1 Preclinical Development of Talimogene Laherparepvec as an Oncolytic Immunotherapy Rafael Ponce.

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Preclinical Development of Talimogene Laherparepvec as an Oncolytic

Immunotherapy

Rafael Ponce

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Overview

• Introduction to talimogene laherparepvec• Design• Proposed mode of action

• Pharmacology data• Clinical data

• Nonclinical assessment• Regulatory guidances• Unique considerations for oncolytic viruses

Immunosurveillance of cancer

Immunosurveillance of cancer – Adaptive immunity

Clinical data on tumor immunology

5 year survival rate

• 38% among patients with CD3+ TIL

• 4.5% among patients without CD3+ TIL

• Survival also correlated with IFN-γ, IL-2 and lymphocyte-attracting chemokines

Clinical data on tumor immunology

Immune status (as characterized by density of CD3+ cells and memory T cells) was a better predictor of survival than histopathological staging

Mechanisms of tumor-mediated immune escape

Clinical data on tumor immunology

~20 months improvement in median survival among 2/3 of patients with highest ratio of CD8 (cytotoxic T cells) to regulatory T cells

~80 months difference to reach 25%ile cumulative survival

Effects of immunity on tumor evolve with the tumor, differ by tumor type

Immune function

Tumor

Overall survival vs Treg in FL

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Immunotherapies in OncologyAntigen Dependent Mechanisms

Specific T cell recruitment and activationBiTE, DART, ImmTac, TCR approaches and Nano-

particles based APC

Adoptive Cell TherapiesAutologous CARs, Allogeneic CARs, Autologous

Dendritic Cell

Vaccine ApproachesViral-based Oncolytic/Immunologic

Cellular/Peptide-based

Antigen Independent Mechanisms Checkpoint Blockade

CTLA-4, PD-1, PD-L1, B7-H3, etc.

Co-ActivationGITR, LAG3, OX40, ICOS, etc.

Inhibition of Immunosuppressive secreted factorsIDO/TDO, TGF-b, M-CSF-R, PI3Kd, etc.

Recombinant Cytokines Peg-IL-2, IL-10, IL-15, IL-21, etc.

TLRTLR-agonists and cross activation

Immune modulation in oncologyDiverse platforms leverage immune system biology

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Talimogene Laherparepvec Nomenclature

• Generic name:• Previously known as OncoVEX or OncoVEXhuGM-CSF

• Herpes Simplex Virus Type 1 (HSV-1)–based, replication competent, gene-modified oncolytic virus immunotherapy

• Name derivation:

talimogene laherparepvec

immuno-modulating

gene therapy herpes simplex virus based

replicating

vector

imo herpatal gene la rep vec

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Two Distinct Mechanisms of Action

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Generation of Talimogene LaherparepvecCharacteristic Rationale

JS1 strain-derived - Improved tumor cell lysis over commonly-used laboratory strains

Deletion of ICP34.5- Provides tumor-selective replication, greatly reduces neurovirulence- Decreases replication

Deletion of ICP47- Prevents block to antigen presentation- Results in earlier/increased US11 expression

Earlier/increased US11 - Restores replication of ICP34.5-deleted HSV-1

Insertion of hGM-CSF (ICP34.5 locus)

- hGM-CSF driven off CMV promoter- Enhances anti-tumor immune response- Increased safety in the event of homologous recombination w/ wild-type HSV-1

US11

ThymidineKinase

ICP47ICP34.5ICP34.5

eIF2α eIF2α- p

PKR- p

PKR

IFNdsRNA (HSV-1)

Dimerization, Autophosphorylation

3, 8

Blocks viral protein synthesis1, 2

Apoptosis of infected cells

Autophagy

7

4,6

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1: He et al (1997) PNAS 94:843; Li et al (2011) JBC 286:247852: Mulvey et al (2003) J Virol 77:109173: Lussignol et al (2013) J Virol 87:859; Xing et al (2012) J Virol 86:3528 4: Esclatine et al (2009) Curr Top Microbiol Immunol 335:336: Talloczy et al (2002) PNAS 99:1907: Dey et al (2005) Cell 122:9018: Gale and Katze (1998) Pharmacol Ther 78:29

12: Peters et al (2002) J Virol 76:11054

The IFN-PKR Response to HSV-1 Infection Normally Protects the Cell Through Host Cell Shut-off (Translational Blockade),

Apoptosis, and Autophagy

ICP34.5(HSV-1)

eIF2α eIF2α- p

PKR- p

PKR

IFNdsRNA (HSV-1)

