Jon Amund Eriksen

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.

Long-term follow-up of patients with resected pancreatic cancer following vaccination against mutant K-ras

K‐ras mutations are frequently found in adenocarcinomas of the pancreas and can elicit mutation‐specific immune responses. Targeting the immune system against mutant Ras may thus influence the clinical course of the disease. Twenty‐three patients who were vaccinated after surgical resection for pancreatic adenocarcinoma (22 pancreaticoduodenectomies, one distal resection), in two previous Phase I/II clinical trials, were followed for more than 10 years with respect to long‐term immunological T‐cell reactivity and survival. The vaccine was composed of long synthetic mutant ras peptides designed mainly to elicit T‐helper responses. Seventeen of 20 evaluable patients (85%) responded immunologically to the vaccine. Median survival for all patients was 27.5 months and 28 months for immune responders. The 5‐year survival was 22% and 29%, respectively. Strikingly, 10‐year survival was 20% (four patients out of 20 evaluable) versus zero (0/87) in a cohort of nonvaccinated patient treated in the same period. Three patients mounted a memory response up to 9 years after vaccination. The present observation of long‐term immune response together with 10‐year survival following surgical resection indicates that K‐ras vaccination may consolidate the effect of surgery and represent an adjuvant treatment option for the future.

T cells recognize a peptide derived from α-gliadin presented by the celiac disease-associated HLA-DQ (α1∗0501, β1∗0201) heterodimer

Abstract CD is unique among the HLA-associated diseases since (a) the disease-promoting agent (gliadin) is known and (b) the disease is precipitated mainly in individuals carrying a particular cis - or trans -encoded HLA-DQ heterodimer; i.e., DQ(α1 ∗ 0501, β1 ∗ 0201). Further, a preponderance of gliadin-specific T cells derived from the small intestinal mucosa of CD patients are restricted by this DQ heterodimer. T-cell recognition of gliadin peptides presented by the DQ(α1 ∗ , β1 ∗ 0201) heterdimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic α-gliadin peptide, when presented by the cis - or trans -encoded CD-associated DQ(α1 ∗ 0501, β1 ∗ 0201) heterdimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31–47 of α-gliadin, which is included in the part of α-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other α-gliadin sequences, one peptide differing by one amino acid was recognized by the T-cell clone, whereas the other peptide differing by two amino acids was not recognized. Our findings demonstrate that the CD-associated DQ(α1 ∗ 0501, β1 ∗ 0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.

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.

A novel human chymotrypsin-like digestive enzyme.

The gene of a novel chymotrypsin-like serine protease has been cloned from human pancreas. The chymotrypsin-like enzyme-1 gene is located on chromosome 16q22.1 in a tight cluster with four unrelated genes. The gene has seven exons with the signal and activation peptide and the three main catalytic residues forming the active site encoded by separate exons. Northern blots of pancreatic mRNA showed a major transcript of 1.0 kilobases and a minor transcript of 1.3 kilobases due to alternative polyadenylation. No transcript was found in other tissues. Its presence in pancreatic tissue, duodenal juice, and urine was demonstrated with antisera raised against synthetic peptides from the derived amino acid sequence of the gene. The peptide sequences were chosen for being most dissimilar to chymotrypsin, and the antisera obtained did not react with purified human chymotrypsin. The proteolytically active CTRL-1 has been identified in pancreatic homogenate, duodenal juice, and urine, and a recombinant CTRL-1 has been characterized. Increased pancreatic secretion of CTRL-1 was induced by protease inhibitors indicating that the enzyme is secreted from pancreas upon feedback stimulation. Both native and recombinant CTRL-1 displayed chymotrypsin- and elastase-2-like activities and hydrolyzed the amide bonds of substrates having tyrosine, phenylalanine, or leucine residues at the P1 position. The enzyme was active over a broad pH range (6.5-9.0), with a maximum at pH 8.0-8.5. CTRL-1 was produced as a zymogen of 264 amino acids as deduced from the gene sequence, with a sequence identity of 54% with human chymotrypsin B. The number and location of intron/exon junctions as well as the sequence similarity to chymotrypsin both at the DNA and protein level and the presence in duodenal juice indicate that this is a novel digestive enzyme of the chymotrypsin superfamily, albeit one with distinct physiological and biochemical features.