Target Discovery and Validation Reviews and Protocols

Overview of Immune System 307
some of these potential problems, we have introduced a subtraction step in
which the undesired antibody-binding phages are subtracted by normal serum
antibodies (62).
After the desired rounds of selection (usually three to four), individual
phages should be tested for binding by, for example, ELISA. To simplify this
time-consuming step, we have used a rapid immunological screening method
(68,75,76). In this method, the immunoselected phage library is transferred to
nitrocellulose membranes, and subsequently the filters are probed by either
serum antibodies from patients or controls (Fig. 13). Therefore, it is possible to
screen many phages in a single step. The data shown in Fig. 13 illustrate the
power of the enrichment achieved by the phage display technology. A strong
reactivity was found with pooled IgG antibodies from breast cancer patients, but
no significant reactivity was found with IgG from normals, patients with lymphoma,
or those with osterosarcoma. Positive clones are clearly distinguishable
from negative clones, confirming the specificity of the immunoreaction.
11.8. Technical Limitation of SEREX and Phage Display Methodologies
The current bacterial expression systems are greatly limited in their ability to
identify antigen that are posttranslationally modified. These modifications play an
important role for proper folding and functioning of many proteins. Moreover,
modifications such as glycosylations are known to affect the immunogenicity of
proteins. As a consequence, several groups have established eukaryotic expression
systems such as baculovirus (77) and retroviral surface display (78).
Recently, the yeast Saccharomyces cerevisiae expression system was applied for
the display and affinity maturation of an scFv antibody via the agglutinin mating
adhesion receptor (79). Yeast display offers the advantage of using flow cytometry
for rapid quantitative isolation of rare binders. The method was used for the
isolation and characterization of tumor antigens recognized by autologous or
allogeneic breast cancer serum (80).
11.9. Identification of Tumor Antigens by Expression Profiling
of Cancer Cells
Another approach to identify tumor antigens is to search for potential T- and
B-cell antigens by mRNA expression profiling, namely, by searching for genes
that are expressed in cancer but not in other normal tissues (81–84). Several
selection and global techniques have been used to monitor gene expression,
including subtractive hybridization, PCR-based subtraction cloning, representational
difference analysis, suppression subtractive hybridization, differential
display, large-scale sequencing of expressed sequence tag (EST) sequences,
serial analysis of gene expression, and DNA microarray array analysis
(see Chapters 2–6, Volume 1). In addition to tumor antigen discovery, these