Development of a hyperimmune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer

Michelle D. Winthrop, Sally J. DeNardo, Gerald L Denardo

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy. Optimizing the approach has proven difficult thus far. Antibody phage display libraries provide unique molecules that could improve RIT. A phage display library of single chain antibody fragments (scFv) against the MUC-1 mucin molecule, which is expressed on 90% of human breast cancers, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice. Increased serum IgG levels, 15 times baseline, were detected following the third immunization. RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions were amplified using PCR technology. The variable heavy and variable light chain gene regions were combined with a flexible linker, ligated into the pCANTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells. A library of 107 initial colonies was compiled. Forty-six of 288 colonies screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 breast cancer cell membrane fragments. Anti-MUC-1 library diversity evaluated by BstNI digest demonstrated that 52% of the anti-MUC-1 scFv binding MCF-7 possessed individual banding patterns representative of approximately 5 x l05 colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers. In summary, the anti-MUC-1 scFv antibody phage library contains diverse scFv molecules, which should provide unique characteristics and epitope recognition. These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer.

Original languageEnglish (US)
JournalClinical Cancer Research
Volume5
Issue number10 SUPPL.
StatePublished - Oct 1999

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Single-Chain Antibodies
Immunoglobulin Fragments
Radioimmunotherapy
Bacteriophages
Libraries
Breast Neoplasms
Epitopes
Immunoglobulin Light Chain Genes
Spleen
Mucin-1
Immunization
Complementary DNA
Immunoglobulin G
Cell Membrane
RNA
Escherichia coli
Technology
Light
Polymerase Chain Reaction
Antibodies

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Development of a hyperimmune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer. / Winthrop, Michelle D.; DeNardo, Sally J.; Denardo, Gerald L.

In: Clinical Cancer Research, Vol. 5, No. 10 SUPPL., 10.1999.

Research output: Contribution to journalArticle

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abstract = "Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy. Optimizing the approach has proven difficult thus far. Antibody phage display libraries provide unique molecules that could improve RIT. A phage display library of single chain antibody fragments (scFv) against the MUC-1 mucin molecule, which is expressed on 90{\%} of human breast cancers, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice. Increased serum IgG levels, 15 times baseline, were detected following the third immunization. RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions were amplified using PCR technology. The variable heavy and variable light chain gene regions were combined with a flexible linker, ligated into the pCANTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells. A library of 107 initial colonies was compiled. Forty-six of 288 colonies screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 breast cancer cell membrane fragments. Anti-MUC-1 library diversity evaluated by BstNI digest demonstrated that 52{\%} of the anti-MUC-1 scFv binding MCF-7 possessed individual banding patterns representative of approximately 5 x l05 colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers. In summary, the anti-MUC-1 scFv antibody phage library contains diverse scFv molecules, which should provide unique characteristics and epitope recognition. These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer.",
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