Article: article from journal or magazin.
Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform.
Journal of Immunological Methods
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov'tPublication Status: ppublish
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.
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
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