Dimerization, Autophosphorylation

3, 8

PP1α

1, 14

ICP34.5(HSV-1)

PP1αAutophagy

ICP34.5(HSV-1)

Beclin

ICP34.5(HSV-1)

Beclin

Blocks anti-HSV-1 CD4+ T-cell response

7

4,6

3, 13

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Viral replication

The HSV-1 ICP34.5 protein overcomes anti-viral responses to promote viral

replication and survival

1: He et al (1997) PNAS 94:843; Li et al (2011) JBC 286:247853: Lussignol et al (2013) J Virol 87:859; Xing et al (2012) J Virol 86:3528 4: Esclatine et al (2009) Curr Top Microbiol Immunol 335:336: Talloczy et al (2002) PNAS 99:1907: Dey et al (2005) Cell 122:901

11: Leib et al (2009) J Virol 83:1216413: Orvedahl (2007) Cell Host Microbe 1:2314: Chou et al (1994) PNAS 91:5247 and (1995) PNAS 92:10516; Bolovan et al (1994) J Virol 68:48; Chou et al (1990) Science 250:1262

ICP34.5(HSV-1)

eIF2α eIF2α- p

PKR- p

PKR

IFNdsRNA (HSV-1)

Dimerization, Autophosphorylation

ICP34.5(HSV-1)

PP1α

Viral replication

Blocks viral protein synthesis

Apoptosis of infected cells

Autophagy

Normal cells can protect themselves from infection of ICP34.5-deficient HSV-1

PP1α

peIF2α eIF2α-

PKR- p

PKR

IFNdsRNA (HSV-1)

Dimerization, Autophosphorylation

Viral replication

Normal translational controls may be dysregulated in tumor cells, enabling active viral replication and cell lysis even in the absence of ICP34.5

IFN/PKR : Haus (2000) Arch Immunol Ther Exper 48:95-100; Meurs et al (1993) PNAS 90:232-236; Farassati et al (2001) 3: 745-750; Leib et al (2000) PNAS 97:6097-6101 MEK: Smith et al (2006) J Virol 80(3) 1110-20 Ras: Farassati et al (2001) Nat Cell Biol 3 745-50, but not Mahller et al (2006) Pediatr Blood Cancer 46:745 PI3k: Sarinella et al (2006) Gene Ther 13(14): 1080-7

PI3k

PI3kRasMEK

IFN/PKR

ICP34.5(HSV-1)

ICP34.5(HSV-1)

PP1αPP1α

Blocks viral protein synthesis

Apoptosis of infected cells

Autophagy

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Two Distinct Mechanisms of Action

• Direct killing of tumor cells mediated by lytic viral replication

• Delivered by intratumoral injection

• Imaging guided administration to deeper SC lesions, nodes and visceral organs

• Selective replication in tumor cells leaves healthy tissue intact

• In-Direct immune-mediated killing of tumor cells not directly infected by virus

• Anti-viral response activates innate immune system, induces migration of immune cells to

tumor

• Tumor-specific antigens released by lysis are presented by APC to effector T cells

• huGM-CSF expression enhances immune response

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- Fully immune-competent female BALB/c mice

- Implant A20 lymphoma cells SC. on both R & L flanks

- Intratumoral injection of virus (50uL) into right flank tumor every 3 days for 3 total doses once tumors

reach ~6mm

A20 Contra-lateral Syngeneic Tumor Model for OncoVEX Studies

LeftCONTRALATERAL

NON-INJECTEDTumor

anti-tumor efficacy mediated by DIRECT lysis and SYSTEMIC immune-

mediated mechanisms

anti-tumor efficacy mediated only by

SYSTEMIC immune-mediated mechanisms

RightINJECTED

Tumor

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Individual Tumor Volumes

Anti-tumor response of OncoVEXmGM-CSF

14 21 28 35 42 490

500

1000

1500

2000

2500

9 of 10tumor-free

Time (days)

14 21 28 35 42 490

500

1000

1500

2000

2500

10 of 10tumor-free

Time (days)

3 Doses 5E+06 pfu/dose

14 21 28 35 42 490

500

1000

1500

2000

2500

1 of 10tumor-free

Time (days)

14 21 28 35 42 490

500

1000

1500

2000

2500

0 of 10tumor-free

Time (days)

INJECTED (R) Tumors

CONTRALATERAL, NON-INJECTED (L) Tumors

Vehicle Control

Tum

or V

olum

e (m

m3 )

Tum

or V

olum

e (m

m3 )

Each line represents tumor progression over time for an individual animal.

