Download Abscopal effect seen with T-cell therapy enhancing oncolytic

Abscopal effect seen with T-cell therapy enhancing oncolytic adenoviruses
armed with TNFα and IL-2
Riikka Havunen1, Suvi Parviainen1,4, Mikko Siurala1,4, Michael Behr1, Dirk Nettelbeck2, Anja Ehrhardt3, Akseli Hemminki1,4
1Cancer
Gene Therapy Group, Medicum, Haartman Institute, University of Helsinki, Helsinki, Finland
2German Cancer Research Center (DKFZ), Heidelberg, Germany
3Institute for Virology and Microbiology, University Witten/Herdecke, Witten, Germany
4TILT Biotherapeutics Ltd, Helsinki, Finland
Background
 Interleukin (IL) -2 and tumor necrosis factor alpha (TNFa) are promising for enhancing antitumor effects of adenoviruses.
 Virotherapy can be further boosted with Tumor-Infiltrating Lymphocyte (TIL) therapy
 With armed viruses, effective and safe cytokine expression can be achieved locally instead of high systemic level administration.
Aims
 To study abscopal effect: Is localized treatment of a tumor with non-oncolytic adenoviruses carrying murine cytokines (Ad5-CMV-mTNFa/mIL2)
able to affect also distant tumor?
 To construct and characterize oncolytic adenoviruses coding for human IL-2 and TNFa (Ad5/3-E2F-d24-hTNFa-IRES-hIL2)
Results
Oncolytic adenoviruses with human IL-2
and TNFa kill tumor cells and produce
active cytokines in vitro.
a.
OVCAR3 - Ovarian cancer
120
120
100
100
Viability (%)
Viability (%)
A549 - Lung cancer
80
60
40
20
80
60
40
20
0
0
0
1
10
100
1000
0
1
10
VP/cell
120
120
100
100
Viability (%)
Viability (%)
Panc1 - Pancreatic cancer
80
60
40
20
80
60
40
20
0
0
0
1
10
100
1000
0
1
10
VP/cell
 The abscopal effect was seen: Treatment of a tumor with adenoviruses also reduced
the growth of untreated tumors.
 Treatment influences distant tumors by activating immune responses and not by
spreading the virus. Virus treatment recruits at least Natural Killer cells to both
tumors and reduces the presence of immunosuppressive M2 macrophages.
 Oncolytic adenoviruses with human cytokines are effective in vitro.
IL2 1:100
TNFa+IL2
TNFa 1:4000
Viability (%)
120
100
80
60
40
20
0
TNFa+IL2
mock
d.
IL-2 activity
Mock
Conclusions
1
0,8
0,6
0,4
0,2
0
c.
hIL2 10
ng/ml
ng/ml
IL-2 and TNFa
expression by the
infected cells
100
1000
VP/cell
Viability (%)
b.
1000
VP/cell
SK-MEL-28 - Melanoma
Figure 3: Mice were sacrificed on day 8. Tumor samples were analyzed with FACS to detect Natural killer cells, M2
macrophages, dentritic cells and CD8+ T cells with melanoma-specific markers. Changes in CD8+ cells were
undetectable. Means +/- SEM are shown. Ns=not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001
100
TNFa activity
120
100
80
60
40
20
0
TNFa+IL2
Virus injections recruit Natural Killer cells to both tumors and
treatment with cytokine-armed viruses reduces the presence of
immunosuppressive M2 macrophages.
Figure 2: Viral DNA was explored from both injected and non-injected
tumors with qPCR. Median amount of viral DNA normalized against
murine β-actin is shown.
hTNFa 10
ng/ml
Figure 1: Two B16-OVA murine melanoma tumors were implanted in C57BL/6 mice. One of the two tumors was
treated intratumorally with cytokine- or Luc1-bearing adenoviruses (10e9 VP/tumor) on days 1 and 4. OT-1 T cells
were administrated intraperitoneally (1.5x10e6 ) on day 1. Tumor growth was followed for 8 days; mock is set as
100%. Mean + SEM are shown. *p<0.05; ***p<0.001; ****p<0.0001
The virus does not spread to distant
tumors.
Mock
Viral treatment induces growth suppression not only at the
injection site but also at distant tumor.
Figure 4 : Cancer cell lines were infected with Ad5/3-E2F-d24hTNFa-IRES-hIL2 or controls. (a) Cell survival was measured after 3
to 8 days. (b) Expression of cytokines was measured with FACSbased bead array and activity (c and d) with indicator cell lines.
Means + StDev are shown. VP=viral particles