Mining the melanosome for tumor vaccine targets: P.polypeptide is a novel tumor-associated antigen.

To identify novel, tumor-specific target antigens for vaccine development, we studied immune responses to P.polypeptide, an M(r) 110,000 integral melanosomal membrane protein associated with the Prader-Willi syndrome. Together with expressed sequence tag (EST) and serial analyses of gene expression (SAGE) library analyses, reverse transcription-PCR and Northern blotting verified that P.polypeptide expression was limited to melanoma and melanocytes. A single dominant epitope corresponding to positions 427-435 (IMLCLIAAV) was identified using allele-specific epitope forecasting combined with work in HLA-A*0201/K(b) transgenic mice. This epitope was then used to generate de novo human P.polypeptide-specific CD8+ T cells capable of recognizing P.polypeptide expressing human tumor cell lines in an HLA-A*0201-restricted fashion. Thus, P.polypeptide may be valuable in the creation of novel therapeutic anticancer vaccines.

Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules.

In previous studies, the shared cancer-testis Ag, NY-ESO-1, was demonstrated to be recognized by both Abs and CD8+ T cells. Gene expression of NY-ESO-1 was detected in many tumor types, including melanoma, breast, and lung cancers, but was not found in normal tissues, with the exception of testis. In this study, we describe the identification of MHC class II-restricted T cell epitopes from NY-ESO-1. Candidate CD4+ T cell peptides were first identified using HLA-DR4 transgenic mice immunized with the NY-ESO-1 protein. NY-ESO-1-specific CD4+ T cells were then generated from PBMC of a patient with melanoma stimulated with the candidate peptides in vitro. These CD4+ T cells recognized NY-ESO-1 peptides or protein pulsed on HLA-DR4+ EBV B cells, and also recognized tumor cells expressing HLA-DR4 and NY-ESO-1. A 10-mer peptide (VLLKEFTVSG) was recognized by CD4+ T cells. These studies provide new opportunities for developing more effective vaccine strategies by using tumor-specific CD4+ T cells. This approach may be applicable to the identification of CD4+ T cell epitopes from many known tumor Ags recognized by CD8+ T cells.

Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice.

CD4+ T cells play a central role in the induction and persistence of CD8+ T cells in several models of autoimmune and infectious disease. To improve the efficacy of a synthetic peptide vaccine based on the self-Ag, gp100, we sought to provide Ag-specific T cell help. To identify a gp100 epitope restricted by the MHC class II allele with the highest prevalence in patients with malignant melanoma (HLA-DRB1*0401), we immunized mice transgenic for a chimeric human-mouse class II molecule (DR4-IE) with recombinant human gp100 protein. We then searched for the induction of CD4+ T cell reactivity using candidate epitopes predicted to bind to DRB1*0401 by a computer-assisted algorithm. Of the 21 peptides forecasted to bind most avidly, murine CD4+ T cells recognized the epitope (human gp10044-59, WNRQLYPEWTEAQRLD) that was predicted to bind best. Interestingly, the mouse helper T cells also recognized human melanoma cells expressing DRB1*0401. To evaluate whether human CD4+ T cells could be generated from the peripheral blood of patients with melanoma, we used the synthetic peptide h-gp10044-59 to sensitize lymphocytes ex vivo. Resultant human CD4+ T cells specifically recognized melanoma, as measured by tumor cytolysis and the specific release of cytokines and chemokines. HLA class II transgenic mice may be useful in the identification of helper epitopes derived from Ags of potentially great clinical utility.

A survey of treatments used in patients with metastatic melanoma: analysis of 189 patients referred to the National Cancer Institute.

