Increased Refolding Yield of Disulfide Bond Bridged Fab-Toxin Homodimers by the Insertion of CH3 Domains

  • Song Jeong-Wha (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

Abstract

Recombinant antibody-toxin is a bifunctional protein that binds and kills a target cell expressing a specific antigen on the surface of the cell, and its structure is chimeric, in which a toxin is fused to an antigen-binding domain such as scFv or Fab. Divalent antibody-toxin molecules showed higher cytotoxicities against cancer cell lines than monovalent molecules. However, the yields of the divalent molecules were very low. In this study, we introduced the CH2, CH3, or CH2-CH3 (=Fc) domain of antibody in the middle of the Fab-toxin between the hinge region of human IgG1 and the toxin domain to increase the yield. The covalently bonded dimer could be formed by three disulfide bridges from cysteine residues in the hinge region. The molecule with the CH3 domain showed about 3-fold higher dimerization yield than previously constructed Fab-toxin molecules, while maintaining the cytotoxic activity comparable to that of scFv-toxin. However, the introduction of CH2 or Fc domain to the same position showed little effect on the dimerization yield. We also observed that the introduction of the CH3 region made it possible to form noncovalently associated dimer molecules.

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 $Le^Y$ antigen expressing tumor cells: Influence of affinity, size, and stability. Bioconjug. Chem. 9: 736-743 https://doi.org/10.1021/bc980028o
  3. Brinkmann, U., L. H. Pai, D. J. FitzGerald, M. Willingham, and I. Pastan. 1991. B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. USA 88: 8616-8620
  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. Buchner, J. and R. Rudolph. 1991. Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli. Bio/Technology 9: 157-162 https://doi.org/10.1038/nbt0291-157
  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. Dall' Acqua, W., A. L. Simon, M. G. Mulkerrin, and P. Carter. 1998. Contribution of domain interface residues to the stability of antibody CH3 domain homodimers. Biochemistry 37: 9266-9273 https://doi.org/10.1021/bi980270i
  9. 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
  10. Hudson, P. J. 1999. Recombinant antibody constructs in cancer therapy. Curr. Opin. Immunol. 11: 548-557 https://doi.org/10.1016/S0952-7915(99)00013-8
  11. 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
  12. 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
  13. 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
  14. Kim, S. H. 2003. Expression and purification of recombinant immunotoxin -- a fusion protein stabilizes a single-chain Fv (scFv) in denaturing condition. Protein Expr. Purif. 27: 85-89 https://doi.org/10.1016/S1046-5928(02)00539-9
  15. Kreitman, R. J. 1999. Immunotoxins in cancer therapy. Curr. Opin. Immunol. 11: 570-578 https://doi.org/10.1016/S0952-7915(99)00005-9
  16. Kreitman, R. J., C. N. Chang, D. V Hudson, C. Queen, P. Bailon, and I. Pastan. 1994. Anti-Tac(Fab)-PE40, a recombinant double-chain immunotoxin which kills interleukin-2-receptor-bearing cells and induces complete remission in an in vivo tumor model. Int. J. Cancer 57: 856-864 https://doi.org/10.1002/ijc.2910570615
  17. Kreitman, R. J., I. Margulies, M. Stetler-Stevenson, Q. C. Wang, D. J. FitzGerald, and I. Pastan. 2000. Cytotoxic activity of disulfide-stabilized recombinant immunotoxin RFB4( dsFv)PE38 (BL22) toward fresh malignant cells from patients with B-cell leukemias. Clin. Cancer Res. 6: 1476-1487
  18. Kuan, C. T. and I. Pastan. 1996. Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation. Biochemistry 35: 2872-2877 https://doi.org/10.1021/bi952047g
  19. 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
  20. Lawrence, L. J., A. A. Kortt, P. Iliades, P. A. Tulloch, and P. J. Hudson. 1998. Orientation of antigen binding sites in dimeric and trim eric single chain Fv antibody fragments. FEBS Lett. 425: 479-484 https://doi.org/10.1016/S0014-5793(98)00292-0
  21. 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
  22. 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
  23. Mansfield, E., P. AmIot, I. Pastan, and D. J. FitzGerald. 1997. Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors. Blood 90: 2020-2026
  24. Miller, K., G. Meng, J. Liu, A. Hurst, V. Hsei, W. L. Wong, R. Ekert, D. Lawrence, S. Sherwood, L. DeForge, J. Gaudreault, G. Keller, M. Sliwkowski, A. Ashkenazi, and L. Presta. 2003. Design, construction, and in vitro analyses of multivalent antibodies. J. lmmunol. 170: 4854-4861
  25. 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
  26. Pai, L. H., R. Wittes, A. Setser, M. C. Willingham, and I. Pastan. 1996. Treatment of advanced solid tumors with immunotoxin LMB-1: An antibody linked to Pseudomonas exotoxin. Nat. Med. 2: 350-353 https://doi.org/10.1038/nm0396-350
  27. 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
  28. Park, S. S., C. J. Ryu, Y. J. Kang, S. V. Kashmiri, and H. J. Hong. 2000. Generation and characterization of a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens. Mol. Immunol. 37: 1123-1130 https://doi.org/10.1016/S0161-5890(01)00027-X
  29. Pastan, I. 2003. Immunotoxins containing Pseudomonas exotoxin A: A short history. Cancer Immunol. Immunother. 52: 338-341
  30. Rajagopal, V., I. Pastan, and R. J. Kreitman. 1997. A form of anti-Tac(Fv) which is both single-chain and disulfide stabilized: Comparison with its single-chain and disulfidestabilized homologs. Protein Eng. 10: 1453-1459 https://doi.org/10.1093/protein/10.12.1453
  31. Reiter, Y., U. Brinkmann, K. O. Webber, S. H. Jung, B. Lee, and I. Pastan. 1994. Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: Improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv. Protein Eng 7: 697-704 https://doi.org/10.1093/protein/7.5.697
  32. Reiter, Y., L. H. Pai, U. Brinkmann, Q. C. Wang, and I. Pastan. 1994. Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Cancer Res. 54: 2714-2718
  33. Reiter, Y. and I. Pastan. 1998. Recombinant Fv immunotoxins and Fv fragments as novel agents for cancer therapy and diagnosis. Trends Biotechnol. 16: 513-520 https://doi.org/10.1016/S0167-7799(98)01226-8
  34. 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
  35. Ridgway, J. B., L. G. Presta, and P. Carter. 1996. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 9: 617 -621 https://doi.org/10.1093/protein/9.7.617
  36. Schmiedl, A., F. Breitling, and S. Dubel. 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
  37. Shim, Y-Y, D.-H. Shon, B.-Y. Kwak, J. Yu, and K.-M. Chee. 2004. Comparison of properties of poly clonal anti-N-acetylchitooligosaccharides and anti-chitooligosaccharides antibodies produced for ELISA. J. Microbiol. Biotechnol. 14: 686-692
  38. Todorovska, A., R. C. Roovers, O. Dolezal, A. A. Kortt, H. R. Hoogenboom, and P. J. Hudson. 2001. Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods 248: 47-66 https://doi.org/10.1016/S0022-1759(00)00342-2