Technical and Scientific information EudraCT: 2014-005386-67 Talimogene laherparepvec Page 1

Talimogene Laherparepvec

Technical and Scientific Information on the GMO Version provided for use in Belgian Public Consultation

June 2016

Amgen Ltd 240 Cambridge Science Park Milton Road, Cambridge CB4 0WD United Kingdom

Technical and Scientific information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 2

CONFIDENTIALITY STATEMENT

Information and data contained herein are proprietary and confidential.

This information should not be disclosed to any third party without the prior written consent of Amgen Inc.

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Table of Contents

I. GENERAL INFORMATION ...... 12 A. NAME AND ADDRESS OF THE NOTIFIER ...... 12 B. NAME, QUALIFICATIONS AND EXPERIENCE OF THE RESPONSIBLE SCIENTIST(S) ...... 12 C. TITLE OF THE PROJECT ...... 12

II. INFORMATION RELATING TO THE GMO ...... 13 A. CHARACTERISTICS OF PARENTAL ORGANISM ...... 13 1. Scientific Name ...... 13 2. Taxonomy ...... 13 3. Other Names (Usual Name, Strain Name, etc.) ...... 13 4. Phenotypic and Genetic Markers...... 13 5. Description of Identification and Detection Techniques ...... 16 6. Sensitivity, Reliability (in Quantitative Terms) and Specificity of Detection and Identification Techniques ...... 17 7. Description of the Geographic Distribution and of the Natural Habitat of the Organism, Including Information on Natural Predators, Preys, Parasites and Competitors, Symbionts and Hosts ...... 17 8. Organisms with Which Transfer of Genetic Material is Known to Occur Under Natural Conditions ...... 18 9. Verification of the Genetic Stability of the Organisms and Factors Affecting it ...... 18 10. Pathological, Ecological and Physiological Traits ...... 18 a) Classification of hazard according to existing Community rules concerning the protection of human health and/or the environment ...... 18 b) Generation time in natural ecosystems, sexual and asexual reproductive cycle ...... 20 c) Information on survival, including seasonability and the ability to form survival structures ...... 22 d) Pathogenicity: infectivity, toxigenicity, virulence, allergenicity, carrier (vector) of pathogen, possible vectors, host range including non-target organism. Possible activation of latent viruses (proviruses). Ability to colonise other organisms ...... 23 e) Antibiotic resistance, and potential use of these antibiotics in humans and domestic organisms for prophylaxis and therapy ...... 26 f) Involvement in environmental processes: primary production, nutrient turnover, decomposition of organic matter, respiration, etc...... 26 11. Nature of Indigenous Vectors ...... 26 12. History of Previous Genetic Modifications ...... 26 B. CHARACTERISTICS OF THE VECTOR ...... 27

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C. CHARACTERISTICS OF THE MODIFIED ORGANISM ...... 28 1. Information Relating to the Genetic Modification ...... 28 a) Methods used for the modification ...... 28 b) Methods used to construct and introduce the insert(s) into the recipient or to delete a sequence; ...... 29 c) Description of the insert and/or vector construction; ...... 30 d) Purity of the insert from any unknown sequence and information on the degree to which the inserted sequence is limited to the DNA required to perform the intended function; ...... 30 e) Methods and criteria used for selection; ...... 30 f) Sequence, functional identity and location of the altered/inserted/deleted nucleic acid segment(s) in question with particular reference to any known harmful sequence...... 30 2. Information on the Final GMO ...... 31 a) Description of genetic trait(s) or phenotypic characteristics and in particular any new traits and characteristics which may be expressed or no longer expressed; ...... 31 b) Structure and amount of any vector and/or donor nucleic acid remaining in the final construction of the modified organism; ...... 32 c) Stability of the organism in terms of genetic traits; ...... 32 d) Rate and level of expression of the new genetic material. Method and sensitivity of measurement; ...... 36 e) Activity of the expressed protein(s); ...... 36 f) Description of identification and detection techniques including techniques for the identification and detection of the inserted sequence and vector; ...... 37 g) Sensitivity, reliability (in quantitative terms) and specificity of detection and identification techniques;...... 37 h) History of previous releases or uses of the GMO; ...... 37 i) Considerations for human health and animal health, as well as plant health: ...... 37 1. Toxic or Allergenic Effects of the GMOs and/or Their Metabolic Products ...... 37 2. Comparison of the modified organism to the donor, recipient or (where appropriate) parental organism regarding pathogenicity; ...... 42 3. Capacity for colonisation; ...... 44 4. If the organism is pathogenic to humans who are immunocompetent: ...... 45 5. Other product hazards...... 45

III. INFORMATION RELATING TO THE CONDITIONS OF RELEASE AND THE RECEIVING ENVIRONMENT ...... 46 A. INFORMATION ON THE RELEASE ...... 46

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1. Description of the Proposed Deliberate Release, Including the Purpose(S) and Foreseen Products ...... 46 2. Foreseen Dates of the Release and Time Planning of the Experiment Including Frequency and Duration Of Releases ...... 46 3. Preparation of the Site Previous to the Release ...... 46 4. Size of the Site ...... 47 5. Method(s) to be Used for the Release ...... 47 6. Quantities of GMOs to be Released ...... 47 7. Disturbance on the Site (Type and Method of Cultivation, Mining, Irrigation, or Other Activities) ...... 47 8. Worker Protection Measures Taken During the Release...... 47 Transportation ...... 50 9. Post-release Treatment of the Site ...... 54 10. Techniques Foreseen for Elimination or Inactivation of the GMOs at the End of the Experiment ...... 54 Contaminated (infectious) “sharps”: ...... 54 Contaminated (potentially infectious) materials for autoclaving and reuse: ...... 55 Contaminated (potentially infectious) materials for disposal: ...... 55 11. Information on, and Results of, Previous Releases of the GMOs, Especially at Different Scales and in Different Ecosystems ...... 55 B. INFORMATION ON THE ENVIRONMENT (BOTH ON THE SITE AND IN THE WIDER ENVIRONMENT) ...... 56 1. Geographical Location and Grid Reference of the Site(S) (In Case of Notifications Under Part C the Site(S) of Release Will be the Foreseen Areas of Use of the Product) ...... 56 2. Physical or Biological Proximity to Humans and Other Significant Biota ...... 56 3. Proximity to Significant Biotopes, Protected Areas, or Drinking Water Supplies ...... 56 4. Climatic Characteristics of the Region(s) Likely to be Affected...... 56 5. Geographical, Geological and Pedological Characteristics ...... 57 6. Flora and Fauna, Including Crops, Livestock and Migratory Species ...... 57 7. Description of Target and Non-target Ecosystems Likely to be Affected ...... 57 8. A Comparison of the Natural Habitat of the Recipient Organism with the Proposed Site(s) of Release ...... 57 9. Any Known Planned Developments or Changes in Land Use in the Region which Could Influence the Environmental Impact of the Release ...... 57

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IV. INFORMATION RELATING TO THE INTERACTIONS BETWEEN THE GMOs AND THE ENVIRONMENT ...... 58 A. Characteristics affecting survival, multiplication and dissemination ...... 58 1. Biological Features Which Affect Survival, Multiplication and Dispersal...... 58 2. Known or Predicted Environmental Conditions which may Affect Survival, Multiplication and Dissemination (Wind, Water, Soil, Temperature, pH, etc.) ...... 58 3. Sensitivity to Specific Agents ...... 58 B. INTERACTIONS WITH THE ENVIRONMENT ...... 60 1. Predicted Habitat of the GMOs ...... 60 2. Studies of the Behaviour and Characteristics of the GMOs and Their Ecological Impact Carried Out in Simulated Natural Environments, Such as Microcosms, Growth Rooms, Greenhouses ...... 60 3. Genetic Transfer Capability ...... 60 4. Likelihood of Postrelease Selection Leading to the Expression of Unexpected and/or Undesirable Traits in the Modified Organism ...... 61 5. Measures Employed to Ensure and to Verify Genetic Stability. Description of Genetic Traits Which May Prevent or Minimise Dispersal of Genetic Material. Methods to Verify Genetic Stability ...... 61 6. Routes of Biological Dispersal, Known or Potential Modes of Interaction with the Disseminating Agent, Including Inhalation, Ingestion, Surface Contact, Burrowing, etc ...... 61 7. Description of Ecosystems to Which the GMOs Could be Disseminated ...... 62 8. Potential for Excessive Population Increase in the Environment ...... 62 9. Competitive Advantage of the GMOs in Relation to the Unmodified Recipient or Parental Organism(s) ...... 62 10. Identification and Description of the Target Organisms if Applicable ...... 62 11. Anticipated Mechanism and Result of Interaction Between the Released GMOs and the Target Organism(s) if Applicable ...... 63 12. Identification and Description of Non-target Organisms Which May be Adversely Affected by the Release of the GMO, and the Anticipated Mechanisms of any Identified Adverse Interaction ...... 63 13. Likelihood of Postrelease Shifts in Biological Interactions or in Host Range ...... 64

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14. Known or Predicted Interactions with Non-target Organisms in the Environment, Including Competitors, Preys, Hosts, Symbionts, Predators, Parasites and Pathogens ...... 64 15. Known or Predicted Involvement in Biogeochemical Processes ...... 64 16. Other Potential Interactions with the Environment ...... 64

V. INFORMATION ON MONITORING, CONTROL, WASTE TREATMENT AND EMERGENCY RESPONSE PLANS ...... 65 A. MONITORING TECHNIQUES ...... 65 1. Methods For Tracing The GMOs, And For Monitoring Their Effects ...... 65 2. Specificity (to identify the GMOs, and to distinguish them from the donor, recipient or, where appropriate the parental organisms), Sensitivity and Reliability of the Monitoring Techniques ...... 65 3. Techniques for Detecting Transfer of the Donated Genetic Material to Other Organisms ...... 65 4. Duration and Frequency of the Monitoring ...... 65 B. CONTROL OF THE RELEASE ...... 66 1. Methods and Procedures to Avoid and/or Minimise the Spread of the GMOs Beyond the Site of Release or the Designated Area for Use ...... 66 2. Methods and Procedures to Protect the Site from Intrusion by Unauthorised Individuals ...... 67 3. Methods and Procedures to Prevent Other Organisms from Entering the Site ...... 67 C. WASTE TREATMENT ...... 68 1. Type of Waste Generated ...... 68 2. Expected Amount of Waste ...... 68 3. Description of Treatment Envisaged ...... 68 D. EMERGENCY RESPONSE PLANS ...... 69 1. Methods and Procedures for Controlling the GMOs in Case of Unexpected Spread ...... 69 2. Methods for Decontamination of the Areas Affected, for example Eradication of the GMOs ...... 69 3. Methods for Disposal or Sanitation of Plants, Animals, Soils, etc. that were Exposed During or After the Spread ...... 70 4. Methods for the Isolation of the Area Affected by the Spread ...... 70 5. Plans for Protecting Human Health and the Environment in Case of the Occurrence of an Undesirable Effect ...... 70

