Epithelial membrane protein 2: a novel biomarker for circulating tumor cell recovery in breast cancer.

PURPOSE: EpCAM is a common marker used in the detection of circulating tumor cells (CTC). Disseminated cancer cells display the characteristics of epithelial-to-mesenchymal transition events. The purpose of this study was to assess the potential of epithelial membrane protein 2 (EMP2) as a novel biomarker for CTC retrieval in breast cancer. METHODS: MCF7 and MDA-MB-231 cells were stained with either anti-EpCAM or anti-EMP2 mAbs, respectively, followed by flow cytometric assay to measure their expression levels. PBMCs isolated from healthy donors were used for breast cancer cell spiking. CD45-depleted PBMCs from breast cancer patients' blood were used for CTC capturing. Immunomagnetic separation was used to enrich breast cancer cells. Cytospin centrifugation was performed to concentrate the captured cells, followed by immunofluorescence staining with anti-CD45 mAb, anti-pan cytokeratin mAb and DAPI. Fluorescent images were taken using a confocal microscope for CTC counts. RESULT: MDA-MB-231 cells had 2.56 times higher EMP2 expression than MCF7 cells, and EMP2 had a significantly higher capture efficiency than EpCAM for MCF7 cells. Furthermore, anti-EMP2 was capable of capturing MCF7 cells that escaped in the flow-through of anti-EpCAM. Likewise, EMP2 had a significantly higher capture efficiency on MDA-MB-231 cells when compared to MCF7 cells. Most importantly, EMP2 biomarker was successfully used for CTC capture in patients with primary breast cancer. CONCLUSIONS: EMP2 is superior to EpCAM for capturing both MCF7 and MDA-MB-231 cells. Additionally, EMP2 is a novel biomarker and capable of capturing breast cancer cells in patient blood samples.

Conjugates of aberrant gangliosides in antiglioma vaccine: toxicological assay.

We studied sterility and toxicity of vaccine LS1 containing aberrant gangliosides isolated from brain bioptates of 48 patients with gliomas of different malignancy and covalently bound to keyhole limpet hemocyanin. The vaccine was safe. This preparation produced no side effects in experimental animals. Our findings substantiated the necessity of father development of this method of vaccination. The vaccine should undergo clinical tests in patients with malignant gliomas.

Cancer vaccines: an update.

This review summarizes the most recent findings and the future directions in designing cancer vaccines. The newest tumor-associated antigens and the most promising approaches to cancer vaccine development are discussed. We categorized them as follows: peptide vaccines, recombinant viral vaccines, DNA vaccines, dendritic cell-based immunotherapy, and the use of heat shock proteins and adjuvants. We focus on their advantages and disadvantages in addition to clinical potential.

A novel melanoma gene (MG50) encoding the interleukin 1 receptor antagonist and six epitopes recognized by human cytolytic T lymphocytes.

We identified a novel 8.1-kb human melanoma gene, MG50, derived from subtractive hybridization with a squamous lung carcinoma cell line, LU-1. 6.8 kb containing an open reading frame were sequenced, and the structure of the encoded 1496 amino acid protein was deduced. With HLA-A2.1-transduced Drosophila cells as antigen-presenting cells, we identified six epitopes restricted by HLA-A2.1 that elicited CTLs in vitro. Reactivity of the CTLs to melanoma cells containing MG50 indicated that the epitopes were displayed naturally. Significant cross-reactivity of CTLs immunized against a melanoma cell line that lacked HLA-A2.1 indicated that at least four of the epitopes were also recognized in a different HLA class I context, most likely HLA-A*6802. By quantitative reverse transcription, MG50 message was found in one of two skin melanoma cell lines, an ocular melanoma cell line, two of four metastatic skin melanomas, two of three mammary carcinomas, one of two colon carcinomas, and an ovarian carcinoma. Of six normal tissues, MG50 was found only in a specimen of normal skin and was absent from a congenital nevus. It is likely that MG50 is the gene for the interleukin 1 receptor antagonist because a reported sequence of cDNA from the latter had a sequence of 528 bases in the 3' region, a long contiguous base sequence, and 176 encoded amino acids identical with those of MG50. MG50 is one of the few melanoma-associated antigens that is not a differentiation antigen or a mutated protein. Because of its nature, it may prove to be important in the pathogenesis of the tumors in which it is found, as well as an immunogen and target for immunotherapy.

Synthetic insertion signal sequences enhance MHC class I presentation of a peptide from the melanoma antigen MART-1.

Cytotoxic T lymphocytes (CTL) recognize minimal peptides of eight to ten residues which are the products of intracellularly processed proteins and are presented at the cell surface by MHC class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane mediated by transporter associated with antigen processing (TAP) proteins, or as an alternative, by endoplasmic reticulum insertion signal sequences. We report here that the addition of synthetic signal sequences at the N terminus, but not at the C terminus, of an epitope from the human melanoma antigen MART-1 greatly enhances its presentation in both TAP-deficient and TAP-expressing cells. A newly designed peptide construct, composed of the epitope replacing the hydrophobic part of a natural signal sequence, was also very effective. Interestingly, an artificial signal sequence containing the same epitope was the most efficient construct for enhancing its presentation. These peptide constructs facilitated epitope presentation when loaded into the cytosol of TAP-deficient T2 cells, TAP-expressing melanoma cells and human dendritic cells. These findings may be of practical significance for the development of synthetic anti-cancer vaccines and in vitro immunization of CTL for adoptive immunotherapy.

