Target Discovery and Validation Reviews and Protocols

302 Sioud
(see Chapter 15, Volume 1). The method does not rely on the availability of T-cell
clones or lines but rather screens antibody response of cancer patients against
autologous (or allogeneic) tumor cDNA expression libraries. Briefly, cDNA
libraries are constructed from tumor tissues or cell lines in a prokaryotic expression
vector such as the lytic phage λ. The library is used to transduce Escherichia
coli. Blotting to a nitrocellulose membrane transfers lytic plaques. The membranes
are then incubated with diluted patient serum antibodies preabsorbed for
removal of E. coli binding antibodies. Positive-reacting clones are detected by an
enzyme-conjugated secondary antibody specific for human IgG. In their initial
application of this method, they identified MAGE-1 and tyrosine, two antigens
originally cloned as CTL targets, indicating that the strategy can identify tumor
antigens that elicit a CTL-mediated immune response (59,60). Subsequent studies
identified an array of TAAs in many human cancers, as demonstrated by more
than 2000 different gene entries of potentially cancer related antigens (see
Chapter 1–15, Volume 1). Notably, strong IgG antibody responses are considered
to depend on the presence of T-cell help.
The SEREX antigens can be divided into several groups (61): (1) cancer-testis
(CT) antigens (e.g., HOM-MEL-40) are selectively expressed in a variety of neoplasms,
but not in normal tissues except for testis; (2) differentiation antigens show
a lineage-specific expression in tumors and in normal cells, but not only at a
defined stage of development (e.g., tyrosinase); (3) antigens encoded by mutated
genes (e.g., p53); (4) splice variants (e.g., restin); (5) viral antigens (e.g., HERV-
K10); (6) overexpressed genes (e.g., Galectin-9) are known to elicit immune
response by overriding thresholds critical for the maintenance of tolerance;
(7) overexpression because gene amplification (e.g., eIF-4γ); (8) cancer-related
autoantigens (e.g., HOM-MEL-2.4); and (9) cancer-independent autoantigens
(e.g., HOM-TES-11).
One of the technical problems with SEREX is that it is based upon a one-step
screening. In contrast, the sensitivity and selectivity are expected to be high
when the selection is performed through iterative, powerful enrichment steps,
thus offering one of the major advantages of phage display method that we have
established in 1993 for the purpose of profiling the immune response in patients
with autoimmune diseases (62). The only prerequisite of this novel approach is
the availability of patient sera. We have extended this method to patients with
cancers, and several tumor antigens have been identified.
11.5. Phage Display
For the generation of peptides or protein collections for the discovery-oriented
method, large numbers of synthetic peptides or recombinant proteins have to
be prepared and purified, which is a major technical challenge. Therefore,
recombinant peptides or proteins displayed on phages are highly recommended.