Literature References ...... 71

APPENDICES ...... 80

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List of Tables Page Table 1. Biosafety Classifications for Wild Type HSV-1 Outside of EU ...... 19 Table 2. Theoretical Stable Genetic Variants of Talimogene Laherparepvec Created by Homologous Recombination ...... 36 Table 3. Summary of Recommended Containment Precautions for BSL-1 Infectious Agents ...... 51 Table 4. Summary of Recommended Containment Precautions for BSL-2 Infectious Agents ...... 52

List of Figures Page Figure 1. Schematic of an HSV-1 Virion with Envelope, Tegument, DNA and Capsid Indicated ...... 14 Figure 2. Structure of HSV-1 Genome ...... 28 Figure 3. Schematic Representation of Talimogene Laherparepvec Genome ...... 30

List of Appendices Page Appendix 1. Responsible Scientists CVs and signature pages ...... 81 Appendix 2. List of the participating study sites (Section III.B.1) ...... 83

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List of Abbreviations

Abbreviation Definition %CV co-efficient of variation °C Degrees Celsius α-TIF alpha-transinducing factor AML Acute Myelogenous Leukemia APCs Antigen Presenting Cells BHK baby hamster kidney BSL-1 Biosafety Level 1 BSL-2 Biosafety Level 2 cDNA complementary deoxyribonucleic acid cGMP Current Good Manufacturing Practices CMV cytomegalovirus CNS Central Nervous System CRO Contract Research Organization CT Computed Tomography DMEM Dulbecco Modified Eagle Medium DNA Deoxyribonucleic Acid DOT US Department of Transport ECACC The European Collection of Cell Cultures ELISA Enzyme-Linked ImmunoSorbent Assay FAM 6-carboxyfluorescein FBS Fetal Bovine Serum FDA Food and Drug Administration of the United States FITC fluorescein isothiocyanate gC, gD, gH, gG, gL Glycoproteins (type C, D, H, G, or L) GCP Good Clinical Practice GFP Green Fluorescent Protein GLP Good Laboratory Practice GM-CSF Granulocyte Macrophage Colony Stimulating Factor GMO Genetically Modified Organism GMP Good Manufacturing Practice GTAC Gene Therapy Advisory Committee GxP Good Practice guidelines, where “x” may be “M” for manufacturing, or “C” for clinical, or “L” for laboratory, etc. hCMV IE Human Cytomegalovirus Immediate Early hGM-CSF Human Granulocyte Macrophage Colony Stimulating Factor Page 1 of 3

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List of Abbreviations

Abbreviation Definition HGMP Human Genome Mapping Project HHV-1 Human Herpes Virus Type 1 HIV Human Immunodeficiency Virus HSV Herpes Simplex Virus HSV-1 Herpes Simplex Virus, Type 1 HSV-2 Herpes Simplex Virus, Type 2 HVEM herpesvirus entry mediator IRL long internal repeated sequence IRS short internal repeated sequence i.l. Intralesional i.t. Intratumoural i.v. Intravenous IATA International Air Transport Association IC50 Inhibitory Concentration At 50% ICH International Conference on Harmonization IEX Ion Exchange Chromatography IgG, IgM Immunoglobulin G or M IPIM Investigational Product Instruction Manual IMP Investigational Medicinal Product LATs Latency-Associated Transcripts LD50 median lethal dose mGM-CSF Murine Granulocyte Macrophage Colony Stimulating Factor MHC I or II Major Histocompatability Complex Type I or Type II MOI Multiplicity of infection miRNA micro ribonucleic acids mRNA Messenger Ribonucleic Acid MVL Micro Virology Laboratories MVSS Master Viral Seed Stock NF12 U.S. National Formulation 12 NSN New Substances Notification OncoVEXGM-CSF OncoVEX virus expressing hGM-CSF OOS Out Of Specification PAP prostatic acid phosphatase PCR Polymerase Chain Reaction PFU Plaque Forming Unit Page 2 of 3

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List of Abbreviations

Abbreviation Definition Ph Eur European Pharmacopoeia PKR Protein Kinase R PPE Personal Protective Equipment QA Quality Assurance QC Quality Control QMS Quality Management System QP Qualified Person qPCR Quantitative Polymerase Chain Reaction RNA ribonucleic acid RT real-time S short s.c. Subcutaneous SCCHN Squamous Cell Cancer of the Head and Neck SDS sodium dodecyl sulfate SEC Size Exclusion Chromatography SEM Skin, Eyes and/or Mouth SOP Standard Operating Procedure TAMRA tetramethylrhodamine TFF Tangential Flow Filtration TK Thymidine Kinase TRL long terminal repeated sequence TRS short terminal repeated sequence UK United Kingdom

UL long unique region USP United States Pharmacopeia

US short unique region U.S. United States VHS virion host shutoff protein VZV Varicella Zoster Virus WCB Working Cell Bank WHO World Health Organization WVSS Working Viral Seed Stock Page 3 of 3

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I. GENERAL INFORMATION A. NAME AND ADDRESS OF THE NOTIFIER Amgen Ltd, (EU Legal Representative of the study sponsor Amgen Inc.) 240 Cambridge Science Park Milton Road, Cambridge CB4 0WD United Kingdom

B. NAME, QUALIFICATIONS AND EXPERIENCE OF THE RESPONSIBLE SCIENTIST(S) This confidential information is provided in the full version submitted to the Belgian Authorities.

C. TITLE OF THE PROJECT Talimogene laherparepvec (JS1/ICP34.5-/ICP47-/hGM-CSF), formerly known as OncoVEXGM-CSF, is a disabled recombinant herpes simplex type 1 virus (HSV-1). Talimogene laherparepvec was generated by modifying the wild type HSV-1 genome (new isolate JS1) to functionally delete both copies of ICP34.5 and the ICP47 gene from the viral backbone and to insert an expression cassette encoding the human granulocyte macrophage colony-stimulating factor (hGM-CSF) gene in both ICP34.5 regions.

Talimogene laherparepvec is intended as an investigational medicinal product in a proposed phase 1, multicenter, open-label, single-arm study to evaluate the safety of the investigational medicinal product when injected into liver tumours (Protocol 20140318). Talimogene laherparepvec is intended for intrahepatic injection into hepatocellular carcinoma (HCC) and metastatic liver tumors (non-HCC) by a trained medical professional in a medical study site facility.

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II. INFORMATION RELATING TO THE GMO A. CHARACTERISTICS OF PARENTAL ORGANISM 1. Scientific Name Herpes simplex virus 1 (HSV-1)

2. Taxonomy Group: Group I (ds DNA)

Order: Herpesvirales

Family: Herpesviridae

Subfamily: Alphaherpesvirinae

Genus: Simplexvirus

Species: Herpes simplex virus 1 (HSV-1).

3. Other Names (Usual Name, Strain Name, etc.) Other name: Human herpes virus 1 (HHV-1)

Strain name: JS1 (ECACC Accession Number 01010209)

4. Phenotypic and Genetic Markers Wild type HSV-1 like all Herpesviridae, consists of a large double-stranded linear DNA genome that is packaged into an icosahedral nucleocapsid approximately 100 nm in diameter. Herpes viruses are enveloped viruses. The phospholipid rich envelope is derived from the nuclear envelope of the infected cell and contains embedded viral glycoproteins which project towards the outside. They are also characterised by an amorphous structure between the nucleocapsid and envelope, known as the tegument, which contains virus proteins important in the early stages of the infection process. The size of the complete virion varies but the average virion diameter is 186nm, or approximately 225nm if the glycoprotein spikes are included (Grünewald et al, 2003).

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Figure 1. Schematic of an HSV-1 Virion with Envelope, Tegument, DNA and Capsid Indicated

Electron micrograph (from Drs. Elisabeth Schraner and Peter Wild, University of Zürich) and diagram of an HSV-1 virion. Envelope is marked with a black arrow and capsid is marked with a white arrow (Jacobs et al, 1999).

In addition to these structural characteristics, the Herpesviridae present the following specific properties:

 During lytic replication, synthesis of the viral DNA and assembly of the nucleocapsids occur in the host cell nucleus. Packaged nucleocapsids bud through the host cell’s Golgi network and plasma membrane. The release of the viral progeny is invariably accompanied by the destruction of the infected cell.  Herpesviridae are capable of establishing latency in their natural host.

HSV-1 is further classified into the Alphaherpesvirinae subfamily of Herpesviridae based on its biological characteristics. The members of the Alphaherpesvirinae subfamily present the following characteristics:

 Broad host cell specificity  Relatively short viral cycle  Rapid spread in cell culture  Effective destruction of the infected cells  Capacity to establish latency in sensorial ganglions

The members of the Alphaherpesvirinae infecting humans are HSV-1, HSV-2 and Varicella Zoster Virus (VZV).

HSV-1 and HSV-2 are over 80% identical at the DNA level. The genetic maps of HSV-1 and HSV-2 are largely co-linear, and they encode the same number of proteins, but the two genomes differ in restriction endonuclease cleavage sites and in the sizes of several viral proteins (Cassai et al, 1975). The determined lengths of the HSV-1 and HSV-2 sequences are 152,261 and 154,746 bp, respectively. The major difference in length is located in the US region, which in HSV-2 is 1,349 bp longer than in HSV-1; with almost all of this difference being found within the US4 gene. UL is 742 bp longer in HSV-2 than in

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HSV-1, and this difference is the net result of many small differences. The sizes of the major repeats are very similar for HSV-1 and HSV-2. The G+C content of the HSV-2 genome is 70.4%, compared with 68.3% for HSV-1. The difference is distributed across the genome, being greatest in RL and least in RS (Dolan et al, 1998).

