Cytotoxic T lymphocytes (CTLs) can play a central role in rejecting tumors (1
). Tumor antigens recognized by CTL generally originate from three sources: (a
) viruses; (b
) self-proteins expressed during development or differentiation; and (c
) mutant or aberrantly expressed proteins (1
). Because many, if not most, tumor antigens are products of normal or altered cellular genes, they are typically not efficient at initiating immune responses. Thus, a central problem in cancer immunotherapy is how to efficiently prime CTLs against poorly immunogenic tumor antigens.
CTLs recognize target antigens in the form of short intracellularly processed peptides, presented by self-MHC– encoded class I molecules (pep:class I). Upon binding of the antigen-specific TCRs on a CTL to its cognate peptide– MHC complex on the tumor cell, the target cell is lysed and the tumor eliminated. To develop into effector CTLs capable of tumor lysis, naive precursor CTLs (pCTLs)1
have to be activated. This pCTL activation requires two signals: the first, or stimulatory signal (signal 1), transmitted via the TCR–CD3 complex, and the second, or costimulatory signal (signal 2), delivered by professional APCs (2
). In the thymus, a strong signal 1 will induce negative selection of immature thymocytes, regardless of signal 2 (5
). In the periphery, the same strong signal 1 will induce immunity (including pCTL → CTL differentiation) or anergy, depending on the presence or absence of signal 2 (2
). By contrast, even a weak signal 1 without signal 2 can be sufficient for target cell lysis by differentiated CTLs (7
). This means that a whole class of antigenic peptides exists that, although poorly immunogenic (i.e., unable to induce CTL immunity), can readily serve as molecular targets for lysis by differentiated effector CTLs. Such antigenic, but poorly immunogenic, peptides remain invisible to the naive pCTL.
One strategy to exploit the presence of such poorly immunogenic or nonimmunogenic peptides at the surface of tumor cells is to design immunogenic variants of these peptides that would prime CTL response that cross-reacts to the original targeting peptide. If successful, this strategy could be attractive, since the relative invisibility of poorly immunogenic self-peptides to the immune system could be advantageous. Namely, unlike self-peptides that provide strong signal 1 (8
), poorly immunogenic peptides would most likely fail to induce tolerance, and the T cell repertoire reactive against them should be intact and available for activation with immunogenic peptide variants. According to the nomenclature used for variants of a pigeon cytochrome C peptide (9
), peptides of higher biological potency than the original peptide were called heteroclitic. Crystal structure analysis revealed that, of the 8–10 amino acid residues of a class I-bound peptide, roughly half point into the solvent and can interact directly with the TCR via their side chains (10
). The other half are buried by class I and are not directly accessible to the TCR (10
). Heterocliticity has been achieved by substituting amino acids that contact class I, the TCR, or both (13
The aim of this study was to produce heteroclitic immunogens that elicit antitumor CTL responses that cross-react to the original poorly immunogenic antigens. Therefore, to design peptides that are heteroclitic for polyclonal CTL responses, one would like to optimize pep:class I binding, since this property correlates with immunogenicity (7
). At the same time, the peptide:TCR contact should not be disturbed, to maximize the potential crossreactivity between the heteroclitic and the original, nonimmunogenic targeting peptide. We sought to test whether this strategy could induce antitumor CTLs reactive to nonimmunogenic targeting peptides. We demonstrate successful in vivo induction of cross-reactive CTLs in two tumor models, using an engineered viral peptide variant expressed as a tumor antigen (a model of a tumor antigen of viral origin) and a self-antigen expressed in melanomas (a model of nonmutated, differentiation antigen). Induced CTLs were biologically active in vivo, and were able to effect rejection of both newly implanted and established day 3 tumors.