Skip to main content

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Abstract

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.

Abbreviations

mAb:

monoclonal antibody

Intrabody:

intracellular antibody

References

  1. Akira, S., Uematsu, S. and Takeuchi, O. Pathogen recognition and innate immunity. Cell 124 (2006) 783–801.

    Article  CAS  PubMed  Google Scholar 

  2. Kawai, T. and Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11 (2010) 373–384.

    Article  CAS  PubMed  Google Scholar 

  3. Lee, B.L., Moon, J.E., Shu, J.H., Yuan L., Newman, Z.R., Schekman, R. and Barton G.M. UNC93B1 mediates differential trafficking of endosomal TLRs. eLife. 00291.

  4. Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K.A., Monks, B.G., Knetter, C.F., Lien, E., Nilsen, N.J., Espevik, T. and Golenbock, D.T. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5 (2004) 190–198.

    Article  CAS  PubMed  Google Scholar 

  5. Trivedi, S. and Greidinger, E.L. Endosomal Toll-like receptors in autoimmunity: mechanisms for clinical diversity. Therapy 6 (2009) 433–442.

    Article  CAS  PubMed  Google Scholar 

  6. Celhar, T., Magalhaes, R., and Fairhust, A.-M. TLR7 and TLR9 in SLE: when sensing self goes wrong. Immunol. Res. 53 (2012) 58–77.

    Article  CAS  PubMed  Google Scholar 

  7. Zeuner, R.A., Verthelyi, D., Gursel, M., Ishii, K.J. and Klinman, D.M. Influence of stimulatory and suppressive DNA motifs on host susceptibility to inflammatory arthritis. Arthritis Rheum. 48 (2003) 1701–1707.

    Article  CAS  PubMed  Google Scholar 

  8. O’Neill, L.A.J. Primer: Toll-like receptor signaling pathways-what do rheumatologists need to know? Nat. Clin. Pract. Rheumatol. 4 (2008) 319–327.

    Article  PubMed  Google Scholar 

  9. Daubeuf, B., Mathison, J., Spiller, S., Hugues, S., Herren, S., Ferlin, W., Kosco-Vilbois, M., Wagner, H., Kirschning, C.J., Ulevitch, R. and Elson, G. TLR4/MD-2 monoclonal antibody therapy affords protection in experimental models of septic shock. J. Immunol. 179 (2007) 6107–6114.

    CAS  PubMed  Google Scholar 

  10. Meng, G., Rutz, M., Schiemann, M., Metzger, J., Grabiec, A., Schwandner, R., Luppa, P.B., Ebel, F., Busch, D.H., Bauer, S., Wagner, H. and Kirschning, C.J. Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes. J. Clin. Invest. 113 (2004) 1473–1481.

    CAS  PubMed  Google Scholar 

  11. Vanags, D., Williams, B., Johnson, B., Hall, S., Nash, P., Taylor, A., Weiss, J. and Feeney, D. Therapeutic efficacy and safety of chaperonin 10 in patients with rheumatoid arthritis: a double-blind randomized trial. Lancet 368 (2006) 855–863.

    Article  CAS  PubMed  Google Scholar 

  12. Hennessy, E.J., Parker, A.E. and O’Neill, L.A.J. Targeting Toll-like receptors: emerging therapeutics? Nat. Rev. Drug. Discov. 9 (2010) 293–307.

    Article  CAS  PubMed  Google Scholar 

  13. Kuznik, A., Bencina, M., Svajger, U., Jeras, M. and Rozman, B. Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. J. Immunol. 186 (2011) 4794–4804.

    Article  CAS  PubMed  Google Scholar 

  14. Klinman, D.M., Zeuner, R., Yamada, H., Gursel, M., Currie, D. and Gursul, I. Regulation of CpG-induced immune activation by suppressive oligodeoxynucleotides. Ann. NY Acad. Sci. 1002 (2003) 112–123.

    Article  CAS  PubMed  Google Scholar 

  15. Barrat, F.J., Meeker, T., Chan, J.H., Guiducci, C. and Coffman, R.L. Treatment of lupus-prone mice with a dual inhibitor of TLR7 and TLR9 leads to reduction of autoantibody production and amelioration of disease symptoms. Eur. J. Immunol. 37 (2007) 3582–3586.

