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G Gaudernack

Publications and source records attributed to G Gaudernack.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

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.

Adjuvants, Immunologic↗
PubMed1997

Different genetic pathways to proximal and distal colorectal cancer influenced by sex-related factors.

Mutations in the k-ras and TP53 genes, as well as microsatellite instability (MIN), are frequent genetic alterations in colorectal carcinomas and represent 3 different mechanisms in the carcinogenic process. Both the incidence of colorectal cancer and the frequency of genetic alterations in such tumours have been related to different clinico-pathological variables, including age and gender of the patient and location of the tumour. A number of studies have also reported associations between different types of genetic alterations. We therefore wanted to explore the relationship between these genetic and clinico-pathological variables using multivariate analysis on material from 282 colorectal carcinomas. Three logistic regression models were constructed: 1) the presence of K-ras mutations was dependent on MIN and age and gender of patient, with an especially low frequency among younger males and in tumours with MIN (overall p = 0.0003); 2) the presence of TP53 mutations was only dependent on tumour location, with a positive association to cancers occurring distally (p = 0.002); and 3) the presence of MIN was dependent on age, gender and K-ras and TP53 mutations, as well as on tumour location. MIN was most frequent among younger male and older female patients, was rare in tumours with K-ras or TP53 mutations and was found almost exclusively in the proximal colon (overall p < 0.0001). Our data confirm that different genetic pathways to colorectal cancer dominate in the proximal and distal segments of the bowel and suggest that the K-ras- and MIN-dependent pathways are influenced by different sex-related factors.

Adult↗
PubMed1997

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.

Adenocarcinoma↗
PubMed1997

CDw78--a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton.

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10% of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90% of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10-20% of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (Ka = 1.5 x 10(8) M(-1)) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-mu suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.

Antibodies, Monoclonal↗
PubMed1997

K-ras mutations and prognosis in large-bowel carcinomas.

BACKGROUND: Colorectal carcinogenesis is regarded as a multistep process involving several genetic alterations, with mutation in the K-ras gene in about half of the tumours. We aimed at clarifying the role of this genetic alteration related to survival and clinicopathologic variables. METHODS: One hundred large-bowel carcinomas operated on between 1978 and 1982 were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene, using enriched polymerase chain reaction amplification, restriction fragment length polymorphism analysis, and direct sequencing. RESULTS: Forty mutations were found (40%): 31 in codon 12 and 9 in codon 13, 7 different types. There was no relationship between tumours with and without K-ras mutations with regard to Dukes' stages, age or sex of the patient, tumour localization, histologic grade, DNA ploidy pattern, HLA-DR staining pattern, or survival. Samples from 5 different localizations in 7 carcinomas showed identical K-ras mutation pattern, as did 19 recurrences/ metastases originating from 11 carcinomas. CONCLUSIONS: When present, the primary tumour shows homogeneous distribution of K-ras mutation, and the mutation follows the carcinoma in the secondary deposit, regardless of lymphogenous or hematogenous spread. The presence of K-ras mutation does not seem to have prognostic significance for the patient, and the precise nucleotide change is furthermore not predictive of tumour behaviour.

Adult↗
PubMed1996

Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells.

In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.

Adenoviruses, Human↗
PubMed1996

Role of the TCR binding region of the HLA class I alpha 2 domain in regulation of cell adhesion and proliferation.

In addition to Ag presentation for T cell surveillance, MHC molecules have been implicated in mediating regulatory signals. We have assessed biologic responses following engagement of the TCR accessible region of the HLA class I alpha 2 domain. mAbs directed to this domain specifically induced cell aggregation of normal hematopoietic and leukemic cells. The functional consequences were unique since other mAbs reactive with HLA class I residues outside the TCR binding domain did not induce cell aggregation. The adhesion response required ATP, mRNA, protein, and actin synthesis and did not depend on LFA-1/ICAM interactions. Cell aggregation was also induced when all but four of the intracytoplasmic residues of the class I molecule were deleted, indicating that transduction of signals leading to cell adhesion does not require this portion of the molecule. mAbs directed to HLA class I alpha 2 amino acid residues within the TCR binding domain were also able to inhibit proliferation of normal mitogen-stimulated T cells. Growth inhibition correlated with down-regulated expression of CD25, CD28, and CD95, suggesting that reduced transduction of costimulatory signals is involved. Although HLA class I signals inducing cell aggregation required engagement of positions within the TCR binding region, growth inhibitory signals could be generated through positions both within and adjacent to this domain. Taken together, engagement of specific positions within the TCR binding domain of the class I alpha 2 helix results in active cellular responses. Thus, this region may be directly involved in signal transduction following CTL recognition of target cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1996

