Post on 24-Jul-2020
Pseudocowpox virus (PCPV), a potent viral vector for both
antigen-dependent and independent cancer immunotherapy
Karola Rittner, Caroline Tosch, Christelle Remy-Ziller, Christine Thioudellet, Marie-Christine Claudepierre, Virginie Nourtier, Chantal Hoffmann, Doris Schmitt, Benoit Grellier, Laurence
Laruelle, Benoit Sansas, Johann Foloppe, Philippe Erbs, Nathalie Silvestre, Kaïdre Bendjama, Eric Quéméneur.
Transgene SA, Illkirch-Graffenstaden, France
CONCLUSIONS AND PERSPECTIVES
Our recent data confirm the potential of PCPV as a new vector for anti-tumor vaccination, in particular for its intrinsic ability to control tumor growth in a tumor antigen-independent manner. Heterologous prime
boost regimen with our individualized vaccine approach myVac or combination with oncolytic virotherapy can be envisaged. Detailed studies in the human immune cells from patients are ongoing to dissect effects
of PCPV on immunosuppressive and immunostimulating cell populations. Patent application filed.
SITC Washington November 7-11, 2018
TAA-INDEPENDENT EFFECTS IN MC38 TUMOR MODEL
MC38 cells (HPV16E7-)(2.106 cells), sc
Tumor control, survival
D1D9 virus, itu D16 virus, ituC57BL/6
D2 virus (1.107 pfu), itu
MVA PCPV buffer
Tumor size evolution MVA vs PCPV: p-value=0.003 at Day 15, and 0.004 at Day 18 PCPV vs MVA p-value=0.006
TAA-DEPENDENT RESPONSES IN NAÏVE AND TC1-BEARING MICE
IV, 1.105 pfu IV, 1.105 pfu Harvest lungs and spleens• MVA
• MVA-HPV16E7 • PCPV-HPV16E7 • buffer D8 D15
Naïve C57BL/6 mice
ELISPOT analysis in splenocytesIntracellular cytokine staining (ICS) in lung cells:
HPV16E7-specific T cells in CD3dimCD8dim population
CD3
CD
8
buffer
PCPV
MVA
BACKGROUND
MC38 tumors - MVA-HPVE7 m
Tu
mo
r vo
lum
e (
mm
3)
Day
5
Day
8
Day
11
Day
15
Day
18
Day
22
Day
25
Day
29
0
500
1000
1500
2000
m 1
m2
m3
m4
m5
m6
m7
m8
m9
m10
MC38 tumors - PCPV-HPVE7 m
Tu
mo
r vo
lum
e (
mm
3)
Day
5
Day
8
Day
11
Day
15
Day
18
Day
22
Day
25
Day
29
0
500
1000
1500
2000
m 1
m2
m3
m4
m5
m6
m7
m8
m9
m10
MC38 tumors - buffer
Tu
mo
r vo
lum
e (
mm
3)
Day
5
Day
8
Day
11
Day
15
Day
18
Day
22
Day
25
Day
29
0
500
1000
1500
2000
m 1
m2
m3
m4
m5
m6
m7
m8
m9
m10
PCPV injected into MC38 tumors reduced tumor size and increased survival
PCPV-HPV16E7 induced strong cellular response against HPV16E7 detectable in spleen and lung
AC
B
cell
nu
mb
er
MVA m
MVA H
PV16E7m
PCPV HPV16E7
buffer
0
200
400
600
CD3dimCD8dim R9F-specific IFN- secreting T cells R9F-specific IFN- secretion
sp
ots
/106 c
ells
MVA m
MVA H
PV16E7m
PCPV HPV16E7
buffer
0
100
200
300
400
R9F (HPV16E7)
K9i-3 (CTRL)
*
*
Viral vectors expressing tumor antigens and/or cytokines have proven to be clinically effective approaches to stimulate anti-tumor immunity and to change the tumor microenvironment. However, novel viral strains
with improved immunogenic properties would be beneficial to expand the scope of virotherapeutic approaches. We identified Pseudocowpox virus (PCPV) from a Poxviridae screening program. Compared to well
established poxvirus strains like MVA or oncolytic Vaccinia virus (VACV), PCPV induced the secretion of 1,000-fold more IFN-alpha in human PBMCs. PCPV treatment induced efficient activation of antigen-
presenting cells, and a less suppressive phenotype in in vitro derived M2-like macrophages and MDSCs [see AACR 2018, poster LB-287]. A recombinant PCPV encoding the tumor associated antigen (TAA)
HPV16E7 was generated to assess the anti-tumor activity in the syngeneic murine tumor models MC38 (HPV16E7-) and TC1 (HPV16E7+).
