Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8+ IFN-γ and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-γ in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30–40 residues (3–4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-γ responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8+ responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8+ T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8+ IFN-γ and HTL responses.

Recent studies have shown the importance of helper CD4 T cells in initiating and sustaining tumor-specific CD8 T-cell immunity. This has paved the way for identifying MHC class II epitopes that could be incorporated into class I-based vaccines.

Purpose: Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell-activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu-specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas.Experimental Design: A combination of algorithms and HLA-DR-binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-γ enzyme-linked immunosorbent spot (ELIspot) assay.Results: Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for ≥11 common HLA-DR molecules. A degenerate pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in ∼84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu 88-102 (p88).Conclusion: The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu-targeting, immune-based therapies such as vaccines. Clin Cancer Res; 16(3); 825-34.

CD4 T cells are important for anti-tumor immune responses. Aside from their role in the activation of CD8 T cells, CD4 T cells also mediate anti-tumor immune responses by recruiting innate immune effectors into the tumor microenvironment. Thus, the search for strategies to boost CD4 T cell immunity is an active area of research. Our goal in this study was to identify HLA-DR epitopes of carcinoembryonic antigen (CEA), a commonly over-expressed tumor antigen. HLA-DR epitopes of CEA were identified using the epitope prediction program, PIC (predicted IC50) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-γ ELIspot assays were used to detect existing immunity against the HLA-DR epitope panel of CEA in breast and ovarian cancer patients. In vitro generated peptide-specific CD4 T cells were used to determine whether the epitopes are naturally processed from CEA protein. Forty-three epitopes of CEA were predicted, 15 of which had high binding affinity for 8 or more common HLA-DR molecules. A degenerate pool of four, HLA-DR restricted 15-amino acid epitopes (CEA.24, CEA.176/354, CEA.488 and CEA.653) consisting of two novel epitopes (CEA.24 and CEA.488) was identified against which 40% of breast and ovarian cancer patients had pre-existent T cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in ~94% of patients. Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. The degenerate pool of CEA peptides may be useful for augmenting CD4 T cell immunity.