Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Oct 1;88(19):8616–8620. doi: 10.1073/pnas.88.19.8616

B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice.

U Brinkmann 1, L H Pai 1, D J FitzGerald 1, M Willingham 1, I Pastan 1
PMCID: PMC52560  PMID: 1924323

Abstract

The genes encoding the heavy- and light-chain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE). The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain. The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coli and the single-chain immunotoxins were purified to near homogeneity. Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active. Furthermore, intravenous administration of B3(Fv)-PE38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice.

Full text

PDF
8616

Images in this article

8618Image
on p.8618

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Batra J. K., FitzGerald D., Gately M., Chaudhary V. K., Pastan I. Anti-Tac(Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interleukin 2 receptor bearing cells. J Biol Chem. 1990 Sep 5;265(25):15198–15202. [PubMed] [Google Scholar]
  2. Batra J. K., Jinno Y., Chaudhary V. K., Kondo T., Willingham M. C., FitzGerald D. J., Pastan I. Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonas exotoxin. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8545–8549. doi: 10.1073/pnas.86.21.8545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird R. E., Hardman K. D., Jacobson J. W., Johnson S., Kaufman B. M., Lee S. M., Lee T., Pope S. H., Riordan G. S., Whitlow M. Single-chain antigen-binding proteins. Science. 1988 Oct 21;242(4877):423–426. doi: 10.1126/science.3140379. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Chaudhary V. K., Batra J. K., Gallo M. G., Willingham M. C., FitzGerald D. J., Pastan I. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1066–1070. doi: 10.1073/pnas.87.3.1066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chaudhary V. K., Gallo M. G., FitzGerald D. J., Pastan I. A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9491–9494. doi: 10.1073/pnas.87.23.9491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaudhary V. K., Jinno Y., FitzGerald D., Pastan I. Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc Natl Acad Sci U S A. 1990 Jan;87(1):308–312. doi: 10.1073/pnas.87.1.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chaudhary V. K., Queen C., Junghans R. P., Waldmann T. A., FitzGerald D. J., Pastan I. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature. 1989 Jun 1;339(6223):394–397. doi: 10.1038/339394a0. [DOI] [PubMed] [Google Scholar]
  10. Hartman A. B., Rudikoff S. VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules. EMBO J. 1984 Dec 1;3(12):3023–3030. doi: 10.1002/j.1460-2075.1984.tb02249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haynes B. F., Hemler M., Cotner T., Mann D. L., Eisenbarth G. S., Strominger J. L., Fauci A. S. Characterization of a monoclonal antibody (5E9) that defines a human cell surface antigen of cell activation. J Immunol. 1981 Jul;127(1):347–351. [PubMed] [Google Scholar]
  12. Huston J. S., Levinson D., Mudgett-Hunter M., Tai M. S., Novotný J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. doi: 10.1073/pnas.85.16.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kondo T., FitzGerald D., Chaudhary V. K., Adhya S., Pastan I. Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain. J Biol Chem. 1988 Jul 5;263(19):9470–9475. [PubMed] [Google Scholar]
  14. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Pai L. H., Batra J. K., FitzGerald D. J., Willingham M. C., Pastan I. Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3358–3362. doi: 10.1073/pnas.88.8.3358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  18. Siegall C. B., Chaudhary V. K., FitzGerald D. J., Pastan I. Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin. J Biol Chem. 1989 Aug 25;264(24):14256–14261. [PubMed] [Google Scholar]
  19. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  20. Taub R., Gould R. J., Garsky V. M., Ciccarone T. M., Hoxie J., Friedman P. A., Shattil S. J. A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen. J Biol Chem. 1989 Jan 5;264(1):259–265. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES