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

J Immunol Methods. 2011 Jan 5;363(2):221-32. doi: 10.1016/j.jim.2010.09.001. Epub 2010 Sep 15.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Single-Chain Antibodies / isolation & purification*
  • Transformation, Genetic / genetics


  • Antigens, CD
  • Antigens, Neoplasm
  • CD248 protein, human
  • Peptide Library
  • Recombinant Proteins
  • Single-Chain Antibodies