Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Increase of Spacer Sequence Yields Higher Dimer $(Fab-Spacer-Toxin)_{2}$ Formation

  • Yoo Mee-Hyeon (College of Life Science and Graduate School of Biotechnology, Korea University) ;
  • Won Jae-Seon (College of Life Science and Graduate School of Biotechnology, Korea University) ;
  • Lee Yong-Chan (College of Life Science and Graduate School of Biotechnology, Korea University) ;
  • Choe Mu-Hyeon (College of Life Science and Graduate School of Biotechnology, Korea University)
  • Published : 2006.07.01
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Abstract

The divalent antibody-toxins are expected to have increased binding avidities to target cells because of the two cell-binding domains. However, previous studies showed that the refolding yield of divalent antibody-toxin is very low, and it is assumed that homodimer formation of antibody-toxin is strongly interfered by the repulsion between the two large toxin domains that come close to each other during dimer formation. In this study, B3 antibody was used as a model antibody, and its Fab domain was used to construct three different kinds of Fab divalent molecules, $[B3(Fab)-toxin]_{2}$. The monomer Fab-toxin molecules were made by fusing the Fab domain of monoclonal antibody B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE), and a connecting sequence that contained spacer amino acid sequence (G4S)n (n=l, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were $[Fab-S\;1,\;2,\;3-PE38]_{2}\;(=[Fab-SKPCIST-KAS(G_{4}S)nGGPE-PE38]_{2}\;(n=1,\;2,\;3))$, and they are derivatives of previously studied $[Fab-H2cys-PE38]_{2}\;(=[Fab-SKPCIST-KASGGPE-PE38]_{2})$. In $[Fab-Sl,\;2,\;3-PE38]_{2}$, two Fab-S1, 2, 3-PE38 monomers were covalently linked by the disulfide bond bridge made from cysteine in the -SKPCIST- sequence. The insertion of spacer amino acids after the disulfide bridge resulted in a 12-18 fold higher yield of dimer formation than previously constructed $[Fab-Hlcys-PZ38]_{2}[7]$, 3-4-fold higher than $[Fab-ext-PZ38]_{2}[25]$. These two molecules have less amino acid spacer sequence between the disulfide bridge and PE38 domain. The design of $[Fab-PE38]_{2}$ in this study gave molecules with a higher refolding yield. The results of cytotoxicity assay showed a higher cytotoxic effect of these divalent molecules than that of the monovalent scFv-PE38 molecule.

Keywords

References

  1. Baluna, R., E. Coleman, C. Jones, V. Ghetie, and E. S. Vitetta. 2000. The effect of a monoclonal antibody coupled to ricin A chain-derived peptides on endothelial cells in vitro: Insights into toxin-mediated vascular damage. Exp. Cell. Res. 258: 417-424 https://doi.org/10.1006/excr.2000.4954
  2. Bera, T. K. and I. Pastan. 1998. Comparison of recombinant immunotoxins against LeY antigen expressing tumor cells: Influence of affinity, size, and stability. Bioconjug. Chem. 9: 736-743 https://doi.org/10.1021/bc980028o
  3. Bera, T. K., J. Viner, E. Brinkmann, and I. Pastan. 1999. Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2. Cancer Res. 59: 4018-4022
  4. Buchner, J., I. Pastan, and U. Brinkmann. 1992. A method for increasing the yield of properly folded recombinant fusion proteins: Single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal. Biochem. 205: 263-270 https://doi.org/10.1016/0003-2697(92)90433-8
  5. Chaudhary, V. K., C. Queen, R. P. Junghans, T. A. Waldmann, D. J. FitzGerald, and I. Pastan. 1989. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 339: 394-397 https://doi.org/10.1038/339394a0
  6. Choe, M., K. O. Webber, and I. Pastan. 1994. B3(Fab)PE38M: A recombinant immunotoxin in which a mutant form of Pseudomonas exotoxin is fused to the Fab fragment of monoclonal antibody B3. Cancer Res. 54: 3460-3467
  7. Choi, S., J. Kim, Y. Lee, Y.-J. Jang, I. Pastan, and M. Choe. 2001. A divalent immunotoxin formed by the disulfide bond between hinge regions of Fab domain. Bull. Kor. Chem. Soc. 22: 1361-1365
  8. Deonarain, M. P. and A. A. Epenetos. 1998. Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins. Br. J. Cancer 77: 537 -546 https://doi.org/10.1038/bjc.1998.87
  9. Hudson, P. J. and A. A. Kortt. 1999. High avidity scFv multimers; diabodies and triabodies. J. Immunol. Methods 231: 177-189 https://doi.org/10.1016/S0022-1759(99)00157-X
  10. Jang, C. H., H. H. Chung, J. Yu, Y. J. Chang, H. B. Kim, S. H. Paek, D. H. Shin, and K. H. Kim. 1999. Single-chain Fv fragment of catalytic antibody 4f4f with glycosidase activity: Design, expression, and purification. J. Microbiol. Biotechnol. 9: 376-380
  11. Jurado, P., D. Ritz, J. Beckwith, V. de Lorenzo, and L. A. Fernandez. 2002. Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli. J. Mol. Biol. 320: 1-10 https://doi.org/10.1016/S0022-2836(02)00405-9
  12. Kihara, A. and I. Pastan. 1995. Cytotoxic activity of chimeric toxins containing the epidermal growth factor-like domain of heregulins fused to PE38KDEL, a truncated recombinant form of Pseudomonas exotoxin. Cancer Res. 55: 71-77
  13. Kim, N. S., K. H. Chang, B. S. Chung, S. H. Kim, J. H. Kim, and G. M. Lee. 2003. Characterization of humanized antibody produced by apoptosis-resistant CHO cells under sodium butyrate-induced condition. J. Microbiol. Biotechnol. 13: 926-936
  14. Kipriyanov, S. M., G. Moldenhauer, M. Braunagel, U. Reusch, B. Cochlovius, F. Le Gall, O. A. Kouprianova, C. W. Von der Lieth, and M. Little. 2003. Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies. J. Mol. Biol. 330: 99-111 https://doi.org/10.1016/S0022-2836(03)00526-6
  15. Kreitman, R. J., Q. C. Wang, D. J. FitzGerald, and I. Pastan. 1999. Complete regression of human B-cell lymphoma xenografts in mice treated with recombinant anti-CD22 immunotoxin RFB4(dsFv)-PE38 at doses tolerated by cynomolgus monkeys. Int. J. Cancer 81: 148-155 https://doi.org/10.1002/(SICI)1097-0215(19990331)81:1<148::AID-IJC24>3.0.CO;2-L
  16. Kreitman, R. J., W. H. Wilson, K. Bergeron, M. Raggio, M. Stetler-Stevenson, D. J. FitzGerald, and I. Pastan. 2001. Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N. Engl. J. Med. 345: 241-247 https://doi.org/10.1056/NEJM200107263450402
  17. Kwak, B.-Y., B.-J. Kwon, C.-H. Kweon, and D.-H. Shon. 2004. Detection of Aspergillus, Penicillium, and Fusarium species by sandwich enzyme-linked immunosorbent assay using mixed monoclonal antibodies. J. Microbiol. Biotechnol. 14: 385-389
  18. Lee, J.- W., I.-J. Jun, H. J. Kwun, K. L. Jang, and J. Cha. 2004. Direct identification of Vibrio vulnificus by PCR targeting elastase gene. J. Microbiol. Biotechnol. 14: 284-289
  19. Lev, A., H. Novak, D. Segal, and Y. Reiter. 2002. Recruitment of CTL activity by tumor-specific antibody-mediated targeting of single-chain class I MHC-peptide complexes. J. Immunol. 169: 2988-2996 https://doi.org/10.4049/jimmunol.169.6.2988
  20. Lim, H.-K., S.-U. Lee, S.-I. Chung, K.-H. Jung, and J.-H. Seo. 2004. Induction of the T7 promoter using lactose for production of recombinant plasminogen Kringle 1-3 in Escherichia coli. J. Microbiol. Biotechnol. 14: 225-230
  21. Mansfield, E., I. Pastan, and D. J. FitzGerald. 1996. Characterization of RFB4-Pseudomonas exotoxin A immunotoxins targeted to CD22 on B-cell malignancies. Bioconjug. Chem. 7: 557-563 https://doi.org/10.1021/bc960043y
  22. Onda, M., S. Nagata, Y. Tsutsumi, J. J. Vincent, Q. Wang, R. J. Kreitman, B. Lee, and I. Pastan. 2001. Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity. Cancer Res. 61: 5070-5077
  23. Onda, M., Q. C. Wang, H. F. Guo, N. K. Cheung, and I. Pastan. 2004. In vitro and in vivo cytotoxic activities of recombinant immunotoxin 8H9(Fv)-PE38 against breast cancer, osteosarcoma, and neuroblastoma. Cancer Res. 15: 1419-1424
  24. Pai, L. H., J. K. Batra, D. J. FitzGerald, M. C. Willingham, and I. Pastan. 1991. Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin [published erratum appears in Proc. Natl. Acad. Sci. USA. 1991. 88: 5066]. Proc. Natl. Acad. Sci. USA 88: 3358-3362
  25. Park, J. H., H. W. Kwon, H. K. Chung, I. H. Kim, K. Ahn, E. J. Choi, I. Pastan, and M. Choe. 2001. A divalent recombinant immunotoxin formed by a disulfide bond between the extension peptide chains. Mol. Cells 12: 398-402
  26. Reiter, Y., U. Brinkmann, R. J. Kreitman, S. H. Jung, B. Lee, and I. Pastan. 1994. Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions. Biochemistry 33: 5451-5459 https://doi.org/10.1021/bi00184a014
  27. Reiter, Y., A. F. Wright, D. W. Tonge, and I. Pastan. 1996. Recombinant single-chain and disulfide-stabilized Fv immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice. Int. J. Cancer 67: 113-123 https://doi.org/10.1002/(SICI)1097-0215(19960703)67:1<113::AID-IJC19>3.0.CO;2-F
  28. Schmiedl, A., F. Breitling, and S. DubeI. 2000. Expression of a bispecific dsFv-dsFv' antibody fragment in Escherichia coli. Protein Eng. 13: 725-734 https://doi.org/10.1093/protein/13.10.725
  29. Shim, Y.-Y., D.-H. Shon, B.-Y. Kwak, J. Yu, and K.-M. Chee. 2004. Comparison of properties of poly clonal anti-Nacetylchitooligosaccharides and anti-chitooligosaccharides antibodies produced for ELISA. J. Microbiol. Biotechnol. 14: 686-692
  30. Studier, F. W. and B. A. Moffatt. 1986. Use of bacteriophage T7 polymerase to direct selective expression of cloned gene. J. Mol. BioI. 189: 113-130 https://doi.org/10.1016/0022-2836(86)90385-2
  31. Tur, M. K., M. Huhn, T. Thepen, M. Stocker, R. Krohn, S. Vogel, E. Jost, R. Osieka, J. G. van de Winkel, R. Fischer, R. Finnern, and S. Barth. 2003. Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells. Cancer Res. 63: 8414-8419
  32. Umetsu, M., K. Tsumoto, M. Hara, K. Ashish, S. Goda, T. Adschiri, and I. Kumagai. 2003. How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system. Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment. J. Biol. Chem. 278: 8979-8987 https://doi.org/10.1074/jbc.M212247200