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.

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.

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.

Selection of a cyclic nonapeptide inhibitor to alpha-chymotrypsin using a phage display peptide library.

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against alpha-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with alpha-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of alpha-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for alpha-chymotrypsin was estimated to be 10(-6) M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified alpha-chymotrypsin than for natural alpha-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to alpha-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe- Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of alpha-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.

Identification of a putative motif for binding of peptides to HLA-DQ2.

To understand the rules determining peptide binding to the celiac disease and type 1 diabetes mellitus associated HLA-DQ2 molecule, we have studies in detail the binding of a peptide OVA 258-276Y (IINFEKLTEWTSSNVMEERY) which exhibits strong binding to DQ2. First we tested a set of N- and C-terminal truncated variants, and found the core binding region to comprise residues 267-276Y. Single alanine substitution analysis of the OVA 267-276Y peptide revealed that replacements of V272, E275 and the C-terminal Y had negative effects whereas the substitution of N271 had a positive effect. A polyalanine analogue of the OVA 267-276Y peptide with V272, E275 and a C-terminal Y bound at least as well as the original peptide. A variant peptide with a deletion of R276 displayed decreased binding, suggesting that the anchor residues were out of frame in this analogue. To further characterize the residues playing a role in the binding of the OVA 267-276Y peptide to DQ2 we tested the binding of several analogues with substitutions for V272, E275 and the C-terminal Y residue. Our results indicate that peptides binding to DQ2 have anchor residues in relative positions 4, 7 and (P4, P7 and P9). Residues with negatively charged or hydrophobic aliphatic but not positively charged side chains are preferred in P4 and P7, whereas residues with bulky hydrophobic side chains are preferred in P9.

Selection of peptides with surface affinity for alpha-chymotrypsin using a phage display library.

Peptides with affinity for the surface of alpha-chymotrypsin (EC 3.4.21.1) were selected from a hexapeptide phage display library consisting of approximately 10(7) different clones. Seven selections were performed and five individual phage clones analysed. Compared to the primary library, the five peptide phage clones all interacted more strongly with alpha-chymotrypsin, and DNA sequencing of the phage clones revealed five different amino acid sequences: Gly-Ala-Val-Ile-Thr-His, Arg-Asp-Ile-Val-Val-Ala, Val-Tyr-Ser-His-Ala-Ser, Gly-Ser-Tyr-Ser-Ala-Gly and Leu-Asp-Ile-Val-Val-Ala. Two of the peptides exhibited 83% identity (i.e. a difference of just one amino acid). The chemically synthesized peptides competitively reduced the binding of the corresponding peptide phage clone to alpha-chymotrypsin. Binding of some of the selected peptide phage clones to alpha-chymotrypsin was also reduced by several of the other non-corresponding synthesized peptides, suggesting that these peptides have common recognition areas on the enzyme. Three of the synthesized peptides were poor substrates of alpha-chymotrypsin and they did not inhibit enzyme activity. Our results suggest that it is possible to select peptides from peptide phage display libraries with affinity for different surface structures on the enzyme, not involved in the biologically active site.

T cells recognize a peptide derived from alpha-gliadin presented by the celiac disease-associated HLA-DQ (alpha 1*0501, beta 1*0201) heterodimer.

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(alpha 1*0501, beta 1*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(alpha 1*0501, beta 1*0201) heterodimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic alpha-gliadin peptide, when presented by the cis- or trans-encoded CD-associated DQ(alpha 1*0501, beta 1*0201) heterodimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31-47 of alpha-gliadin, which is included in the part of alpha-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other alpha-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(alpha 1*0501, beta 1*0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.

Binding of peptides to HLA-DQ molecules: peptide binding properties of the disease-associated HLA-DQ(alpha 1*0501, beta 1*0201) molecule.

Peptide binding to DQ molecules has not previously been described. Here we report a biochemical peptide-binding assay specific for the DQ2 [i.e. DQ(alpha 1*0501, beta 1*0201)] molecule. This molecule was chosen since it shows a strong association to diseases such as celiac disease and insulin-dependent diabetes mellitus. Initially we radiolabelled some selected peptides and tested them for binding to affinity-purified DQ2 molecules. One of the peptides, a Mycobacterium bovis (MB) 65 kDa 243-255Y peptide, displayed a good signal-to-noise ratio and was thus chosen as an indicator peptide in the DQ2 binding assay. The MB 65 kDa 243-255Y peptide bound to DQ2 in a strictly pH-dependent fashion, with optimal binding around pH 5 and only weak binding at pH 7.4. The association of the MB 65 kDa 243-255Y peptide to DQ2 was slow, but once formed, the peptide-HLA complexes were very stable. The binding of peptides to DQ2 was specific, as shown in inhibition experiments with a panel of 47 peptides, differing in length, sequence, and origin. The binding of peptides to DR3 was tested in a similar assay with a Mycobacterium tuberculosis 65 kDa 3-13 peptide as the binding indicator. DQ2 and DR3 molecules bound to different sets of peptides. However, the peptide binding to DQ2 and DR3 showed, in general, similar characteristics with respect to pH dependence and kinetic parameters, indicating that the overall rules for peptide binding to DQ molecules are the same as those previously shown for human DR and murine I-A and I-E molecules.

p21-ras-peptide-specific T-cell responses in a patient with colorectal cancer. CD4+ and CD8+ T cells recognize a peptide corresponding to a common mutation (13Gly-->Asp).

Peptides derived from mutated ras are immunogenic in mice and humans, and represent a group of specific tumor antigens that are potential targets for immunotherapy. T-cell responses against mutant p21 ras can be initiated in vitro by repeated stimulation of peripheral-blood mononuclear cells with mutant ras-derived peptides. Patients with tumors commonly harbouring ras mutations may therefore show evidence of in vivo reactivity against such mutations. Peripheral-blood mononuclear cells from 10 patients with colorectal adenocarcinoma were screened for reactivity against synthetic ras-derived peptides corresponding to the most commonly found mutations in this type of cancer. In one patient, T-cell reactivity against the 1-25,13Gly-->Asp peptide was detected. From this patient, both CD4+ and CD8+ T-cell clones specific for the 1-25,13Gly-->Asp mutation could be raised. We were not, however, able to detect the corresponding mutation in the cancer. The 13Gly-->Asp mutation in the ras oncogene is frequent and constitutes 9 to 27% of all K ras mutations found in biopsies from patients with colorectal carcinomas. Our study demonstrates a mutant ras-specific T-cell response of both the CD4+ and the CD8+ phenotype in a cancer patient. We speculate that in this patient a specific T-cell response resulted in eradication of tumor cells harboring the 13Gly-->Asp mutation.

Overlapping epitopes encompassing a point mutation (12 Gly-->Arg) in p21 ras can be recognized by HLA-DR, -DP and -DQ restricted T cells.

Mutations in ras genes which result in transforming gene products carrying amino acid substitutions in position 12, 13 or 61 are common in human cancer. Peptides encompassing these mutations in ras are shown to be immunogenic in both mice and humans. The potential usefulness of such peptides in cancer therapy, depends on their ability to bind to HLA molecules. We therefore stimulated T cells from healthy donors with mutated ras-derived peptides. By repeated in vitro stimulation of peripheral blood mononuclear cells, several T cells clones could be generated which recognized a p21 ras derived peptide carrying a position 12 Gly-->Arg substitution. This peptide (1-25,12 Arg) could be specifically recognized by T cells restricted by either HLA-DQ7 or -DP3. Previously, we showed that this peptide is also recognized by a T cell clone restricted by HLA-DR2. The core region of the peptide was determined to span positions 9-16 for all three HLA restriction elements, and accordingly contains the mutational hot spots in position 12 and 13. The observation that the mutant 1-25,12 Arg ras-derived peptide may contain a promiscuous epitope encompassing the Gly-->Arg mutation in position 12 indicates that lack of peptide presentation by given HLA molecules may not be a major constraint in responsiveness against ras mutations.

T cell clones specific for p21 ras-derived peptides: characterization of their fine specificity and HLA restriction.

Peptides corresponding to the mutated regions of the oncoprotein p21 ras are immunogenic and capable of eliciting HLA class II-restricted T cell responses. Here we report studies on the fine specificity of four T lymphocyte clones (TLC) from a single donor, using various truncated peptides derived from the residues 6-19 of p21 ras and a panel of well-characterized HLA homozygous cells as antigen-presenting cells. Putative minimum peptides of nine or ten amino acids could be defined for each TLC. Two of the TLC recognized peptides presented by DR2, and the two others recognized peptides presented by DQ6. Some notable differences in the requirement for certain amino acids were seen between the DR- and DQ-restricted TLC. Thus, Ser at residue 17 was required for stimulation of the DQ6-but not the DR2-restricted TLC. Val at residue 8 was essential for stimulation of all TLC, whereas one of the DR2-restricted TLC also required Val at residue 7. Some peptides which were nonstimulatory were still capable of binding to DQ6 molecules in peptide competition experiments. The results may be of importance for potential immunotherapy of cancer where transforming ras oncoproteins are involved.

