Target Discovery and Validation Reviews and Protocols

Overview of Immune System 301
demonstrate the specificity of binding. Using this method, a few antigens have
been identified. A major impediment to progress in this approach was the general
inability to grow primary human cells in tissue culture.
11.2. Biochemical Approach
This method relies on the purification and analysis of peptides eluted from
MHC molecules of tumor cells. Acid elution of antigenic peptides bound to MHC
class I molecules from tumor cells (55). Subsequently, tumor peptides are fractionated
by high-performance liquid chromatography, and then the different fractions
are tested for their ability to sensitize tumor cells for lysis by autologous tumorspecific
cytotoxic CD8+ T-cell clones. Subsequently, peptide fractions conferring
sensitivity to lysis by tumor-specific T-cells are sequenced. Although this method
led to the identification of several human tumor antigens eliciting cellular immune
responses (56), it also relies on the generation of autologous specific T-cell clones.
11.3. T-Cell-Based Identification of Recombinant Tumor Antigens
Several studies indicated that T-cells are a critical mediator of tumor immunity.
Establishment of T-cell clones from tumor-infiltrated T-cells was important for
this method. The first experimental approach is based on the transfection of
recombinant cDNA libraries into cells expressing the MHC-presenting molecules
(57,58). Autologous tumor-specific cytotoxic CD8+ T-cell clones (CTL)
are then used to test for recognition of the transfected cells. In the case of positive
response, cDNA fragments of the isolated gene are subcloned to define the
region encoding the antigenic peptide. Subsequently, synthetic peptides are
tested in a target cell sensitization assay for their ability to sensitize target cells
(tumor cells) to lysis by the original tumor-specific CTLs. Using this approach,
Boon and colleagues (57) reported the first successful cloning of human tumor
antigen, termed melanoma antigen-1 or MAGE-1, which elicited a spontaneous
CTL response in the autologous melanoma patient (57). Further studies showed
MAGE-1 to be expressed exclusively in normal testis among normal tissues.
The use of this “genetic” approach of T-cell epitope cloning also led to the identification
of the BAGE and GAGE antigens, both of which are recognized by
CTL of melanoma patients (58). Despite the identification of some tumor antigens,
this approach is technically challenging in that it requires the establishment
of autologous CTL lines/clones and tumor cell lines from the same patient, a
task not achievable for most epithelial tumor types (58).
11.4. Serological Analysis of Autologous Tumor Antigens
by Recombinant cDNA Expression Cloning (SEREX)
To overcome some of the technical problems related to the generation of T-cell
clones, in 1995 Sahin and colleagues (59) developed the SEREX technique