title: Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform.
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.
authors.affiliation: Penn Ovarian Cancer Research Center, Center for Research on Reproduction and Women's Health (CRRWH), Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Aizhi
authors.initials: A
authors.lastname: Zhao
authors.forename: Selene
authors.initials: S
authors.lastname: Nunez-Cruz
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Li
authors.forename: George
authors.initials: G
authors.lastname: Coukos
authors.forename: Don L
authors.initials: DL
authors.lastname: Siegel
authors.forename: Nathalie
authors.initials: N
authors.lastname: Scholler
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology*; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D010051:Ovarian Neoplasms / Q000276:immunology*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014170:Transformation, Genetic / Q000235:genetics
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; C078203:CD248 protein, human; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies