ONCOLYTIC VIRUSES BIOTHERAPEUTICS AGAINST CANCER.

ONCOLYTIC VIRUSES

BIOTHERAPEUTICS AGAINST CANCER

73,000 CANADIANS WILL DIE THIS YEAR

GENES CONTROL ALL ASPECTS OF GROWTH

Hours Weeks Decades

Normal Cell Growth and Death

Cell Divides

Cell Death (controlled by “cell death” genes)

Tumour Cell Growth

Cell Divides

Cell Death Genes Mutated

Cigarette smoke, UV, pollutants, bad luck

And Divides

And Divides

And Divides…….

Immortalized cells form tumour

CHEMOTHERAPY

CANCER TARGETED THERAPIES

u

Genetic Material

Virus

Cell

VIRUS MULTIPLIES

Our bodies are made of billions and billions of cells

Viruses are parasites and can only reproduce inside of cells

Cellular Antiviral Programs… neighbourhood blockwatch!

• Interferon (IFN) is secreted by infected cells• Alerts neighbouring cells to presence of virus• Leads to death of infected cells, prevents growth

of surrounding cells and raises their defenses against infection

IFN

IFN

IFN

Immunology 101

Note : The immune system can also be trained to recognize tumors!

Normal Cell Tumour Cell

Mutations in individual genes

Some of the same genes that control cell growth/death are also involved in cellular

anti-viral programs

Antiviral defense: The “Achilles Heel” of Cancer

~70% of Cancer cells have defects in anti-viral programs !!!

Cancer

Virus

Oncolytic Viruses : A targeted approach to Cancer therapy

Because cancers have defects in antiviral responses, this makes it possible to create viruses that replicate in and specifically kill cancer cells!

In addition to antiviral defects, the typical tumor cell…

• Grows rapidly and generates tumors with leaky vasculature

• Expresses high levels of enzymes involved in nucleic acid metabolism (eg. Thymidine kinase or TK)

• Has Hyperactive growth receptor pathways (eg. EFGR, Ras)

Vaccinia Virus

1.Large double-stranded DNA poxvirus ~ 200 kbp

2.Replicates exclusively in the cytoplasm of cells

3.Can’t recombine with cellular DNA in the nucleus

4.Large amount of genes which can be removed or replaced to accomodate transgenes

5.One of the best studied viruses known to man

Vaccinia Virus engineered from live vaccine

Given to > 100 million healthy children world-wide

1800 1980

The JX-594 Oncolytic Vaccinia strain

vaccine straingenome

thymidine kinase

engineered product:JX-594

lac-Zmarker

GMGM-CSFpayload

lac-Z

B18R mutation

1. Viral gene expressing the cellular equivalent of Thymidine kinase has been removed => dependence on high TK levels provided by tumor cells

2. Mutation in B18R gene required to overcome IFN-mediated antiviral response => no consequence in tumor cells have defects in this pathway

3. Added GM-CSF transgene to stimulate an anti-tumor immune response

NORMAL

CANCER

JX

IFN EGFR

JX

JX

TK

E2F

E 2F

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JXJX

JX

JX JX

JXJX

JX

JX

JX

GMGM

GM

GM

JX

JX

JX

JX

JX

JXJX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

JX

GM

GM

GM

GM

GM

GM

GM

JX

JXJXJX

JX

IFN EGFR

X

GM

GM

JX

JX

ras

Vaccinia Virus (JX-594) cancer targeting & three-pronged MOA

T KX

GROW IN AND KILL TUMOURS

ONCOLYTIC VIRUSES

Phase 1: RECIST tumor response in HCC patient

Baseline 4 cycles

Phase 2: RECIST responses in HCC tumors located

at periphery of cirrhotic liver

Baseline Week 8

Baseline Day 12

Baseline Week 8

ONCOLYTIC VIRUSES

SELF AMPLIFYING DOSING

JX-594 activity

Stanford Bio-Imaging Center: (S Thorne - Jennerex virus labeled green)

Amplification, spread, cell killing within human tumors

Days, Post JX594 injection

0 2 4 6 8 10 12 14 16 18 20 22 24

Gen

omes

/ 5L

Who

le B

lood

105

106

107

108

Hours, post administration Hours, post administration

4.5 x 107 genome

01201C1

Days, Post JX594 injection

0 2 4 6 8 10 12 14 16 18 20 22 24

Ge

no

mes/

5L

Wh

ole

Blo

od

105

106

107

108

109

1010

1011

8.8 x 109 genome

Pharmacokinetic Profile: Waves of 1˚+ 2˚ Vaccinia Spread in Human Cancer Patients

Days post tumour injection with vaccinia virus

MINIATURE “BIOLOGICAL BATTLESHIPS” ATTACK TUMOURS IN MULTIPLE WAYS

ONCOLYTIC VIRUSES

VIRUS ENTERS THROUGH LEAKY VASCULATURE (24 Hr PI)

Oncolytic Virus Initial Sites of Infection in Mouse Tumour

CD31-red Virus-GFP-green

TumorVasculature

Steve Thorne University of Pittsburgh

Tumour Vasculature Infection in Patients Treated IV in Ottawa

VV positive tumor

VV infected vessel

VV positive tumor

VV negative stroma

Necrotic response in distant non-injected tumor

Baseline Day 5 Week 8

Pt 1304

Immunity and OV therapy

IV VSV

Colon Cancer tumour Challenge with

Colon Cancer cells

“Cured” mouse wait 7 months Mice reject

Tumours!