Despite the fact that HSV-1 and HSV-2 are closely related at both the DNA and amino acid level, some significant functional differences have been identified. Most notably, although both HSV-1 and HSV-2 can infect either oral or genital sites, HSV-1 is more likely to reactivate frequently from oral sites and HSV-2 is more likely to reactivate from genital sites (Lafferty et al, 1987). This may be attributable to differences in the cellular entry mechanisms of the two viruses. For example, in HSV-1 glycoprotein C is the major viral function responsible for virus attachment to cells and glycoprotein B mediates penetration, while in HSV-2 glycoprotein B is the major protein involved in both binding and penetration. Additionally, HSV-1 and HSV-2 also show significant differences in virulence. HSV-2 is more efficient at causing necrotizing stromal keratitis and encephalitis and grows to higher titers than HSV-1 in animal models (Stanberry et al, 1997). The differences in virulence may be attributable, at least in part, to the virion host shutoff (vhs) genes of HSV-1 and HSV-2. Although the vhs proteins are 87% identical at the amino acid level, their host shutoff activities differ significantly with the shutoff activity of HSV-2 vhs being more than 40 times faster than that of HSV-1 (Fenwick & Everett,

1990; Fenwick et al, 1979; Hill et al, 1983).

Neither latent infection or lytic replication of HSV-1 results in integration with host cell DNA, as demonstrated by Mellerick & Fraser, 1987, using a technique which exploits the large difference in G + C content of cellular and HSV-1 DNA. This technique (cesium chloride isopycnic density gradient centrifugation) compares the density of latent HSV-1 DNA to incoming virion DNA and host cell chromosomal DNA. It was observed that HSV-1 latent DNA banded at the same density as virion DNA, demonstrating that it is free and not integrated into the cellular DNA. As a control in this experiment, a cell line known to contain one integrated copy of another G + C rich herpesvirus (Gammaherpesviridae; Epstein Barr Virus), was used. In this case, the single integrated copy of the Epstein Barr Virus genome banded at the density of cellular DNA, demonstrating the sensitivity of the technique.

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5. Description of Identification and Detection Techniques The diagnosis of HSV-1 infection is usually made by the appearance of the lesions and the patient’s history. However, if the clinical pattern of the lesions is not specific to HSV, its diagnosis can be made by viral culture, Polymerase Chain Reaction (PCR), viral antigen detection, Tzanck test or serology.

Viral culture: Cells or fluid from a fresh lesion or cold sore are collected with a cotton swab and cultured on a permissive cell line in vitro. Viral culture is the most common method and is considered the gold standard, but can lead to false-negative results (see Section II.A.6).

Polymerase chain reaction (PCR) test: A PCR test can be performed on cells or fluid from a sore or on blood or on other fluid, such as cerebrospinal fluid. PCR testing is more sensitive than viral culture and is specific for HSV-1 (see Section II.A.6). However, due to cost it is not usually performed unless the infection is severe such as in the case of encephalitis. Detection of viral DNA sequences does not necessarily indicate infectious HSV-1, since infectivity relies on the presence of an intact virion (see Section II.A.10 (b)).

Antigen detection: Cells from a fresh cold sore are analysed for the presence of viral antigens on the surface of cells infected with the herpes virus, using a direct fluorescent antibody test system.

Tzanck test: A scraping of the floor of the herpetic vesicle is stained and examined microscopically for multinucleated giant cells. Its results do not specify the type of HSV infection.

Serology: Immunoglobulin G (IgG) antibodies that are type-specific to HSV develop during the first 2-3 weeks of an initial infection and persist indefinitely. As such they cannot differentiate between a current active herpes infection and a herpes infection that occurred in the past. In addition, the IgG test may be negative if the infection has occurred recently. Some IgG antibody tests specifically test for HSV serostatus as they are able to distinguish between HSV-1 and HSV-2 on the basis of differences in the patient's immune response to HSV glycoprotein G (gG).

Testing for the presence of HSV-1 or HSV-2 immunoglobulin M (IgM) antibodies can be used to indicate an active or recent infection.

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6. Sensitivity, Reliability (in Quantitative Terms) and Specificity of Detection and Identification Techniques PCR is the most sensitive and specific method in the laboratory diagnosis of HSV infection (96% sensitive and 99% specific for HSV-1; Whitley et al, 1998)

Serology testing is also very sensitive and specific (Annunziato & Gersho, 1996). For example, commercially available IgG tests claim to have a sensitivity of 91-100% and specificity of 92-95% for HSV-1 and HSV-2 infection, even in the absence of symptoms (Ashley, 2002).

In general, viral culture for all types of HSV has a sensitivity of approximately 70-80% (Whitley et al, 1998).

Direct fluorescent antibody testing may be performed from air-dried specimens, and can detect 50-70% of true HSV-positive cases compared with culture results (Clark et al, 1995).

A Tzanck test is inexpensive, however it is difficult to perform correctly without specific training in its use and its sensitivity is 50-70% for acute herpetic gingivostomatitis (Clark et al, 1995).

7. Description of the Geographic Distribution and of the Natural Habitat of the Organism, Including Information on Natural Predators, Preys, Parasites and Competitors, Symbionts and Hosts HSV-1 is a globally endemic pathogen of humans, which is usually initially transmitted in childhood via nonsexual contact, though it may be acquired in young adulthood through sexual contact.

The seroprevalence in adults is estimated to be 70% in developed countries and 100% in developing countries (Gupta et al, 2007). Orolabial herpes has an infection rate of approximately 33% in developing countries and 20% in developed countries (Chayavichitsilp et al, 2009).

The natural habitat of HSV-1 is humans, with no known alternative natural host. There are no known predators, preys, parasites, competitors or symbionts associated with HSV-1.

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8. Organisms with Which Transfer of Genetic Material is Known to Occur Under Natural Conditions HSV-1 is not known to transfer genetic material to organisms other than humans under natural conditions. The virus is not known to be zoonotic or reverse zoonotic. DNA replication occurs in the cell nucleus. No integration of the viral genome with the cellular genome occurs during replication or latency.

9. Verification of the Genetic Stability of the Organisms and Factors Affecting it In general, DNA viruses have greater genetic stability than RNA viruses. Firstly, DNA is more thermodynamically stable than RNA; secondly, replication of DNA is a much less error-prone process than the replication of RNA; and thirdly, more mechanisms exist in the host cell for repairing errors in DNA than in RNA.

The overall mutation rate for HSV-1 is low and has been estimated to be 1.8 × 10−8 mutations per nucleotide, per genomic replication (Duffy et al, 2008).

However, homologous genomic recombination may occur spontaneously in nature between the viral genomes of HSV-1 strains. For this to occur, it would be essential for a (human) cell to be infected simultaneously by two different strains. There is evidence that this may be a common occurrence in natural infections with wild type viruses (Bowden et al, 2004, Norberg et al, 2006). However, all strains of HSV-1 which were investigated and considered to have potentially evolved by this mechanism remained recognisably HSV-1 in their familial characteristics and pathogenicity.

10. Pathological, Ecological and Physiological Traits a) Classification of hazard according to existing Community rules concerning the protection of human health and/or the environment Wild type Herpes Simplex virus Type 1 is classified in Risk Group 2 in the European Union (EU) according to Directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work. A Risk Group 2 biological agent is defined in the EU as ‘one that can cause human disease and might be a hazard to workers; it is unlikely to spread to the community; there is usually effective prophylaxis or treatment available’.

It should be noted that this classification does not consider genetically modified micro- organisms which are attenuated or have lost known virulence genes.

Conversely, this classification is based on the effects on healthy workers, and does not consider effects on individuals with altered susceptibility which may be as a result of pre-existing disease, medication, compromised immunity, pregnancy or breast feeding.

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Similar classifications of hazard have been assigned to HSV-1 by the World Health Organisation (WHO), and in the US, Canada and Australia as summarised in Table 1.

Table 1. Biosafety Classifications for Wild Type HSV-1 Outside of EU Territory Category Definition Reference WHO Risk Group 2 A pathogen that can cause human WHO Laboratory (moderate or animal disease but is unlikely to Biosafety Manual, 3rd Ed individual risk, be a serious hazard to laboratory (2004) low workers, the community, livestock community or the environment. Laboratory risk). exposures may cause serious infection, but effective treatment and preventative measures are available and the risk of spread of infection is limited. US Risk Group 2 Agents associated with human NIH Recombinant DNA disease that is rarely serious and Guidelines (USA, 2011) for which preventive or therapeutic Appendix B-II-D. interventions are often available. CDC/NIH Guidelines (2009) "Biosafety in Microbiological and Biomedical Laboratories" 5th Edition, 2009. Section VIII-E.

Not listed under 42CFR73.3 – Select Agents and Toxins Canada Risk Group 2 Any pathogen that can cause Canadian Laboratory (moderate human disease, but under normal Safety Guidelines (2004) individual risk, circumstances is unlikely to be a limited serious hazard to laboratory Human Pathogens and community workers, the community, livestock Toxins Act. S.C. 2009, risk). or the environment. Laboratory c. 24. Schedule II. exposures rarely cause infection leading to serious disease, effective treatment and preventive measures are available and the risk of spread is limited. Australia/ Group 2 A pathogen that can cause human, Standard AS/NZS NZ (moderate animal or plant disease but is 2243.3:2010. Safety in individual risk, unlikely to be a serious hazard to laboratories Part 3: limited laboratory workers, the community, Microbiological aspects community livestock or the environment. and containment facilities. risk). Laboratory exposures may cause Standards Association of infection, but effective treatment Australia, Sydney. and preventive measures are available and the risk of spread is limited.

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 20 b) Generation time in natural ecosystems, sexual and asexual reproductive cycle HSV-1 does not persist in natural ecosystems, relying on its host organism for asexual replication with a short reproductive cycle (~18 hr.). Replication outside the host organism (human) does not occur and non –human infection is rare.

Infection of the host cell (cell entry) involves interactions of several glycoproteins (gC, gD, gH and gL) on the surface of the enveloped virus, with receptors (heparan sulfate, HVEM, nectin-1 and 3-O sulfated heparan sulfate) on the surface of the host cell. The envelope covering the virus particle, when bound to specific receptors on the cell surface, fuses with the host cell membrane to create a pore through which the contents of the virus envelope enter the host cell.

Following infection of a cell, immediate-early, early, and late genes are expressed in a sequential temporal manner, co-ordinating viral replication.

The virion host shutoff protein (vhs, encoded by UL41) is expressed in the cell cytoplasm and shuts off protein synthesis in the host, degrades host mRNA and regulates expression of viral proteins.

The viral capsid is transported to the cell nucleus. Once attached to the nucleus at a nuclear entry pore, the capsid ejects its DNA contents in a single linear segment via the capsid portal. The linear viral genome circularises shortly after infection and this circular DNA is the template for replication and remains as an extrachromosomal episome, not integrated into the host genome (Efstathiou et al, 1986; Mellerick & Fraser, 1987; Rock & Fraser, 1985). Circularization likely results from direct ligation mediated by pre-existing cellular factors or components of the incoming virion (Mocarski & Roizman, 1982).

