arming the oncolytic virus enadenotucirev to...
TRANSCRIPT
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Arming the Oncolytic Virus Enadenotucirev to Develop Tumor-Localized Combination
Immunotherapeutics
Charles Q. Morris MBChB MRCP(UK)
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Start with a very large randomly created
library of chimeric
adenoviruses
Passage the viruses
repeatedly on human carcinoma
cells
Screencandidate viruses for
loss of activity in a variety of normal human
cell types
Enadenotucirev (EnAd):Developed using directed evolution
Select the most
potent,selective
and blood stable
tumor killing virus
EnAd
Select only the
most potenttumorkilling viruses
Selectonly the
most selective
and potenttumorkilling viruses
Screencandidate viruses on
human carcinomacells in the presence of
fresh human blood
EnAd = Ad11/Ad3 group B chimeric adenovirus
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The key components of EnAd
Ad 11 capsid:
Group B virus (vs C for Ad5)
CD46 & DSG2 receptors
No CAR receptor involvement
Low pre-existing immunity against Ad11 surface proteins in humans
EnAd = Ad11p with chimeric Ad3 E2B region and deletions in E3 and E4
Three deleted or mutated gene regions
E3 and E4 deletions and a chimeric Ad3/Ad11p E2B region
• E3 & E4: both regions normally involved in immune modulation by adenoviruses
• E2B: adenovirus replication in the nucleus of normal cells
Reduced genome size capacity for armingEnAd is human tumor-specific
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Clinical PoC: Tumor-selective expression of nuclear hexon staining after IV dosing
“Normal” TumorMargin
TumorNormal
Dark brown intra-nuclear staining of hexon demonstrates positive viral replication. Representative IV patient from the MoA study.4
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Clinical PoC: Virus uptake into tumors is associated with CD8+ T-cell infiltrates
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MoA CRC IV Cohort: 4/5 with high numbers of CD8+ cells amongst tumorcells (all CRC MSI low)
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Next Generation Viruses:Tumor-Specific Immuno-Gene therapy (T-SIGn)
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Armed Enadenotucirev to Deliver Immuno-Therapeutics to Local Tumor Sites of Action
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Difference between armed and parental
enadenotucirev
Armed virus particles are structurally the same as enadenotucirev
EnAd NG-EnAd
E1 E2 L1-3 E2A L4 E3 L5 E4
E1 E2 L1-3 E2A L4 E3 L5 E4
E1 E2 L1-3 E2A L4 E3 L5 E4TG
Ad11p
EnAd
ArmedEnAd
Transgene cassetteEncoded therapeutics expressed from virus major late promoter, products only made in cells supporting virus replication (i.e. tumor)
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Properties of exemplifier antibody-armed EnAd NG-135 (full IgG1 antibody)
Virus Replication in Carcinoma Cell Lines Anti-VEGF Antibody Expression
Antibody ‘armed’ viruses efficiently replicate and express antibody in lung, colon and ovarian carcinoma cells
Time (hrs)24 48 72 96 120
1
10
102
103
An
ti-V
EGF
Ab
Pro
du
ctio
n (
ng/
ml p
er 1
06
cells
)
HT29 DLD HCT SKOV A549
Ovary
Virus Input
103Gen
om
e co
pie
s p
er c
ell Colon
HT29 DLD HCT116 A549 SKOVLung
104
105
106
107
EnAd NG-135
Log[ppc]
-1 0 1 2 30
50
100 EnAd
NG-135
35kD25kD
40kD
55kD
70kD
100kD
15kD
10kD
Heavy Chain
Light Chain
Antibody expressionVirus Potency
% C
ell
Surv
ival
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NG-135: Uptake, replication and antibody expression in tumor cells in vitro
1ppc
Antibody Production (72 hrs)
10ppc
10ppc
1ppc
1ppc 10ppc 100ppc0.1
1
10
100
0.1
1
10
0.1
1
10
100
24h 48h 72h24h 48h 72h
106
Infectivity
105
104
103
103
106
105
104
103
103
Vir
us
gen
om
es/c
ell
Vir
us
gen
om
es/c
ell
An
ti-V
EGF
Ab
(n
g/m
l)
An
ti-V
EGF
Ab
(n
g/m
l)
Virus replication
Anti-VEGF antibody production
24h 48h 72h24h 48h 72h
ND ND ND
Virus replication is required for antibody production
HT-29 colon carcinoma cell line infected with NG-135 in vitro
