Although the peptide variants used in our study effectively stimulated a tumor-specific T cell clone in vitro, they elicited variable responses from the endogenous T cell population. In this study, we found that effective cancer peptide vaccines stimulate T cells with hypervariable regions similar to those that respond to the natural tumor antigen, not a de novo T cell repertoire. These results suggest that the heterogeneity in the repertoire of AH1-specific T cells contributes to the ineffectiveness of some of the peptide-variant vaccines.
Similar to our results, a study of the T cell response to the Melan-A/MART-126–35
TAA showed that the TCRβ gene usage of the T cells responding to the native peptide was broader than that responding to the peptide variant [21
]. They also identified a CDR3β motif that was shared among several patients, a so-called public TCR, although these motif-containing T cells were not among the dominant clones identified in each patient and did not have a higher functional avidity [21
]. They concluded that the repertoires responding to the peptide variants and the native tumor antigen were subtly different but over-lapping, and that the observed functional differences were due to subtle structural changes in the TCR. In contrast, the T cells responding to the ineffective peptide variants in our study express a unique repertoire of TCRs that do not overlap with the repertoire responding to the native tumor antigen and that do not contain a significant fraction of the shared CDR3β motif. Therefore, in our study, only the effective peptide variants elicited a repertoire of T cells that over-lapped with those responding to the native tumor antigen.
The most effective peptide variants (A5 and F1A5) have minimal amino acid sequence changes relative to the AH1 peptide. It is, therefore, not surprising that these vaccines raise similar T cell repertoires. These data suggest that peptides with conservative amino acid changes may be more effective in stimulating antitumor immunity by eliciting a repertoire of T cells that mimics the response to the native tumor antigen. In agreement with this premise, another study concluded that substitutions at MHC-binding positions create immunogenic peptide variants with an overall similar structure to the native peptide [4
]. However, the opposite has also been shown in these studies. Peptides specifically designed to encode conservative amino acid changes were ineffective vaccines and raised a repertoire of T cells different from that of the native tumor antigen [10
]. These discrepancies suggest that even subtle amino acid substitutions may cause unpredictable changes in the repertoire and binding kinetics of the responding endogenous T cells.
The T cells responding to the F1A5 peptide express higher-affinity T cell receptors that functionally recognize the AH1 TAA better than the T cells responding to the ineffective peptide, WMF. Furthermore, soluble TCRs encoding the common CDR3β motif do not bind efficiently to the WMF peptide, providing a mechanistic explanation for the lack of these TCRs in the responding repertoire. We predict from these data that the structure of the WMF peptide prevents productive interactions with TCRs containing the common CDR3β motif. Specifically, the tryptophan residue at position 7 of the WMF peptide may affect the position of the adjacent tyrosine residue at position 6, precluding its predicted interaction with the tyrosine residue encoded by the Jβ2.6 gene segment of the motif-containing TCRs. Important for peptide vaccine development, these results imply that T cells responding to the ineffective variants (like WMF) cannot be “rescued” or forced to respond to the tumor antigen with better adjuvants; they are not anergic or improperly activated, they simply express T cell receptors that interact poorly with the native tumor antigen.
A clinically relevant strategy in the design of effective peptide vaccines is to optimize the T cells used in the identification of peptide variants. We selected our peptide variants based on improved binding to and activation of the CT clone, an AH1-specific T cell clone that was in vitro expanded and is not similar to those analyzed ex vivo (Fig. ). The T cell clones that were found in the endogenous repertoire bound to the peptides that elicit better tumor-specific responses (Fig. ). Therefore, we predict that peptide variants selected with an ex vivo tumor-specific repertoire, rather than an in vitro selected T cell clone, may be the most effective peptides for cancer vaccines.
Due to the unpredictable nature of the responding T cell repertoire, even if optimal T cells are used for peptide selection, ineffective peptides may be unavoidable in peptide-variant vaccine development. Therefore, a general strategy to improve the response to peptide variants such as the WMF peptide should be considered. In this particular case, rather than providing additional adjuvants or cytokines during T cell activation, the repertoire of the T cell response needs to be re-focused to those with high affinity for the native tumor antigen. Although strategies to engineer T cells with receptors that have high affinity for tumor antigens have been developed and employed in the clinical treatment for cancer [42
], effective antitumor immunity may be more easily achieved by “boosting” the peptide-variant responses with native tumor antigens [14
]. This strategy may increase the number of high-affinity T cells that cross-react with native tumor antigens and reduce competition with T cells that only bind to peptide variants.
In summary, peptide-variant vaccines improve the proliferation and activation of T cells that respond to native tumor antigens. Because the T cell response is unpredictable and different for every antigen, optimal peptide variants may be most successfully identified using a representative TAA-specific T cell population to select preferred residue changes from a large and random set of peptides.