BACKGROUND: Few effective treatments exist for patients with metastatic melanoma. The United States Food and Drug Administration has approved the use of interferon alfa-2b after the resection of locoregional disease, and dacarbazine or interleukin-2 for the treatment of patients with metastatic melanoma beyond the locoregional area, although many additional agents and combinations of agents are currently in use. METHODS: Between January 1997 and June 1998, the Surgery Branch of the National Cancer Institute conducted a prospective analysis of 226 consecutive patients with metastatic melanoma referred for protocol evaluation. The previous systemic treatments these patients received both before and after the development of metastatic disease were tabulated, along with the association of these treatments with formal institutional protocols. Only the identity of the agents and not the dose or the schedule of treatments was considered in this analysis. Complete information could be obtained from 189 of the 226 patients. RESULTS: Of the 189 patients evaluated for this study, 135 (71%) received some form of systemic therapy before referral to the National Cancer Institute. Before the development of metastatic disease, 80 patients were administered 25 different systemic treatments, including 23 different agents. After the development of metastatic disease, 53 patients were administered 57 different systemic and regional treatments, including 37 different agents. After the resection of all metastatic sites, 23 patients were administered nine different systemic adjuvant treatments. Overall, 78 different systemic treatments were administered to these patients. The majority of treatments in each group were not associated with formal institutional protocols. CONCLUSIONS: This study has demonstrated that a large number of agents and different combinations of agents are currently being administered to patients before and after the development of metastatic melanoma, and frequently not within the context of an approved institutional protocol. These results indicate a need for more formal evaluation of treatments in prospective protocols and greater standardization of the treatment of patients with melanoma.

Recombinant virus vaccination against "self" antigens using anchor-fixed immunogens.

To study the induction of anti-"self" CD8+ T-cell reactivity against the tumor antigen gp100, we used a mouse transgenic for a chimeric HLA-A*0201/H-2 Kb molecule (A2/Kb). We immunized the mice with a recombinant vaccinia virus encoding a form of gp100 that had been modified at position 210 (from a threonine to a methionine) to increase epitope binding to the restricting class I molecule. Immunogens containing the "anchor-fixed" modification elicited anti-self CD8+ T cells specific for the wild-type gp100(209-217) peptide pulsed onto target cells. More important, these cells specifically recognized the naturally presented epitope on the surface of an A2/Kb-expressing murine melanoma, B16. These data indicate that anchor-fixing epitopes could enhance the function of recombinant virus-based immunogens.

Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes.

Many human and mouse tumor antigens are normal, nonmutated tissue differentiation antigens. Consequently, immunization with these "self" antigens could induce autoimmunity. When we tried to induce immune responses to five mouse melanocyte differentiation antigens, gp100, MART-1, tyrosinase, and tyrosinase-related proteins (TRP) 1 and TRP-2, we observed striking depigmentation and melanocyte destruction only in the skin of mice inoculated with a vaccinia virus encoding mouse TRP-1. These mice rejected a lethal challenge of B16 melanoma, indicating the immune response against TRP-1 could destroy both normal and malignant melanocytes. Cytotoxic T lymphocytes specific for TRP-1 could not be detected in depigmented mice, but high titers of IgG anti-TRP-1 antibodies were present. Experiments with knockout mice revealed an absolute dependence on major histocompatibility complex class II, but not major histocompatibility complex class I, for the induction of both vitiligo and tumor protection. Together, these results suggest that the deliberate induction of self-reactivity using a recombinant viral vector can lead to tumor destruction, and that in this model, CD4(+) T lymphocytes are an integral part of this process. Vaccine strategies targeting tissue differentiation antigens may be valuable in cancers arising from nonessential cells and organs such as melanocytes, prostate, testis, breast, and ovary.

Lotus lectin labels subpopulation of olfactory receptor cells.

Experiments were performed to test the hypothesis that subsets of olfactory receptor cells could be recognized based on their lectin binding and that mapping of their projections onto the olfactory bulb would reveal details of anatomic organization of the olfactory nerve projection to the olfactory bulb. The results from one lectin, Lotus, were examined in detail. Olfactory receptor cells in the lateral part of the main epithelium were labeled, as well as scattered cells in the remainder of the epithelium. Glomeruli labeled by Lotus were concentrated primarily in the region of the olfactory bulb that receives its input from the lateral epithelium, although scattered glomeruli could be identified in other regions. Within the terminal field of these axons there was a mosaic pattern, with some glomeruli densely labeled, some lightly labeled and others unlabeled. These findings support the notion that there are biochemically distinct populations of olfactory receptor cells having localized distributions in the epithelium, with axons that coalesce to terminate in specific glomeruli, rather than diffusely over their projection field.