Production of camelid heavy-chain antibodies as specific enzyme inhibitors using new immunization strategies

• Antragsteller: Professor Dr. Friedrich Koch-Nolte
• Fachliche Zuordnung: Immunologie
• Förderung: Förderung von 2002 bis 2010
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5395981

• Erstellungsjahr: 2011

Zusammenfassung der Projektergebnisse

The goal of the proposed project was to generate single domain antibodies (sdAbs) from immunized new world camelids (llamas) against lumazine synthase (LS) and ADP- ribosyltransferases (ARTs) as model antigens. Single domain antibodies derived from llama heavy chain antibodies display several useful features including high solubility, stability, low production costs, favorable in vivo biodistribution, and a propensity to bind to functional crevices on proteins. LS, a polymeric, highly immunogenic protein from Brucella abortus, can be used as carrier and molecular adjuvant. ARTs, such as the actin-ADP- ribosylating toxin SpvB from Salmonella entericae and the toxin-related murine T cell ectoenzyme ART2 regulate important functions - including bacterial pathogenicity and lymphocyte migration - by transferring the ADP-ribose moiety from NAD+ onto arginine side chains in target proteins. In the project, single domain antibodies were raised successfully against LS, the T cell ecto-enzyme ART2, and the Salmonella toxin SpvB. The LS-specific sdAbs were used to aid binding of genetically fused proteins to LS in order to enhance their immunogenicity. The ART2-specific antibodies were useful tools for blocking ART2 activity and cytotoxicity both, in vitro and in vivo. The SpvB-specific nanobodies proved useful for protecting cells against the cytotoxic effects of recombinant SpvB and of SpvB-expressing strains of Salmonella. The antibodies generated in this project provide a basis for the development of new experimental and therapeutic intervention tools. The project has strengthened the protein engineering capacities of the cooperating partners and has promoted the use of llamas as a source for producing enzyme inhibitors.

Projektbezogene Publikationen (Auswahl)

• Engineering of a polymeric bacterial protein as a scaffold for the multiple display of peptides. Proteins 57:820-828. 2004
  Laplagne, D. A., V. Zylberman, N. Ainciart, M. W. Steward, E. Sciutto, C. A. Fossati, and F. A. Goldbaum
  
• Sequence determinants of quaternary structure in lumazine synthase. Mol Biol Evol 21:97. 2004
  Fornasari, M. S., D. A. Laplagne, N. Frankel, A. A. Cauerhff, F. A. Goldbaum, and J. Echave
  
• Brucella spp. lumazine synthase: a novel antigen delivery system. Vaccine 23:2784. 2005
  Sciutto, E., A. Toledo, C. Cruz, G. Rosas, G. Meneses, D. Laplagne, N. Ainciart, J. Cervantes, G. Fragoso, and F. A. Goldbaum
  
• Llama single domain antibodies as a tool for molecular mimicry. J Mol Biol 349:814. 2005
  Zarebski, L. M., M. Urrutia, and F. A. Goldbaum
  
• Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation. Cell Immunol 236:66. 2005
  Koch-Nolte, F., G. Glowacki, P. Bannas, F. Braasch, G. Dubberke, E. Ortolan, A. Funaro, F. Malavasi, and F. Haag
  
• Brucella spp. lumazine synthase: a novel adjuvant and antigen delivery system to effectively induce oral immunity. Microbes Infect 8:1277. 2006
  Rosas, G., G. Fragoso, N. Ainciart, F. Esquivel-Guadarrama, A. Santana, R. J. Bobes, O. Ramirez-Pliego, A. Toledo, C. Cruz-Revilla, G. Meneses, P. Berguer, F. A. Goldbaum, and E. Sciutto
  
• "Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation". Purinergic Signal 3, 359–66. 2007
  S. Moller, C. Jung, S. Adriouch, G. Dubberke, F. Seyfried, M. Seman, F. Haag, F. Koch-Nolte
  
• Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo. FASEB J. 21:3490-3. 2007
  Koch-Nolte F, Reyelt J, Schössow B, Schwarz N, Scheuplein F, Rothenburg S, Haag F, Alzogaray V, Cauerhff A, Goldbaum FA
  
• Mammalian ADP-ribosyltransferases and ADP-ribosylhydrolases. Front Biosci.13:6716-29. 2008
  Koch-Nolte F, Kernstock S, Mueller-Dieckmann C, Weiss MS, Haag F
  
• Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 198: 157-174. 2009
  Wesolowski J, V Alzogaray, J Reyelt, M Unger, K Juarez, M Urrutia, A Cauerhff, W Danquah, B Rissiek, F Scheuplein, N Schwarz, S Adriouch, O Boyer, M, Seman, A Licea, D V. Serreze, F A. Goldbaum, F Haag and F Koch-Nolte
  
• "A recombinant heavy chain antibody approach blocks ART2-mediated deletion of an iNKT cell population that upon activation inhibits autoimmune diabetes". J Autoimmun. 34, 145–54. 2010
  Scheuplein F, Rissiek B, Driver JP, Chen YG, Koch-Nolte F, Serreze DV
  
• "Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway". J Exp Med. 207:2561-8. 2010
  Hubert S, Rissiek B, Klages K, Huehn J, Sparwasser T, Haag F, Koch-Nolte F, Boyer O, Seman M, Adriouch S
  
• Toward a unified nomenclature for mammalian ADP-ribosyltransferases.Trends Biochem Sci. 35:208-19. 2010
  Hottiger MO, Hassa PO, Lüscher B, Schüler H, Koch-Nolte F
  
• Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium. FASEB J. 25:526-34. 2011
  Alzogaray V, Danquah W, Aguirre A, Urrutia M, Berguer P, García Véscovi E, Haag F, Koch- Nolte F, Goldbaum FA