A potential pitfall of the use of single class I binding peptides is illustrated in two similar studies vaccinating patients with an HLA-A2-binding peptide, p369-377, derived from the protein sequence of HER-2/neu, a well-defined tumor antigen. In an initial clinical study, HLA-A2-positive patients with metastatic HER-2/neu-overexpressing breast, ovarian, or colorectal carcinomas were immunized with 1 mg of p369-377 admixed in incomplete Freund's adjuvant (IFA) every 3 wk (38). Peptide-specific CTLs were isolated and expanded from the peripheral blood of patients after two or four immunizations. The CTLs could lyse HLA-matched peptide-pulsed target cells but could not lyse HLA-matched tumors expressing the HER-2/neu protein. Even when tumors were treated with IFN-y to upregulate class I, the CTL lines generated from the patients would not respond to the peptide presented endogenously on tumor cells. More recently a similar study was performed, immunizing patients with p369-377 using GM-CSF as an adjuvant (39). GM-CSF is a recruitment and maturation factor for skin dendritic cells (DCs), Langerhans cells (LCs), and theoretically may allow more efficient presentation of peptide epitopes than standard adjuvants such as IFA. Six HLA-A2 patients with HER-2/neu-overexpressing cancers received six monthly vaccinations with 500 ^g of HER-2/neu peptide, p369-377, and mixed with 100 ^g of GM-CSF. The patients had either stage III or IV breast or ovarian cancer. Immune responses to the p369-377 were examined using an IFN-y ELIspot assay. Prior to vaccination, the median precursor frequency, defined as precursors/106 PBMC, to p369-377 was not detectable. Following vaccination, HER-2/neu peptide-specific precursors developed to p369-377 in just two of four evaluable subjects. The responses were short-lived and not detectable at 5 mo after the final vaccination. Immunocompetence was evident as patients had detectable T-cell responses to tetanus toxoid and influenza. These results demonstrate that HER-2/neu MHC class I epitopes can induce HER-2/neu peptide-specific IFN-y-producing CD8+ T cells. However, the magnitude of the responses were low, as well as short-lived. Theoretically, the addition of CD4+ T-cell help would allow the generation of lasting immunity.
A successful vaccine strategy for generating peptide-specific CTLs capable of lysing tumor expressing HER-2/neu and resulting in durable immunity involved immunizing patients with putative Th epitopes of HER-2/neu that had, embedded in the natural sequence, HLA-A2-binding motifs of HER-2/neu. Thus, both CD4+ T-cell help and CD8+-specific epitopes were encompassed in the same vaccine. In this trial, 19 HLA-A2 patients with HER-2/neu-overexpressing cancers received a vaccine preparation consisting of putative HER-2/neu helper peptides (40). Contained within these sequences were the HLA-A2-binding motifs. Patients developed both HER-2/neu-specific CD4+ and CD8+ T-cell responses. The level of HER-2/neu immunity was similar to viral and tetanus immunity. In addition, the peptide-specific T cells were able to lyse tumor. The responses were long-lived and detectable for greater than 1 yr after the final vaccination in selected patients. These results demonstrate that HER-2/neu MHC class II epitopes containing encompassed MHC class I epitopes are able to induce long-lasting HER-2/ neu-specific IFN-y-producing CD8 T cells. Currently, several groups are focusing on the identification of peptides that are suited for binding to class II and contain within their natural sequence class I epitopes for the same antigen. A peptide with dual class I and II specificities has recently been identified for NY-ESO (41) as well as the melanoma antigen, gp100 (42).
Stimulating an effective Th response, even without concomitant CD8+ peptide vaccination, is a way to boost antigen-specific immunity as CD4+ T cells generate the cytokine environment required to support an evolving immune response. Vaccinating patients with HER-2/neu Th peptides, patients with advanced-stage HER-2/neu-overexpressing breast, ovarian, and non-small cell lung cancer were enrolled. Thirty-eight patients finished the planned course of six immunizations (2). Patients received 500 ^g of each peptide admixed in GM-CSF (43). Over 90% of patients developed T-cell immunity to HER-2/neu peptides and over 60% to a HER-2/neu protein domain. Thus, immunization with peptides resulted in the generation of T cells that could respond to protein processed by APCs. Furthermore, at 1-yr follow-up, immunity to the HER-2/neu protein persisted in over a third of patients. Immunity elicited by active immunization with CD4+ T-helper epitopes was durable. An additional finding of this study was that epitope spreading was observed in the majority of patients and significantly correlated with the generation of HER-2/neu protein-specific T-cell immunity. Epitope, or determinant spreading, is a phenomenon first described in autoimmune disease (44) and represents the generation of an immune response to a particular portion of an immunogenic protein and then the natural spread of that immunity to other areas of the protein or even to other antigens present in the environment. The same phenomenon, epitope spreading, was reported recently in a vaccine trial immunizing breast and ovarian cancer patients with autologous DCs pulsed with MUC-1 or HER-2/neu peptides (45). Epitope spreading may represent the ability of the initial immune response to create a microenvironment at the site of the tumor that enhances endogenous local immunity (46). Despite the generation of detectable immunity against the self-protein HER-2/neu and the development of epitope spreading, none of the patients in the studies described above developed any evidence of autoimmunity directed against tissues expressing basal levels of HER-2/neu such as skin, liver, and digestive tract epithelium.
4. modifications of peptides to improve immunogenicity
Initial clinical trials of peptide-based vaccines indicated that patients can be immunized, but immune responses were generally not to the levels of a vaccinated antigen such as tetanus toxoid. Therefore, more recent investigations are focused on improving the immunogenicity of peptide immunization either by modifying the route, adjuvant, or dose of the vaccine or via the modification of the peptide itself.
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