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Phase III OPTiM Study

• Primary endpoint• Durable response rate

(Rate of objective response lasting continuously for 6 months, beginning at any point within 12 months of initiating therapy)

• Secondary endpoint• Overall survival

• Exploratory endpoint• Quality of life (FACT-BRM)

Untreated or previously treated, unresectable stage IIIb, IIIc, and IV

melanoma

4 mL talimogene laherparepvec

intratumoral every 2 weeks for up

to 18 months

Subcutaneous GM-CSF 14 days every

28-day cycle

n = 4362:1 randomization

EndpointsPrimary:

6-month durableresponse rate

(CR + PR)Secondary: survival

Clinicaltrials.gov. NCT00769704. www.clinicaltrials.gov

Top-Line Results• Talimogene laherparepvec met its primary endpoint• A statistically significant difference was observed in DRR

• 16% in the talimogene laherparepvec arm vs. 2% in the GM-CSF arm (ASCO, June 1, 2013)

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Local And Distant Lesion Response

Patient had 2 liver lesions at baseline with a combined size of 2 cm, which were never injected

Screening Screening

Screening

Cycle 3 Cycle 8 Cycle 8

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P3 Talimogene Laherparepvec in Melanoma

Safety: AEs

No treatment-related, fatal AEs were observed. Of 10 total fatal AEs on the talimogene laherparepvec arm, 8 were due to PD. The only 2 fatal AEs on the talimogene laherparepvec arm not associated with PD were sepsis (in the setting of cholangitis) and myocardial infarction.

AEs of All Grades Occurring in 20% of Talimogene Laherparepvec–Treated

Patients

Grade 3/4 AEs Occurring in 5 Patients in Either Arm

AE = adverse event; GM-CSF = granulocyte-macrophage colony-stimulating factor; PD = progressive disease; TVEC = talimogene laherparepvec. Andtbacka RHI, et al. J Clin Oncol. 2013;31(suppl). Abstract LBA9008.

Preferred Term, % All Grade AEs

GM-CSF (N = 127)

TVEC (N = 292)

Fatigue 36.2% 50.3%

Chills 8.7% 48.6%

Pyrexia 8.7% 42.8%

Nausea 19.7% 35.6%

Influenza-like illness 15.0% 30.5%

Injection site pain 6.3% 27.7%

Vomiting 9.4% 21.2%

Preferred Term, % All Grade AEs

GM-CSF (N = 127)

TVEC (N = 292)

Cellulitis < 1% 2.1%

Fatigue < 1% 1.7%

Vomiting 0 1.7%

Dehydration 0 1.7%

Deep vein thrombosis

0 1.7%

Tumor pain 0 1.7%

Oncolytic viruses• International Conference on Harmonisation (ICH) Considerations: Oncolytic

viruses, November 2008

• ICH Considerations: General principles to address virus and vector shedding, June 2009

Gene therapies• Gene therapy clinical trials: Observing subjects for delayed adverse events

[Center for Biologics Evaluation and Research, United States Food and Drug Administration (US FDA)], November, 2006

• Guideline on the non-clinical studies required before first clinical use of gene therapy medicinal products [EMEA/CHMP/GTWP/125459/2006], May 2008

• Guideline on scientific requirements for the environmental risk assessment of gene therapy medicinal products [EMEA/CHMP/GTWP/125491/2006], May 2008

Anti-cancer therapeutics and biotechnology-derived therapeutics• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals, October 2009

• ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, June 2011

Regulatory Guidances for Nonclinical Development

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General principles for nonclinical evaluation of an oncolytic virus

• Evaluate selectivity of tumor cell killing in vivo and in vitro

• Use relevant animal model• Viral tropism, infectivity/permissiveness, replication ability, cytopathic

potential, and anti-tumor effect

• Use of both tumor-bearing and normal animals, genetically-modified (humanized) animals• Proof-of-concept, PK/PD, viral shedding, safety (normal vs tumor-bearing)• Ideally, use relevant tumor model• If virus contains transgene, evaluate in pharmacologically-responsive

model/use analogous species-specific transgene• Model intended clinical dosing route

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Concerns with oncolytic viruses• Biodistribution/shedding

• Distribution of virus in the body, potential for secondary transmission (shedding tissues/excreta)• Latency, clearance• Is virus in target tissues (and not in non-target tissues)?

