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Su.68. CD95 and CD95L in Gastrointestinal Cancer Patients Peripheral Blood
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Su.67. Establishing a Model for Examination of HER-2 Specific IgG and IgE Mediated Tumour Cell Killing Josef Singer,1 Panos Karagiannis,1 Sophia Karagiannis,2 Samuel Gan,2 James Hunt,2 Regina Knittelfelder,1 Andrew Beavil,2 Manuel Penichet,3 Erika Jensen-Jarolim,1 Hannah Gould. 2 1Medical University of Vienna, Vienna, Austria; 2 King's College London, London, United Kingdom; 3 University of California, Los Angeles, Los Angeles, CA Background: Although there are 9 antibody subclasses in man, all of the clinically used monoclonal antibodies are of the IgG subclass, e.g. Trastuzumab (Herceptin®) for the treatment of breast cancer. Recent studies have shown that IgE has presumably a natural tumor surveillance function, which would suggest that tumor specific IgE antibodies could be therapeutical tools. We aimed to produce an anti-HER-2 IgE antibody to show its tumoricidic effects in a breast cancer model in comparison with Herceptin®. Antibody-dependent tumour cell killing via cytotoxicity (ADCC) and via phagocytosis (ADCP) could be measured by establishing a 3-colour flow cytometry method. Methods: To get the DNA sequence of the variable parts of Herceptin®, we applied its amino acid sequence, reverse-translated it, cloned it in two vectors for heavy and light chain and HEK-293 cells were transfected. Furthermore a 3-colour flow cytometry assay developed by Bracher M et al. was modified by using monocytic U937 as effector cells and the HER-2 overexpressing cell line TAUF-1.1 as target for analyzation of HER-2 specific IgG and IgEmediated tumour cell killing. Results: The anti-HER-2-IgE antibody was characterized in immunological and molecular biological assays which showed similar binding properties to HER-2 compared to Herceptin®. Using the 3-colour assay, our results indicate that IgG1 antibodies do not just kill tumour cells via ADCC which was known so far, but are also able to mediate ADCP of tumour cells. Conclusion: In addition to the well-documented cytotoxic killing mechanisms of Herceptin®, our in vitro data clearly show that this IgG1 antibody also induces monocyte-mediated tumour killing via ADCP. The created anti-HER-2 antibody of the IgE subclass will serve as a tool for comparison studies in this 3-colour ADCC/ADCP assay. doi:10.1016/j.clim.2008.03.418
Su.68. CD95 and CD95L in Gastrointestinal Cancer Patients Peripheral Blood Inta Jaunalksne,1 Simona Donina,2 Ludmila Engele,2 Tatjana Romanova. 1 1Paula Stradina KUS, Riga, Latvia; 2RAKUS Latvian Oncology Center, Riga, Latvia Immunity is one of the key factors in oncological processes. Cell immune answer play crucial role in balance between cell proliferation and apoptosis.Immune cell apoptosis is one of the factors which leads to malignant process spread. With the aim to check CD95 , CD95L level in 37 histologically verified gastrointestinal cancer patients we also analyzed CD4+, CD38+, CD25+ cell level by laser flow cytofluorimeter (Becton Dickenson, USA) and TNFalfa, SIL-2R level in patients serum (Immulite 2000,USA) and sTNF alfa IR by ELISA method Biosource,Belgium).Patients were divided
Abstracts in two groups- with local (I+II stage) and with advanced ( III+ IV stage)disease. We observed that there is a difference in CD95 cell absolute count, it is increased in more advanced patiets group, but there is no difference in CD95L, CD38+, CD25+, TNFalfa, sTNFRI level in patients with local and more advanced process. sIL-2R level was increased in advanced gastrointestinal cancer patients serum, but CD4+ cell absolute count was decreased if patients had III+IV stage of disease. Therefore CD4 cells are more sensitive to FAS mediated apoptosis and increased sIL-2R level may lead to decreased immune answer in advanced stages of gastrointestinal cancer patients. doi:10.1016/j.clim.2008.03.419
