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

of B lymphocytes and plasma cells in the tumor specimens used for the cDNA library,
may represent more than 90 % of all ªpositiveº clones in libraries derived from
certain tissues. They can be identified by a modified initial screening procedure
whereby the nitrocellulose membrane is incubated with enzyme-conjugated anti-human
IgG followed by visualization with the appropriate enzymatic color reaction
prior to the incubation of the autologous patient's serum [8]. Screening of tumor cell
lines rather than fresh tumor specimen circumvents this problem and additionally
provides a pure RNA source which is not contaminated with normal stroma [9]. Subtractive
approaches allowenriching the cDNA library for tumor-specific transcripts
[10]. cDNA libraries may also be prepared from sources of specific interest, such as
amplified chromosomal regions obtained by microdissection [11].
2.3
Searching for Human Antigens by SEREX
Expression libraries were constructed and analyzed by SEREX from a variety of different
neoplasms, including three different renal cell carcinomas of the clear cell
type, two melanomas, one ovarian carcinoma, one hepatocarcinoma, 10 gliomas,
two colorectal cancers, one pancreatic cancer, two breast cancers, two Hodgkin's lymphomas,
two acute T cell leukemias and two acute myelogenous leukemias. Primary
libraries with at least 1 × 10 6 independent clones were established. The screening of
at least 1 × 10 6 clones per library revealed multiple reactive clones in each library.
Some transcripts were detected repeatedly, indicating that they were multiply represented
in the library. In order to bias for the detection of antigens of the cancer testis
class, libraries were constructed from normal testis tissue and screened with allogeneic
tumor patients' sera.
2.4
Molecular Characterization of SEREX Antigens
2.4 Molecular Characterization of SEREX Antigens
Clones were selected for more in-depth analysis on the basis of:
1. Their sequence data and comparison with databases to reveal identity or homologies
with known genes and to identify domains or motifs informative for a putative
function or cellular localization.
2. The analysis of the expression pattern of the respective antigen in normal tissues
and tumors by RT-PCR, by Northern blot hybridization with specific probes and
by analysis in expressed sequence tag-containing databases.
3. An initial survey for antibodies in the sera from healthy controls and allogeneic tumor
patients to evaluate the incidence of serum antibodies to the respective antigen.
Four different groups of genes coding for antigens were identified. The first group
codes for known tumor antigens such as the melanoma antigens MAGE-1, MAGE-4a
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