On entering the nucleus, the VP16 (α-TIF) protein aids in immediate-early transcription. The immediate early and early proteins transcribed are used in the regulation of replication of the virus.

Neurovirulence is mediated by the virally encoded ICP34.5 gene product, which overcomes two important host anti-viral defenses: the shutdown of protein synthesis (Chou et al, 1990) and autophagy (Orvedahl et al, 2007). ICP34.5 also allows the efficient egress of newly formed viral particles to be released from the nucleus of infected cells (Brown et al, 1994a).

ICP34.5 prevents shutdown of protein synthesis by reversing the actions of the cellular anti-viral protein, protein kinase R (PKR). When PKR senses that a cell is infected, it phosphorylates and thereby inactivates the eukaryotic translation initiation factor eIF2,

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 21 which then prevents the infected cell from making new proteins. ICP34.5 recruits a phosphatase that reverses this effect of PKR, allowing protein synthesis to continue in the face of viral infection (He et al, 1997). ICP34.5 overcomes autophagy by binding to the mammalian autophagy protein, Beclin, and inhibiting its function (Orvedahl et al, 2007). Beclin is required for the initiation of the formation of the autophagasome in autophagy. Beclin also functions downstream of PKR, although the regions of ICP34.5 that recruit the phosphatase and bind Beclin are distinct. Deletion of the Beclin binding domain of ICP34.5 in HSV-1 completely blocks the ability of HSV-1 to cause lethal encephalitis in mice, without affecting the ability of ICP34.5 to dephosphorylate eIF2 or block protein synthesis (Orvedahl et al, 2007). This demonstrates that it is the Beclin-binding ability of ICP34.5, not its ability to antagonise host cell shutoff that is essential for neurovirulence. This distinction is important because the host cell shutoff function of ICP34.5 is partially redundant with another viral protein, US11. In this role, US11 binds PKR, blocking the phosphorylation of eIF2 and, if provided prior to PKR activation, precludes PKR autophosphorylation without affecting neurovirulence (Cassady et al, 1998a; Cassady et al, 1998b; Mohr & Gluzman, 1996; Mohr et al, 2001).

Wild type HSV-1 evades the immune system through interference of viral antigen presentation to MHC class I and II molecules. The virally encoded protein ICP47 blocks the transporter associated with antigen processing (TAP1 and TAP2; Hill et al, 1995).

Following DNA replication in the cellular nucleus, the late proteins are involved in the formation of the capsid and the receptors on the surface of the virus progeny. Packaging of the viral particles including the genome, core and the capsid occurs in the nucleus of the cell. HSV-1 DNA undergoes a process of primary and secondary envelopment. The primary envelope is acquired by budding into the inner nuclear membrane of the cell. This then fuses with the outer nuclear membrane releasing a naked nucleocapsid into the cytoplasm. Packaged nucleocapsids bud through the Golgi network and the plasma membrane, which are rich in virus glycoproteins targeted to these membranes.

HSV-1 may also enter sensory neurons via their termini and retrograde transport takes the genome to the neuronal nuclei in the sensory ganglia that innervate the infected dermatome. Immediately after infection, virus replication occurs in these neurons but within a few days no virus can be detected. However, the genome persists in a latent state from which it reactivates periodically to resume replication and produce infectious virus, usually at the site of the initial infection. This reactivation event may be spontaneous or provoked by stressful stimuli that act on the neuron, or at a peripheral

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 22 site innervated by the infected ganglion, or systemically. HSV-1 latency consists of three distinct stages. Firstly, establishment of latency occurs immediately following the initial infection. Although virus replication can be detected in a subset of neurons during this phase, the initiation and normal progression of productive infection and cell death is arrested in those neurons destined to become latently infected. However, the point of arrest in neurons is not currently known. Secondly, the maintenance of latency is characterised by the lifelong retention of the HSV genome in a silent state, characterised by repression of all viral lytic genes and absence of viral protein production. One region, encoding the latency-associated transcripts (LATs), remains active during latency and this may encode regulatory RNAs or miRNAs. Quantification of viral DNA load has demonstrated that latently infected neurons contain, on average, 1-100 viral genomes per cell. Thirdly, during the reactivation phase the quiescent viral genome responds to signals that provoke the re-initiation of viral gene expression. The molecular basis for this dramatic functional reversal is poorly understood, but since viral proteins cannot be detected in latently infected neurons, it is generally accepted that cellular mechanisms must be important for reactivation. Reactivation appears to be triggered by illnesses such as colds and influenza, eczema, emotional and physical stress, gastric upset, fatigue or injury, by menstruation and exposure to bright sunlight. It is often symptomatic, but may be asymptomatic (reviewed in Preston & Efstathiou, 2007). c) Information on survival, including seasonability and the ability to form survival structures Wild type HSV-1 survives in the environment as a persistent infection in the host species (humans) or as a latent infection in the nucleus of some infected cells (principally neurons of the trigeminal ganglion), where it may remain inactive indefinitely, or be reactivated giving rise to secretion of virus and sometimes (though not always) clinical symptoms.

HSV-1 does not exhibit seasonality; HSV-1 infections occur worldwide without any specific seasonal distribution (Jerome & Morrow, 2007).

Outside of the host, HSV-1 is an enveloped virus which is sensitive to and rapidly inactivated by both physical inactivation (dehydration, heat, low pH) and disinfectants (lipid solvents and mild detergents). It does not form survival structures and its survival outside the host organism is limited to short periods of time (Chayavichitsilp et al, 2009).

A review by Kramer et al (2006) reports that HSV-1 can survive on dry inanimate surfaces for periods ranging from a few hours to 8 weeks (the latter citing survival on a dried surface, Mahl & Sadler (1975)). However, individual publications report much

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 23 lower survival times under conditions of higher humidity. Nerurkar et al (1983) reports survival times of 4 hours in tap water, and 4.5 hours on plastic surfaces at high humidity. A series of publications by Bardell (1989, 1990, 1993, 1994) report a marked (2-3 log) reduction in viral titer of HSV-1 within 1 hour on plastic surfaces (doorknobs) and chrome plated surfaces (faucet/tap handles), though infectious virus was still recoverable after 2 hours (the maximum timepoint studied). Infectious virus could also be recovered from human skin at least 2 hours after it was introduced. d) Pathogenicity: infectivity, toxigenicity, virulence, allergenicity, carrier (vector) of pathogen, possible vectors, host range including non-target organism. Possible activation of latent viruses (proviruses). Ability to colonise other organisms Humans are the only natural host for HSV-1 infection. Amgen performed a literature search to identify reports on HSV-1 infections in non-human species. The literature search revealed that non-human infection is rare, but identified that HSV-1 infection has been reported in a variety of species including rodents, rabbits, hedgehogs, and non-human primates (Weissenbock et al, 1997; Grest et al, 2002; Huemer et al, 2002; Wohlsein et al, 2002; Allison et al, 2002; Lefaux et al, 2004; Muller et al, 2009; Longa et al, 2011).

Most viruses encode strong transcriptional activators. HSV-1 encodes several of these transactivators which have been shown in tissue culture systems to activate transcription from quiescent viral genomes if present in the same cell as the latent virus. For example, HSV-1 or individual HSV-1 proteins can activate transcription from a number of quiescent viruses, although only in the case of AAV, HSV-1, and HSV-2 has this increase in transcription been shown to result in viral replication or the formation of new infectious particles (Geoffroy et al, 2004; Harris et al, 1989; Russell et al, 1987).

The mode of transmission of wild type HSV-1 is through direct contact with infected secretions or mucous membranes/skin with lesions from an asymptomatic or symptomatic patient shedding the virus (Jerome & Morrow, 2007; Chayavichitsilp, 2009; Whitley, 2006). Transmission of HSV-1 can also occur by respiratory droplets (Whitley, 2006).

The incubation period for orolabial HSV-1 infection is 2 to 12 days, with an average of 4 days (Miller & Dummer, 2007).

During one study of orolabial HSV-1 infection, the median duration of HSV-1 shedding was 60 hours when measured by polymerase chain reaction (PCR) and 48 hours when

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 24 measured by viral culture. Peak viral DNA load occurred at 48 hours, with no virus detected beyond 96 hours of onset of symptoms (Boivin et al, 2006).

HSV-1 also travels to the sensory ganglia, where latency is established. Oral HSV-1 infections reactivate from the trigeminal sensory ganglia, affecting the facial, oral, labial, oropharyngeal, and ocular mucosa (Usatine & Tinitigan, 2010).

Recurrent infections may be precipitated by various stimuli, such as stress, fever, exposure to sunlight, extremes in temperature, ultra-violet radiation, immunosuppression, or trauma. The virus remains dormant for a variable amount of time. On reactivation, generally the duration of symptoms is shorter and the symptoms are less severe (Usatine & Tinitigan, 2010).

Several wild type HSV-1 mediated conditions may occur, as summarised below.

Herpes labialis/cold sores: Primary infections with HSV-1 are acquired usually in childhood and may be asymptomatic or subclinical (Drew, 2004; Jerome & Morrow, 2007; Kimberlin 2005). Symptomatic primary infections present mainly as gingivostomatitis, with fever, sore throat, fetor oris, anorexia, cervical adenopathy, and mucosal edema and vesicular and ulcerative painful lesions involving the buccal mucosa, tongue, gums, and pharynx (Drew, 2004; Jerome & Morrow 2007; Kimberlin, 2005; Miller & Dummer, 2007). Ulcers heal without scarring within 2-3 weeks (Drew, 2004; Jerome & Morrow, 2007). Recurrent infections have generally milder symptoms and clinical course (Jerome & Morrow, 2007). Recurrent lesions due to HSV-1 occur mainly on a specific area of the lip (vermillion border of the lip), and are called “cold sores” or “fever blisters” (Drew, 2004, Kimberlin, 2005). The lesions heal in approximately 8-10 days (Kimberlin, 2005).

Herpetic whitlow: Characterised by formation of painful vesicular lesions on the nail or finger area (Drew, 2004), and more commonly seen in healthcare professionals (eg. dentists).

Infections of the eye: Characteristic dendritic ulceration occurs on conjunctiva, and cornea (Drew, 2004). HSV infection may cause other ocular diseases, including blepharitis/dermatitis, conjunctivitis, dendritic epithelial keratitis, and corneal ulceration (Green & Pavan-Langston, 2006).

Encephalitis: Serious infections of the CNS, affecting both children and adolescents (Whitley, 2006). Encephalitis is a rare complication, affecting approximately 1 in 500,000 people per year (Rozenberg et al, 2011). It may occur due to primary or latent

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 25 infection with HSV-1 virus (Drew, 2004, Whitley, 2006). HSV encephalitis affects one temporal lobe, leading to focal neurologic signs and edema. The disease can be fatal (mortality rate of 70%), if left untreated (Drew, 2004, Whitley, 2006).

Genital herpes: Genital herpes is caused mainly by HSV-2, although HSV-1 has become as common as HSV-2 in primary genital infections in developed countries. It is transmitted sexually through genital-genital or oro-genital contact.

Effects in special populations (neonates and immunocompromised individuals) are discussed below.

Neonatal HSV infection causes significant morbidity and mortality despite significant advances in treatment (reviewed in Kimberlin, 2004; Thompson & Whitley, 2011). The current estimated rate of occurrence of neonatal HSV disease in the United States is approximately 1 in 3,200 deliveries. HSV infections in newborns can be classified into three patterns, which occur with roughly equal frequency. These comprise disseminated disease involving multiple visceral organs, including lungs, liver, adrenal glands, skin, eyes, and the brain; central nervous system (CNS) disease, with or without skin lesions; and disease limited to the skin, eyes, and/or mouth (SEM disease). Patients with disseminated disease and SEM disease present earliest, generally at 10–12 days of life, whereas CNS disease presents during the second or third week of life. Since the advent of antiviral therapy the prognosis of neonatal HSV has improved. Prior to antiviral therapy, 85% of patients with disseminated HSV disease and 50% of patients with CNS disease died within 1 year. With the use of high-dose acyclovir, 12-month mortality has reduced to 29% for disseminated neonatal HSV disease and to 4% for CNS HSV disease (reviewed in Kimberlin, 2004; Thompson & Whitley, 2011). The majority of neonatal HSV infections are caused by HSV-2, but approximately 15 to 30 percent are thought to be caused by HSV-1 Neonatal Herpes Simplex Virus Infections (Rudnick & Hoekzema, 2002).

In the immunocompromisedhost, including those with HIV infection, HSV disease can be particularly severe, resulting in chronic, persistent, active infection in some cases, life-threatening disease (Stewart et al, 1995). Immunosuppressed patients, especially those with impaired T-cell immunity, develop severe lesions that persist longer than those in the normal host; these lesions can progress to visceral disease. As a result of this, almost all examples of serious complications of wild type HSV infections in humans occur in immunocompromised individuals. In these cases, the immune system fails to control the infection, and it becomes disseminated. Susceptible immunocompromised

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 26 individuals include patients receiving cytotoxic therapy, transplant recipients, and patients with human immunodeficiency virus (HIV) (reviewed in Brady & Bernstein, 2004). The emergence of HSV resistance to acyclovir, a phenomenon which is mainly observed among immunocompromised patients due to the long-term treatment they receive, is also a concern. However, no case of encephalitis due to an acyclovir-resistant HSV strain has been reported to date (Rozenberg et al, 2011). e) Antibiotic resistance, and potential use of these antibiotics in humans and domestic organisms for prophylaxis and therapy Antibacterials are not effective in the treatment of HSV-1 infection, nor does the virus present specific resistance to antibacterials. The virus does not contain any gene that confers resistance to antibacterials of interest in terms of health.

Antiviral medicinal products like acyclovir, valacyclovir, and famciclovir can be used to inhibit wild type HSV-1 replication (Drew, 2004; Usatine & Tinitigan, 2010). The standard antiviral drug used against HSV-1 is acyclovir. Inhibition of viral replication by acyclovir depends on the viral thymidine kinase (TK) gene, which catalyzes the first step necessary to convert acyclovir from an inactive to an active form. Valacyclovir and famciclovir can be used to inhibit wild type HSV-1 replication (Usatine & Tinitigan, 2010). In rare cases, HSV can mutate its viral kinases to gain resistance to acyclovir. In these cases, the anti-viral drug Foscarnet (phosphonoformic acid) which does not require activation by viral kinases can be used. Foscarnet directly inhibits the viral DNA polymerase. f) Involvement in environmental processes: primary production, nutrient turnover, decomposition of organic matter, respiration, etc. Wild type HSV-1 is a human pathogen which is not known to be involved in environmental processes. It does not respire and does not contribute to primary production or decomposition processes. In its virion form, it does not display any metabolic activity.

11. Nature of Indigenous Vectors There are no known indigenous vectors of HSV-1, other than human beings. The presence of natural mobile genetic elements such as proviruses, transposons or plasmids related to HSV-1 has not been reported.

12. History of Previous Genetic Modifications The parental strain of HSV-1 used in the construction of talimogene laherparepvec was named JS1. This strain was a new isolate taken from a healthy individual and subsequently banked (ECACC Accession Number 01010209) . There are no known previous genetic modifications of this strain of HSV-1.

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B. CHARACTERISTICS OF THE VECTOR There is no mobile genetic vector in talimogene laherparepvec. Shuttle vectors were used to construct the recombinant virus, and are described below (Section II.C).

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C. CHARACTERISTICS OF THE MODIFIED ORGANISM 1. Information Relating to the Genetic Modification Talimogene laherparepvec is an attenuated replication competent HSV-1 that has been developed for the treatment of solid tumours. The intended therapeutic strategy is to produce a direct oncolytic effect by replication of the virus within the tumour, and induction of an anti-tumour immune response, enhanced by the local expression of hGM-CSF.

The clinical effects are intended to include the destruction of injected tumours, the destruction of local locoregional and distant un-injected tumours, a reduction in the development of new metastases, a reduction in the rate of overall progression and of the relapse rate following the treatment of initially present disease, and prolonged overall survival of patients. a) Methods used for the modification The HSV-1 genome (Figure 2) consists of linear, double-stranded DNA that is divided into two components: L (long) and S (short) (Wadsworth et al, 1975). Each component contains a unique region [long unique region (UL) and short unique region (US)] flanked by inverted repeat regions, both internally [long internal repeated sequence (IRL) and short internal repeated sequence (IRS)] and at the termini [long terminal repeated sequence (TRL) and short terminal repeated sequence (TRS)] (Wadsworth et al, 1976). The ICP47 gene is situated in the US region and the ICP34.5 gene is situated in the long inverted repeats (IRL and TRL). Therefore, there are two copies of the ICP34.5 gene in the HSV-1 genome.

Figure 2. Structure of HSV-1 Genome

Features of HSV-1 include: long unique region (UL) and short unique region (US); long internal repeated sequence (IRL) and short internal repeated sequence (IRS); long terminal repeated sequence (TRL) and short terminal repeated sequence (TRS); two copies of the ICP34.5 gene; one copy of the ICP47 gene; and the viral gene US11 that has some functional redundancy with ICP34.5 (Mohr and Gluzman, 1996).

Talimogene laherparepvec was generated by modifying the wild type HSV-1 genome (new isolate JS1) to functionally delete both copies of ICP34.5 and the ICP47 gene from the viral backbone and insert an expression cassette encoding the human GM-CSF gene in both ICP34.5 regions.

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The modification method used was a sequential series of in vivo homologous recombination events on cells in culture co-transfected with non-enveloped viral DNA and appropriate shuttle vectors.

New Isolate HSV Type 1 Strain JS1 The virus was isolated by taking a swab from a cold sore of an otherwise healthy volunteer, and serially expanding in BHK cells (baby hamster kidney fibroblasts, ECACC no. 85011433). Virus strain JS1 was confirmed to be HSV-1 by an HSV-1 specific culture test. JS1 itself has not been sequenced, but restriction profiling of strain JS1 compared to HSV-1 strain 17syn+ and HSV-2 strain HG52 has been conducted. The restriction profile for JS1 was very similar to HSV-1 strain 17syn+ and vastly different to those of HSV-2 strain HG52.

Growth characteristics of JS1 on various cell types were determined and compared to the laboratory strain 17syn+ obtained from University of Glasgow, UK. Whilst growing to only slightly higher titer, the JS1 virus was shown to kill the various tumour cell lines tested more rapidly and at a lower dose than 17syn+ (Liu et al, 2003a).

Human GM-CSF Gene The gene was cloned from an IMAGE clone obtained from the Human Genome Mapping Project (HGMP) Resource Centre, UK.

The Producer Cell Line Virus construction was performed using the BHK cell line. b) Methods used to construct and introduce the insert(s) into the recipient or to delete a sequence; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

The construction of talimogene laherparepvec (JS1/ICP34.5-/ICP47-/hGM-CSF) is described briefly in Liu et al (2003a).

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 30 c) Description of the insert and/or vector construction; The neurovirulence factor ICP34.5 and the ICP47-encoding genes are functionally deleted from the virus. The hGM-CSF expression cassette is inserted into the two sites of ICP34.5 deletion. The genome of the talimogene laherparepvec is presented schematically in Figure 3.

Figure 3. Schematic Representation of Talimogene Laherparepvec Genome

d) Purity of the insert from any unknown sequence and information on the degree to which the inserted sequence is limited to the DNA required to perform the intended function; The modification in the final product (ie, insertion of hGM-CSF gene) is confirmed by performing hGM-CSF ELISA (Study 1182-00009, BioVex, 2011). Further details on construction and characterization results, which confirm the modification, are on file (Study 4647-00041).

The region of talimogene laherparepvec viral DNA containing the insert (hCMV IE promoter-hGM-CSF-BGHpA) has been completely sequenced at least 3 times between 2001 and 2012 and found to only contain the intended DNA. The cassette has also been shown to be genetically stable (see Section II.C.2(c)). e) Methods and criteria used for selection; Selection of recombinants at each stage was determined by gain or loss of the reporter gene GFP by homologous recombination between the shuttle plasmid and the viral genome. Recombinants were plaque purified at least 3 times after each homologous recombination step. f) Sequence, functional identity and location of the altered/inserted/deleted nucleic acid segment(s) in question with particular reference to any known harmful sequence. Sequence coverage across the talimogene laherparepvec genome has been obtained. The region of talimogene laherparepvec viral DNA containing the insert (hCMV IE promoter-hGM-CSF-BGHpA) has been completely sequenced at least 3 times between 2001 and 2012 and found to only contain the intended DNA.