Su.69. Immunotherapy of Adenocarcinoma Patients with Gc Protein-Derived Macrophage Activating Factor, GcMAF Nobuto Yamamoto,1 Masumi Ueda,1 Kazuya Hashinaka,1 Theodore Sery,1 Charles Benson. 2 1Socrates Institute for Therapeutic Immunology, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA Intratumor BCG administration eradicates local as well as metastasized tumors. Administration of BCG into noncancerous tissues results in no effect on the tumors. Inflammation induced by BCG in normal tissues releases lysophospholipids that activate macrophages. Because cancerous tissues contain alkylphospholipids, BCG-induced inflammation of cancerous tissues produces alkyl-lysophospholipids that activate macrophages approximately 400 times more effective than lysophospholipids, implying that highly activated macrophages are tumoricidal. Inflammation-primed macrophage activation is the principal macrophage activation process that requires serum vitamin Dbinding protein (known as Gc protein) which is the precursor for the macrophage activating factor (MAF). Stepwise treatment of purified Gc protein with immobilized βgalactosidase and sialidase generates the most potent MAF (GcMAF) that produces no side effect in humans. Intramuscular administration of 100 ng GcMAF activate systemic macrophages at maximal level with 30-fold increased ingestion index and 15-fold increased superoxide generating capacity in 3.5 hr. GcMAF also has a potent mitogenic activity on myeloid progenitor cells that generate systemically 40-fold increase in the activated macrophages in 4 days. When adenocarcinoma (metastatic breast and prostate cancer) patients (n = 32) were intramuscularly administered with 100 ng GcMAF/week, their tumors were eradicated in 16-25 weeks. These patients were tumor free for more than six years after GcMAF therapy. Since intravenous administration of GcMAF allows rapid interaction of GcMAF with myeloid progenitor cells in bone marrow, the systemic cell counts of the activated macrophages increased to 200-fold. Weekly intravenous administration of 100 ng GcMAF to adenocarcinoma patients eradicates tumors in 11-16 weeks. doi:10.1016/j.clim.2008.03.420
Su.67. Establishing a Model for Examination of HER-2 Specific IgG and IgE Mediated Tumour Cell Killing Josef Singer,1 Panos Karagiannis,1 Sophia Karagiannis,2 Samuel Gan,2 James Hunt,2 Regina Knittelfelder,1 Andrew Beavil,2 Manuel Penichet,3 Erika Jensen-Jarolim,1 Hannah Gould. 2 1Medical University of Vienna, Vienna, Austria; 2 King's College London, London, United Kingdom; 3 University of California, Los Angeles, Los Angeles, CA Background: Although there are 9 antibody subclasses in man, all of the clinically used monoclonal antibodies are of the IgG subclass, e.g. Trastuzumab (Herceptin®) for the treatment of breast cancer. Recent studies have shown that IgE has presumably a natural tumor surveillance function, which would suggest that tumor specific IgE antibodies could be therapeutical tools. We aimed to produce an anti-HER-2 IgE antibody to show its tumoricidic effects in a breast cancer model in comparison with Herceptin®. Antibody-dependent tumour cell killing via cytotoxicity (ADCC) and via phagocytosis (ADCP) could be measured by establishing a 3-colour flow cytometry method. Methods: To get the DNA sequence of the variable parts of Herceptin®, we applied its amino acid sequence, reverse-translated it, cloned it in two vectors for heavy and light chain and HEK-293 cells were transfected. Furthermore a 3-colour flow cytometry assay developed by Bracher M et al. was modified by using monocytic U937 as effector cells and the HER-2 overexpressing cell line TAUF-1.1 as target for analyzation of HER-2 specific IgG and IgEmediated tumour cell killing. Results: The anti-HER-2-IgE antibody was characterized in immunological and molecular biological assays which showed similar binding properties to HER-2 compared to Herceptin®. Using the 3-colour assay, our results indicate that IgG1 antibodies do not just kill tumour cells via ADCC which was known so far, but are also able to mediate ADCP of tumour cells. Conclusion: In addition to the well-documented cytotoxic killing mechanisms of Herceptin®, our in vitro data clearly show that this IgG1 antibody also induces monocyte-mediated tumour killing via ADCP. The created anti-HER-2 antibody of the IgE subclass will serve as a tool for comparison studies in this 3-colour ADCC/ADCP assay. doi:10.1016/j.clim.2008.03.418
