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Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.

Publication ,  Journal Article
Lorimer, IA; Keppler-Hafkemeyer, A; Beers, RA; Pegram, CN; Bigner, DD; Pastan, I
Published in: Proc Natl Acad Sci U S A
December 10, 1996

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

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Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

December 10, 1996

Volume

93

Issue

25

Start / End Page

14815 / 14820

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Mutation
  • Molecular Sequence Data
  • Mice
  • Immunotoxins
  • Immunoglobulin Variable Region
  • Humans
  • Glioblastoma
  • Gene Library
  • ErbB Receptors
 

Citation

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Lorimer, I. A., Keppler-Hafkemeyer, A., Beers, R. A., Pegram, C. N., Bigner, D. D., & Pastan, I. (1996). Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display. Proc Natl Acad Sci U S A, 93(25), 14815–14820. https://doi.org/10.1073/pnas.93.25.14815
Lorimer, I. A., A. Keppler-Hafkemeyer, R. A. Beers, C. N. Pegram, D. D. Bigner, and I. Pastan. “Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.Proc Natl Acad Sci U S A 93, no. 25 (December 10, 1996): 14815–20. https://doi.org/10.1073/pnas.93.25.14815.
Lorimer IA, Keppler-Hafkemeyer A, Beers RA, Pegram CN, Bigner DD, Pastan I. Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14815–20.
Lorimer, I. A., et al. “Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display.Proc Natl Acad Sci U S A, vol. 93, no. 25, Dec. 1996, pp. 14815–20. Pubmed, doi:10.1073/pnas.93.25.14815.
Lorimer IA, Keppler-Hafkemeyer A, Beers RA, Pegram CN, Bigner DD, Pastan I. Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14815–14820.
Journal cover image

Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

December 10, 1996

Volume

93

Issue

25

Start / End Page

14815 / 14820

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Mutation
  • Molecular Sequence Data
  • Mice
  • Immunotoxins
  • Immunoglobulin Variable Region
  • Humans
  • Glioblastoma
  • Gene Library
  • ErbB Receptors