D3 m D8 m Karola Rittner, Caroline Tosch, Christelle Remy ...€¦ · Pseudocowpox virus (PCPV), a...
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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 - ) (2.10 6 cells), sc Tumor control, survival D1 D9 virus, itu D16 virus, itu C57BL/6 D2 virus (1.10 7 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, 1.10 5 pfu IV, 1.10 5 pfu Harvest lungs and spleens • MVA • MVA-HPV16E7 • PCPV-HPV16E7 • buffer D8 D15 Naïve C57BL/6 mice ELISPOT analysis in splenocytes Intracellular cytokine staining (ICS) in lung cells: HPV16E7-specific T cells in CD3dimCD8dim population CD3 CD8 buffer PCPV MVA BACKGROUND MC38 tumors - MVA-HPVE7 m Tumor volume (mm3) 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 Tumor volume (mm3) 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 Tumor volume (mm3) Day 5 Day 8 Day 11 Day 15 Day18 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 A C B cell number MVA m MVA HPV16E7 m PCPV HPV16E7 buffer 0 200 400 600 CD3 dim CD8 dim R9F-specific IFN- secreting T cells R9F-specific IFN- secretion spots/106 cells MVA m MVA HPV16E7 m 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 CD3 dim CD8 dim T cells anti CD8 PCPV Tumor volume (mm3) 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 5000 m2 m 1 m3 m4 m5 m6 m7 m8 m9 m10 m12 m13 buffer Tumor volume (mm3) 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 5000 m2 m 1 m3 m4 m5 m6 m7 m8 m9 m10 m12 m13 Depletion of CD8+ T cells reverts effects of PCPV ELISPOT analysis on splenocytes Stimulation Spots/10 6 cells medium MC38 mitoC i8L (irrelevant) 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 IFN gamma 0 50 100 150 200 IP-10 (CXCL10) pg/ml 0 50 100 150 TNF- 0 20 40 60 80 100 IL-12p70 pg/ml 0 10 20 30 40 IL-1 beta 0 20 40 60 RANTES (CCL5) 0 500 1000 1500 IL-18 pg/ml MVA PCPV buffer 400 600 800 1000 1200 MIP-1 beta (CCL4) MVA PCPV buffer 0 100 200 300 400 MCP-1(CCL2) MVA PCPV buffer 0 1000 2000 3000 4000 5000 % gated in CD45 + TILs MVA PCPV buffer 0 5 10 15 20 25 neutrophils % gated in CD45 + TILs MVA PCPV buffer 0 10 20 30 40 50 MHCII lo TAMs D9: intratumoral injection D10: take tumor, dissociate cells 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 were enriched using CD45+ magnetic beads. Subpopulations were identified by flow cytometry according to Brauner et al. (AACR 2017 poster N°1672). PCPV ( itu ) increased frequencies of neutrophils and decreased those of MHCII lo 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 5.10 5 pfu/flank. After 16h, skin samples were 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 individual survivors (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 CD3 dim CD8 dim 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 PCPV itu, 1x10 6 pfu • MVA-HPV16E7 • PCPV-HPV16E7 • buffer D14 D21 C57BL/6 mice D1 MVA IV, 1x10 6 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 + ) (5.10 5 cells), sc Tumor control, survival buffer TC1 tumors - MVA-HPVE7 m 2x (itu, iv) Tumor volume (mm3) 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 2000 m2 m 1 m3 m4 m5 m6 m7 m8 m9 m10 TC1 tumors - PCPV-MVA-HPVE7 m (itu, iv) Tumor volume (mm3) 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 2000 m 1 m2 m3 m4 m5 m6 m7 m8 m9 m10 TC1 tumors buffer (itu, iv) Tumor volume (mm3) 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 2000 m2 m 1 m3 m4 m5 m6 m7 m8 m9 m10
Transcript of D3 m D8 m Karola Rittner, Caroline Tosch, Christelle Remy ...€¦ · Pseudocowpox virus (PCPV), a...
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
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r vo
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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
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nu
mb
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MVA m
MVA H
PV16E7m
PCPV HPV16E7
buffer
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CD3dimCD8dim R9F-specific IFN- secreting T cells R9F-specific IFN- secretion
sp
ots
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ells
MVA m
MVA H
PV16E7m
PCPV HPV16E7
buffer
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R9F (HPV16E7)
K9i-3 (CTRL)
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*
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
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r vo
lum
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mm
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m13
Depletion of CD8+ T cells reverts
effects of PCPVELISPOT analysis on splenocytes
Stimulation
Sp
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/10
6 c
ell
s
med
ium
MC38
mito
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i8L (i
rrel
evan
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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
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IP -10 (C X C L 10)
pg
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pg
/ml
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IL -1 b eta
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R ANT E S (C C L 5)
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pg
/ml
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PCPV
buffe
r400
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M IP-1 b eta (C C L 4)
MVA
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M C P-1 (C C L 2)
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buffe
r0
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% g
ate
d in
CD
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+ T
ILs
MVA
PCPV
buffer
0
5
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neutrophils
% g
ate
d in
C
D4
5+ T
ILs
MVA
PCPV
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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)
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r vo
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m 1
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TC1 tumors - PCPV-MVA-HPVE7 m (itu, iv)T
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