Download Oncolytic Activity of Coxsackievirus A21 (CAVATAKTM) in Human

Oncolytic Activity of Coxsackievirus A21 (CAVATAKTM) in Human Lung Cancer : A
Novel Targeted Anti-Cancer Strategy
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examined by flow cytometry for the
expression of ICAM-1 (red histogram)
and DAF (blue histograms) with a control
comparison.
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One step viral growth curve of CAVATAKTM in lung cells:
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Figure 2B. Quantitation of ICAM-1
and DAF levels on the surface of
lung cells. Quantitative analysis were
performed to obtain the approximate
number of antibodies specific for
ICAM-1 and DAF expression bound per
cell.
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Figure 4. Dose response curve of CAVATAKTM on a panel of lung cell lines.
Quantification of CAVATAKTM-mediated cell lysis revealed that significant cell death was induced
in four out of five lung cancer cell lines, in a dose-dependant manner. H157 appeared to be the most
sensitive cell line following CAVATAKTM treatment, with no viable cells at concentrations greater
than . Normal lung cells BEAS-2B and MRC5 exhibited cytopathic effects following CAVATAKTM
treatment as well. Only normal lung cells VAI3 appeared to be insensitive towards CAVATAKTM
infections. SK-Mel-28 melanoma cells were included as it was observed to be the most sensitive
cell line towards CAVATAKTM infections based on previous studies.
Intratumoural injection of a single dose of CAVATAKTM in subcutaneous
xenografts of the NSCLC line H157 in SCID Balb/C mice resulted in rapid
cancer cell destruction and subsequent reductions in tumour burden.
Viral-mediated oncolysis of the H157 xenografts was accompanied by the
release of progeny virus as observed by increases in the systemic load of
infectious CAVATAKTM
Coxsackievirus A21 displays significant pre-clinical oncolytic activity against
human lung cancer cells both within in vitro and in vivo environments. The
presented pre-clinical findings establish proof of concept for the potential
application of oncolytic virotherapy with CVA21 as a novel targeted anti-lung
cancer therapeutic withini the clinical environment.
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enteroviruses. Fields Virology, ed. D.M. Knipe, et al. 2001, Philadelphia, PA: Lippincott
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Chemother, 1999. 43(9): p. 2109-15.
3.  Xiao, C., et al., The crystal structure of coxsackievirus A21 and its interaction with ICAM-1.
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4.  Mullen, J.T. and K.K. Tanabe, Viral oncolysis. Oncologist, 2002. 7(2): p. 106-19.
5.  Brooks, K.J., E.J. Coleman, and E.S. Vitetta, The antitumor activity of an anti-CD54
antibody in SCID mice xenografted with human breast, prostate, non-small cell lung, and
pancreatic tumor cell lines. Int J Cancer, 2008. 123(10): p. 2438-45.
6.  Shafren, D.R., et al., Systemic therapy of malignant human melanoma tumors by a common
cold-producing enterovirus, coxsackievirus a21. Clin Cancer Res, 2004. 10(1 Pt 1): p. 53-60.
7.  Skelding, K.A., R.D. Barry, and D.R. Shafren, Systemic targeting of metastatic human
breast tumor xenografts by Coxsackievirus A21. Breast Cancer Res Treat, 2009. 113(1): p.
21-30.
8.  Berry, L.J., et al., Potent oncolytic activity of human enteroviruses against human prostate
cancer. Prostate, 2008. 68(6): p. 577-87.
Acknowledgement
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High ICAM-1 expressing NSCLC and SCLC cells displayed significant
susceptibility to rapid multi-cycle replication and cell cytolysis following in
vitro challenge with CAVATAKTM. NSCLC H157 lung cells were found to be
the most sensitive lung cell line.
References
Moderate to high levels of cell surface ICAM-1 expression were observed
following flow cytometric analysis of a panel of both human NSCLC and
SCLC in vitro cultures.
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Figure 2A. Expression of surface
cellular receptors on human lung
cell lines utilized for CAVATAKTM
infections. A panel of lung cells were
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Immunohistochemical staining showed low or no detection of ICAM-1
expression in biopsies of normal lung tissues, while significant levels of
ICAM-1 were detected in numerous lung cancer biopsies from varying stages of
disease.