Su.68. CD95 and CD95L in Gastrointestinal Cancer Patients Peripheral Blood Inta Jaunalksne,1 Simona Donina,2 Ludmila Engele,2 Tatjana Romanova. 1 1Paula Stradina KUS, Riga, Latvia; 2RAKUS Latvian Oncology Center, Riga, Latvia Immunity is one of the key factors in oncological processes. Cell immune answer play crucial role in balance between cell proliferation and apoptosis.Immune cell apoptosis is one of the factors which leads to malignant process spread. With the aim to check CD95 , CD95L level in 37 histologically verified gastrointestinal cancer patients we also analyzed CD4+, CD38+, CD25+ cell level by laser flow cytofluorimeter (Becton Dickenson, USA) and TNFalfa, SIL-2R level in patients serum (Immulite 2000,USA) and sTNF alfa IR by ELISA method Biosource,Belgium).Patients were divided
Abstracts in two groups- with local (I+II stage) and with advanced ( III+ IV stage)disease. We observed that there is a difference in CD95 cell absolute count, it is increased in more advanced patiets group, but there is no difference in CD95L, CD38+, CD25+, TNFalfa, sTNFRI level in patients with local and more advanced process. sIL-2R level was increased in advanced gastrointestinal cancer patients serum, but CD4+ cell absolute count was decreased if patients had III+IV stage of disease. Therefore CD4 cells are more sensitive to FAS mediated apoptosis and increased sIL-2R level may lead to decreased immune answer in advanced stages of gastrointestinal cancer patients. doi:10.1016/j.clim.2008.03.419
Su.69. Immunotherapy of Adenocarcinoma Patients with Gc Protein-Derived Macrophage Activating Factor, GcMAF Nobuto Yamamoto,1 Masumi Ueda,1 Kazuya Hashinaka,1 Theodore Sery,1 Charles Benson. 2 1Socrates Institute for Therapeutic Immunology, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA Intratumor BCG administration eradicates local as well as metastasized tumors. Administration of BCG into noncancerous tissues results in no effect on the tumors. Inflammation induced by BCG in normal tissues releases lysophospholipids that activate macrophages. Because cancerous tissues contain alkylphospholipids, BCG-induced inflammation of cancerous tissues produces alkyl-lysophospholipids that activate macrophages approximately 400 times more effective than lysophospholipids, implying that highly activated macrophages are tumoricidal. Inflammation-primed macrophage activation is the principal macrophage activation process that requires serum vitamin Dbinding protein (known as Gc protein) which is the precursor for the macrophage activating factor (MAF). Stepwise treatment of purified Gc protein with immobilized βgalactosidase and sialidase generates the most potent MAF (GcMAF) that produces no side effect in humans. Intramuscular administration of 100 ng GcMAF activate systemic macrophages at maximal level with 30-fold increased ingestion index and 15-fold increased superoxide generating capacity in 3.5 hr. GcMAF also has a potent mitogenic activity on myeloid progenitor cells that generate systemically 40-fold increase in the activated macrophages in 4 days. When adenocarcinoma (metastatic breast and prostate cancer) patients (n = 32) were intramuscularly administered with 100 ng GcMAF/week, their tumors were eradicated in 16-25 weeks. These patients were tumor free for more than six years after GcMAF therapy. Since intravenous administration of GcMAF allows rapid interaction of GcMAF with myeloid progenitor cells in bone marrow, the systemic cell counts of the activated macrophages increased to 200-fold. Weekly intravenous administration of 100 ng GcMAF to adenocarcinoma patients eradicates tumors in 11-16 weeks. doi:10.1016/j.clim.2008.03.420