A degenerate HLA-DR epitope pool of HER-2/neu reveals a novel in vivo immunodominant epitope, HER-2/neu88-102.

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-gamma 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 > or =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 approximately 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.

Identification of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen.

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 IC(50)) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-gamma 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 approximately 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.

An HLA-DR-degenerate epitope pool detects insulin-like growth factor binding protein 2-specific immunity in patients with cancer.

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. In this study, the goal was to identify an HLA-DR-degenerate epitope pool derived from insulin-like growth factor binding protein 2 (IGFBP-2). IGFBP-2, a regulator of insulin-like growth factor action, is overexpressed in the majority of breast and ovarian cancers. Using algorithms, we predicted 29 HLA-DR1-binding epitopes. Binding assays targeting 15 different HLA-DRs revealed that 10 epitopes were degenerate, binding to at least four different HLA-DR variants. An IFN-gamma enzyme-linked immunosorbent spot assay was used to assess immunity to these 10 epitopes in 48 patients with either breast or ovarian cancer and 18 controls. Elevated T-cell immunity in patients was detected in 4 of the 10 epitopes (IGFBP2.17, IGFBP2.22, IGFBP2.249, and IGFBP2.293). The cumulative T-cell frequency of these four epitopes was elevated in patients relative to controls. All four peptides are naturally processed and presented to CD4 T-cells. The degenerate pool of peptides covers nearly 80% of patients and may be useful for augmenting CD4 T-cell immunity in patients undergoing immunization.

Design of multi-epitope, analogue-based cancer vaccines.

The current objective of our cancer programme is to develop an effective vaccine based on rationally designed T cell epitope analogues, for use in the adjuvant setting for non-small cell lung cancer (NSCLC) and colon cancer. Analogue epitopes, enhanced for either human leukocyte antigen (HLA) binding or T cell receptor (TCR) signalling, have been shown to be more effective at breaking immunological tolerance than cognate wild-type epitopes. Although encouraging early-phase clinical data has been obtained by others using a limited number of HLA-A2-restricted epitope analogues, the clinical benefits and immune correlates for vaccines comprised of multiple epitope analogues restricted by multiple HLA supertypes remains to be investigated. Clinical studies are currently being conducted on EP-2101, a prototype vaccine that delivers multiple HLA-A2-restricted analogue epitopes. In parallel, fixed anchor and heteroclitic analogues restricted by three other commonly expressed HLA supertypes are being identified. These analogues will be incorporated into future vaccines including optimised minigenes (epigenes) and tested in preclinical and clinical studies addressing various different cancer indications.