    Article  CAS  PubMed  Google Scholar 

  16. Marshak-Rothstein, A. Toll-like receptors in systemic autoimmune disease. Nat. Rev. Immunol. 6 (2006) 823–835.

    Article  CAS  PubMed  Google Scholar 

  17. Graham, K.L., Lee, L.Y., Higgens, J.P., Steinmann, L., Utz, P.J. and Ho, P.P. Treatment with a toll-like receptor inhibitory CpG oligonucleotide delays and attenuates lupus nephritis in NZB/W mice. Autoimmunity 43 (2010) 140–155.

    Article  CAS  PubMed  Google Scholar 

  18. Böldicke, T. Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER. J. Cell Mol. Med. 11 (2007) 54–70.

    Article  PubMed  Google Scholar 

  19. Böldicke, T., Somplatzki, S., Sergeev, G. and Mueller, P.P. Functional inhibition of transitory proteins by intrabody-mediated retention in the endoplasmatic reticulum. Methods 56 (2012) 338–350.

    Article  PubMed  Google Scholar 

  20. Bilanges, B. and Stokoe, D. Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi. Biochem. J. 388 (2005) 573–583.

    Article  CAS  PubMed  Google Scholar 

  21. Qiu, S., Adema, C.M. and Lane, T. A computational study of off-target effects of RNA interference. Nucleic Acids Res. 33 (2005) 1834–1847.

    Article  CAS  PubMed  Google Scholar 

  22. Cao, T. and Heng, B.C. Intracellular antibodies (intrabodies) versus RNA interference for therapeutic applications. Ann. Clin. Lab. Sci. 35 (2005) 227–229.

    CAS  PubMed  Google Scholar 

  23. Kirschning, C.J., Dreher, S., Maaß, B., Fichte, S., Schade, J., Köster, M., Noack, A., Lindenmaier, W., Wagner, H. and Böldicke, T. Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation. BMC Biotechnol. 10 (2010) 31.

    Article  PubMed  Google Scholar 

  24. Ahmad-Nejad, P., Häcker, H., Rutz, M., Bauer, S., Vabulas, R.M. and Wagner, H. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32 (2002) 1958–1968.

    Article  CAS  PubMed  Google Scholar 

  25. Böldicke, T., Weber, H., Mueller, P.P., Barleon, B. and Bernal, M. Novel highly efficient intrabody mediates complete inhibition of cell surface expression of the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR). J. Immunol. Meth. 300 (2005) 146–159.

    Article  Google Scholar 

  26. Böldicke, T., Tesar, M., Griesel, C., Rohde, M., Gröne, H.-J., Waltenberger J., Kollet, O., Lapidot, T., Yayon, A. and Weich, H. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2, flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood. Stem Cells 19 (2001) 24–36.

    Article  PubMed  Google Scholar 

  27. Mayer, H., Bertram, H., Lindenmaier, W., Korff, T., Weber, H. and Weich, H. Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation. J. Cell Biochem. 95 (2005) 827–839.

    Article  CAS  PubMed  Google Scholar 

  28. Ospelt, C. and Gay, S. TLRs and chronic inflammation. Int. J. Biochem. Cell Biol. 42 (2010) 495–505.

    Article  CAS  PubMed  Google Scholar 

  29. Thomas, C.E., Ehrhardt, A. and Kay, M.A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4 (2003) 346–358.

    Article  CAS  PubMed  Google Scholar 

  30. Swan, C.H., Bühler, B., Steinberger, P., Tschan, M.P., Barbas III, C.F. and Torbett, B.E. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther. 13 (2006) 1480–1492.

    Article  CAS  PubMed  Google Scholar 

  31. Cerullo, V., Seiler, M.P., Mane, V., Brunetti-Pierri, N., Clarke, C., Bertin, T.K., Rodgers, J.R. and Lee, B. Toll-like receptor 9 triggers an innate immune response to helper-dependent adenoviral vectors. Mol. Ther. 15 (2007) 378–385.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Thomas Böldicke.

Additional information

These authors contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Reimer, E., Somplatzki, S., Zegenhagen, D. et al. Molecular cloning and characterization of a novel anti-TLR9 intrabody. Cell Mol Biol Lett 18, 433–446 (2013). https://doi.org/10.2478/s11658-013-0098-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-013-0098-8

Key words

  • Recombinant antibodies
  • Recombinant adenovirus
  • Protein knockdown
  • Intracellular toll-like receptors
  • TLR9
  • ER intrabodies
  • Macrophage activation