Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice

Abstract

Vascular leak syndrome is a major and often dose-limiting side effect of immunotoxins and cytokines. We postulated that this syndrome is initiated by damage to vascular endothelial cells. Our earlier studies identified a three–amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. We have now generated a panel of recombinant ricin A chains with mutations in this sequence or in amino acids flanking it in the three-dimensional structure. These have been evaluated alone and as immunotoxins for activity, ability to induce pulmonary vascular leak in mice, pharmacokinetics, and activity in tumor-xenografted mice. One mutant was comparable to the ricin A chain used before in all respects except that it did not cause vascular leak at the same dose and, when used as an immunotoxin, was more effective in xenografted SCID mice.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Ribbon diagram of RTA.
Figure 2: In vivo PVL of RFB4-RTA immunotoxins.
Figure 3: Effect of equitoxic doses of immunotoxins prepared with N97A vs. dgRTA on the survival of SCID-Daudi mice.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Farah, R.A., Clinchy, B., Herrera, L. & Vitetta, E.S. The development of monoclonal antibodies for the therapy of cancer. Crit. Rev. Eukaryotic Gene Expr. 8, 321–356 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Sausville, E.A. & Vitetta, E.S. Monoclonal Antibody-Based Therapy of Cancer (Marcel Dekker, Boston, 1997).

    Google Scholar 

  3. 3

    Frankel, A.E., Kreitman, R.J. & Sausville, E.A. Targeted toxins. Clin. Cancer Res. 6, 326–334 (2000).

    CAS  PubMed  Google Scholar 

  4. 4

    Amlot, P.L. et al. A phase I study of an anti-CD22-deglycosylated ricin A chain immunotoxin in the treatment of B-cell lymphomas resistant to conventional therapy. Blood 82, 2624–2633 (1993).

    CAS  PubMed  Google Scholar 

  5. 5

    Vitetta, E.S. et al. A phase I immunotoxin trial in patients with B-cell lymphoma. Cancer Res. 51, 4052–4058 (1991).

    CAS  PubMed  Google Scholar 

  6. 6

    Stone, M.J. et al. A Phase I study of bolus versus continuous infusion of the anti-CD19 immunotoxin, IgG-HD37-dgA, in patients with B-cell lymphoma. Blood 88, 1188–1197 (1996).

    CAS  PubMed  Google Scholar 

  7. 7

    Engert, A. et al. A Phase I study of an anti-CD25 ricin A-chain immunotoxin (RFT5- SMPT-dgA) in patients with refractory Hodgkin's lymphoma. Blood 89, 403–410 (1997).

    CAS  PubMed  Google Scholar 

  8. 8

    Messmann, R.A. et al. A phase I study of combination therapy with immunotoxins IgG-HD37-deglycosylated ricin A chain (dgA) and IgG-RFB4-dgA (Combotox) in patients with refractory CD19+, CD22+ B-cell lymphoma. Clin. Cancer Res. 6, 1302–1313 (2000).

    CAS  PubMed  Google Scholar 

  9. 9

    Schnell, R. et al. A Phase I study with an anti-CD30 ricin A chain immunotoxin (Ki-4.dgA). Clin. Cancer Res. 8, 1779–1786 (2003).

    Google Scholar 

  10. 10

    Sausville, E.A. et al. Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study. Blood 85, 3457–3465 (1995).

    CAS  PubMed  Google Scholar 

  11. 11

    Rosenberg, S.A. et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N. Engl. J. Med. 316, 889–897 (1987).

    CAS  Article  Google Scholar 

  12. 12

    Schindler, J., Sausville, E., Messmann, R., Uhr, J.W. & Vitetta, E.S. The toxicity of deglycosylated ricin A chain-containing immunotoxins in patients with non-Hodgkin's lymphoma is exacerbated by prior radiotherapy: a retrospective analysis of patients in five clinical trials. Clin. Cancer Res. 7, 255–258 (2001).

    CAS  PubMed  Google Scholar 

  13. 13

    Senderowicz, A.M. et al. Complete sustained response of a refractory, post-transplantation, large B-cell lymphoma to an anti-CD22 immunotoxin. Ann. Intern. Med. 126, 882–885 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Soler-Rodriguez, A.M., Ghetie, M.A., Oppenheimer-Marks, N., Uhr, J.W. & Vitetta, E.S. Ricin A-chain and ricin A-chain immunotoxins rapidly damage human endothelial cells: implications for vascular leak syndrome. Exp. Cell Res. 206, 227–234 (1993).

    CAS  Article  Google Scholar 

  15. 15

    Baluna, R., Rizo, J., Gordon, B.E., Ghetie, V. & Vitetta, E.S. Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome. Proc. Natl. Acad. Sci. USA 96, 3957–3962 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Coulson, B.S., Londrigan, S.L. & Lee, D.J. Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells. Proc. Natl. Acad. Sci. USA 94, 5389–5394 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Baluna, R. & Vitetta, E.S. An in vivo model to study immunotoxin-induced vascular leak in human tissue. J. Immunother. 22, 41–47 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Baluna, R., Coleman, E., Jones, C., Ghetie, V. & Vitetta, E.S. 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 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Press, O.W., Vitetta, E.S., Farr, A.G., Hansen, J.A. & Martin, P.J. Evaluation of ricin A-chain immunotoxins directed against human T cells. Cell. Immunol. 102, 10–20 (1986).