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.

Adult↗
PubMed1996

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.

Adenocarcinoma↗
PubMed1996

K-ras mutations and HLA-DR expression in large bowel adenomas.

A total of 72 sporadic colorectal adenomas in 56 patients were studied for the presence of point mutations in codons 12 and 13 of the K-ras gene and for HLA-DR antigen expression related to clinicopathological variables. Forty K-ras mutations in 39 adenomas were found (54%): 31 (77%) in codon 12 and nine (23%) in codon 13. There was a strong relationship between the incidence of K-ras mutations and adenoma type, degree of dysplasia and sex. The highest frequency of K-ras mutations was seen in large adenomas of the villous type with high-grade dysplasia. Fourteen out of 15 adenomas obtained from 14 women above 65 years of age carried mutations. HLA-DR positivity was found in 38% of the adenomas, large tumours and those with high-grade dysplasia having the strongest staining. Coexpression of K-ras mutations and HLA-DR was found significantly more frequently in large and highly dysplastic adenomas, although two-way analysis of variance showing size and grade of dysplasia to be the most important variable. None of the adenomas with low-grade dysplasia showed both K-ras mutation and HLA-DR positivity (P = 0.004). K-ras mutation is recognised as an early event in colorectal carcinogenesis. The mutation might give rise to peptides that may be presented on the tumour cell surface by class II molecules, and thereby induce immune responses against neoplastic cells.

Adenoma↗
PubMed1996

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).

B-Lymphocytes↗
PubMed1996

Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blast cells.

The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes whose expression may be related to the maturation or function of the cells. Monoclonal antibodies specific for different epitopes have been reported to detect different numbers of CD34+ leukaemic blast cells. We wanted to confirm this observation and study whether parallel findings could be observed for normal haemopoietic progenitor cells. The cells were immunophenotyped by flow cytometry with a series of monoclonal antibodies reactive with different CD34 epitopes. Class III epitopes (resistant to enzymatic cleavage with neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica) showed a broader distribution on normal haemopoietic progenitor cells and leukaemic blast cells than class I epitopes (sensitive to cleavage with all three enzymes) and class II epitopes (sensitive to degradation with glycoprotease and chymopapain, and resistant to neuraminidase). The subpopulation of normal progenitor cells which exclusively expressed class III epitopes had flow cytometric characteristics compatible with mature myeloid progenitor cells whereas class I, II and III epitopes were equally expressed on cells enriched for immature subsets. No discordant epitope expression could be observed for the more immature leukaemias (AML-M0/1) and a higher percentage of the more mature leukaemic blast cells (AML-M3 and AML-M4/5) expressed class III epitopes compared to the percentage expressing class I and II epitopes. These data indicate that CD34 class III epitopes are more broadly distributed on normal haemopoietic progenitor cells and leukaemic blast cells than class I and II epitopes, and that class I and II epitopes may be down-regulated prior to class III epitopes during normal haemopoietic progenitor cell differentiation. These findings should be considered when selecting CD34 mabs for quantification and positive selection of haemopoietic progenitor cells for research and clinical purposes.

Acute Disease↗
PubMed1995

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.

Adenocarcinoma↗
PubMed1995

CD8+ T cells from a patient with colon carcinoma, specific for a mutant p21-Ras-derived peptide (Gly13-->Asp), are cytotoxic towards a carcinoma cell line harbouring the same mutation.