An
ti-V
EGF
Ab
(n
g/m
l)
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Lack of infectious virus or antibody production in stromal cells (non-transformed fibroblasts)
Lack of detectable antibody < 0.33fg/cell/24hr
HT29 WI38 MRC5
Vir
al g
eno
me
cop
ies
per
cel
l
106
105
104
103
102
101
1ND ND
HT29 WI38 MRC5
103
102
101
1
An
ti-V
EGF
Ab
(n
g/m
l)
ND ND
106
105
104
103
102
101
1
108
107
HT29 WI38 MRC5
Infe
ctio
us
par
ticl
es (
TCID
50)
Replication Anti-VEGF Ab Infectious Virus
ND = not detected
Cell Type
Over 2000-fold less antibody made by non-transformed
cells than by cancer cells
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EnAd and NG-135 replication and transgene production in primary hepatocytes*
Genome replication and antibody expression: cells (3x105) infected with EnAd or NG-135 at 1ppc, cultured for 72hr
Virus genome levels measured by qPCR . Antibody expression measured by IgG1 ELISA
* Hepatocytes from peri-tumoral liver tissue
ND = Not Detectable
Ge
no
me
co
pie
s/ce
ll
10 0
10 1
10 2
10 3
10 4
10 5
ND ND
HT-29 Hepatocytes
NG-135
EnAd
[An
tib
od
y] n
g/m
l
0
5
10
15
20
ND ND ND
NG-135
EnAd
HT-29 Hepatocytes
Replication & antibody production not detectable in human hepatocytes
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Next Generation VirusesT-SIGn Examples
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Cytokine Armed Enadenotucirevcytokines and / or chemokines
Tumorcell
T cell
NextGenvirus
Anti-tumor immune response
Recruitment & activation signals
Cytokine X, Y, ZCytokine X, YCytokine X
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NG-345 encodes three different
cytokines/chemokines
IFNa MIP-1a Flt3L
TDC
NG-345
Three different human cytokines/chemokines produced
by A549 tumor cells infected with a single T-SIGn virus
Time post-infection (hrs)
500
1000
1500
2000
2500
100
200
300
24 48 72
MIP
-1a
(ng
/1
e6
ce
lls)
500
1000
IFNa
(ng
/1
e6
ce
lls)
Flt
3L (
ng
/1
e6
ce
lls)
24 48 7224 48 72
Time post-infection (hrs)Time post-infection (hrs)
IFNa MIP-1a Flt3L
Cytokine production by NG-345 infected A549 lung carcinoma cells
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Membrane-integrated T-cell Engagers (MiTe):T-cell Activating Ligands
Tumorcell
T cell
NextGenvirus
Anti-tumor immune response
Activation signals
MiTe 1 MiTe 2 MiTe 1 MiTe 2
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NG-348 PsiOxus’ lead T-SIGn product
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• NG-348 is a first in class immune-gene therapy product for the treatment of carcinomas.
• Forces membrane expression of T-cell activating ligands on tumor cells
• In situ activation of T-cells leading to antigen independent tumor cell killing
• Significant advantages over CAR-T and TCR technologies:• Off the shelf product: not personalized• Mass produced: no autologous manufacturing• Antigen independent: not dependent on CD19 or other antigens• Directed to solid tumors: not restricted to hematological malignancies
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NG-348: Lead T-SIGn Therapy
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NG-348 Selective expression of MiTe ligand
on tumor cell surface
10
60
80
None EnAd NG-348 None EnAd NG-348
% A
54
9 c
ells e
xp
ressin
g M
iTe
Tumor Cells(A549 lung carcinoma)
Non-Tumor Cells(MRC5 Fibroblasts)
Virus treatment:
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Only tumor cells infected with NG-348 express the MiTe
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NG-348 Mediates T-cell expansion in the context of tumor
cells
20 fold increase in T cell number post NG-348 stimulation20
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NG-348 Infected A549 tumor cells
polyclonally activate human CD8+ T-cells
Infection and expression of NG-348 payload on human tumor cells
leads to a direct and potent activation of CD8 cytotoxic effector T-cells
A549
only
+ EnAd + NG-348
5
10
15***
% C
D8
T-c
ells e
xp
ressin
g C
D1
07
a
IFNγ Secretion
A549
only
+ EnAd + NG-3480
300
400
500
IFNg
(ng
/m
L)
Treatment