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and H157 tumours in SCID Balb/C mice
#6 CAVATAKTM oncolytic virotherapy of A549, H1395
#4 Oncolytic activity of CAVATAKTM on a panel of
human lung cells
Virus yields of CAVATAKTM were examined in a range of lung cell types inoculated with
CAVATAKTM at a MOI of 10 TCID50/cell for a period of 48 hours. Replication kinetics of
CAVATAKTM from each lung cancer type showed that the maximal replication rate peaks at
approximately 6 hours post-infection. Lung cancer cells were observed to generate a higher viral
replication yield compared to the normal lung cells, with H157 expressing the highest viral yield.
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panel E-H show the lung cells 24 hours after CAVATAKTM infection. Cellular images from four
different lung cell monolayers (BEAS-2B; A549; H1395; H157) are as seen. Presence of rounded/
shrivelled dead cells were observed in the CAVATAKTM infected cells and not in the uninfected
cells, indicating the existence of virus induced cytopathic effect.
H157
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#2 ICAM-1 surface expression on a panel of lung cancer cells
H1395
A549
Figure 5. Viral growth curves of CAVATAKTM in normal lung and lung cancer cells.
tissues and lung cancer biopsies. Representative data from each lung cancer types are shown. The control
stained tissue (in the top panel) was included for comparison. Lung cancer samples were stained strongly
for ICAM-1 as shown by the dark brown staining, whereas in normal lung tissues, weaker or no ICAM-1
staining was seen.
BEAS-2B
Figure 3. Microscopic examination of cell morphologies in uninfected lung cells and
CAVATAKTM infected lung cells. Panel A-D show the uninfected lung cell controls whereas
Figure 1A and B. Immunohistochemical staining (IHC) of ICAM-1 on normal lung tissues
(A) and lung cancer biopsies (B). ICAM-1 expression was investigated in a range of normal lung
X 40
X 100
Analysis of virus induced cytopathic effect (CPE):
All animal work was performed under the approval of our institutional Animal Care and Ethics Committee.
Female SCID (severe combined immunodeficient) mice (n=28) were subcutaneously injected with either
A549, H1395 and H157 cells (0.1mL containing 2x106 cells) in the hind flank. Two weeks post-injection,
mice were treated weekly with a single intratumoural (i.t) injection of either 0.1mL saline (n=4 per group) or
0.1mL of CAVATAKTM (1x108 TCID50) (n= 4 per group) for 3 weeks. Monitoring of tumour development
was performed weekly using electronic calipers. Viremia levels were measured from mouse serum samples
collected from the saphenous vein at weekly intervals. The levels of virus in the serum was assessed using a
standard virological endpoint dilution assay.
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1. Squamous cell carcinoma Stage II
2. Small cell carcinoma Stage II
3. Atypical carcinoid Stage III
4. Combined small cell carcinoma Stage III
5. Adenocarcinoma Stage III
6. Large cell carcinoma Stage IV
7. Adenosquamous carcinoma Stage IV
8. Papillary adenocarcinoma Stage IV
Pre-clinical testing of CAVATAKTM in A549, H1395 and H157 xenograft mouse
models:
24 hours
postCAVATAKTM
infection
Receptor levels of ICAM-1 and DAF (Decay Accelerating Factor) were analysed using quantitative flow
cytometry with QuantiBRITETM PE beads (BD #340495) as a calibrator to establish the number of
antibodies bound per cell. Lung cell lines and SK-Mel-28 cells were harvested and stained with PEconjugated antibodies against ICAM-1 (ab18222) and DAF (ab25540)[Abcam, Sapphire Bioscience].
To investigate the capacity of CAVATAKTM to replicate within lung cells, viral growth curves were
established by inoculating confluent lung cell monolayers in 24-well plates with virus at a multiplicity of
infection (MOI) of 10 TCID50/cell in 2% FCS media. Following incubation at 37°C for 1 hour, cell
monolayers were washed three times with PBS. The infection was interrupted at time intervals of 0, 6, 24
and 48 hours, the virus supernatants collected and subjected to three freeze-thaw cycles. The concentration
of virus or the yield of virus in these samples was determine by performing an endpoint titration on SKMel-28 cells.
