Effective targeted cytotoxicity of neuroblastoma cells

Patrick B. Thomas, Stephen J. Delatte, Aimee Sutphin, Arthur E. Frankel, Edward P. Tagge

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Background/Purpose: Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DTA) linked to a single chain antibody (sc5F11) targeting the GD2 antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11. Methods: Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD2 targeting antibody (sc5F11) and truncated diphtheria toxin (DTA). DH5α Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD2-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD2-negative (HeLa) cells. Results: Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DTA antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD2 positive cells. Conclusions: This study demonstrates creation of a novel fusion toxin with effective GD2-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.

Original languageEnglish (US)
Pages (from-to)539-544
Number of pages6
JournalJournal of Pediatric Surgery
Volume37
Issue number3
DOIs
StatePublished - 2002

Fingerprint

Neuroblastoma
Diphtheria Toxin
Western Blotting
Local Area Networks
Immunotoxins
Single-Chain Antibodies
Antibodies
Gene Fusion
Fluorescent Dyes
HeLa Cells
Electrophoresis
Digestion
Neoplasms
Plasmids
Radiotherapy
Therapeutics
Gels
Antigens
Drug Therapy
Polymerase Chain Reaction

Keywords

  • Fusion toxin
  • GD-targeting
  • Neuroblastoma

ASJC Scopus subject areas

  • Surgery

Cite this

Effective targeted cytotoxicity of neuroblastoma cells. / Thomas, Patrick B.; Delatte, Stephen J.; Sutphin, Aimee; Frankel, Arthur E.; Tagge, Edward P.

In: Journal of Pediatric Surgery, Vol. 37, No. 3, 2002, p. 539-544.

Research output: Contribution to journalArticle

Thomas, Patrick B. ; Delatte, Stephen J. ; Sutphin, Aimee ; Frankel, Arthur E. ; Tagge, Edward P. / Effective targeted cytotoxicity of neuroblastoma cells. In: Journal of Pediatric Surgery. 2002 ; Vol. 37, No. 3. pp. 539-544.
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AB - Background/Purpose: Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DTA) linked to a single chain antibody (sc5F11) targeting the GD2 antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11. Methods: Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD2 targeting antibody (sc5F11) and truncated diphtheria toxin (DTA). DH5α Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD2-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD2-negative (HeLa) cells. Results: Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DTA antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD2 positive cells. Conclusions: This study demonstrates creation of a novel fusion toxin with effective GD2-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.

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