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Functional deletion of ICP34.5, addition of hGM-CSF, and deletion of ICP47 were confirmed by both Southern blotting and sequencing. To show that talimogene laherparepvec expressed hGM-CSF, several clones generated during the construction of talimogene laherparepvec were used to infect individual wells of BHK cells. The infected cells were incubated for 24 hours and the supernatant was then harvested and assayed for hGM-CSF using a commercially available ELISA kit. All clones produced hGM-CSF and one clone was selected at random for further amplification. In order to determine that the hGM-CSF produced by talimogene laherparepvec was functional, a cell proliferation assay was performed using TF-1 cells, which are growth dependent on hGM-CSF (Kitamura et al, 1989). Addition of the hGM-CSF supernatant to TF-1 cells caused proliferation in a dose-dependent manner indicating that the hGM-CSF produced by the selected clone of talimogene laherparepvec was active.

There are no known harmful DNA sequences introduced in the construction process. The potential toxicity of the intended insertion (hGM-CSF) is discussed in Section II.C.2(i)1.

2. Information on the Final GMO a) Description of genetic trait(s) or phenotypic characteristics and in particular any new traits and characteristics which may be expressed or no longer expressed; Talimogene laherparepvec is based on a newly isolated strain of HSV-1 that has been engineered to replicate selectively in tumours, killing tumour cells by viral lysis, followed by spread of talimogene laherparepvec within the tumour and further tumour cell lysis. Additionally, the oncolysis of tumour cells by talimogene laherparepvec also releases and exposes an array of antigens to initiate a systemic immune response, and this is augmented through the expression of an immune stimulatory protein, hGM-CSF from the virus (Study 4647-00041). Released tumour antigens are expected to be taken up by antigen presenting cells (APCs) which then traffic to lymph nodes and present to T cells, inducing an immune response. hGM-CSF increases the activity of APCs, enhancing the immune responses. This immune response is intended to provide a systemic anti-tumour effect, including the shrinkage of tumours which do not come into direct contact with talimogene laherparepvec, reduction of micrometastatic disease, and protection against future relapse.

The key features of talimogene laherparepvec are:

 HSV-1 is a non-integrating virus, so treatment with talimogene laherparepvec does not result in any change to the patient’s DNA.

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 Talimogene laherparepvec is based on a newly isolated strain of HSV-1 (strain JS1) that has been shown to kill human tumour cells more effectively than laboratory strains of HSV-1 (Liu et al, 2003a).  The HSV-1 protein ICP34.5 normally promotes neurovirulence by overcoming host defence pathways and allowing the virus to replicate in non-dividing cells such as neurons (see Section II.A.10(b)). Both copies of ICP34.5 are functionally deleted from talimogene laherparepvec, preventing the virus from replicating efficiently in non-dividing cells. In tumour cells, these host defence pathways are often impaired, so ICP34.5 is dispensable for replication (Aita et al, 1999; Farassati et al, 2001; Liang et al, 1999).  The HSV-1 protein ICP47 normally inhibits antigen processing in infected cells, allowing the virus to hide from the immune system (Hill et al, 1995). ICP47 is deleted from talimogene laherparepvec in order to improve the presentation of viral and tumour antigens following tumour-selective virus replication, enhancing any anti-tumour immune response (Liu et al, 2003a).  Removal of ICP47 from talimogene laherparepvec causes the increased expression of another viral protein, US11 that has some functional redundancy with ICP34.5 (Mohr and Gluzman, 1996). Increased US11 expression enhances the replication of ICP34.5-deleted HSV-1 in tumour cells without loss of tumour selectivity (Mohr et al, 2001).  Talimogene laherparepvec expresses the immune stimulatory protein hGM-CSF to augment the immune response to released tumour antigens by aiding the differentiation and proliferation of dendritic cell precursors in and around the injected tumour (Dranoff et al, 1993).  The HSV thymidine kinase (TK) gene remains intact which renders talimogene laherparepvec susceptible to anti-viral agents such as acyclovir. Therefore, acyclovir could be used to block replication of talimogene laherparepvec. b) Structure and amount of any vector and/or donor nucleic acid remaining in the final construction of the modified organism; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

There are no known harmful DNA sequences introduced unintentionally in the construction process, as verified by genome sequencing. c) Stability of the organism in terms of genetic traits; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Generation of wild type during manufacture is not possible as none of the gene deletions in talimogene laherparepvec require complementation for growth in tissue culture. This means that the cells used to manufacture talimogene laherparepvec do not contain the DNA sequences encoding the deleted genes, preventing repair of the mutations during

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 33 virus production. Each batch of the medicinal product is strictly controlled throughout production and rigorously tested to confirm its identity prior to release.

The genetic stability of talimogene laherparepvec is expected to be the same as wild type HSV-1. ie. stable in isolation but with the potential for homologous recombination with other HSV-1 viruses if they simultaneously infect the same (human) cell (see Section II.A.9).

Genetic Stability in Isolation The genetic stability of talimogene laherparepvec in isolation (ie. in the absence of a co-infecting different strain of HSV-1) has been demonstrated and continues to be monitored. Genetic stability testing has been achieved through repeat sequencing of small areas of the talimogene laherparepvec genome from 2001 to 2012.

Potential for Recombination with Wild Type-HSV-1 A spontaneously occurring genetic variant of talimogene laherparepvec would require an initial recombination event(s) leading to the creation of the genetic variant itself. It is unlikely that a wild type virus would be in the same tissue as talimogene laherparepvec in subjects since the latter is directly injected into tumour cells and cannot spread effectively into normal tissue, while the pre-existing HSV-1 would be in the mucosal tissues or neuronal ganglia of the patient. The possibility of the creation of stable genetic variants with unintended characteristics is also minimised by the design of the talimogene laherparepvec genetic construct.

Experimentally, non-homologous recombination (recombination between different regions of two viral genomes) has been shown not to occur at detectable levels between replication incompetent and replication competent viruses (Smith et al, 2003). The possible products of homologous recombination between talimogene laherparepvec and wild type HSV-1 are summarised in Table 2 and discussed below.

In the unlikely event that homologous recombination between wild type HSV-1 and talimogene laherparepvec occurred and was in the region of ICP34.5, the insertion site of the hGM-CSF cassette (which is in place of ICP34.5) dictates that the transfer of the hGM-CSF gene would result in functional deletion of the ICP34.5 gene of the wild type HSV-1 during the recombination process. The result of this recombination event would therefore be a wild type virus (ICP47+) expressing hGM-CSF but functionally deleted for ICP34.5 and therefore only capable of tumour-selective replication. Expression of US11 would be equivalent to wild type since the natural US11 regulatory regions are located within ICP47. The variant of talimogene laherparepvec simultaneously produced in this

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 34 scenario would have ICP34.5 restored but would not encode or express hGM-CSF. ICP47 (and US11 regulatory regions located within it) would remain deleted, such that US11 expression remained upregulated. Recombinant viruses that contain the US11 upregulation mutation in a wild-type ICP34.5 genetic background have been generated (Mohr et al, 2001). These data demonstrated that deletion of the ICP34.5 gene is completely responsible for the attenuated phenotype of the US11 upregulated/ICP34.5 deleted virus and there is little contribution of US11 upregulation in the presence of ICP34.5. This suggests that should ICP47 be deleted in the context of an ICP34.5 positive virus, the contribution of the US11 upregulation to replication of such a virus would be negligible. In terms of replication competency, the resulting viruses would therefore be the same as the starting materials.

In the unlikely event that homologous recombination between wild type HSV-1 and talimogene laherparepvec occurred in the region of ICP47, a version of talimogene laherparepvec containing hGM-CSF in place of both copies of ICP34.5 but with a repaired copy of ICP47 could be produced. As ICP47 deletion does not significantly affect replication in non-tumour cells, this virus would be expected to be similarly impaired to talimogene laherparepvec, however, the virus would be less able to replicate in tumour cells, as the recombination event would replace the natural US11 regulatory regions (which are located within ICP47). The variant of wild-type HSV-1 simultaneously produced in this scenario would be deleted for ICP47 (and US11 regulatory regions located within it) and therefore much less able to block antigen presentation and evade the immune system. Indeed, a virus lacking ICP47 but still expressing US11 was found to be equivalent to wild type in terms of replication and virulence at the inoculation site and surrounding skin. However, the virus lacking ICP47 was less neurovirulent than wild type following corneal injection, and this was found to be due to an inability to inhibit a protective CD8+ T cell response (Goldsmith et al, 1998).

Homologous recombination at both ICP34.5 and ICP47 regions would result in either wild-type HSV-1 or talimogene laherparepvec.

It is theoretically possible that individual recombinant virions containing DNA with one copy of ICP34.5 and one copy of GM-CSF could be generated (since ICP34.5 is present in two copies in the wild type HSV-1 genome, one in each of the long repeat regions). While heterozygosity within the repeat regions has been observed, these heterozygote species are not stable and revert to homozygotes at a high frequency.

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One example of this is with the gene encoding ICP4, located in the short repeat region of HSV-1. ICP4 is an immediate-early polypeptide essential for the expression of delayed-early and late viral genes. Temperature sensitive mutations affecting this diploid gene, which are necessarily present in both copies of the gene, are recovered at a reasonable frequency (Schaffer et al, 1978). As it is extremely improbable that both copies of ICP4 acquired the same mutation independently, this observation suggests that a mechanism exists which generates mutant homozygotes from heterozygotes at a high frequency. This was confirmed by propagating virus stocks descended from a heterozygotic repeat mutant of HSV-1 (where the two inverted repeats flanking the short segment of viral DNA differed in length by approximately 60 base pairs). It was observed that the progeny of this heterozygote contained approximately 1/3 heterozygotes and 2/3 homozygotes after growth from 1 to 107 PFU, confirming that a mechanism exists which generates homozygotes from heterozygotes at a high frequency (Varmuza & Smiley, 1984). The heterozygote can therefore be viewed as an intermediate in a chain of events starting with the introduction of a change in one of the diploid sequences, followed by later acquisition of this change in the other diploid sequence. This instability is likely due to the ongoing recombination between the HSV repeat regions within that individual viral genome and those of the other virions which initiated the infection, since more than one virion is needed for productive replication to proceed. Further evidence that heterozygotic repeat viruses do not persist within a population comes from analysis of ICP4 deletion mutants isolated on a cell line providing ICP4 in trans. These mutants all contain the same deletion within both copies of ICP4 and are therefore homozygotes (DeLuca et al, 1985).

