Marianne Klemp Gjertsen

Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose escalating phase I/II study

Patients with inoperable pancreatic cancer have a dismal prognosis with a mean life expectancy of 3–6 months. New treatment modalities are thus urgently needed. Telomerase is expressed in 85–90% of pancreas cancer, and immunogenic telomerase peptides have been characterised. A phase I/II study was conducted to investigate the safety, tolerability, and immunogenecity of telomerase peptide vaccination. Survival of the patients was also recorded. Forty-eight patients with non-resectable pancreatic cancer received intradermal injections of the telomerase peptide GV1001 at three dose levels, in combination with granulocyte–macrophage colony-stimulating factor. The treatment period was 10 weeks. Monthly booster vaccinations were offered as follow-up treatment. Immune responses were measured as delayed-type hypersensitivity skin reaction and in vitro T-cell proliferation. GV1001 was well tolerated. Immune responses were observed in 24 of 38 evaluable patients, with the highest ratio (75%) in the intermediate dose group. Twenty-seven evaluable patients completed the study. Median survival for the intermediate dose-group was 8.6 months, significantly longer for the low- (P=0.006) and high-dose groups (P=0.05). One-year survival for the evaluable patients in the intermediate dose group was 25%. The results demonstrate that GV1001 is immunogenic and safe to use. The survival data indicate that induction of an immune response is correlated with prolonged survival, and the vaccine may offer a new treatment option for pancreatic cancer patients, encouraging further clinical studies.

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.

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 TGFβRII 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 TGFβRII, 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.

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.

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. Int. J. Cancer 72:784–790, 1997.

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

In a pilot phase 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.

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

Abstract. 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 β receptor type II (TGFβRII), are targets for mutations. The TGFβRII 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 a new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA ), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGFβRII. 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 TGFβRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.

Mutated ras peptides as vaccines in immunotherapy of cancer

Mutations in codon 12 and 13 of K‐RAS are frequently found in human cancer, including pancreatic‐ and colorectal adenocarcinomas. T cell responses specific for individual RAS mutations can be elicited in vitro by stimulation with synthetic peptides and in vivo following vaccination with antigen presenting cells pulsed ex vivo with synthetic peptides. The peptide‐responding T cells are capable of responding to intact p21 ras, and can recognise and kill tumour cell lines and isolated tumour cells harbouring the corresponding RAS mutation. The responding cells can be of both CD4+ and CD8+ phenotype, and these T cell subsets recognise nested epitopes within the vaccine peptides. Mutant ras peptides are therefore possibly an important vaccine for specific immunotherapy in patients with pancreatic and colorectal carcinomas, and are currently being tested in vivo together with GM‐CSF as an adjuvant in these cancer patients.

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).

HLA-A3 restricted mutant ras specific cytotoxic T-lymphocytes induced by vaccination with T-helper epitopes

Abstract. Cytotoxic T-lymphocytes are one of the most important elements of the antitumor defense. Stimulation of cytotoxic T-lymphocytes outgrowth after immunization with mutant ras peptides is a desired goal since these cells may kill tumor cells in vivo. In this study we tested responding peripheral mononuclear cells from a patient with pancreatic adenocarcinoma who had received intradermal peptide vaccination with a mixture of 17-mer mutant ras peptides and granulocyte-macrophage colony-stimulating factor as an adjuvant. Responding peripheral T-cells were cloned by limiting dilution and several CD8+ cytotoxic T-lymphocytes, specific for the K-RAS 12-Cys mutation were obtained. By using a panel of nonamer peptides containing the 12-Cys mutation and covering position 4–21 in the ras molecule, the 9-mer peptide which was actually recognized by the cytotoxic T-lymphocytes could be identified. HLA-A*0302 could be identified as the antigen-presenting molecule, and the amino acid sequence of the T-cell epitope carries the previously identified HLA-A*0302 binding motif. The nonamer peptide was contained within the vaccine peptide originally used for intradermal immunization of the patient. The cytotoxic T-lymphocytes were capable of killing target cells expressing HLA-A*0302 that coexpressed the K-RAS 12-Cys mutation after transfection. These data demonstrate that the peptide used for vaccination (17-mer) is processed and presented in vivo, and that generation of cytotoxic T-lymphocytes by vaccination with T-helper epitopes may be important for further development of specific immunotherapy of cancer patients.