Pre-treatmentPre-treatment After vaccinia After vaccinia

75 year-old man:• Multiple met sites (n=24)

• Complete tumor regressions:

• Injected

• Distant dermal

• Disease-free 3+ years

32 year-old woman:

• Refractory, widespread met

• Complete tumor regressions:

•Injected

•Distant dermal, chest (surg)

• Disease-free 1.5+ years

Long-term Survivors Disease-Free after Vaccinia

Phase I Clinical Trial Metastatic Melanoma

1. Infection & cell lysis

2. Immune response stimulation

3. Vascular disruption

JX-594: novel 3-pronged mechanism-of-actionReplication & GM-CSF expression dependent

primary MOA:

leads to complementary:

JX 594 Clinical Activity

Survival (years)

pati

ents

3-4 months life expectancy

renal

melanoma

lung

liver

melanoma

melanoma

colon

+

+

+

+

>/= 8 monthssurvival

melanoma

thyroid

thymic

melanoma

colon

colon

melanoma

melanoma

15long-term

Survivors(1)

7

cancer types

(1) Based on data from ~43 8-mo evaluable patients to date.

Ph 1 long-term survival: many cancer types

Intra-hepatic injection of JX-594 for Phase II clinical trial

Promising Ph 2 survival in advanced liver cancerSuperior to internal & historical controls, including sorafenib

Lancet Oncology 2009 (n=226)

Hep0072010 (n=22)

IV response summary

% p

atie

nts

(n=6) (n=4) (n=4) Full dose(n=9)*

*Note: Response rate assessment incomplete

IV delivery: biopsy-proven cancer-specific targeting

Colon cancerglandular structures = infected (IHC+)

& evolving necrotic tumor tissue

But What do we do when tumors resist infection with OVs?

Clinical data so far with JX-594 and other oncolytic viruses suggest that significant therapeutic responses can be obtained in a subset of patients

1. Negative single strand RNA virus of the Rhabdovirus family

2. Small genome, 5 gene products (N, P, M, G, L)

3. Potent cytolytic

VSV M protein

-plays a role in virion budding

-causes cell rounding and induces cell death

-interacts with nuclear export machinery prevent expression of cellular antiviral genes

VSV51 : Mutation in M protein at methionine 51 prevents interaction with nuclear machinery

=> Sensitivity to antiviral signaling eg. Interferon

V

VV V

V

V

V

V

= Antiviral defense pathway

VVV

V

V

V

V

VVV

V

V

V

V

VVV

V

V

V

V

Normal Cell

HighlySensitive

Moderately Sensitive

Tumor Cells

Resistant

V

VV V

V

V

V

V

= Antiviral defense pathway

VVV

V

V

V

V

VVV

V

V

V

V

VVV

V

V

V

V

Normal Cell

HighlySensitive

Moderately Sensitive

Tumor Cells

Resistant

Can we complement the defects of VSV51 in resistant tumors using a chemical complementation strategy?

Drugs?

Resistant

Screen design

4T1 mouse breast cancer cells

Controls (SAHA/DMSO)

Library Compounds

4h pre-treat

Add control (media) Add VSV51at low MOI (0.03)

40h incubation

Add Alamar Blue (fluorescent viability dye)

2h incubation

Measure fluorescence

Calculate normalized viability ratio (VSV-treated/Control) for each drug => Low ratio indicates viral sensitizer

High Throughput screen identifies novel “virus sensitizer” or VSe drugs

In vitro validation of VSe compounds

Identification of VSe candidates

VSe1 increases viral replication in cancer but not normal cells

Up to > 1000-fold increase in virus Up to > 1000-fold increase in virus in cancer cells !in cancer cells !

VSe1 represses VSV51-induced genes

Overall # of genes affected by VSe1 = 111Overall # of genes affected by VSe1 = 111

VSe1 enhances VSVd51 efficacy in a resistant CT26 syngenic colon tumor model

VSe1 enhances VSVd51 replication in human clinical samplesP

BS

VS

e1

+ V

SV5

1 V

SV5

1

immune cells

virus infection & cell lysis

tumor-targeting antibodies

shutting off tumor blood supply

AcknowledgementsBell/Atkins labDr. John BellDr. Harry AtkinsDr. Fabrice LeBoeufDr. Markus Vaha-KoskelaHeather MacTavishTheresa FallsJulie CoxAlanah KempNicayla KeathJad Farah

Jennerex BiotherapeuticsDavid KirnCaroline BreitbachTae Ho HwangTheresa HickmanAdina PelusoKelley ParatoAnn MoonManijeh Daneshmand

HTS Facility (McMaster)Jenny WangJan BlanchardRyan BrownDr.Eric Brown

Lichty LabDr. Brian LichtyFrances Lai

Auer LabDr.Rebecca AuerLisa Mackenzie

InstitutionsOHRIUniversity of OttawaMcMaster University

Funding AgenciesFRSQOICR