Construction of human monoclonal single-chain Fv antibodies against small-cell lung cancer by phage display libraries derived from cell-immunized SCID mice engrafted with human peripheral blood lymphocytes

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Abstract

In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new mole- cular targets for immunotherapy and markers for diagnosis.

Original languageEnglish
Pages (from-to)1265-1272
Number of pages8
JournalProteomics - Clinical Applications
Volume3
Issue number11
DOIs
Publication statusPublished - 2009 Dec

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Single-Chain Antibodies
Bacteriophages
SCID Mice
Lymphocytes
Small Cell Lung Carcinoma
Libraries
Blood
Display devices
Monoclonal Antibodies
Cells
Membrane Proteins
Dihydrolipoyllysine-Residue Acetyltransferase
Biotinylation
Neoplasms
Antibodies
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Immunoprecipitation
Proteomics
Immunotherapy
Immunization

Keywords

  • PDC-E2
  • Phage display
  • SCID mice engrafted with human peripheral blood lymphocytes
  • Single-chain Fv
  • Small-cell lung cancer

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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title = "Construction of human monoclonal single-chain Fv antibodies against small-cell lung cancer by phage display libraries derived from cell-immunized SCID mice engrafted with human peripheral blood lymphocytes",
abstract = "In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new mole- cular targets for immunotherapy and markers for diagnosis.",
keywords = "PDC-E2, Phage display, SCID mice engrafted with human peripheral blood lymphocytes, Single-chain Fv, Small-cell lung cancer",
author = "Junya Douguchi and Akinori Hashiguchi and Michiie Sakamoto",
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AU - Douguchi, Junya

AU - Hashiguchi, Akinori

AU - Sakamoto, Michiie

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N2 - In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new mole- cular targets for immunotherapy and markers for diagnosis.

AB - In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new mole- cular targets for immunotherapy and markers for diagnosis.

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KW - Small-cell lung cancer

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