• HSV- is a neurotropic virus• Guides clinical monitoring, safety precautions (close contacts, HCP)

• Use sensitive test (qPCR)• Note that this detects viral DNA, but doesn’t address infectivity, challenging to relate qPCR

data to infectivity risk

• Recombination: Generation of novel viral strains with unique properties

• Genomic integration: Insertional mutagenesis, germ-line transmission

• Effect of prior infection/immunity (↑ disease,↓ activity)

• Effect of immunosuppression/immunodeficiency

• HSV-1 specific• Neurovirulence risk• Perinatal risk• Activity of anti-viral therapy

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Non-clinical development

• Generally conducted in mice, which is well accepted as a model of human herpes

virus infection and disease pathogenesis

• Limitation: Low rate of latency and spontaneous reactivation

• Dosing denominated in terms of plaque-forming units (PFU)/animal

• PFU is a measure a measure of the number of viral particles capable of forming plaques in

an in vitro assay with permissive cells

• Allometric scaling of dose based on body weight, used dose range spanning approx. max

therapeutic dose to 60-fold max therapeutic dose

• Evaluated

• General safety of talimogene laherparepvec and OncoVEXmGM-CSF

• Note that hGM-CSF is not active in mice, so a surrogate virus was constructed

• Tolerability/biodistribution in normal mice, tumor-bearing mice, genetically immunodeficient

(SCID, BALB/c nude) mice

• Intratumoral, SC, IV, and intra-cranial, and intra-nasal dosing routes

• Embryo-fetal development, maternal-fetal biodistribution

• Susceptibility to acyclovir, latency/reactivation

Nonclinical safety programRepeat-dose Studies

Safety in tumor bearing mice‑ Vehicle, talimogene laherparepvec 0, 5 x 106 (3 doses); IT

Tolerability and biodistribution in tumor-bearing mice

Vehicle, talimogene laherparepvec 0, 105, 5 x 105 (3 doses); IT

Comparative safety in mice with OncoVEXmGM-CSF

OncoVEXmouseGM-CSF, talimogene laherparepvec

106, 107 (5 doses); SC

General safety in mice Vehicle, talimogene laherparepvec 0, 105, 106, 0.8x107 (5 doses); SC

General safety in mice Vehicle, talimogene laherparepvec 0, 105, 106, 107 (5 doses); SC

General safety in mice Vehicle, talimogene laherparepvec 0, 105, 106, 107 (once weekly for 12 weeks); SC

Reproductive and Developmental ToxicityEmbryo-fetal toxicity in mice Vehicle, talimogene laherparepvec 0, 105, 106, 107 (4 doses, GD 6-

15); IV

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Key findings – nonclinical safety• Reversible inflammation at the injection site

• Reversible increase in lymphocytes and neutrophils, and evidence of transient immune activation• enlargement and increased germinal centers in the spleen• lymphoid hyperplasia in spleen and bone marrow

• Other minor findings, variably observed across studies,• minimal, transient decreases in circulating red blood cell mass• variably increased (minimal to moderate) or decreased (minimal) bone

marrow erythroid cell production• minimal to mild splenic extramedullary hematopoiesis

• 60-fold dose margin (based on body weight) using NOAEL in mice (≤12 weeks) vs maximum proposed clinical dose

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Other key nonclinical findings

• No effects on embryo-fetal development• Negligible viral transfer from mother to fetus

• <0.001% viral DNA in fetal blood:maternal blood in one pooled fetal sample in the high dose group

• Susceptible to acyclovir

• No neurovirulence following SC/IV/IT dosing

• No unique effects of administering virus encoding mGM-CSF

• No unusual biodistribution (distinct from wtHSV-1)

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Discussion

• Oncolytic viruses provide a novel modality for oncology therapy• Currently in evaluation as combination with immunotherapies

• Vector-specific issues can be addressed adequately in non-clinical models, supplemented by ex vivo/in vitro studies

• Non-traditional safety considerations• Recombination (generation of novel strains with unique properties)• Latency/reactivation• Shedding/infection of close contacts• Unusual biodistribution• Heightened attention with immune impairment• Traditional PK not useful with replicating virus

• qPCR for viral detection (sensitive) vs plaque assay (intact virus)

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Acknowledgements

CBSSIan PyrahJim RottmanBarb ThomasCameron Zimmerman

Therapeutic Innovation UnitCourtney BeersBill FanslowTiep LeJulia Piasecki

Medical SciencesMike Chastain

Amgen (Biovex, Inc.)Rob CoffinColin LoveSuzanne Thomas

PSTJen GansertCaroline LillyAri VanderWaldeAnnie Woodland