Dr. Brendan Mowery Matt Windsor Dr. Felix Friere-Iribarne Brooke Richardson Dr. Myung-Ryul Lee Emily English Becca Splain Lisa Johnson Aaron Almeida Lexie Dillon Dmitri Svetlov Ryan Drake Melissa Hardy

  43. Questions?

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