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12 Lévy et al.
of tumor cells by CD8 þ T cells, it inherently ensures that the target peptide antigen
is correctly processed by the tumor cell. It also guarantees the presence of effector
T cells capable of recognizing this target antigen. A variation of this method has
been used to identify CD8 þ T-cell epitopes derived from mutated tumorassociated
antigens (96). Tumor-infiltrating lymphocytes from melanoma patients
were screened for recognition of target cells expressing the HLA molecules of the
patients and transfected with the mutated gene. However, these T-cell-based
approaches present several technical limitations, including the need for sufficient
tumor-specific lymphocytes, the capacity of T-cell clones to expand in vitro, and
the need for autologous tumor cell lines. Given these limitations, other methods
have been developed and implemented.
Among them, the so-called reverse immunology approach has been most
frequently used. The first step of this method includes the selection of a target
protein, which may be a protein directly involved in tumorigenesis
(e.g., mutated p53, survivin, telomerase). Next, the target protein is analyzed
for the presence of potential MHC class I ligands. This is facilitated by the
extensive characterization of MHC class I ligands and the discovery of specific
pairs of highly conserved amino acids, so-called anchor residues, present in the
majority of peptides associated with defined MHC class I alleles. These anchor
residues are specific for each MHC class I allele, mediate the binding of
peptide to the MHC molecules, and are generally located at the subaminoterminal
(i.e., position 2) and C-terminal position of the peptide. The
search and predicted affinity score of peptides potentially binding to given
MHC class I molecules is nowadays automated, thanks to several web-based
software (e.g., http://www.syfpeithi.de/, http://www-bimas.cit.nih.gov/molbio/
hla_bind/). After selection of the top candidates, peptides are generally synthesizedandusedtoinducespecificcytotoxicTlymphocytes(CTLs),which
are then tested on peptide-pulsed target cells to confirm specificity and/or on
cells expressing the targeted gene product. However, several peptides eliciting
CTLs are inefficiently processed by tumor cells and are not presented when
expressed at physiological levels (97–100). On the basis of these limitations,
modified “reverse immunology” has been developed. The procedure remains
similar to the one described above, except that in vitro proteasome degradation
is included. Active 20 S proteasomes are easily purified and retain their
original specificities in vitro. Depending on the purification scheme, 20 S
proteasomes may have to be activated by adding minute amounts of SDS or
purified PA28. Active proteasomes are then incubated with long synthetic
peptides encompassing the antigenic peptide of interest plus several N- and Cterminal
amino acids and the fragmented products are quantified by HPLC and
identified by mass spectrometry (13,55). With this procedure, the processing of
candidate peptides is monitored in an easily tractable manner. Several tumorspecific
peptide antigens have been identified with this refined “reverse
immunology” approach and have been shown to be naturally processed by
tumor cells (16,101,102). This procedure can be performed with any type of