Target Discovery and Validation Reviews and Protocols

308 Sioud
molecular techniques have proven to be a powerful approach for the discovery
of novel therapeutic genes. So far, microararys are the most used methods for
generating quantitative information about the expression of thousands of genes
with relative rapidity. However, novel transcripts cannot be discovered by this
method. To uncover new tumor antigens and therapeutic targets, we have combined
PCR-based cDNA subtraction technique and Northern blotting (85).
After cloning and sequencing, several potential known and new targets were
identified, including ADP/ATP carrier protein, ErbB2, and ribosomal protein
RPL19.
A key in tumor antigen discovery before proceeding to vaccine development
is demonstrating the immunoreactivity of the new target antigens. A
variety of in vitro and in vivo assays are used to identify peptide epitopes
capable of inducing CD8+, CD4+, or both (see Chapter 1–15, Volume 1).
Serum IgG antibody reactivity can be considered as a good indicator for activated
humoral and cellular immunity. Significant antibody reactivity against
several antigens was found in patients with breast cancer compared with
controls (72,75,76,85).
Public access to the human genome sequences, cDNAs derived from normal
and malignant tissues, together with the EST database has facilitated the
discovery of tumor antigens through a bioinformatics approach ([81,84]; see
Chapter 2, Volume 1). The data can be searched for overexpressed genes,
which should be considered as candidate tumor antigens. However, as overexpression
does not always imply that there will be an immune response
against the protein in question; additional steps have to be integrated to direct
the screening efforts toward identifying the best candidate antigen or therapeutic
target for immunologic or therapeutic intervention. Differentially
expressed genes need to be identified systematically (perhaps by the application
of rapidly improving gene array technologies), and their immunogenicity
needs to be determined. Ideally, candidate genes would only be expressed in
tumor cells but not in any normal cells.
11.10. Proteomics and Tumor Antigen Identification
It is anticipated that RNA levels of many genes do not reflect protein contents;
therefore, transcriptome analysis might be misleading for target discovery.
Moreover, posttranscriptional modifications cannot be detected by this technique,
and dynamic changes on protein level would not be revealed on the transcriptome
level. Proteomics, the study of proteins and protein pathways, is a promising technique
for tumor antigen discovery (86). It depends on a combination of methods,
including two-dimensional gel electrophoresis, mass spectrometry, peptide
sequencing, image analysis, and bioinformatics. The promise of this technology to
identify novel antigen is described in Chapter 16, Volume 1. Using this method and