Novel Monoclonal Antibodies Against the Proximal (Carboxy-Terminal) Portions of MUC16
ABSTRACT The CA125 antigen, recognized by the OC125 antibody, is a tissue-specific circulating antigen expressed in ovarian cancer. The CA125 antigen is encoded by the MUC16 gene cloned by Yin and Lloyd. The full-length gene describes a complex tethered mucin protein present primarily in a variety of gynecologic tissues, especially neoplasms. OC125 and other related antibodies react with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. These antibodies are not useful as screening tools, nor can they detect the proximal residual MUC16 protein fragment after cleavage. This has limited its diagnostic and therapeutic applications. Using synthetic peptides, we raised novel-specific antibodies to the carboxy-terminal portion of MUC16 retained by the cell proximal to the putative cleavage site. These antibodies were characterized using fluorescence-activated cell-sorting analysis, enzyme-linked immunoassay, Western blot analysis, and immunohistochemistry. Each of the selected monoclonal antibodies was reactive against recombinant GST-ΔMUC16 protein and the MUC16-transfected SKOV3 cell line. Three antibodies, 4H11, 9C9, and 4A5 antibodies showed high affinities by Western blot analysis and saturation-binding studies of transfected-SKOV3 cells and displayed antibody internalization. Immunohistochemical positivity with novel antibody 4H11 was similar to OC125 but with important differences, including diffuse positivity in lobular breast cancer and a small percentage of OC125-negative ovarian carcinomas that showed intense and diffuse 4H11. Development of such antibodies may be useful for the characterization of MUC16 biology and allow for future studies in targeted therapy and diagnostics.
- SourceAvailable from: Isabelle MatteOvarian Cancer - Basic Science Perspective, 02/2012; , ISBN: 978-953-307-812-0
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ABSTRACT: The tumour-associated antigen CA125 (mucin 16, MUC16) is commonly expressed in ovarian cancer, and can also be detected in other tumour of epithelial origin, but its physiological role is largely unknown. The aim of the present study was to investigate the impact of MUC16 gene silencing on the growth properties of ovarian and breast cancer cells. We analysed cellular effects linked to oncogenesis, such as proliferation, cell cycle and apoptosis, after transient and stable transfection with MUC16 short hairpin RNA (shRNA) in diverse epithelial cancer cell lines with different MUC16 expression. Furthermore, alterations in cell adhesion, migration and invasion were evaluated in stable MUC16 knockdown clones. The growth of all tested MUC16(+) tumour cells was significantly suppressed by induction of caspase-dependent apoptosis after transient transfection with MUC16 shRNA, irrespective of the initial MUC16 expression level and cancer origin. Growth inhibition could be confirmed in stable MUC16 knockdown clones, albeit caspase-dependent death pathways seemed no longer be activated. In MUC16(low+) ovarian cancer cells, stable MUC16 gene silencing resulted in a substantial blockade of colony formation, cell adhesion, migration and invasiveness associated with reduced activation of metalloproteinases-2 (MMP-2). By contrast, the tested MUC16(high+) cell lines displayed a non-motile and non-invasive phenotype which was not affected by MUC16 knockdown, probably due to the expression of different MUC16 isoforms with divergent functions in individual cell lines. Our results provide evidence for a central role of MUC16 in cancer cell survival pathways. Additionally, MUC16 might also be involved in adhesion, migration and invasion depending on the type of cancer cell.European journal of cancer (Oxford, England: 1990) 08/2011; 48(10):1558-69. DOI:10.1016/j.ejca.2011.07.004 · 4.82 Impact Factor
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ABSTRACT: Recurrent platinum-resistant ovarian cancer has no curative options, necessitating the development of novel treatments, including immunotherapy. Patient-derived T cells can be genetically modified to express chimeric antigen receptors (CARs) specific to tumor-associated antigens in an HLA-independent manner, with promising preclinical results. MUC16(ecto) is highly expressed on most epithelial ovarian carcinomas but at low levels on normal tissues, offering an excellent immunotherapeutic target for this cancer. CAR T cells further modified to secrete IL-12 show enhanced cytotoxicity, persistence, and modulation of the tumor microenvironment. We propose a dose escalation phase I clinical trial for patients with recurrent MUC-16(ecto+) ovarian cancer to test the safety of intravenous and intraperitoneal administration and the preliminary efficacy of autologous IL-12 secreting, MUC-16(ecto) CAR T cells containing a safety elimination gene. This trial targets MUC-16(ecto), a novel and promising tumor-associated antigen. This will be the first time CAR T cells are injected intraperitoneally directly into the site of the tumor within the abdomen in humans. Furthermore, the ability of genetically modified cells to secrete IL-12 will potentially enhance CAR T cell persistence and modulate the tumor microenvironment. For safety purposes, an elimination gene has been incorporated into the CAR T cells to mitigate any on-target, off-tumor or other unforeseen toxicity.Journal of Translational Medicine 03/2015; 13(1):102. DOI:10.1186/s12967-015-0460-x · 3.99 Impact Factor