Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer.

The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch-repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as TGF beta RII and BAX. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in TGF beta RII, in TIL and peripheral blood lymphocytes from patients with MSI(+) tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI(+) colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC.

Successful induction of immune responses against mutant ras in melanoma patients using intradermal injection of peptides and GM-CSF as adjuvant.

The rapidly increasing incidence and mortality rate of malignant melanoma, together with the lack of efficient treatment of the late stages, makes it a serious threat to public health. Innovative new treatments are needed. The proteins of the ras-family of proto-oncogenes, functioning as relay switches for signalling pathways between cell surface and nucleus, are involved in cell proliferation, differentiation, apoptosis and transformation. If over-expressed or mutated they can induce and/or maintain a transformed state of a cell. Codon 61 mutations of N-ras seem to be involved in melanoma development on sun exposed sites. In order to induce an immune response towards mutated N-ras proteins we performed a phase 1 feasibility study. Ten melanoma patients were immunized intradermally 6 times with N-ras peptides (residue 49-73) with 4 codon 61 mutations using GM-CSF as adjuvant. HLA typing was not used as an inclusion criterion. Eight patients responded with strong delayed type hypersensitivity reactions. In 2 of the patients an in vitro response to the vaccine could also be detected. The specificity of the reaction could be confirmed by cloning of peptide-specific CD4 positive T cells from peripheral blood of the patients. Intradermal injection of ras peptides using GM-CSF as adjuvant is simple to perform and seems to be efficient in inducing cellular immune responses. Since a majority of the patients showed positive skin reactions and 2 of the patients analysed showed a T-helper response to this melanoma specific antigen, these promiscuous HLA class II binding mutant ras peptides may be candidates for inclusion into vaccine cocktails containing various established CTL epitopes.

Intradermal ras peptide vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: Clinical and immunological responses in patients with pancreatic adenocarcinoma.

K-RAS mutations are frequently found in adenocarcinomas of the pancreas, and induction of immunity against mutant ras can therefore be of possible clinical benefit in patients with pancreatic cancer. We present data from a clinical phase I/II trial involving patients with adenocarcinoma of the pancreas vaccinated by i.d. injection of synthetic mutant ras peptides in combination with granulocyte-macrophage colony-stimulating factor. Forty-eight patients (10 surgically resected and 38 with advanced disease) were treated on an outpatient basis. Peptide-specific immunity was induced in 25 of 43 (58%) evaluable patients, indicating that the protocol used is very potent and capable of eliciting immune responses even in patients with end-stage disease. Patients followed-up for longer periods showed evidence of induction of long-lived immunological memory against the ras mutations. CD4(+) T cells reactive with an Arg12 mutation also present in the tumor could be isolated from a tumor biopsy, demonstrating that activated, ras-specific T cells were able to selectively accumulate in the tumor. Vaccination was well tolerated in all patients. Patients with advanced cancer demonstrating an immune response to the peptide vaccine showed prolonged survival from the start of treatment compared to non-responders (median survival 148 days vs. 61 days, respectively; p = 0.0002). Although a limited number of patients were included in our study, the association between prolonged survival and an immune response against the vaccine suggests that a clinical benefit of ras peptide vaccination may be obtained for this group of patients.

A TGF betaRII frameshift-mutation-derived CTL epitope recognised by HLA-A2-restricted CD8+ T cells.

Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences caused by defects in the DNA mismatch repair machinery. Such mutation patterns have been found in almost all analysed malignancies from patients with hereditary non-polyposis colorectal cancer, and in approximately 15% of sporadic colorectal cancers. In cancers with the MSI phenotype, microsatellite-like sequences in coding regions of various cancer-related genes, including transforming growth factor beta receptor type II (TGF betaRII), are targets for mutations. The TGF betaRII gene harbours a 10-bp polyadenine tract, and mutations within this region are found in 90% of colorectal cancers with MSI. The frameshift mutations result in new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. In this study we have defined new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGF betaRII. A CTL line and several CTL clones were generated from an HLA-A2+ normal donor by repeated stimulation of T cells with dendritic cells pulsed with the peptide. One of the CTL clones was able to kill an HLA-A2+ colon cancer cell line harbouring mutant TGF betaRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.

Detection and characterization of alpha-beta-T-cell clonality by denaturing gradient gel electrophoresis (DGGE).

Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.

Generation and characterization of gp100 peptide-specific NK-T cell clones.

MHC-restricted cytotoxic T lymphocytes (CTLs) specific for antigens expressed by malignant cells are important components of immune responses against human cancer. Peripheral blood monocytes of HLA-A2+ healthy donors were used to induce dendritic cells (DCs) by granulocyte-macrophage colony-stimulating factor and interleukin-4 and loaded with a gp100 peptide (YLEPGPVTA). By applying these peptide-loaded DCs, a CTL line that displayed high cytotoxic reactivity with peptide-loaded target cells was generated. A total of 11 gp100 peptide-specific CTL clones were generated from this cell line. Several of these CTL clones were studied in detail. Of particular interest was clone CTL-45, which, contrary to the parental cell line, displayed strong NK activity and, by flow-cytometric analysis, revealed a CD3+, TCR BV17, CD8+ and CD56+ phenotype. This clone was strictly peptide-specific and effectively killed a panel of melanoma cells expressing HLA-A2 and gp100. Tumor-specific T cells with this kind of dual function are potentially of great clinical importance as they have a backup mechanism that may go into action when tumor cells escape specific killing by losing their HLA-class I molecules.

Cytotoxic CD4+ and CD8+ T lymphocytes, generated by mutant p21-ras (12Val) peptide vaccination of a patient, recognize 12Val-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation.

Mutant p21-ras proteins contain sequences that distinguish them from normal ras, and represent unique epitopes for T-cell recognition of antigen-bearing tumour cells. Here, we examined the capacity of CD4+ and CD8+ T cells, generated simultaneously by mutant-ras-peptide vaccination of a pancreatic-adenocarcinoma patient, to recognize and lyse autologous tumour cells harbouring corresponding activated K-ras epitopes. The patient was vaccinated with a purified 17mer ras peptide (KLVVVGAVGVGKSALTI), containing the Gly12 --> Val substitution. Responding T cells were cloned following peptide stimulation, and CD4+ and CD8+ peptide-specific cytotoxic T lymphocytes(CTL) were obtained. Transient pancreatic-adenocarcinoma cell lines(CPE) were established in cell culture from malignant ascites of the patient, and were shown to harbour the same K-ras mutation as found in the primary tumour. These cells were efficiently killed by the T-cell clones and CD8+-mediated cytotoxicity was HLA-class-I-restricted, as demonstrated by inhibition of lysis by anti-class-I monoclonal antibodies. By employing as targets different class-I-matched tumour cell lines expressing a 12Val mutation, we were able to demonstrate HLA-B35 as the restriction molecule, and further use of peptide-sensitized EBV-B cells as target cells identified VVVGAVGVG as the nonamer peptide responsible for CD8+-T-cell recognition. These data demonstrate that peptide vaccination with a single mutant p21-ras-derived peptide induces CD4+ and CD8+ CTL specific for nested epitopes, including the Gly --> Val substitution at codon 12, and that both these T-cell sub-sets specifically recognize tumour cells harbouring the corresponding K-ras mutation.

Characterisation of immune responses in pancreatic carcinoma patients after mutant p21 ras peptide vaccination.

This is a study of immune responses generated by mutant ras peptide vaccination of patients with pancreatic adenocarcinoma. Responding T cells from one patient were cloned and two CD4+ T-lymphocyte clones (TLC) specific for the 12 Val peptide and restricted by HLA-DR6 or DQ2 were obtained. These class II molecules have not previously been found to bind or present mutant ras peptides to T cells. The DR6-restricted TLC showed marked cytotoxicity against autologous target cells pulsed with the 12 Val peptide. Target cells pulsed with the control peptide were not killed. Responding T cells from another patient showed cross-reactivity towards the homologous ras peptides. Investigation by limiting dilution analysis (LDA) revealed different T-cell precursor frequencies for the immunising, mutant ras peptide (1:28000), compared with the normal ras peptide (1:110000).

Antigen-presenting function of human peritoneum mesothelial cells isolated from a pancreatic carcinoma patient after mutant Ras peptide vaccination.

Mesothelial cells obtained from ascites fluid of a Ras-peptide vaccinated pancreatic adenocarcinoma patient were cultured in vitro. Fresh isolated cells expressed HLA class II molecules, which were lost upon culture, but could be up-regulated after coculture with human recombinant interferon-gamma. The antigen-presenting capacity of these mesothelial cells was tested with allogeneic peripheral blood mononuclear cells (PBMC) in a mixed lymphocyte mesothelial cell culture and by stimulating autologous PBMC with purified protein derivative of Mycobacterium tuberculosis. Cloned T cells from the same patient allowed us to test the ability of mesothelial cells to present a mutant Ras-derived peptide to specific T cells in a DLA-DR-restricted manner. Mesothelial cells effectively stimulated allogeneic resting T lymphocytes to proliferate and presented soluble protein antigen or a mutant Ras-derived peptide to specific T cells, indicating that they display processing and presenting capabilities. Since mesothelial cells are in close anatomical relationship with intraabdominal malignancies, they may contribute to stimulation of specific T cells by endocytosing tumour-specific antigens and presenting them to T lymphocytes. This could be a possible mechanism in which mesothelial cells participate in maintaining local specific immunity in patients already primed.

Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase I/II study.

In a pilot I/II study we have tested synthetic ras peptides used as a cancer vaccine in 5 patients with advanced pancreatic carcinoma. The treatment principle used was based on loading professional antigen-presenting cells (APCs) from peripheral blood with a synthetic ras peptide corresponding to the ras mutation found in tumour tissue from the patient. Peptide loading was performed ex vivo and the next day APCs were re-injected into the patients after washing to remove unbound peptide. Patients were vaccinated in the first and second week and thereafter every 4-6 weeks. In 2 of the 5 patients treated, an immune response against the immunising ras peptide could be induced. None of the patients showed evidence of a T-cell response against any of the ras peptides before vaccination. The treatment was well tolerated and could be repeated multiple times in the same patient. Side effects were not observed even if an immunological response against the ras peptide was evident. We conclude that ras peptide vaccination according to the present protocol is safe and may result in a potentially beneficial immune response even in patients with advanced malignant disease.

Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation.

Mutations in codon 12 of K-RAS are frequently found in pancreatic adenocarcinomas. T-cell responses specific for individual RAS mutations can be elicited in vitro by stimulation of peripheral blood mononuclear cells with synthetic peptides. Mutant ras peptides are therefore a candidate vaccine for specific immunotherapy in pancreatic carcinoma patients. When vaccinated with a synthetic ras peptide representing the K-RAS mutation in their tumours, a transient ras-specific T-cell response was induced in two of five patients treated. The vaccination protocol involved multiple infusions of large amounts of peptide-pulsed antigen-presenting-cells obtained by leucapheresis. These results indicate that specific T-cell responses against mutations uniquely harboured in tumour cells can be induced in cancer patients by vaccination.