Identification of New Epitopes from Four Different Tumor-Associated Antigens: Recognition of Naturally Processed Epitopes Correlates with HLA-A∗0201-Binding Affinity

Abstract

Forty-two wild-type and analogue peptides derived from p53, carcinoembryonic Ag, Her2/neu, and MAGE2/3 were screened for their capacity to induce CTLs, in vitro, capable of recognizing tumor target lines. All the peptides bound HLA-A*0201 and two or more additional A2 supertype alleles with an IC50 of 500 nM or less. A total of 20 of 22 wild-type and 9 of 12 single amino acid substitution analogues were found to be immunogenic in primary in vitro CTL induction assays, using normal PBMCs and GM-CSF/IL-4-induced dendritic cells. These results suggest that peripheral T cell tolerance does not prevent, in this system, induction of CTL responses against tumor-associated Ag peptides, and confirm that an HLA class I affinity of 500 nM or less is associated with CTL epitope immunogenicity. CTLs generated by 13 of 20 of the wild-type epitopes, 6 of 9 of the single, and 2 of 5 of the double substitution analogues tested recognized epitopes generated by endogenous processing of tumor-associated Ags and expressed by HLA-matched cancer cell lines. Further analysis revealed that recognition of naturally processed Ag was correlated with high HLA-A2.1-binding affinity (IC50 = 200 nM or less; p = 0.008), suggesting that high binding affinity epitopes are frequently generated and can be recognized as a result of natural Ag processing. These results have implications for the development of cancer vaccines, in particular, and for the process of epitope selection in general.

Improved Immunogenicity of an Immunodominant Epitope of the Her-2/neu Protooncogene by Alterations of MHC Contact Residues

“…Previously, we reported the identification of 11 new epitopes derived from HER-2, CEA, p53, and MAGE2/3, which included the three epitopes analyzed in this study (22). These epitopes were produced by modifying both the main anchor positions by introducing L, V, or T at position 2 and V at the C terminus, and were demonstrated to have improved HLA-A2.1 binding and/or supertype cross-reactive binding compared with the wild-type epitope.…”

“…Three HER-2.369 analogue peptides, produced by modifying both main anchor positions by introducing L, V, or T at position 2 and V at the C terminus, were previously demonstrated to have improved HLA-A2.1 binding and/or supertype cross-reactive binding compared with the wild-type epitope (22). We first analyzed to what extent these HER-2/neu.369 (HER-2.369) variants would be able to sensitize HLA-A2-expressing T2 cells for recognition by HER-2.369 wild-type-specific CTLs, as measured in ELISPOT IFN-␥ production assays.…”

“…3, tumor-specific CTL lines from HLA-A2-positive healthy donors were generated with some variations on the protocol given above as described previously (22).…”

Predicting T cell recognition of MHC class I restricted neoepitopes

“…However, this tenet was challenged by several studies showing that cancer and autoantigen associated epitopes also bound with affinities similar to those of their microbial counterparts. 16,17 The controversy was renewed in the setting of mutated cancer epitopes, with some studies suggesting that HLA binding is a crucial factor in selecting them 18 with others indicating that binding is of marginal importance 19 and that the MHC binding affinity of the non-mutated sequence from which the mutation is derived is also of importance.…”

High-Affinity HLA-A(*)02.01 Peptides from Parathyroid Hormone-Related Protein Generate In Vitro and In Vivo Antitumor CTL Response Without Autoimmune Side Effects

“…All these trials are based on the use of dominant epitopes with a high affinity for HLA, derived from self protein expressed in tumors and in normal tissues. Despite some positive results, increasing evidence indicates that the lack of clear efficacy is probably due to tolerance to self-Ags that concerns dominant determinants for self Ags (15)(16)(17)(18).…”