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MUC4: monocolanal antibodies for
recognition of cancer stem cell-specific
surface antigens
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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
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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.
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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
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