Target Discovery and Validation Reviews and Protocols

Protein Arrays 341
combinatorial screening in which phage display random peptide libraries are
screened on patient antibodies; when successful, such approach can lead to epitope
mapping as well as the identification of the corresponding native tumor antigens.
Because cancer cells are expected to express several different antigens,
fingerprinting might yield markers of disease aggressiveness or targets for therapy.
Hansen and colleagues identified several consensus peptide motifs that mimic
some potential tumor antigens, such as nuclear autoantigen sp100 and an uncharacterized
66-kDa protein, which has recently been identified as dihydrolipoamide
S-acetyltransferase. Interestingly, serum reactivity to the identified peptides correlated
with patient prognosis. Recently, Vidal and colleagues identified a consensus
peptide motif that is selectively recognized by antibodies derived from ascites from
ovarian cancer patients and identified the corresponding native antigen mimicked
by the motif: heat-shock protein of 90 kDa (HSP90). Furthermore, they showed
that although HSP90 is widely expressed in ovarian cancer, the humoral response
against HSP90 is tumor associated and stage specific (44).
In addition to peptides, the cDNA repertoires from cancer cells can be displayed
on phage and screened with patient sera (15,19,20,45). Compared with random
peptide phage libraries, phage-displaying cDNA repertoires from cells of interest
may have an advantage for the identification of the parental antigens, because
they represent naturally expressed antigens. In this respect, cDNA repertoires
from breast cancer cell lines T47D and MCF-7 were cloned in the three open
reading frames as fusion with the M13 pVI protein (20). When the libraries were
screened with serum antibodies from patients with breast cancer, phage-encoded
cDNA products were selected. Sequence analysis of the selected clones identified
important antigens, including p53, centromere-F, int-2, pentraxin I, integrin β5,
cathepsin L2, and S3 ribosomal protein. The selected phage-displayed cDNA
products were recognized by a significant number of breast cancer sera compared
with sera from normal individuals.
In addition to fingerprinting immune responses in patients, phage display
libraries are a valuable tool for the selection of cancer cell binding peptides. In
this respect, we have explored the possibility of selecting small peptides that
bind specifically to breast cancer cell lines. Phage displaying an LTVSPWY
peptide sequence exhibited a specific binding to breast cancer cells. None of the
selected peptides bound to human primary cells from different tissue origin
(e.g., epithelial, endothelial, and hematopoetic) (45). In vivo screening of a
peptide library in a patient also was performed by Arap and coworkers (46,47).
They selected 47,160 motifs that localized to different organs. This largescale
screening indicates that the tissue distribution of circulating peptides is
Fig. 1. (Opposite page) Hierarchical clustering of patient immunoreactivity against
phage-displayed peptides (23).