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.

Figure 1. Antigen immunoprecipitation using soluble scFv-3GA5, scFv-3GA9 and scFv-C3.2

(A) Single chain Fvs coupled to Ni-NTA-agarose were used to precipitate SK-BR-3 biotinylated cell lysates. Immune complexes were eluted using imidazole and eluted fractions were tested (upper band) by Western Blot in reducing conditions using HRP conjugated-streptavidin or stained with Ponceau Red (lower band); lane 1 (biotinylated crude extract), lanes 2 to 6 (eluted immune complexes from Ni-NTA agarose column) (B) Immunoblotting of scFv Bot, 3GH7, 3GA5, 3GA9 and C3.2 immunoprecipitates of SK-BR-3 cell extracts with anti-TfR mAb.

Figure 2. LC-MS/MS identification of 3GA5 cognate antigen

(A) Western Blot of scFv 3GA5 immunoprecipitated biotinylated SK-BR-3 cell lysates. The immune complexes were precipitated either with Protein A-agarose (lane 1), or with Ni-NTA-agarose (lane 2), and were tested by Western Blot, using HRP conjugated-streptavidin. (B) The purified band from scFv-3GA5 immunoprecipitation using Protein A-agarose was subjected to an in-gel digestion and subsequently analyzed by LC-MS/MS.. Each peptide was sequenced by tandem mass spectrometry. (C) Alignment of the 11 peptides identified on the sequence of CD9P-1.

Figure 3. Confirmation of specificity of 3GA5 to its cognate antigen CD9P-1

(A) Wild-type CHO cells and CHO cells transfected with human CD9P-1 cDNA were stained with Ph-Ab-3GA5. The binding of Ph-Ab-3GA5 is specific to CHO-CD9P-1. (B) Immune complexes from the immunoprecipitation of CHO, CHO-CD9P-1, SK-BR-3 and MCF7 cell lysates with 3GA5 were tested by Western Blot using mAb 1F11, specific to human CD9P-1.

Figure 4. Internalization of scFv 3GA5

(A) SK-BR-3 cells were incubated with 3GA5-DiI liposomes for 3 h either at 4°C or 37°C, washed with PBS and analyzed by confocal microscopy. (B) SK-BR-3 cells were incubated with 3GA5-NiNTA liposomes for 3 h either at 4°C or at 37°C, after trypsinization the cells were washed either with PBS or with imidazole and analyzed by flow cytometry.

Figure 5. Analysis of CD9P-1 and associated CD9 and CD81 tetraspanins

(A) CHO-CD9P-1, CHO, SK-BR-3, Hs-578Bst and Hs-578T cell lysates were subject to Western Blot analysis using anti CD9P-1, anti-CD9 and anti-CD81 mAbs. (B) The cell surface expression of CD9P-1, CD9 and CD81 on CHO-CD9P-1, CHO, SK-BR-3, Hs-578Bst and Hs-578T cells was analyzed by flow cytometry, by staining the cells with mAb 1F11, anti-CD9 and anti-CD81 Ab, MFI (Mean Fluorescence Intensity).

Figure 6. Phenotypic knock out of CD9P-1 in CHO cells

(A) CHO cells were transfected with pCMV-CD9P-1 and/or pKDEL-scFv plasmids as indicated. Two days later, cell lysates were prepared, immunoprecipitated with anti-CD9P-1 mAb 1F11 and analyzed by Western Blot in non-reducing conditions using biotinylated 1F11 or anti-myc tag mAbs. (B) The transfected cells were also stained with biotinylated 1F11 and anti-myc-Tag mAbs and analyzed by confocal microscopy (C) CHO-CD9P-1 cells were transfected with pKDEL-scFv plasmids and analyzed for CD9P-1 membrane expression two days later by flow cytometry using anti-CD9P-1 mAb 1F11. Labeling is revealed using PE conjugated anti-mouse Abs. The negative control is performed using the secondary antibody alone The pourcentage of the CD9P-1 positive cell population is indicated as well as the mean fluorescence intensity (MFI) of this positive cell population.