    CAS  Article  Google Scholar 

  20. 20

    Shen, G.-L. et al. Evaluation of four CD22 antibodies as ricin A chain-containing immunotoxins for the in vivo therapy of human B-cell leukemias and lymphomas. Int. J. Cancer 42, 792–797 (1988).

    CAS  Article  Google Scholar 

  21. 21

    Ghetie, M.A. et al. Antitumor activity of Fab′ and IgG-anti-CD22 immunotoxins in disseminated human B lymphomas grown in mice with severe combined immunodeficiency disease: effect on tumor cells in extranodal sites. Cancer Res. 51, 5876–5880 (1991).

    CAS  PubMed  Google Scholar 

  22. 22

    Soler-Rodriguez, A.M., Uhr, J.W., Richardson, J. & Vitetta, E.S. The toxicity of chemically deglycosylated ricin A-chain in mice. Int. J. Immunopharm. 14, 281–291 (1992).

    CAS  Article  Google Scholar 

  23. 23

    Rosenstein, M., Ettinghausen, S.E. & Rosenberg, S.A. Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin-2. J. Immunol. 137, 1735–1742 (1986).

    CAS  PubMed  Google Scholar 

  24. 24

    Ghetie, M.-A., Tucker, K., Richardson, J., Uhr, J. & Vitetta, E.S. The antitumor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD19 antibody or an anti-CD19 immunotoxin. Blood 80, 2315–2320 (1992).

    CAS  PubMed  Google Scholar 

  25. 25

    Baluna, R., Ghetie, V., Oppenheimer-Marks, N. & Vitetta, E.S. Fibronectin inhibits the cytotoxic effect of ricin A chain on endothelial cells. Int. J. Immunopharm. 18, 355–361 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Yan, X. et al. Structure-based identification of a ricin inhibitor. J. Mol. Biol. 266, 1043–1049 (1997).

    CAS  Article  Google Scholar 

  27. 27

    Simpson, J.C., Lord, J.M. & Roberts, L.M. Point mutations in the hydrophobic C-terminal region of ricin A chain indicate that Pro250 plays a key role in membrane translocation. Eur. J. Biochem. 232, 458–463 (1995).

    CAS  Article  Google Scholar 

  28. 28

    O'Hare, M. et al. Expression of ricin A chain in Escherichia coli. FEBS Lett. 216, 73–78 (1987).

    CAS  Article  Google Scholar 

  29. 29

    Sambrook, J., Maniatis, T. & Fritsch, E.F. Molecular Cloning: A Laboratory Manual edn. 2 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

    Google Scholar 

  30. 30

    Ghetie, V. et al. The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond. J. Immunol. Methods 142, 223–230 (1991).

    CAS  Article  Google Scholar 

  31. 31

    Knowles, P.P. & Thorpe, P.E. Purification of immunotoxins containing ricin A-chain and abrin A-chain using Blue Sepharose CL-6B. Anal. Biochem. 160, 440–443 (1987).

    CAS  Article  Google Scholar 

  32. 32

    Fraker, P.J. & Speck, J.C.J. Protein and cell membrane iodinations with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849–857 (1978).

    CAS  Article  Google Scholar 

  33. 33

    Kalbfleisch, J.D. & Prentice, R.L. The Statistical Analysis of Failure Time Data (Wiley, New York, 1980).

    Google Scholar 

  34. 34

    Shah, S.A. et al. Anti-B4-blocked ricin immunotoxin shows therapeutic efficacy in four different SCID mouse tumor models. Cancer Res. 53, 1360–1367 (1993).

    CAS  PubMed  Google Scholar 

  35. 35

    Shumaker, R.C. PKCALC: a basic interactive program for statistic and pharmacokinetic analysis of data. Drug Metab. Rev. 17, 331–348 (1986).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank Lien Le, Ming-Mei Liu, Yuen Chinn, Ana Firan, Steve Ruback, and Stephanie Tuggle for exceptional technical assistance. We thank Shannon Flowers and Linda Owens for secretarial assistance and M. Lord for providing the rRTA clone. We are indebted to Jonathan Uhr and John Schindler for helpful comments concerning the manuscript. This work was supported by US National Institutes of Health grant CA-77701 and a grant from the Higher Education Coordinating Board of the state of Texas.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ellen S. Vitetta.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Smallshaw, J., Ghetie, V., Rizo, J. et al. Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice. Nat Biotechnol 21, 387–391 (2003). https://doi.org/10.1038/nbt800

Download citation

Further reading

Search

Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing