Target Discovery and Validation Reviews and Protocols

328 Le Naour
dependent on CD8+ cytotoxic T-lymphocytes. Thus, attention has been focused
on tumor-specific CD8+ T-cells. Since the cloning of MAGE-1, the first reported
antigen to encode a human tumor antigen recognized by autologous CTLs (5),
an array of autoimmunogenic tumor antigens have been identified (6). Although
the characterization of tumor antigen recognized by cytotoxic T-cells is crucial,
a limitation stems from this approach requiring the establishment of autologous
target cells in culture and the isolation of stable lines of autologous CTLs
that recognize antigens. In addition, some epithelial cell types are poorly susceptible
to CTLs in vitro. In addition, the B-cell response (antibodies) also has
been demonstrated in cancer patients. Recently, a method called serological
analysis of recombinant tumor cDNA expression libraries (SEREX) has been
used for the identification of tumor antigens (7). SEREX is based on screening
of autoantibodies in sera from patients with cancer against an expression library
made with mRNAs from the autologous tumor (see Chapter 15, Volume 1). By
applying this strategy to human tumors of different origin, an unexpected frequency
of tumor antigens that elicit specific immune responses in the autologous
host has been demonstrated (7–10). However, SEREX is limited by the
necessity to construct expression libraries, and the analysis is usually restricted
to one or a few patients (7–11).
The recent progress of proteomics, allowing large-scale analysis of proteins
within a single experiment, has opened new avenues for TAA discovery. This
chapter focuses the utility of proteomics for identification of tumor-associated
antigens with emphasis on proteins eliciting humoral response in cancer patients.
2. Proteomics: The State of the Art
Proteomics designates the study of the proteome. It involves a variety of
approaches combining high-resolution separation techniques with identification
methods such as mass spectrometry. For many years, two-dimensional (2D)
polyacrylamide gel electrophoresis (PAGE) has been the primary technique for
proteomic-based biomarker discovery. Thus, 2D electrophoresis (2DE) allows
the separation of the proteins according to their charge in a first dimension where
they stop their migration after reaching their isoelectric point (pI) and then in a
second dimension according to their molecular mass. The proteins can be visualized
by different staining, such as Coomassie blue or silver, and digitalized.
2DE is a highly resolutive technique leading to the visualization of at least 1000
protein spots in one experiment. Therefore, 2DE allows comparative analysis of
protein expression levels and the visualization of various protein isoforms. The
identification of the proteins can be performed after enzymatic digestion of
the proteins in-gel followed by analysis of the resulting peptides by mass spectrometry.
This process can be performed by mass fingerprinting, usually using
matrix-assisted laser desorption ionization/time of flight mass spectrometry or