HAGE, a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides.

There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence. However, only the longer peptide was found to be both endogenously processed and immunogenic for T cells in transgenic mice in vivo, as well as for human T cells in vitro. A different class I-binding peptide, not contained within a longer class II sequence, was subsequently found to be both immunogenic and endogenously processed in transgenic mice, as was a second class II epitope. These novel HAGE-derived epitopes may contribute to the range of immunotherapeutic targets for use in cancer vaccination programs

HAGE, a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides

There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence. However, only the longer peptide was found to be both endogenously processed and immunogenic for T cells in transgenic mice in vivo, as well as for human T cells in vitro. A different class I-binding peptide, not contained within a longer class II sequence, was subsequently found to be both immunogenic and endogenously processed in transgenic mice, as was a second class II epitope. These novel HAGE-derived epitopes may contribute to the range of immunotherapeutic targets for use in cancer vaccination programs

Analysis of the oligomeric requirement for signaling by CD40 using soluble multimeric forms of its ligand, CD154

We describe the construction of a novel soluble dodecameric form of CD154 (CD40 ligand) that is more effective than trimeric tCD154 in triggering B cell activation. Dodecameric surfactant protein (SP)-D-CD154 was more potent than tCD154 in inducing B cell proliferation over a wide range of concentrations. At saturating concentrations, the level of proliferation triggered by SP-D-CD154 was fourfold higher than that achieved with tCD154. Moreover, stimulation with dodecameric CD154 induced higher levels of the costimulatory molecules ICAM-1 and CD86. The higher activity of dodecameric CD154 when compared to trimeric CD154 is unlikely to be due to differences in their avidity for CD40, since both forms bound to CD40 strongly. Therefore, the extent of receptor clustering directlyregulates signaling by CD40

High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects.

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01(+) healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP(+)HLA-A(*)02.01(+) breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies

Determination of cellularly processed HLA-A2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE

Purpose: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two "cancer germ line" genes, MAGE-A4 and SAGE. Experimental Design: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8(+) T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8(+) T cells sensitized with autologous CD4(+) PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. Results: After DNA vaccination, murine CD8(+) T cells recognizing MAGE-A4(143-15), or SAGE(715-723) in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402- positive CD8(+) T cells with autologous CD4(+) PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE(715-723)-specific precursors was observed in HLA-A2402- positive healthy individuals. Conclusions: Two novel HLA-A2402- restricted CTL epitopes, MAGE-A4(143-15), and SAGE(715-723), were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes