1

Arming the Oncolytic Virus Enadenotucirev to Develop Tumor-Localized Combination

Immunotherapeutics

Charles Q. Morris MBChB MRCP(UK)

2

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

3

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

5

Clinical PoC: Virus uptake into tumors is associated with CD8+ T-cell infiltrates

5

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

7

8

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