Mariana Bucur

Tumor selective replication of Newcastle disease virus: Association with defects of tumor cells in antiviral defence

To investigate tumor‐selective viral replication, we compared several tumorigenic human cell lines to nontumorigenic human cells from the blood for the sensitivity to become infected by a recombinant lentogenic strain of Newcastle Disease Virus (NDV) with incorporated transgene EGFP (NDFL‐EGFP). Although fluorescence signals in nontumorigenic cells were only weak or missing completely, a massive and long‐lasting transgene‐expression was observed in all tumor cell lines. The majority of tumor cells (50–95%) could be infected, and viral replication was associated with an increase in the cell surface density of viral antigens. To clarify the underlying mechanism of the observed difference in virus susceptibility we examined the kinetics of interferon‐induced antiviral enzymes because NDV is a strong type‐I interferon inducer. This analysis revealed several defects of tumor cells in their antiviral defence responses: They showed no response to UV‐inactivated NDV, whereas nontumorigenic cells reacted with induction of high‐levels of the antiviral enzymes PKR and MxA. Upon coincubation with live NDV, tumor cells showed a delayed response in the increased expression of the antiviral enzymes in comparison with PBMC. In nontumorigenic cells the replication cycle of NDV stopped after the production of positive‐strand RNA, while tumor cells continued in the replication cycle and copied viral genomes 10–50 hr after infection. These results can explain the tumor selective replication behavior of this interesting antineoplastic virus.

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAOhigh and NAOlow, respectively). Sorted NAOhigh cells were found to survive in culture whereas sorted NAOlow cells died. Moreover, NAOhigh cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long‐term culture. These cells showed a similar cardiolipin content and expressed the same level of anti‐apoptotic proteins Bcl‐2 and Bcl‐xL as APO‐S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine‐sulfoximine (BSO) correlated with a significant stimulation of NO‐mediated apoptosis whereas the exposure of NO‐sensitive APO‐S cells to the glutathione precursor N‐acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO‐induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role. J. Cell. Biochem. 78:578–587, 2000.

Heparanase: A New Metastasis-Associated Antigen Recognized in Breast Cancer Patients by Spontaneously Induced Memory T Lymphocytes

Increased expression and secretion of heparanase (Hpa) by tumor cells promotes tumor invasion through extracellular matrices, tissue destruction, angiogenesis, and metastasis. Here, we show the existence in breast cancer patients of Hpa-specific T lymphocytes by fluorescence-activated cell sorting flow cytometry using Hpa peptide-MHC class I tetramers. We furthermore show memory T-cell responses in a high proportion of breast cancer patients to Hpa-derived HLA-A2-restricted peptides, leading to production of IFN-gamma and to generation of antitumor CTLs lysing breast cancer cells. Such CTLs recognized endogenously processed respective Hpa peptides on Hpa-transfected and Hpa-expressing untransfected breast carcinoma cells. According to these results and to the fact that such cells were not found in healthy people, Hpa seems to be an attractive new tumor-associated antigen and its HLA-A2-restricted peptides ought to be good candidates for peptide vaccination to reactivate memory immune responses to invasive and metastatic cancer cells.

Heparanase expression at the invasion front of human head and neck cancers and correlation with poor prognosis.

Purpose: Head and neck squamous cell carcinomas (HNSCC) are characterized by a poor prognosis due to aggressive, recurrent tumor growth. Expression of the extracellular matrix–degrading enzyme heparanase was associated with poorer prognosis in several cancers. We analyzed the presence of heparanase in HNSCC tissues and tumor cells and its potential prognostic significance. Experimental Design: We analyzed the expression of the active form of heparanase in HNSCC tissues in corresponding tumor cell cultures and after xenotransplantation of tumor cell cultures into NOD/Scid mice by immunohistochemistry, Western blot analysis, and reverse transcription-PCR in altogether 25 patients and did a comparison with clinicopathologic data of the patients. Results: Heparanase expression in situ was detected in all tumor biopsies in the tumor stroma and in tumor cells from 13 of 19 primary tumors and 9 of 12 lymph node metastases. Heparanase was localized in disseminated tumor cells, in tumor cell clusters invading adjacent stromal tissues, and in tumor cells at the tumor invasion front. Lymph node metastases expressed higher levels of heparanase compared with corresponding primary tumors. In contrast to a heterogeneous expression pattern in tumor tissues, all corresponding HNSCC tumor cell cultures showed a rather homogeneous heparanase expression on the mRNA and protein levels. Comparison of heparanase expression in situ and in corresponding tumor cell cultures in vitro or after xenotransplantation into NOD/Scid mice revealed that heparanase expression was regulated in vivo. Lack of heparanase in tumor cells from primary tumors or lymph node metastases was correlated with prolonged disease-free survival and overall survival. Conclusion: Heparanase expression seems to be involved in the invasiveness and aggressiveness of HNSCC.