Memory T cells of a patient with follicular thyroid carcinoma recognize peptides derived from mutated p21 ras (Gln-->Leu61).

Point mutations in ras genes resulting in substitutions of amino acid Gly in positions 12 and 13, and Gln in position 61 of the ras gene product p21, are commonly found in human tumors. Peptides derived from aberrant p21 may elicit a tumor specific T cell response, provided that these peptides can bind to HLA molecules of the tumor and the patient has T cells able to recognize the corresponding peptide-HLA complex. Here we report that CD4+ T cells of memory type (CD45RO+) from a patient with a follicular thyroid carcinoma respond against a synthetic peptide derived from aberrant p21 ras having a Gln-->Leu substitution at position 61. Such responses were not observed when T cells from healthy volunteers or cancer patients where this mutation does not usually occur were stimulated with this peptide. The responding T cells did not cross-react with the corresponding peptide derived from native p21 ras nor did they recognize peptides carrying other substitutions in position 61. T cells clones were generated which recognized this Leu61 peptide when presented by HLA-DQ8 molecules. These T cell clones also recognized the corresponding intact p21 ras protein. By using several different synthetic peptides, a peptide with optimal stimulatory capacity was defined. Performing polymerase chain reaction and oligonucleotide probing we were, however, not able to detect the p21 ras gene encoding the Gln-->Leu substitution in DNA from tumor biopsies from the patient. This may indicate that tumor cells harboring the mutation leading to the Gln-->Leu substitution had been eliminated and that tumor progression was due to cells that had deleted the mutated ras gene. The finding that ras derived peptides and recombinant mutated p21 ras are immunogenic in man may form the basis for the development of cancer immunotherapy based on synthetic oncogene derived peptides.

T-cell responses against products of oncogenes: generation and characterization of human T-cell clones specific for p21 ras-derived synthetic peptides.

Peptides derived from mutated human proto-oncogenes bound to HLA may represent a novel type of tumor-specific antigen. Mutated ras genes are the oncogenes most frequently identified in human cancer. The transforming genes carry a mutation in codons 12, 13, or 61. We have investigated whether the T-cell repertoire of healthy individuals contains T cells capable of recognizing and responding to oncogene-derived peptides. Synthetic peptides derived from mutated p21 ras proto-oncogenes, covering mutations at codons 12 or 13 were selected. It was feasible to elicit T-cell responses and isolate several new T-cell clones (TCC) with specificity for a number of different mutated ras peptides after repeated in vitro immunization. Four TCC were characterized with respect to fine specificity and HLA restriction. TCC B and I were restricted by HLA-DR molecules, and recognized the mutated p21 ras-derived peptide carrying Arg and Lys at residue 12, respectively. TCC E and F were restricted by HLA-DQ molecules, the former being specific for a mutated p21 ras-derived peptide with Val in position 13 and the latter more broadly reactive. Peptide competition experiments with a panel of ten peptides derived from p21 ras indicated that all could bind to HLA-DQ molecules of the T-cell donor, while several were also able to bind his HLA-DR molecules. These results show that several p21 ras mutations resulting in aa substitutions at residues 12 or 13 could be recognized by T cells derived from precursor T cells of relatively low frequency present in the normal repertoire of a single donor.

The dimer of hemoregulatory peptide (HP5B) stimulates mouse and human myelopoiesis in vitro.

A synthetic analogue of a pentapeptide associated with mature granulocytes has been described earlier and shown to suppress myelopoietic colony formation in vitro in concentrations from 10(-13) to 10(-6) M. By oxidation of the peptide, a dimer will rapidly occur by formation of disulfide bridges between cysteine residues. We here demonstrate that this dimer has the opposite effects of the monomer. For both mouse and human granulocyte-macrophage colony-forming units (CFU-GM), a dose-dependent enhancement of colony formation was observed in the dose range 10(-16) to 10(-5) M, where a saturation level was reached above 10(-8) M. At low doses of colony-stimulating activity (CSA) and in the linear stimulating phase, an up to ten times increase of colony formation was seen, whereas at higher doses the effect was less pronounced. Also at the plateau level of CSA stimulation an increased colony yield was seen. All types of colonies were stimulated. The dimer itself had no colony-stimulating factor activity and was not toxic to bone marrow cells in suspension cultures up to 24 h. Upon reduction of the dimer by use of sulfhydryl compounds, inhibitory effects on CFU-GM were restored. The peptide had no effect on the phagocytic process in human granulocytes, including attachment and internalization of bacteria or Zymosan particles. The monomerdimer equilibrium of hemoregulatory peptide may constitute a new mechanism for proliferative regulation of myelopoietic cells.