Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform.

Détails

ID Serval
serval:BIB_8D0290737376
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform.
Périodique
Journal of Immunological Methods
Auteur(s)
Zhao A., Nunez-Cruz S., Li C., Coukos G., Siegel D.L., Scholler N.
ISSN
1872-7905 (Electronic)
ISSN-L
0022-1759
Statut éditorial
Publié
Date de publication
2011
Volume
363
Numéro
2
Pages
221-232
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov'tPublication Status: ppublish
Résumé
Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10⁹ members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer.
Mots-clé
Animals, Antibody Affinity/genetics, Antibody Affinity/immunology, Antigens, CD/genetics, Antigens, CD/immunology, Antigens, Neoplasm/genetics, Antigens, Neoplasm/immunology, Cell Line, Tumor, Enzyme-Linked Immunosorbent Assay, Female, Flow Cytometry, Genetic Vectors/genetics, Humans, Mice, Ovarian Neoplasms/immunology, Peptide Library, Recombinant Proteins/genetics, Recombinant Proteins/immunology, Saccharomyces cerevisiae/genetics, Saccharomyces cerevisiae/immunology, Single-Chain Antibodies/genetics, Single-Chain Antibodies/immunology, Transformation, Genetic/genetics
Pubmed
Web of science
Création de la notice
14/10/2014 12:43
Dernière modification de la notice
03/03/2018 19:14
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