**
D1
Lung CD3dimCD8dim T cells
anti CD8 PCPV
Tu
mo
r vo
lum
e (
mm
3)
Day
2
Day
6
Day
8
Day
12
Day
14
Day
16
Day
20
Day
22
Day
27
Day
29
Day
34
0
1000
2000
3000
4000
5000m2
m 1
m3
m4
m5
m6
m7
m8
m9
m10
m12
m13
buffer
Tu
mo
r vo
lum
e (
mm
3)
Day
2
Day
6
Day
8
Day
12
Day
14
Day
16
Day
20
Day
22
Day
27
Day
29
Day
34
0
1000
2000
3000
4000
5000m2
m 1
m3
m4
m5
m6
m7
m8
m9
m10
m12
m13
Depletion of CD8+ T cells reverts
effects of PCPVELISPOT analysis on splenocytes
Stimulation
Sp
ots
/10
6 c
ell
s
med
ium
MC38
mito
C
i8L (i
rrel
evan
t)
0
200
400
600
PCPV survivor pool
PCPV survivor N°1
PCPV survivor N°2
Pool of naïves mice
PCPV treatment induced MC38-specific
responses in survivors (Day 34)
PARTICULARITIES OF PCPV VS MVA
IF N g am m a
0
50
100
150
200
IP -10 (C X C L 10)
pg
/ml
0
50
100
150
T N F -
0
20
40
60
80
100
IL -12p 70
pg
/ml
0
10
20
30
40
IL -1 b eta
0
20
40
60
R ANT E S (C C L 5)
0
500
1000
1500
IL -18
pg
/ml
MVA
PCPV
buffe
r400
600
800
1000
1200
M IP-1 b eta (C C L 4)
MVA
PCPV
buffe
r0
100
200
300
400
M C P-1 (C C L 2)
MVA
PCPV
buffe
r0
1000
2000
3000
4000
5000
% g
ate
d in
CD
45
+ T
ILs
MVA
PCPV
buffer
0
5
10
15
20
25
neutrophils
% g
ate
d in
C
D4
5+ T
ILs
MVA
PCPV
buffer
0
10
20
30
40
50
MHCIIlo TAMs
D9: intratumoral injection
D10: take tumor, dissociatecells and enrich CD45+ TILs, characterize cell subsets.
D1: sc injection of MC38 cells
Heterologous prime boost PCPV (itu) / MVA (iv) superior to homologous MVA (itu) / MVA (iv) vaccination
The day after virus injection, tumors were enzymatically dissociated and TILs wereenriched using CD45+ magnetic beads. Subpopulations were identified by flow cytometryaccording to Brauner et al. (AACR 2017 poster N°1672).
PCPV (itu) increased frequencies of neutrophils
and decreased those of MHCIIlo TAMs in MC38 tumors
MVA itu – MVA iv PCPV itu – MVA iv Buffer itu – buffer iv
4 mice/group were injected in both flanks applying 5.105 pfu/flank. After 16h, skin sampleswere taken and mechanically dissociated. Cleared supernatant was analyzed (Luminex).
PCPV increased local of Th1
Cytokines and Chemokines release
13 mice per group were injected with buffer of PCPV as indicated above. Left: mice were treated D-2, D-1, D6 and D13 with anti CD8 antibody (clone 53-6-7, 200 µg/injection, ip). N=1.Depletion of CD8+ T cells was confirmed in blood and spleen Day 14.
PCPV itu + anti CD8 antibody ip
Detection of MC38-specific T cells in splenocytes from pooled or individualsurvivors (D34), after stimulation with MC38 cells treated with Mitomycin C or irrelevant peptide IRL. Control: splenocytes from naïve mice.
A) Detection of HPV16E7-specific T cells in pooled splenocyes from 6 mice (ELISPOT), after stimulation with R9F peptide, specific for HPV16E7. B) Detection of T lymphocytes in dissociated lung tissue from 6 mice / group, according to Remy-Ziller et al, 2017. Appearance of CD3dimCD8dim population after repeated virus treatment. C) Detection of HPV16E7-specific IFN-γ secreting T cells within the CD3dimCD8dim population in the lung of iv treated mice.
ABOUT PCPV AND THE GENERATION OF ITS RECOMBINANTS
Pseudocowpox PCPV (Parapoxvirus)
o PCPV strain TJS (ATCC, VR-634) was isolated from a human case of “Milker’s nodules”.
o Patients with Milker’s nodules did not develop immunity to vaccinia et vice versa.
o Recombinant PCPV were generated by insertion of genes into the non-essential VEGF loci.
o PCPV has detectable but low oncolytic potential in human and murine cells (Ricordel et al., OncoTarget,
paper under revision). Lower oncolytic activity in vitro observed than for the prototype Parapoxvirus ORF.
o Less than 80% pairwise identity with ORFvirus.
From Friedman and Kien, 1963
MVA or PCPVitu, 1x106 pfu• MVA-HPV16E7
• PCPV-HPV16E7 • buffer
D14 D21C57BL/6 mice D1
MVAIV, 1x106 pfu
TC1 cells were sc injected into C57BL/6 mice (10 mice /group). After 14 days of tumor growth, mice were randomized and first intratumorally injected with MVA or PVPV encoding HPV16E7, and one week later intravenously with MVA HPV16E7. Tumor growth and survival were followed.
• MVA-HPV16E7 • PCPV-HPV16E7 • buffer
TC1 cells (HPV16E7+)(5.105 cells), sc
Tumor control, survival
buffer
TC1 tumors - MVA-HPVE7 m 2x (itu, iv)
Tu
mo
r vo
lum
e (
mm
3)
Day
8
Day
10
Day
13
Day
16
Day
18
Day
21
Day
23
Day
25
Day
28
Day
30
Day
32
Day
35
Day
37
Day
39
0
500
1000
1500
2000m2
m 1
m3
m4
m5
m6
m7
m8
m9
m10
TC1 tumors - PCPV-MVA-HPVE7 m (itu, iv)T
um
or
vo
lum
e (
mm
3)
Day
8
Day
10
Day
13
Day
16
Day
18
Day
21
Day
23
Day
25
Day
28
Day
30
Day
32
Day
35
Day
37
Day
39
0
500
1000
1500
2000m 1
m2
m3
m4
m5
m6
m7
m8
m9
m10
TC1 tumors buffer (itu, iv)
Tu
mo
r vo
lum
e (
mm
3)
Day
8
Day
10
Day
13
Day
16
Day
18
Day
21
Day
23
Day
25
Day
28
Day
30
Day
32
Day
35
Day
37
Day
39
0
500
1000
1500
2000m2
m 1
m3
m4
m5
m6
m7
m8
m9
m10