Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

18 2 Serological Determinants On Tumor Cells
2.2
SEREX: The Approach
To identify tumor antigens recognized by the antibody repertoire of cancer patients
we developed a serological cloning approach, termed SEREX (serological analysis of
tumor antigens by recombinant cDNA expression cloning). It allows a systematic
and unbiased search for antibody responses against proteins and the direct molecular
definition of the respective tumor antigens based on their reactivity with autologous
patient serum. For SEREX, cDNA expression libraries are constructed from
fresh tumor specimens, cloned into l phage expression vectors and phages are used
to transfect Escherichia coli. Recombinant proteins expressed during the lytic infection
of the bacteria are transferred onto nitrocellulose membranes, which are then
incubated with diluted (1:500±1:1000) and, most important, extensively pre-absorbed
serum from the autologous patient. Clones reactive with high-titered antibodies
are identified using an enzyme-conjugated second antibody specific for human
IgG. Positive clones are subcloned to monoclonality thus allowing the direct molecular
characterization by DNA sequencing.
The SEREX approach is technically characterized by several features [5, 6]:
1. There is no need for established tumor cell lines and pre-characterized CTL
clones.
2. The use of fresh tumor specimens restricts the analysis to genes that are expressed
by the tumor cells in vivo and circumvents in vitro artifacts associated
with short- and long-term tumor cell culture.
3. The use of the polyclonal (polyspecific) patient's serum allows for the identification
of multiple antigens with one screening course.
4. The screening is restricted to clones against which the patient's immune system
has raised high-titered IgG or/and IgA antibody responses indicating the presence
of a concomitant T helper lymphocyte response in vivo.
5. As both the expressed antigenic protein and the coding cDNA are present in the
same plaque of the phage immunoscreening assay, identified antigens can be sequenced
immediately. Sequence information of excised cDNA inserts can be directly
used to determine the expression spectrum of identified transcripts by
Northern blot and reverse transcription polymerase chain reaction (RT-PCR).
6. The release of periplasmatic proteins involved in protein folding during phage-induced
bacterial lysis allows at least partial folding of recombinant proteins and
provides the basis for the identification of linear as well as non-linear epitopes.
This has been confirmed by the expression of transcripts that code for enzymatically
active proteins [7]. In contrast, epitopes derived from eukaryotic post-translational
modification (e.g. glycosylation) are not detected by the phage immunoscreening
assay.
Meanwhile, a number of modifications of the original method have been implemented.
Immunoglobulins, which are also recombinantly expressed due to the presence