Flow cytometry evaluation of ICAM-1 & DAF receptor levels:
Incubation of lung cells with serially diluted CAVATAKTM inocula for 72 hours at 37°C with 5% CO2 was
performed and cell viability examined using a XTT cell proliferation assay. At 24 hours post-infection with
CVA21 or media alone, photomicrographs of cell morphologies (BEAS-2B; A549; H1395; H157) were
taken using the Olympus IX70 microscope with DP-90 digital camera (at X40 magnification)
Uninfected
controls
ICAM-1 expression was examined on a microscopic array of formalin-fixed lung tissues containing both
normal specimens and cancerous tissue. The primary monoclonal antibody ICAM-1 G5 (Santa Cruz) was
used at a ratio of 1:1250. A Vectastain peroxidase Mouse IgG ABC Kit (Vector laboratories) was used for the
immunohistochemical staining, according to manufacturer’s protocol.
in normal lung and lung cancer cells
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Tissue arrays and immunohistochemical analysis of ICAM-1 levels:
CAVATAKTM infection
Viralytics Ltd
levels in biopsy samples from lung cancer patients compared
to normal lung tissues
Virus: Coxsackievirus A21 (CVA21) Kuykendall strain (product name: CAVATAKTM) was supplied by
#5 Evaluation of viral replication yield of CAVATAKTM
Normal lung cells MRC5 were obtained from the American Type Culture Collection (ATCC); BEAS-2B
were obtained from Dr Nicole Hansbro (University of Newcastle, Australia); VAI3 were obtained from Dr
Linda Berry (Peter MacCallum Cancer Centre, Australia).
Adherent lung cancer cells H157, H1395, A549 and suspension lines H82 and LX22cl were obtained from
Prof Neil Watkins (Monash University, Australia). SK-Mel-28 melanoma cells were obtained from Dr S.
Ralph (Monash University, Australia) and were used in the virological assays due to their high sensitivity to
CVA21 infection.
Normal lung cells and SK-Mel-28 cells were maintained in DMEM containing 10% FCS, while lung cancer
cells were maintained in RPMI with 10% FCS . All cell lines were incubated at 37°C with 5% CO2.
#3 Cell morphology of a panel of lung cells post-
Methods
Cells:
Conclusion
Results
#1 Immunohistochemical staining of ICAM-1 receptor
Oncolytic virotherapy is a novel targeted therapeutic approach that utilises direct
tumour lysis and generation of secondary anti-tumour immune responses.
Coxsackievirus A21 (CVA21) is a naturally occuring “common cold” virus that
within pre-clinical studies displays both in vitro and in vivo oncolytic activity across
a wide spectrum of cancers1,2. Natural infection by CVA21 is usually self-limiting
within the upper respiratory tract1,2. CVA21 targets human cancerous cells by
binding to the N-terminal domain of surface expressed human intercellular adhesion
molecule-1 (ICAM-1)3. Subsequent infection, viral replication and rapid cytolysis of
the targeted cell results in systemic release of progeny virus4. ICAM-1 is highly
expressed on the surface of numerous human cancers including lung, pancreatic,
breast, prostate, head/neck cancer and melanoma.
ICAM-1 expression levels often correlate with the degree of metastatic spread of the
malignancy5. CAVATAKTM is the name of a proprietary formulation of
Coxsackievirus A21 and it is currently being evaluated in Phase I trials in patients
with late stage melanoma6, prostate cancer7, breast cancer8 and recurrent Head/Neck
cancer.
Figure 6A and B. Tumour volumes (mm3) following treatment with either saline or
CAVATAKTM injections (i.t) (A) and corresponding viremia levels analyzed from the
mouse serum samples (B). Mice were treated each week with a single intratumoural (i.t)
injection of either 0.1mL saline (n=4 per gourp) or 0.1mL CAVATAKTM (1x108 TCID50) (n=4 per
group) for 3 weeks. At 43 and 47 days post tumour inoculation, a significant difference in H157
tumour volumes was observed between i.t CAVATAKTM injections and i.t saline injections (p<0.05
and p<0.001respectively) using the two-way analysis of variance (ANOVA). However, no significant
tumour remission was seen in the H1395 and A549 tumours. Corresponding serum samples from
CAVATAKTM treated mice were analyzed from dilutions of 1:102 to 108 in triplicates to determine
the levels of CAVATAKTM RNA present. Increasing viremia levels observed in all three lung cell
lines indicate successful CAVATAKTM infection and replication in the cells.
We would like to thank Professor Neil Watkins and colleagues Anette Szczepny and
Luciano Martelotto from Monash University for their kind assistance with providing
the lung tumour cell lines. We would also like to thank Dr Nicole Hansbro and Dr
Linda Berry for their contribution in providing us with normal lung cell lines.