MUC4: monocolanal antibodies for recognition of stem cell-specific surface antigens the science of innovation

MUC4 Is A Known Surface Antigen In A Variety Of Cancer Cells And Tumors

Figure 1: MUC4 colocalizes with surface antigens Figure 2: MUC4 colocalizes in pancreatic cancer cells with known surface antigens in sectioned tissue samples MUC4 HER2 Merge MUC4

A

HER2

B Merge

Executive Summary Figure 1: Colocalization of MUC4 and HER2, a surface antigen, in pancreatic cancer cells. A, CD18/HPAF 4 (MUC4) is a high molecular weight, membrane-bound, and highly glycosylated (A) and CaPan1 (B) cells were grown at low density on sterilized coverslips, washed, and fixed with methanol, and that is overexpressed in a variety of . Dr. Surinder Batra at the University of Nebraska permeabilized before incubation with antibodies against Medical Center has found MUC4 expression to be surface localized in both ovarian and pancreatic MUC4 and HER2. Fluorescence signals from each scan cancers and cancer stem cells derived from ovarian cancer stem cells. Furthermore, Dr. Surinder were acquired sequentially. MUC4 (green) was detected Batra has developed and continues to engineer antibodies to MUC4. UNeMed is seeking com- by using a fluorescein-conjugated secondary antibody; HER2 was identified with CY3-conjugated secondary panies to partner to develop and license new technologies exploiting MUC4 as a biomarker and antibody (red). Dual-merge of MUC4/HER2 (green and immunological target for cancer and cancer stem cell therapies. red) showed colocalization (white arrows).

Figure 2: Immunohistofluorescence analysis was carried out on formalin-fixed and paraffin-embedded n multiple pancreatic cancer cell lines, MUC4 is localized to both the cytoplasm and the cell pancreatic tumor tissue sections after rehydration and Imembrane (Fig 1). MUC4 is surface localized in sections of pancreatic cancer tumors (Fig 2). antigen-retrieval utilizing mouse monoclonal anti-MUC4 When MUC4 is over expressed, 90 percent of ovarian cancer cells showed localization of MUC4 antibody. H&E staining was also performed on the serial in both cytoplasm and membranes. Vector-transfected cells lacked localized MUC4 expression sections to study the cellular morphology. Digital merging of the immunostained tissue section clearly showed the colocalization of MUC4

MUC4 In Cancer Stem Cells | Dr. Surinder Batra 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 Figure 4: MUC4-expressing surface antigen in cancer stem cells, in isolated colonies isolated colonies are like cancer stem cells MUC4 CD133 MUC4 / CD133 / DAPI A B

MUC4

HER2

ALDH1

ancer stem cells express numerous universal markers like: CD133, CD44, CD24, ESA and ALDH1. CD133 CCells 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 self- renewal 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). Figure 3 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 Confocal analysis (methods comparable to Figure 3) showed significant expression of CD133 (Red) in iso- cells. In addition, the Shh self-renewal protein expression was observed only in isolated colonies, lated colonies compared to SKOV3-MUC4 cells. MUC4 while there was no expression in SKOV3-MUC4 cells. Increased Shh expression suggests that the (Green) expression was seen in both isolated colonies isolated colonies from MUC4 overexpressed cells behave like cancer stem cells which are capable and SKOV3 MUC4 cells. of maintaining self-renewal (Fig 4). Figure 4 DR. Batra has shown that MUC4 is expressed on the membranes of mature cancer cells and is also Western blot analysis showed MUC4, HER2, ALDH1, highly expressed in phenotypic cancer stem cells. MUC4 is a compelling target antigen present on CD133 and Shh expression in SKOV3-MUC4 and isolated cancer stem cells. colonies from MUC4 overexpressed SKOV3 cells. b-actin served as a loading control.

MUC4 In Cancer Stem Cells | Dr. Surinder Batra 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 Figure 6: K2G6 is a promising, surface-specific MUC4 antibody A B

MUC4

A = Pane 1 B = CD18/HPAF

long with Dr. Batra’s work into MUC4 biology, he engineered a monocolonal antibody, 8G7, Figure 5 Athat is strongly reactive against the MUC4 peptide and native MUC4 from human tissues or Protein lysates from the MUC4 nonexpressing Panc 1 pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis (Fig 5). cells and the MUC4-expressing CD18/HPAF cells were immunoprecipitated using 4µg/ml of 8G7 MUC4-specific Dr. Batra continued to innovate and identify new MUC4 antibodies. Where 8G7 targeted the variable 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). Figure 6 Cells were harvested non-enzymatically, fixed with para- formaldehyde and incubated with the indicated antibodies. Following incubation with secondary antibody, cells were UNeMed Corporation, the technology transfer partner for the University of Nebraska Medical analyzed using BD FACSCalibur. The mean fluorescence Center, is seeking a commercialization partner to build a therapeutic platform around biological intensities (MFI) values obtained with each antibody is targeting of MUC4. In order to develop new oncology products for therapeutics, drug delivery 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