The instability of heterozygotic ICP34.5 regions was investigated when two different strains of HSV-1 were used to generate a heterozygous virus containing different long repeat regions (where ICP34.5 is located). The resultant heterozygotic virus segregated to both classes of homozygotes equally and at a high frequency (Umene, 1987). In order to artificially assess the virulence of virus containing a single copy of ICP34.5, the gene was cloned under the control of a different promoter into a unique region of an ICP34.5 deleted virus (Holman & MacLean, 2008). This virus, despite expressing more ICP34.5 than wild type virus, did not have its virulence restored to wild type levels.

Oncolytic viruses expressing only one copy of ICP34.5 have been artificially generated. These viruses are only able to stably exist due to extensive genomic deletions that prevent homologous recombination. For example, in the oncolytic virus NV1020, the joint region of the long (L) and short (S) regions is deleted, meaning one copy of ICP34.5

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 36 is lost (Meignier et al, 1988). The deleted region was replaced with a fragment of HSV-2 DNA from the unique short region. This fragment does not contain any sequences from the deleted repeat region and therefore does not provide a template for homologous recombination.

The theoretical stable genetic variants which may be created by homologous recombination between talimogene laherparepvec and wild-type HSV-1 are summarised in Table 2.

Table 2. Theoretical Stable Genetic Variants of Talimogene Laherparepvec Created by Homologous Recombination

US11 Gene ICP34.5 GM-CSF ICP47 Upregulation Function Virulence Immune Immune Increases stimulation evasion replication of ICP34.5 null viruses in tumours Genetic variant Talimogene laherparepvec -/- +/+ - + ICP47 restoration -/- +/+ + - Homozygous ICP34.5 restoration +/+ -/- - + ICP47 restoration and homozygous +/+ -/- + - ICP34.5 restoration (wild type HSV-1) d) Rate and level of expression of the new genetic material. Method and sensitivity of measurement; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Expression is expected to be greatest where the virus replicates (ie. infected tumour cells). e) Activity of the expressed protein(s); A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

The activity of the virally expressed hGM-CSF in humans is unknown. In clinical trials, no dose limiting toxicity attributed to hGM-CSF has been observed when talimogene laherparepvec has been administered to tumour bearing patients.

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 37 f) Description of identification and detection techniques including techniques for the identification and detection of the inserted sequence and vector; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

The same techniques used to detect the parental organism can be used to detect talimogene laherparepvec.

Viral culture (plaque assay; Dulbecco, 1952) is routinely used to detect talimogene laherparepvec in swabs or other samples, although this assay only detects live virus and does not discriminate between talimogene laherparepvec and wild type HSV-1.

A qPCR assay has been developed which can be used to detect talimogene laherparepvec specifically. The qPCR assay does not detect whether the talimogene laherparepvec detected is viable, but it does distinguish between talimogene laherparepvec and wild type HSV-1. Wild type HSV-1 is not detected by this test as the primers for the PCR amplification are within the CMV-hGM-CSF-BGHpA expression cassette inserted in place of ICP34.5 in talimogene laherparepvec. The qPCR assay allows highly sensitive and specific detection of DNA sequences as well as quantification of the target sequence. g) Sensitivity, reliability (in quantitative terms) and specificity of detection and identification techniques; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities. h) History of previous releases or uses of the GMO; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Fifteen clinical studies have been or are being conducted in several advanced tumour types (advanced solid tumours, melanoma, squamous cell cancer of the head and neck (SCCHN), and pancreatic cancer) with a total of 486 patients treated with talimogene laherparepvec as of 22 September 2015 i) Considerations for human health and animal health, as well as plant health: 1. Toxic or Allergenic Effects of the GMOs and/or Their Metabolic Products A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

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Virulence and Toxicity of the Virus The functional deletion of ICP34.5 in talimogene laherparepvec significantly decreases virulence compared to wild type HSV-1. Talimogene laherparepvec is therefore able to replicate in tumour cells but is significantly attenuated in normal cells. Virus mediated toxicity is therefore likely to be minimal.

Strains of HSV-1 lacking ICP34.5 have been extensively utilised without incident in a variety of animal models and also in several human clinical trials as detailed below.

In any eventuality, toxicity would be no greater than that observed for wild-type HSV-1 (see Section II.A.10 (d)) and talimogene laherparepvec remains sensitive to anti-viral agents such as acyclovir (see Section II.C.2(i).2).

Non-clinical Safety Data Obtained with HSV-1 ICP34.5 Null Mutants Intracerebral inoculation of mice has been used to investigate the degree of attenuation conferred by the deletion of ICP34.5. Estimates range from 25 to 90 fold more attenuated than wild type (Bolovan et al, 1994; although the mutant used in this reference contained an inserted stop codon in the gene encoding ICP34.5, such that some level of read-through may have occurred) to an impairment of over 100,000 fold (Chou et al, 1990; MacLean et al, 1991).

Based on the observations from animal studies, ICP34.5 null viruses are anticipated to have enhanced safety over wild-type HSV-1 strains in therapy of a number of human tumours (Chambers et al, 1995; Kesari et al, 1998; Martuza et al, 1991; Miller et al, 2001; Thomas & Fraser, 2003). It has been demonstrated that ICP34.5 deleted HSV-1 is unable to replicate or cause disease in non-dividing cells (Brown et al, 1994b) and is attenuated for causing encephalitis (Whitley et al, 1993). Additionally, ICP34.5 deleted HSV-1 may have reduced virulence in certain immune-deficient mice (Valyi-Nagy et al, 1994); this issue is discussed in further detail in “Safety in immunodeficient mice”, below. Furthermore, the safety of ICP34.5 deleted viruses has also been demonstrated in a variety of species, including mice (McKie et al, 1998), rabbits (Perng et al, 1995) and non-human primates (Hunter et al, 1999; Varghese et al 2001).

Non-clinical Safety Data Obtained with Talimogene Laherparepvec A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Clinical Safety Data Obtained with HSV-1 ICP34.5 Null Mutants Several strains of HSV-1 lacking ICP34.5 have been extensively utilised without incident in several published human clinical trials.

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No patients in any of these clinical studies developed HSV encephalitis or required treatment with acyclovir. Using viruses deleted only for ICP34.5, no toxicity was observed following i.t. administration of up to 105 PFU of virus into 21 patients with malignant glioma (Papanastassiou et al, 2002; Rampling et al, 2000) and 5 patients with metastatic melanoma (MacKie et al, 2001). None of the patients in these studies experienced any adverse symptoms attributable to virus administration. The same virus was later injected into the resection cavity rim in 12 patients with surgically treated glioma (Harrow et al, 2004), or intratumourally in 20 patients with oral squamous cell carcinoma (Mace et al, 2008). Again, there was no clinical evidence of toxicity associated with the virus administration in any of these patients.

There have also been several clinical trials with several related ICP34.5 null versions of HSV. One of these viruses, G207, also has the gene encoding ribonucleotide reductase deleted so results are not directly comparable to an ICP34.5 null virus. Nonetheless, up to 109 PFU of G207 has also been found to be safe in 27 patients with malignant glioma (Markert et al, 2000; Markert et al, 2009).

In addition to the functional deletion of ICP34.5, talimogene laherparepvec is deleted for ICP47 and has a hGM-CSF expression cassette inserted in place of ICP34.5. As described above, the hGM-CSF expression cassette is an immune stimulatory modification and will therefore not affect virulence or further attenuate the virus. Deletion of ICP47 indirectly affects the ability of the virus to replicate in tumour cells, but does not reverse the attenuation. This distinction is possible because in addition to allowing antigen presentation to occur in infected cells, deletion of ICP47 causes upregulation of the neighboring gene, US11. US11 is normally regulated as a late gene, but deletion of ICP47 places US11 under the control of the ICP47 regulatory sequences, causing it to be expressed as an immediate early gene (Mohr & Gluzman, 1996). This is an important aspect of the mechanism of action of talimogene laherparepvec, as US11 upregulation allows talimogene laherparepvec to replicate more efficiently in tumours. However, it has been proven that upregulation of US11 in the background of an ICP34.5 deleted virus does not affect virulence in non-tumour tissue – in immune competent or immune compromised mice an ICP34.5 null virus with US11 upregulated is equally attenuated as an ICP34.5 null virus alone. In one study examining this issue, 100% of immune compromised mice that received 106 PFU of wild type HSV-1 virus were dead by 8 days post intraperitoneal injection. In contrast, the ICP34.5 null virus with US11 upregulated was indistinguishable from the ICP34.5 null virus, with 100% of the mice surviving administration of 106 PFU of either virus for at least 21 days post injection (Mohr et al, 2001).

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Clinical Safety Data Obtained with Talimogene Laherparepvec A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

At the time of the study-specific data cutoff dates for the IB Edition 13, dated 22 September 2015, 486 subjects have received talimogene laherparepvec and have provided safety data across 15 studies. Amgen has published clinical data from a number of these including safety data on 292 subjects with unresected stage IIIB to IV melanoma, in the Phase 3 study (005/05) (Andtbacka et al 2015), 30 subjects with relapsed disease from various solid tumour cancers (Hu et al, 2006), 50 subjects with metastatic melanoma (Senzer et al, 2009) and 17 subjects with squamous cell cancer of the head and neck (Harrington et al, 2010). No subjects in any of these studies or any of the on-going studies have developed HSV encephalitis. Overall, most adverse events reported in subjects administered talimogene laherparepvec are non-serious and primarily include flu-like symptoms and injection site reactions. Most fatal adverse events reported in subjects administered talimogene laherparepvec were reported in the setting of disease progression. The concurrent use of talimogene laherparepvec, cisplatin, and external beam radiation therapy, in the 17 subjects with squamous cell cancer of the head and neck (Harrington et al, 2010), did not result in more frequent or more severe adverse events beyond those typically encountered with these other anti-cancer therapies alone.

Further details of the safety profile of talimogene laherparepvec are provided in the current Investigator's Brochure.

Potential to Cause Encephalitis To thoroughly assess the risk of HSV encephalitis, studies have been performed to assess the ability of ICP34.5 null viruses to replicate in ependymal cells, which are dividing cells that line the ventricles of the brain, but are non-tumour in origin. Despite still being greatly attenuated compared to wild type virus, ICP34.5 null viruses can replicate to a low level in ependymal cells (Kesari et al, 1998; Markovitz et al, 1997; Mehta et al, 2010). Exposure of these particular cell types to virus would only occur if the virus leaked or was inadvertently inoculated into the cerebral ventricle, so is highly unlikely to occur as a result of i.t injection. However to assess the potential consequences of this, a high dose of ICP34.5 null virus (G207, in which ribonucleotide reductase is also deleted) was injected into the cerebral ventricles of mice. 100% of mice injected with the deleted virus survived for over 20 weeks with no apparent symptoms of disease, where as 80% of mice injected with 10,000 times less wild type

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 41 virus died within 10 days (Sundaresan et al, 2000). Furthermore, in one human clinical using the same virus, G207, for malignant gliomas an inadvertent protocol deviation resulted in inoculation of virus into the cerebral ventricle rather than directly into the glioma for one clinical trial subject (Markert et al, 2009). This patient required treatment with dexamethasone, but an MRI found no changes indicative of encephalitis, and acyclovir was not required.

