Target Discovery and Validation Reviews and Protocols

304 Sioud
display if the foreign protein cannot be secreted through the E. coli membrane.
Additionally, the chemical characteristics of the periplasmic environment may
affect the folding, processing, and stability of the protein to be displayed. In part
because of such limitations, systems using lytic phage such as λ, T4, and T7 have
been developed (69). Phage assembly takes place in the cytoplasm, and mature
phages are released by cell lysis. This sequence implies that the protein included
in the phage capsids is folded and assembled in the cytoplasm and does not
require secretion through the membrane.
The T7 select TM display system exploits the product of gene 10 to display
foreign peptides on the T7 capsid (70). The cDNA inserts are placed at
the carboxy-terminal to the coat protein, so the expression of intact fusion
protein is not affected by the presence of stop codon (71). The T7 bacteriophage
has a very short life cycle and as a consequence, the time needed to
perform multiple panning cycles is decreased compared with other types of
display. Moreover, phage particles are extremely robust and resistant to
harsh conditions that can be used for phage elution. We implemented this
system in our laboratory and showed its feasibility in identifying tumor antigens
(72).
11.6. Application of Phage Display to Study Immune Responses
in Patients
The characterization of B- and T-cell specificities is crucial for the understanding
of the immune response mechanism and its role in the prevention
and cause of human diseases. However, the determination of B- and T-cell
specificities requires access to the parental antigen(s) that initiate the immune
response, a major limiting step if such antigens are not available. We have
shown that random peptide libraries can identify serum antibody specificities
whether or not the parental antigens are known (62,73,74), the rationale being
that antibodies from patient sera will bind to the phage containing the peptide
initiating the immune response against self- or nonself proteins. Thus,
through this strategy, it is possible to probe any specific immune response
in patients. Sera from patients, in addition to disease-specific antibodies, contains
antibodies of irrelevant specificities. To enrich for the disease-specific
epitopes, different approaches for subtraction or counterscreening have been
developed (62,68).
An important application of the phage display technology will be the
analysis of immune responses in patients with cancer, because it has been difficult
to identify immunogenic antigens capable of inducing immune responses
in patients to a level high enough for tumor rejection (68). Although, most of
the studies carried out with phage display libraries have been applied to
homogenous proteins, we have shown for the first time that peptide phage