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MUC4: monocolanal antibodies for recognition of cancer stem cell-specific surface antigens the science of innovation MUC4 Is A Known Surface Antigen In A Variety Of Cancer Cells And Tumors Figure 2: MUC4 colocalizes with known surface antigens in sectioned tissue samples Figure 1: MUC4 colocalizes with surface antigens in pancreatic cancer cells MUC4 HER2 Merge MUC4 A HER2 B Merge Executive Summary Mucin 4 (MUC4) is a high molecular weight, membrane-bound, and highly glycosylated protein that is overexpressed in a variety of cancers. Dr. Surinder Batra at the University of Nebraska Medical Center has found MUC4 expression to be surface localized in both ovarian and pancreatic cancers and cancer stem cells derived from ovarian cancer stem cells. Furthermore, Dr. Surinder Batra has developed and continues to engineer antibodies to MUC4. UNeMed is seeking companies to partner to develop and license new technologies exploiting MUC4 as a biomarker and immunological target for cancer and cancer stem cell therapies. I Figure 1: Colocalization of MUC4 and HER2, a surface antigen, in pancreatic cancer cells. A, CD18/HPAF (A) and CaPan1 (B) cells were grown at low density on sterilized coverslips, washed, and fixed with methanol, and permeabilized before incubation with antibodies against MUC4 and HER2. Fluorescence signals from each scan were acquired sequentially. MUC4 (green) was detected by using a fluorescein-conjugated secondary antibody; HER2 was identified with CY3-conjugated secondary antibody (red). Dual-merge of MUC4/HER2 (green and red) showed colocalization (white arrows). Figure 2: n multiple pancreatic cancer cell lines, MUC4 is localized to both the cytoplasm and the cell membrane (Fig 1). MUC4 is surface localized in sections of pancreatic cancer tumors (Fig 2). When MUC4 is over expressed, 90 percent of ovarian cancer cells showed localization of MUC4 in both cytoplasm and membranes. Vector-transfected cells lacked localized MUC4 expression MUC4 In Cancer Stem Cells | Dr. Surinder Batra Immunohistofluorescence analysis was carried out on formalin-fixed and paraffin-embedded pancreatic tumor tissue sections after rehydration and antigen-retrieval utilizing mouse monoclonal anti-MUC4 antibody. H&E staining was also performed on the serial sections to study the cellular morphology. Digital merging of the immunostained tissue section clearly showed the colocalization of MUC4 UNeMed Corporation 402-559-2171 the science of innovation MUC4 Is A Surface Expressed Antigen And Biomarker For Cancer Stem Cells Figure 3. MUC4 colocalizes with CD133, a known surface antigen in cancer stem cells, in isolated colonies MUC4 CD133 MUC4 / CD133 / DAPI Figure 4: MUC4-expressing isolated colonies are like cancer stem cells A B MUC4 HER2 ALDH1 C CD133 ancer stem cells express numerous universal markers like: CD133, CD44, CD24, ESA and ALDH1. Cells from circular colonies from MUC4-transfected SKOV3 cells were isolated and grown on cover slips for cancer stem cell marker analysis by confocal microscopy. The confocal results showed SHH immunofluorescence staining of CD133 marker expression (Red) Both the isolated circular colonies from MUC4 overexpressed SKOV3 cells and SKOV3-MUC4 cells ß- actin were analyzed for MUC4, HER2, ALDH1 and CD133 for the cancer stem cells and Shh for the selfrenewal pathway. MUC4 expression was observed at an almost equal level in both SKOV3-MUC4 and A = SKOV3-MUC4 B = SKOV3-MUC4-Colonies isolated colonies although there was a minor molecular weight change in isolated colonies. Interestingly, increased expression of HER2 was seen in isolated colonies compared to SKOV3-MUC4 cells (Fig 4). Expression of CD133 was also shown in both SKOV3-MUC4 and isolated colonies, whereas ALDH1 showed an increased expression in isolated colonies compared to MUC4 overexpressed SKOV3 cells. In addition, the Shh self-renewal protein expression was observed only in isolated colonies, while there was no expression in SKOV3-MUC4 cells. Increased Shh expression suggests that the isolated colonies from MUC4 overexpressed cells behave like cancer stem cells which are capable Figure 3 Confocal analysis (methods comparable to Figure 3) showed significant expression of CD133 (Red) in isolated colonies compared to SKOV3-MUC4 cells. MUC4 (Green) expression was seen in both isolated colonies and SKOV3 MUC4 cells. of maintaining self-renewal (Fig 4). DR. Batra has shown that MUC4 is expressed on the membranes of mature cancer cells and is also highly expressed in phenotypic cancer stem cells. MUC4 is a compelling target antigen present on cancer stem cells. MUC4 In Cancer Stem Cells | Dr. Surinder Batra Figure 4 Western blot analysis showed MUC4, HER2, ALDH1, CD133 and Shh expression in SKOV3-MUC4 and isolated colonies from MUC4 overexpressed SKOV3 cells. b-actin served as a loading control. UNeMed Corporation 402-559-2171 the science of innovation Dr. Batra Has Unique Expertise In The Development Of MUC4 Antibodies Figure 5: 8G7 is highly specific to MUC4 A Figure 6: K2G6 is a promising, surface-specific MUC4 antibody B MUC4 A = Pane 1 B = CD18/HPAF A long with Dr. Batra’s work into MUC4 biology, he engineered a monocolonal antibody, 8G7, that is strongly reactive against the MUC4 peptide and native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis (Fig 5). Dr. Batra continued to innovate and identify new MUC4 antibodies. Where 8G7 targeted the variable Figure 5 Protein lysates from the MUC4 nonexpressing Panc 1 cells and the MUC4-expressing CD18/HPAF cells were immunoprecipitated using 4µg/ml of 8G7 MUC4-specific MAb and were analyzed by Western blotting. tandem repeat region of MUC4, Dr. Batra identified several candidate monoclonal antibodies that target the less variable non-tandem repeat of MUC4. One candidate, K2G6, is highly effective at cell surface binding (Fig 6). UNeMed Corporation, the technology transfer partner for the University of Nebraska Medical Center, is seeking a commercialization partner to build a therapeutic platform around biological targeting of MUC4. In order to develop new oncology products for therapeutics, drug delivery Figure 6 Cells were harvested non-enzymatically, fixed with paraformaldehyde and incubated with the indicated antibodies. Following incubation with secondary antibody, cells were analyzed using BD FACSCalibur. The mean fluorescence intensities (MFI) values obtained with each antibody is indicated in parantheses. and diagnostics, UNeMed offers the expertise of Dr. Batra, the world expert in MUC4 biology, the antibodies he is engineering and his extensive network of academic collaborators UNeMed seeks to build on its existing intellectual property portfolio of therapeutic targets, novel therapeutic methods and antibodies. For the appropriate partner, UNeMed Corporation is offering the opportunity to develop novel oncology products with the world expert on MUC4. MUC4 In Cancer Stem Cells | Dr. Surinder Batra UNeMed Corporation 402-559-2171