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Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2011 Jan 05; Vol. 363 (2), pp. 221-32. Date of Electronic Publication: 2010 Sep 15. - Publication Year :
- 2011
-
Abstract
- 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.<br /> (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Antibody Affinity genetics
Antigens, CD genetics
Antigens, Neoplasm genetics
Cell Line, Tumor
Enzyme-Linked Immunosorbent Assay
Female
Flow Cytometry
Genetic Vectors genetics
Humans
Mice
Peptide Library
Recombinant Proteins genetics
Recombinant Proteins immunology
Saccharomyces cerevisiae genetics
Single-Chain Antibodies genetics
Single-Chain Antibodies immunology
Transformation, Genetic genetics
Antibody Affinity immunology
Antigens, CD immunology
Antigens, Neoplasm immunology
Ovarian Neoplasms immunology
Saccharomyces cerevisiae immunology
Single-Chain Antibodies isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7905
- Volume :
- 363
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 20837020
- Full Text :
- https://doi.org/10.1016/j.jim.2010.09.001