Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies

Abstract

To generate a panel of antibodies binding human breast cancers, a human single chain Fv phage display library was selected for rapid internalization into the SK-BR-3 breast cancer cell line. Thirteen unique antibodies were identified within the 55 cell binding antibodies studied, all of them showing specific staining of tumor cells compare to normal epithelial cells. Two of the antibodies bound the ErbB2 oncogene while 6 bound the tumor marker transferrin receptor (TfR). By developing a scFv immunoprecipitation method, we were able to use LC-MS/MS to identify the antigen bound by one of the antibodies (3GA5) as FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1 (CD9 partner 1) an Ig superfamily member that has been described to interact directly with CD9 and CD81 tetraspanins and to be overexpressed in adherent cancer cell lines. Although the 3GA5 scFv had no direct anti-proliferative effect, intracellular expression of the scFv was able to knockdown CD9P-1 expression and could be used to further define the role of the tetraspanin system in proliferation and metastasis. Moreover, the 3GA5 scFv was rapidly internalized into breast tumor cells and could have potential for the targeted delivery of cytotoxic agents to breast cancers. This study is the proof of principle that the direct selection of phage antibody libraries on tumor cells can effectively lead to the identification and functional characterization of relevant tumor markers.

Development of Human Single-Chain Antibodies to the Transferrin Receptor that Effectively Antagonize the Growth of Leukemias and Lymphomas

“…Following this process, we obtained a panel of six distinct TfR specific scFvs, namely, 3TF2, 3TF12, 3TG9, 3GH9, 3GH7, and C3.2, which were characterized as TfR specific based on their ability to immunoprecipate human TfR (20).…”

“…In this work, we characterized six human scFvs specific for the human TfR, previously isolated from a phage display library (20). Because the library had been selected for binding and internalization into living cells, we hypothesized that many of these scFvs would function as ligand mimetics.…”

“…The colon carcinoma ErbB2-positive cell line LS174T, which expresses high levels of TfR (20), was used to design a competitive binding assay between phage-scFvs and human holo-Tf (Fig. 1A).…”

“…For this work, we investigated the growth inhibitory potential of six human anti-TfR single-chain variable antibody fragments (scFv) selected by phage display for their ability to internalize rapidly into living cancer cells via receptormediated endocytosis (19,20). We reasoned that the stringency of a functional cell-based phage display selection would allow the isolation of functional ligand-like antibodies with agonistic or antagonistic activities.…”

Screening for antibodies against intact cancer cells with a na�ve large phage antibody library

“…Compared with intact antibodies, multivalent small molecular antibodies have shown the best balance of tumor targeting, tissue penetration and blood clearance rate. Thus, they are a promising vehicle for immunotherapy (24)(25)(26)(27), and they may play important roles in site-specific imaging diagnosis and targeted tumor therapy.…”