Mutated p21 ras proteins (muRas) are present in f90% of pancreatic adenocarcinomas and express mutants which can function as cancer-specific antigens. To evaluate the frequency and magnitude of the natural T-cell response against muRas in 19 HLA-A2-positive patients with muRas-positive pancreatic carcinomas, antigen-experienced T lymphocytes in fresh peripheral blood mononuclear cells were shown by IFN-g enzyme-linked immunospot using muRas peptides (5-21) that encompass both HLA class I (HLA-A2)^and class II^restricted (HLA-DRB1) epitopes. Six of 19 patients (32%) were found to have a specific T-cell response against individual mutation-specific ras 5-21 but not against other ras mutations or wild-type ras. In contrast, none of 19 healthy subjects had T cells specifically secreting IFN-g (P = 0.004). The T-cell response consisted of both CD8 + and CD4 + T cells but was dominated by CD8 T cells in three of four patients. MuRas 5-14 and muRas 6-14 were shown to specifically induce CD8 + T-cell mediated cytotoxicity against HLA-A2-positive, muRas-bearing pancreatic carcinoma cells. The T-cell response was not correlated with prognostic or clinical variables such as tumor-node-metastasis status, stage, or survival. In conclusion, a natural T-cell response against muRas proteins that could be exploited for immunostimulatory therapeutic approaches has been shown in a significant proportion of patients with pancreatic cancer.The ras proto-oncogenes (H-ras, K-ras, and N-ras) encode 21-kDa proteins with intrinsic GTPase activity that are involved in cell proliferation and differentiation. Point mutations in the ras proto-oncogenes have been identified in a wide range of human solid tumors, in particular carcinomas of the pancreas, colon, lung, and thyroid as well as myeloid malignancies. The majority of point mutations of the ras gene reside at codons 12, 13, and 61 (1). These mutations result in the production of aberrant proteins, which are structurally and functionally distinct from normal endogenous ras and, due to their strict tumor specificity, can function as targets for cellular and humoral immune responses in cancer patients (1-5) and healthy donors (6-8).In pancreatic adenocarcinomas where ras point mutations occur in 80% to 90% of cases, it is the K-ras gene at codon 12 that is found frequently mutated. Replacement of the normal glycine (Gly) residue by an aspartatic acid (Asp), valine (Val), cysteine (Cys), or arginine (Arg) residue accounts for f98% of all ras mutations in pancreatic carcinomas, rendering pancreatic cancer an ideal model for the investigations of T-cell responses against ras mutations at position 12. Previous studies have shown that CD4 + T-cell immunity to position 12 mutated Ras (muRas) can be detected in the majority of patients with pancreatic cancer (3, 5). The proof of this T-cell response and the early presence of muRas in the tumoral process have prompted the implementation of clinical trials aimed at inducing CTL responses against defined muRas antigens (9-11). In these st...

Genetically modified lymphoblastoid cell lines (LCL) have been shown to be an attractive alternative source of antigen-presenting cells for cancer vaccination in vitro. We tested their application in patients with pancreatic cancer in a phase I clinical trial. As a model tumor antigen, we selected the point-mutated (codon 12) Ki-Ras p21 oncogene (muRas) frequently (∼85%) present in pancreatic adenocarcinoma. Autologous LCLs were established in vitro by spontaneous outgrowth from peripheral blood lymphocytes of seven pancreatic carcinoma patients and were genetically modified with an episomal Epstein-Barr virus (EBV)-based expression vector to express muRas (muRas-LCL). Weekly vaccinations with subcutaneous injection of 5×10(6) muRas-LCL were done. In six of seven patients, therapeutic vaccination elicited a T-cell response with an increase in the frequency of muRas-specific precursor cytotoxic T lymphocytes in the peripheral blood and positive delayed-type hypersensitivity reactions at the injection site. Besides local reactions and flu-like symptoms, there were no signs of toxicity and no acute EBV infection, onset of EBV-associated lymphoma, or other severe complications. A clinical response (stable disease) was observed for a short time period (2-4 months) in four of seven patients (57%), mostly in earlier tumor stages. Our results indicate that LCL presenting genetically modified antigen represent a valuable and easily available tool for in vivo autologous tumor vaccination. LCL can be transfected with any known tumor antigen and therefore should be further clinically investigated.

Spontaneous Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (SP-LCLs) can be easily obtained from latently EBV-infected cancer patients and used as a source of antigen-presenting cells (APCs) for immunotherapy. Using point-mutated (codon 12) p21(ras) (muRas) as a model tumor antigen, we evaluated the practicability of using genetically modified SP-LCLs as cancer vaccines for patients with pancreatic cancer expressing mutated Ras (muRas). The repeated stimulation of peripheral blood mononuclear cells (PBMCs) from patients with muRas-LCLs elicited a strong, muRas-specific T cell response. A significant cytotoxic activity against EBV virus proteins or components of the expression vector was not observed. The T cells were able to recognize naturally presented muRas, as shown by their cytotoxicity against muRas (Gly-12 to Val-12 or Asp-12)-expressing tumor cells. The T cell response was mainly MHC class I restricted, and peptides containing amino acids 5 to 14 of muRas-Val-12 and muRas-Asp-12 were identified as immunogenic peptides for HLA-A2. In contrast to the situation in patients with putatively muRas-primed T cells, muRas-LCLs were not able to prime naive T lymphocytes from healthy controls. Vaccination of a pancreatic cancer patient with muRas-LCL induced muRas-specific T cells in PBMCs after 4 weeks. We conclude that genetically modified muRas-LCLs can efficiently present tumor antigens to the immune system and induce antigen-specific cytotoxic T cell responses in vitro and in vivo.