Several T lymphocyte clones (TLC), specific for a p21-Ras-derived peptide expressing a Gly13-->Asp mutation and of the CD8+ subtype, were generated from peripheral blood of a colon carcinoma patient. The TLC exerted cytotoxicity against an interferon-gamma (IFN gamma)-pretreated colon carcinoma cell line, HCT116, which harbours the Gly13-->Asp mutation and shares both HLA-A2 and HLA-B12(44) with the patient. This cytotoxic effect could be blocked by a monoclonal antibody (mAb) against CD8 molecules, as well as with a mAb against HLA class I molecules and a polyclonal antiserum against HLA-B12, identifying B12(44) as the antigen-presenting molecule. In growth-inhibition experiments, the growth of both IFN gamma-pretreated and untreated target cells were strongly inhibited by the presence of the CD8+ TLC. Together these data indicate that human cancer cells harbouring a spontaneous ras mutation can process aberrant p21 Ras and express peptide/HLA-class-I complexes on their surface in sufficient density to be recognized by Ras-specific cytotoxic T lymphocytes.

Adenocarcinoma↗
PubMed1995

Characterization of an HLA-DQ2-specific monoclonal antibody. Influence of amino acid substitutions in DQ beta 1*0202.

The HLA-DQ(alpha 1*0501, beta 1*0201) and-DQ(alpha 1*0501, beta 1*0202) (i.e., DQ2) heterodimers are probably involved in the pathogenesis of celiac disease and several other HLA-DQ-associated diseases. To obtain a tool for studies of these molecules, a mAb of the IgG1 isotype, 2.12.E11, was produced by immunization with purified DQ(alpha 1*0501, beta 1*0201) molecules and murine NIH 3T3 cells transfected with both DQA1*05011 and DQB1*0202. Panel cell studies with HLA homozygous B-lymphoblastoid cells and HLA-transfected murine cells demonstrated that 2.12.E11 bound only to cells expressing HLA-DQ beta 1*0201 or 0202, irrespective of the accompanying DQ alpha chain (i.e., DQ alpha 1*0501 or DQ alpha 1*0201). Another DQ2-specific mAb (XIII 358.4) and the broadly HLA class-II-reactive mAb Tü39 strongly inhibited binding of 2.12.E11. Epitope mapping employing mutants with single aa substitutions of DQ beta 1*0202 indicated that position 37 may be of some importance for 2.12.E11 binding. A triple mutant (45G-->E, 46E-->V, 47F-->Y) failed to bind 2.12.E11, suggesting a crucial role for one or more of these residues in the epitope. However, the expression of the mutant beta chain could not be formally proved, as none of the DQ2-reactive mAbs recognized this transfectant.

3T3 Cells↗
PubMed1995

Influence on antibody recognition of amino acid substitutions in the cleft of HLA-DQ2 molecules. Suggestive evidence of peptide-dependent epitopes.

The purpose of this study was to identify polymorphic residues of DQ2 beta chains which are involved in the epitopes recognized by DQ2-reactive mAbs. Binding studies were performed on a panel of DQ2 mutant cells made by transfection of a DQ (alpha 1*0501, beta 1*0301)-positive B-LCL with DQB1 genes encoding either wild-type or mutated DQ beta 1 *0202. Several aa substitutions were found to influence the binding of DQ2 mAbs. All but one of the mAbs were clearly sensitive to the introduced aa substitutions, but individual mAbs were differentially influenced, suggesting that they recognized different epitopes on the DQ2 molecule. Substitutions in positions 30 and 37 of the peptide-binding cleft were also found to influence the binding of some mAbs. Second, two mAbs, NFLD.M71 and NFLD.M102, which bound to DQ(alpha 1*0501, beta 1*0202) expressed in human cells and were sensitive to the introduction of aa substitutions in the cleft, bound weakly to the DQ(alpha 1*0501, beta 1*0202) heterodimer when expressed in a transfected murine NIH 3T3 cell line. Together this indicates that some DQ2-reactive mAbs may recognize peptide-dependent epitopes.

3T3 Cells↗