200
100
Treatment
Cytotoxic T-cell
degranulation
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NG-348Selectivity of T cell activation: CD8 degranulation
CD107a
CD107a
Uninfected EnAd NG-348
MRC-5
A549
No T-cell activation (CD8 effector function) induced by NG-
348 treated non-transformed cells (MRC5 fibroblasts)
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Antibody Armed EnadenotucirevAntibodies, Antibody Fragments, or BiTes
Tumorcell
T cell
NextGenvirus
Anti-tumor immune response
Specific Ab binding and activity
Full length antibody A Antibody (Ab) fragments A, B, C BiTe
Target A’Target A’ Target B’ Target C’
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Bi-specific T-cell Engagers (BiTEs)Targeting Cancer Associated Fibroblasts
Fibroblast-targeted T-cell activation
CancerAssociatedFibroblast
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0 20 40 60 80 1000
10
20
30
40
50
60
70
***
***
***
Time (h)
% C
D2
5+
0 20 40 60 80 1000
10
20
30
40
50
60
70
EnAd-ControlBiTE
EnAd-FAPBiTE
EnAd
Uninfected******
******
% C
D69
+
Bi-specific T-cell Engagers (BiTEs)Infection with EnAd-FAP-BiTE induce T-cell activation
Activation
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EnAd-FAP-BiTE induces rapid killing of stromal fibroblasts
Bi-specific T-cell Engagers (BiTEs)Infection with EnAd-FAP-BiTE kills stromal fibroblasts
20 40 60 80
-0.5
0.0
0.5
1.0
1.5
2.0 Uninfected
EnAd
EnAd-ControlBiTE
EnAd-FAPBiTE
Time (h)
Cell in
dex
Virus infection
Activation
Kill
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Bi-specific T-cell Engagers (BiTEs)Ovarian malignant ascites as an ex-vivo human model
Rich content of tumour-associated lymphocytes, fibroblasts, tumour cells, macrophage
Immunosuppressive T-cells are:• PD1-positive• Attenuated activation markers + proliferation of T-cells• IL-2 non-producers
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Untr
eate
d
Contr
ol BiT
E
FAP B
iTE
EnA
d
EnA
d-Contr
olBiT
E
EnA
d-FAPBiT
E
0
20
40
60
80
100
***
***
CD
25-p
osit
ive (
%)
Untr
eate
d
Contr
ol BiT
E
FAP B
iTE
EnA
d
EnA
d-Contr
olBiT
E
EnA
d-FAPBiT
E
0
10000
20000
30000
40000
FA
P-p
osit
ive c
ells
***
***
Untr
eate
d
Contr
ol BiT
E
FAP B
iTE
EnA
d
EnA
d-Contr
olBiT
E
EnA
d-FAPBiT
E
0
50000
100000
150000
200000
250000
300000
Lym
ph
ocyte
s (
tota
l)
***
***
Bi-specific T-cell Engagers (BiTEs)Ovarian Malignant Ascites “treated” with EnAd-FAP-BiTE
EnAd-FAP-BiTE treatmentof unseparated ovariancancer ascites cells leads toactivation of T-cells anddepletion of FAP+ cells
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Combination Therapies Combinations to Address Multiple Pathways
MiTe 1 MiTe 1 MiTe 1MiTe 2
Cytokine Z
Tumorcell
T cell
NextGenvirus
Anti-tumor immune response
Combined recruitment & activation signals
Cytokine X, YAb fragment A
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PipelineR&D programs and progress
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Summary
1. EnAd is a potent & selective oncolytic virus, broadly active against epithelial cancer cells
2. Can be delivered to patients IV (clinically demonstrated) and can be efficiently armed with multiple therapeutic genes without disrupting oncolytic and tumor-selectivity properties of parental EnAd virus
• Viruses encoding of variety of transgenes (e.g. antibodies, cytokines, tumorantigens) have been made and characterized
3. Excellent platform for tumor-specific immunogene therapy (T-SIGn)
• Delivery and local production of immunotherapeutic combinations locally and selectively within tumours
• Exemplified with multiple viruses expressing different classes and combinations of transgenes
4. NG-348, lead candidate selective expression of ligands on tumorcells to drive localized activation of T-cells
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Acknowledgements
All PsiOxus, particularly:
Alice BrownSam IllingworthMatt BesneuxNalini MarinoDarren Plumb
Prithvi KodialbailRochelle Lear
Hugo CalderonKerry Fisher
Brian ChampionJohn Beadle
Oxford Univeristy
Josh FreedmanLen SeymourKerry Fisher