Cytotoxic T cell immunity against telomerase reverse transcriptase in humans.

Telomerase is a ribonucleoprotein enzyme which has been linked to malignant transformation in human cells. Telomerase activity is increased in the vast majority of human tumors, making its gene product the first molecule common to all human tumors. The generation of endogenously processed telomerase peptides bound to Class I MHC molecules could therefore target cytotoxic T lymphocytes (CTL) to tumors of different origins. This could advance vaccine therapy against cancer provided that precursor CTL recognizing telomerase peptides in normal adults and cancer patients can be expanded through immunization. We demonstrate here that the majority of normal individuals and patients with prostate cancer immunized in vitro against two HLA-A2.1 restricted peptides from telomerase reverse transcriptase (hTRT) develop hTRT-specific CTL. This suggests the existence of precursor CTL for hTRT in the repertoire of normal individuals and in cancer patients. Most importantly, the CTL of cancer patients specifically lysed a variety of HLA-A2(+) cancer cell lines, demonstrating immunological recognition of endogenously processed hTRT peptides. Moreover, in vivo immunization of HLA-A2.1 transgenic mice generated a specific CTL response against both hTRT peptides. Based on the induction of CTL responses in vitro and in vivo, and the susceptibility to lysis of tumor cells of various origins by hTRT CTL, we suggest that hTRT could serve as a universal cancer vaccine for humans.

Cancer vaccines: novel approaches and new promise.

Cancer vaccines are a promising tool in the hands of the clinical oncologist. We have summarized the most recent findings and achievements in this exciting field. Tumor-associated antigens, as a basis for the new cancer vaccines, are reviewed. We emphasize novel approaches for the design of safe and more effective vaccines for cancer. We also discuss the possible clinical applications and the future prospects for vaccine development.

New trends in the development of cancer vaccines.

Recent advances in understanding of the molecular mechanisms of antigen processing and presentation, and the identification of tumor-associated antigens in melanoma and other cancers, have stimulated the development of a new generation cancer vaccines. This review summarizes the most recent approaches for the design of safe and more effective vaccines for cancer. Peptide-based vaccines are safe and can be synthesized with high purity and reproducibility. Recombinant viruses encoding tumor-associated antigens allow efficient delivery and precise control over the form and the quantity of the delivered antigens. DNA-based vaccines induce long-lasting immune responses and are considered very safe. Antigen-loaded dendritic cells, and the use of newly developed adjuvants are also very promising new approaches. In this review, we also discuss the possible clinical applications and future directions for vaccine development.

Insertion signal sequence fused to minimal peptides elicits specific CD8+ T-cell responses and prolongs survival of thymoma-bearing mice.

CD8+ T-lymphocytes (TCD8+) recognize minimal peptides of 8-10 residues which are the products of intracellularly processed proteins and are presented at the cell surface by major histocompatibility complex class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane, mediated by transporter associated with antigen-processing proteins or alternatively by endoplasmic reticulum-insertion signal sequences located at the NH2-terminus of the precursor molecules. We report here that the addition of an endoplasmic reticulum-insertion signal sequence at the NH2-terminus of TCD8+ epitopes from chicken ovalbumin (amino acids 257-264) or a naturally occurring tumor antigen expressed by the murine mastocytoma P815 (P1A amino acids 35-43) significantly enhanced the priming of specific TCD8+ in vivo. The signal sequence did not enhance peptide immunogenicity by merely increasing the hydrophobicity of the peptide, since ovalbumin amino acids 257-264 peptide with the signal sequence at its COOH-terminus did not demonstrate enhanced efficacy. The signal sequence did not act as a helper epitope, since TCD8+ responses were not diminished in class II-deficient transgenic mice or in mice depleted of CD4+ T-cells in vivo. Importantly, a single immunization with the fusion peptide significantly prolonged survival of mice challenged with E.G7OVA, a thymoma transfected with the complementary DNA of chicken ovalbumin.

Enhancing the recognition of tumour associated antigens.

Activated CD8+ T cells (TCD8+) can directly recognize malignant cells because processed fragments of tumour associated antigens (TAA), 8-10 amino acids in length and complexed with MHC class I molecules, are displayed on tumour cell surfaces. Tumour cells have been genetically modified in a variety of ways in efforts to enhance the immune recognition of TAA. An alternative strategy is the expression of TAA in recombinant or synthetic form. This has been made possible by the recent cloning of TAA recognized by TCD8+. In this communication we review recent work in our own laboratory on the expression of TAA as synthetic peptide, by "naked" plasmid DNA injected intramuscularly or transdermally, and by recombinant viruses including vaccinia (rVV), fowlpox (rFV) and adenovirus (rAd). The expression of TAA in recombinant and synthetic forms allows increased control over the quantity, location, and kinetics of TAA presentation and can result in powerful, specific, anti-tumour immune responses.