Characterisation of mouse monoclonal antibodies against rhesus macaque killer immunoglobulin-like receptors KIR3D.

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.

Combining forces to hit cancer stem cells: TRAIL-lymphocytes and EpCAMxCD3 bispecific antibody show efficacy against pancreatic cancer.

TRAIL selectively kills cancer cells while bispecific antibody EpCAMxCD3 guides effector lymphocytes to cancer cells. Arming of ex vivo constructed TRAIL-lymphocytes with EpCAMxCD3 enhances contact time and affinity between lymphocytes and tumor cells and enforces tumor elimination. This boosts endogenous immune responses and augments the effect of cytotoxic tumor therapy.

Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment.

Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide.

New gene-immunotherapy combining TRAIL-lymphocytes and EpCAMxCD3 Bispecific antibody for tumor targeting.

PURPOSE: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA. EXPERIMENTAL DESIGN: Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells. RESULTS: Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced. CONCLUSIONS: This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.

Evaluation of porcine mesenchymal stem cells for therapeutic use in human liver cancer.

Mesenchymal stem cell (MSC) transplantation is suggested for therapy of end-stage liver disease, due to e.g. liver cancer and metastasis. Liver transplantation is the only therapeutic option so far but donor organs are short. Also, the availability of allogeneic human MSCs for liver regeneration is limited. Therefore, we evaluated the suitability of porcine bone marrow MSCs from semi-adult pigs and found that morphology, surface expression pattern and multilineage differentiation are similar to those of human MSCs. Porcine MSCs differentiated to a hepatocyte-like phenotype and expressed porcine mRNA of typical liver proteins. However, hepatocyte-like MSCs failed to express the corresponding proteins and did not produce glycogen and urea as primary porcine hepatocytes do. Porcine MSCs were immunotolerated, since they did not activate resting human PBMCs, and were not attacked by human activated PBMCs. However, porcine MSCs led to enhanced proliferation of human pre-activated PBMCs suggesting that immunotoleration of porcine MSCs in the human system has limitations. Together, the potential of porcine MSCs for xenogenous use in human liver therapy is promising but needs further evaluation prior to clinical use.

Mechanisms of tumor and viral immune escape from natural killer cell-mediated surveillance.

Human natural killer (NK) cells recognize and efficiently eliminate MHC class I low or negative malignant targets and virally infected host cells, without requirement for prior sensitization. However, viruses and various tumor cells display elaborate adaptations to evade and overcome immunosurveillance. The current review focuses on escape mechanisms of viruses and malignantly transformed 'stressed' cells to evade from NK cell cytotoxicity. A general overview of recent clinical studies using allogeneic donor NK cells is given, summarizing first data about a possible benefit for patients suffering from high-risk leukemia and solid tumors. Finally, the review discusses the future perspectives and hypotheses aiming to improve therapeutic NK cell strategies against tumor immune escape mechanisms.

Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium.

Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.

Targeting of cancer stem cell marker EpCAM by bispecific antibody EpCAMxCD3 inhibits pancreatic carcinoma.

Patients with pancreatic cancer have a poor survival rate, and new therapeutic strategies are needed. Epithelial cell adhesion molecule (EpCAM), suggested as a marker for cancer stem cells, is over-expressed on most pancreatic tumour cells but not on normal cells and may be an ideal therapeutic target. We evaluated the anti-tumour efficiency of bispecific EpCAMxCD3 antibody linking tumour cells and T lymphocytes. In NOD SCID mice, EpCAMxCD3 had a long serum half-life (t(1/2) approximately 7 days). EpCAMxCD3 significantly retarded growth of BxPC-3 pancreatic carcinoma xenografts. For mimicking a pancreatic cancer microenvironment in vitro, we used a three-dimensional tumour reconstruct system, in which lymphocytes were co-cultured with tumour cells and fibroblasts in a collagen matrix. In this in vivo-like system, EpCAMxCD3 potently stimulated production of the effector cytokines IFN-gamma and TNF-alpha by extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3 antibody, EpCAMxCD3 more efficiently activated the production of TNF-alpha and IFN-gamma by non-stimulated peripheral blood mononuclear cells. Most excitingly, we demonstrate for the first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumour cells, which may be the main reason for the observed anti-tumour effects. As an important prerequisite for future use in patients, EpCAMxCD3 did not alter lymphocyte migration as measured by time-lapse video microscopy. Our data may open a way to improve the immune response and treatment outcome in patients with pancreatic cancer.

Adult stem cells in progression and therapy of hepatocellular carcinoma.

Hepatocellular carcinoma is one of the most aggressive solid tumours associated with poor prognosis. Despite its significance, there is only an elemental understanding of the mechanisms that drive disease pathogenesis, and there are just limited therapy options. The medical community is currently experiencing a wave of enthusiasm for clinical trials, in which adult stem/progenitor cells are used for liver regeneration. This is based on promising results in animal models and encouraging reports from some initial clinical studies. On the other hand, several essential precautions are not being fully addressed. Stem cells may contribute to fibrosis or give rise to hepatic cancer stem cells as a source of hepatocellular carcinoma. This review outlines the current state of knowledge in progression of liver disease and highlights the function of adult stem cells in disease and therapy.