Melanoma-Reactive T Cells in the Bone Marrow of Melanoma Patients: Association with Disease Stage and Disease Duration

Using IFN-gamma enzyme-linked immunospot, we investigated reactivity of T cells from bone marrow and peripheral blood to melanoma lysate-pulsed autologous dendritic cells in 40 melanoma patients. Melanoma-reactive T cells were present in the bone marrow of seven patients and in peripheral blood of four patients. In the bone marrow, melanoma-reactive T cells were present in 6 of 21 stage IV patients and in 1 of 10 stage III patients, whereas none were detected in stage I to II patients (0 of 9). The occurrence of tumor-reactive bone marrow T cells in melanoma patients was associated with advanced disease stage, disease duration and tumor load, and independent of treatment. These findings provide new insights into the generation of T-cell responses in melanoma patients.

Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Anti-angiogenic activity of VXM01, an oral T-cell vaccine against VEGF receptor 2, in patients with advanced pancreatic cancer: A randomized, placebo-controlled, phase 1 trial.

VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points – pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

Breaking tolerance to a tumor-associated viral superantigen as a basis for graft-versus-leukemia reactivity.

A major goal in tumor immunotherapy consists of breaking potential tumor‐specific T‐cell unresponsiveness (tolerance), which may explain tumor growth in cancer patients. We report that immunological tolerance to a tumor‐associated viral superantigen (SAg) is overcome in a mouse lymphoma model by transfer of allogeneic T cells expressing SAg‐reactive Vβ6 T‐cell receptor chains. Surprisingly, upon contact with SAg‐expressing lymphoma cells, Vβ6 T cells became activated rather than tolerized (as reported previously). They also developed SAg‐specific cytotoxic T‐lymphocyte activity and secreted IL‐2 and IFN‐γ. The grafted T cells infiltrated liver metastases, formed close contact with SAg‐expressing tumor cells, and caused significant graft‐vs.‐leukemia (GvL) effects. Selection for tumor resistance among the progeny from a cross between SAg‐negative donor and SAg‐ positive recipient strains revealed a strict correlation between loss of the endogenous SAg tolerogen, rescue of Vβ6 T cells from SAg‐mediated deletion, and leukemia resistance. These findings suggest that immune responses to SAg can be exploited to break tolerance and augment immune responses to tumors. Int. J. Cancer 87:695–706, 2000.

A phase 1 trial extension to assess immunologic efficacy and safety of prime-boost vaccination with VXM01, an oral T cell vaccine against VEGFR2, in patients with advanced pancreatic cancer

ABSTRACT VXM01 is a first-in-kind orally applied tumor vaccine based on live attenuated Salmonella typhi carrying an expression plasmid encoding VEGFR2, an antigen expressed on tumor vasculature and a stable and accessible target for anti-angiogenic intervention. A recent randomized, placebo-controlled, phase I dose-escalation trial in advanced pancreatic cancer patients demonstrated safety, immunogenicity and transient, T-cell response-related anti-angiogenic activity of four priming vaccinations applied within one week. We here evaluated whether monthly boost vaccinations are safe and can sustain increased frequencies of vaccine-specific T cells. Patients with advanced pancreatic cancer were randomly assigned at a ratio of 2:1 to priming with VXM01 followed by up to six monthly boost vaccinations, or placebo treatment. Vaccinations were applied orally at two alternative doses of either 106 colony-forming units (CFU) or 107 CFU, and concomitant treatment with standard-of-care gemcitabine during the priming phase, and any treatment thereafter, was allowed in the study. Immunomonitoring involved interferon-gamma (IFNγ) ELIspot analysis with long overlapping peptides spanning the entire VEGFR2 sequence. A total of 26 patients were treated. Treatment-related adverse events preferentially associated with VXM01 were decreases in lymphocyte numbers in the blood, increased frequencies of neutrophils and diarrhea. Eight out of 16 patients who received at least one boosting vaccination responded with pronounced, i.e. at least 3-fold, increase in VEGFR2-specific T cell response over baseline levels. In the VXM01 vaccination group, VEGFR2-specific T cells peaked preferentially during the boosting phase with an average 4-fold increase over baseline levels. In conclusion, prime/boost vaccination with VXM01 was safe and immunogenic and increased vaccine specific T cell responses compared with placebo treatment.