Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Disulfide Bond Bridged Divalent Antibody-Toxin, $(Fab-PE38fl)_2$ with the Toxin PE38 Fused to the Light Chain

  • Won, Jae-Seon (College of Life Sciences and Graduate School of Biotechnology, Korea University) ;
  • Choe, Mu-Hyeon (College of Life Sciences and Graduate School of Biotechnology, Korea University)
  • Published : 2008.08.31
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Abstract

B3 antibody specifically binds the $Lewis^Y$-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, $(Fab-PE38fl)_2$. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences hi, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers $(Fabh1-PE38fl)_2$, $(Fabh2-PE38fl)_2$, and $(Fabh3-PE38fl)_2$. The refolding yields of these dimers were 5-16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment $(Fabh2-PE38)_2$ [8]. Our data suggest that the steric repulsion between the two PE38s in $(Fabh1-PE38)_2$ during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.

Keywords

References

  1. Allured, V. S., R. J. Collier, S. F. Carroll, and D. B. McKay. 1986. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0 Angstrom. Proc. Natl. Acad. Sci. USA 83: 1320-1324
  2. Benhar, I. and I. Pastan. 1995. Characterization of B1(Fv)PE38 and B1(dsFv)PE38: Single-chain and disulfide-stabilized Fv immunotoxins with increased activity that cause complete remissions of established human carcinoma xenografts in nude mice. Clin. Cancer Res. 1: 1023-1029
  3. Brinkmann, U., J. Buchner, and I. Pastan. 1992. Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: Influence of interdomain connections. Proc. Natl. Acad. Sci. USA 89: 3075-3079
  4. 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
  5. 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
  6. Buchner, J. and R. Rudolph. 1991. Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli. Biotechnology (NY) 9: 157-162 https://doi.org/10.1038/nbt0291-157
  7. 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
  8. Choi, S. H., J. E. Kim, Y. C. Lee, Y. J. Jang, I. Pastan, and M. H. Choe. 2001. A divalent immunotoxin formed by the disulfide bond between hinge regions of Fab domain. Bull. Kor. Chem. Soc. 22: 1361-1365
  9. Colcher, D., R. Bird, M. Roselli, K. D. Hardman, S. Johnson, S. Pope, et al. 1990. In vivo tumor targeting of a recombinant single-chain antigen-binding protein. J. Natl. Cancer Inst. 82:1191-1197 https://doi.org/10.1093/jnci/82.14.1191
  10. Derocq, J. M., P. Casellas, G. Laurent, S. Ravel, H. Vidal, and F. Jansen. 1988. Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins. J. Immunol. 141:2837-2843
  11. Horio, M., E. Lovelace, I. Pastan, and M. M. Gottesman. 1991. Agents which reverse multidrug-resistance are inhibitors of [3H]vinblastine transport by isolated vesicles. Biochim. Biophys. Acta 1061: 106-110 https://doi.org/10.1016/0005-2736(91)90274-C
  12. Hwang, J., D. J. FitzGerald, S. Adhya, and I. Pastan. 1987. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 48: 129-136 https://doi.org/10.1016/0092-8674(87)90363-1
  13. 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
  14. 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. 64: 1419-1424 https://doi.org/10.1158/0008-5472.CAN-03-0570
  15. 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. Proc. Natl. Acad. Sci. USA 88: 3358-3362
  16. 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
  17. Pastan, I., E. T. Lovelace, M. G. Gallo, A. V. Rutherford, J. L. Magnani, and M. C. Willingham. 1991. Characterization of monoclonal antibodies B1 and B3 that react with mucinous adenocarcinomas. Cancer Res. 51: 3781-3787
  18. Reiter, Y., U. Brinkmann, S. H. Jung, B. Lee, P. G. Kasprzyk, C. R. King, and I. Pastan. 1994. Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment. J. Biol. Chem. 269:18327-18331
  19. 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
  20. Saito, T., R. J. Kreitman, S. Hanada, T. Makino, A. Utsunomiya, T. Sumizawa, et al. 1994. Cytotoxicity of recombinant Fab and Fv immunotoxins on adult T-cell leukemia lymph node and blood cells in the presence of soluble interleukin-2 receptor. Cancer Res. 54: 1059-1064