Potential Toxicity of the Inserted Gene The only gene inserted into talimogene laherparepvec is hGM-CSF, the product of which is not a known toxin and is a United States (US) Food and Drug Administration (FDA) approved pharmaceutical. hGM-CSF is also a naturally occurring, endogenous and well-characterised protein that stimulates the production and maturation of macrophages and dendritic cells. The expression of hGM-CSF mediated by talimogene laherparepvec is intended to stimulate immune cells to attack tumour cells and display tumour antigens, thereby alerting the immune system and resulting in tumour-specific immunity (Liu et al, 2003b).

Administration of hGM-CSF has been extensively tested and found to be safe in a variety of species (Baiocchi et al, 2001; Davis et al, 1990; Liu et al, 2003a; Liu et al, 2003b; Nemunaitis et al, 1991; Rowe et al, 1995; Soiffer et al, 2003; Wang et al, 2002). Human GM-CSF has also been widely used in several clinical trials and has been found to be safe and effective against several malignancies (Bendandi et al, 1999; Daud et al, 2008; Jager et al, 1996; Sato et al, 2008; Schmittel et al, 1999; Spitler et al, 2009). Most recently, a clinical trial involving over 800 patients concluded that hGM-CSF was safe and effective, as measured by disease free survival of patients with resected high-risk melanoma (Lawson et al, 2010).

Recombinant human GM-CSF (Leukine; sargramostim) was approved by the FDA in 1991, and again in a different formulation in 1996, for stimulation of white blood cell recovery following chemotherapy or bone marrow transplantation. The doses of hGM-CSF delivered by this therapy are typically much higher than would be expected to be generated following treatment with talimogene laherparepvec. Adverse effects resulting from intravenous infusion or subcutaneous injection of hGM-CSF in humans are usually mild, consisting primarily of fever, muscle pain, and injection site reaction. Provenge®, autologous peripheral blood mononuclear cells activated with PAP-GM-CSF (sipuleucel-T) for use in ex-vivo maturation and loading of dendritic cells with prostate-specific antigens for use in prostate cancer patients, was granted marketing

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 42 authorisation in the EU on 6 September 2013. On 6 May 2015, the European Commission withdrew the marketing authorisation for Provenge® at the request of the marketing authorisation holder, Dendreon UK Ltd, for commercial reasons. Provenge® has also been approved in the USA since April 2010.

Recently, EmbryoGen®, a medium for in vitro fertilization containing GM-CSF was found to aid embryo implantation in women who are at high risk for miscarriage. EmbryoGen® was CE-marked in the European Union in 2011 and received FDA 510k clearance in late 2012.

Potential Oncogenicity of the Inserted Gene hGM-CSF (Leukine®; sargramostim) is a US licensed pharmaceutical product that is in routine clinical use in the US. GM-CSF is not a known oncogene, but it does inhibit apoptosis and induce proliferation of specific short-lived target cells which express the hGM-CSF receptor. When certain cells that do not normally express the hGM-CSF receptor were engineered to overexpress it, and then exposed to hGM-CSF, some of the cells underwent transformation (Areces et al, 1993; Lang et al, 1985). However, in these studies the recipient cells were already immortalised, suggesting that they had already undergone at least one major step in the transformation process. hGM-CSF has been found to be well tolerated clinically (see above). However, in one trial assessing long-term treatment with hGM-CSF, two out of 98 subjects developed acute myelogenous leukemia (AML) (Spitler et al, 2009). It is important to note that these patients were treated with much higher doses of hGM-CSF than would be expressed from talimogene laherparepvec. Furthermore, it is unclear from this study whether these cases resulted from a hGM-CSF induced uncontrolled growth of myeloblasts or pre-existing conditions (one of the two patients had a chromosomal translocation and the other was a sheet metal worker with heavy industrial exposure to carcinogens). No cases of AML have been reported in patients treated with talimogene laherparepvec in clinical trials to date.

2. Comparison of the modified organism to the donor, recipient or (where appropriate) parental organism regarding pathogenicity; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Virulence and Toxicity of the Virus The virulence and toxicity of talimogene laherparepvec is highly attenuated by the functional deletion of both copies of the neurovirulence factor, ICP34.5.

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Strains of HSV-1 lacking ICP34.5 have been extensively utilised without incident in a variety of animal models and also in several human clinical trials as detailed in Section II.C.2 (i) 1.

The inserted gene, hGM-CSF, has no effect on virulence. The potential effects of hGM-CSF expression from talimogene laherparepvec are also discussed in detail in Section II.C.2 (i) 1.

Susceptibility to Antiviral Agents The gene deletions in talimogene laherparepvec do not affect the functioning of the virally encoded thymidine kinase (TK), and the TK gene in talimogene laherparepvec has been repeatedly sequenced and found to be very well conserved. Unsurprisingly, therefore, talimogene laherparepvec is equally susceptible to acyclovir as the unmodified parental strain of HSV-1 (see Part II.A.10 (d)).

Potential for Integration of Viral DNA into the Host Genome Wild type HSV-1 DNA circularises as an extrachromosomal episome, and is not integrated into the host cell genome (see Section II.A.4). The ability of the HSV-1 genome to circularise does not require the viral terminal repeat sequences, synthesis of any viral proteins, or viral replication. The efficiency and kinetics of talimogene laherparepvec circularization would therefore be expected to be the same as wild type HSV-1, with no integration into the host cell genome.

Potential to Establish Latency/Reactivation or Reactivate Existing Viral Infections It was initially thought that viruses lacking ICP34.5 were able to establish latency but were deficient for reactivation (Perng et al, 1995; Robertson et al, 1992; Spivack et al, 1995). However, further work revealed that this apparent difference was actually due to impaired levels of establishment of latency, likely due to deficient replication of the ICP34.5 deleted virus in the peripheral tissues innervated by the neurons. Wild type levels of reactivation can be achieved from an ICP34.5 deleted virus if 1000 times more virus than wild type is used (Perng et al, 1996). In these experiments, the ICP34.5 null virus was still avirulent, even at the 1000-fold higher infectious dose, indicating that latency/reactivation and virulence are separable.

Thus, none of the genetic modifications made to talimogene laherparepvec prevent it from entering latency or, subsequently, to reactivate. However, because ICP34.5 deletion markedly reduces the ability of the virus to replicate in normal tissues in general, and also greatly reduces neurovirulence, should talimogene laherparepvec enter latency in nerve cells and then reactivate, clinical signs/symptoms of viral infection would not be

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 44 expected. For any clinical signs or symptoms to be seen, replication in normal host tissue (ie, non-tumour tissue) would be required. A review of available biodistribution/toxicity studies confirmed low persistence of virus in tissues, including central nervous system (CNS), and no clinical signs or histopathology indicative of replication in normal tissues or neurovirulence associated with talimogene laherparepvec was reported in any study.

Therefore, while talimogene laherparepvec may be able to establish a latent infection, there would be no expected evidence or consequence of any potential reactivation since efficient replication is needed for symptoms to be apparent. In the 427 patients dosed so far there have been no clinical cases documenting latent viral infection of talimogene laherparepvec.

3. Capacity for colonisation; A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Replicative Capacity of Talimogene Laherparepvec Talimogene laherparepvec an attenuated version of HSV-1, modified so that replication occurs selectively in tumour cells. The potential for recombination with wild type HSV-1 is low, as this could only occur if both strains were present and replicating in the same cell, which is unlikely in patients following i.t administration. If this was to happen, however, and homologous recombination occurred, any resulting virus would itself be expected to be less pathogenic than the wild type HSV-1 due to the deletion of the neurovirulence factor (see Section II.C.2 (c)).

Conclusions Based on the low level of virus shedding observed and the attenuated nature of talimogene laherparepvec, the likelihood for persistent or systemic infection of personal contacts of the patients and health care personnel is considered low. If this were to occur it is not expected that any significant clinical manifestations would be evident as effects in non-tumour tissue in talimogene laherparepvec treated patients have not been noted.

Further, talimogene laherparepvec is known to be susceptible to acyclovir, and any unexpected clinical side effects thought to be caused by talimogene laherparepvec replication would be anticipated to respond to treatment with the typical clinical dosing regimen of acyclovir (see Section II.C.2.(i) 2).

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4. If the organism is pathogenic to humans who are immunocompetent: Wild type HSV-1 is a universally prevalent virus that rarely causes severe problems (see Section II.A.10 (d)). Talimogene laherparepvec an attenuated version of HSV-1, modified so that replication occurs selectively in tumour cells. Because the virus is attenuated in normal cells, no adverse effects would be expected for immunocompetent individuals who i inadvertently come in contact with it. Considerable literature shows that HSV-1 deleted for ICP34.5 has been extensively utilized without incident in animals and humans (see Section II.C.2 (i) 1).

5. Other product hazards. None known or anticipated.

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III. INFORMATION RELATING TO THE CONDITIONS OF RELEASE AND THE RECEIVING ENVIRONMENT A. INFORMATION ON THE RELEASE 1. Description of the Proposed Deliberate Release, Including the Purpose(S) and Foreseen Products The purpose of the release is to conduct a phase 1, multicenter, open-label, single-arm study to evaluate the safety of the investigational medicinal product when injected into liver tumours (Protocol 20140318).

Talimogene laherparepvec is intended for intrahepatic injection into hepatocellular carcinoma (HCC) and metastatic liver tumors (non-HCC) by a trained medical professional in a medical study site facility.

2. Foreseen Dates of the Release and Time Planning of the Experiment Including Frequency and Duration Of Releases A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

The use of talimogene laherparepvec will commence as soon as possible following receipt of the necessary local approvals.

Investigators are encouraged to use the maximum amount whenever lesions allow.

3. Preparation of the Site Previous to the Release All principal investigators and sub-investigators participating in the study will be qualified by education, training and experience to assume responsibility for the proper conduct of the trial according to the guidelines outlined in International Conference on Harmonisation (ICH) E6 - Good Clinical Practices.

Participating study sites will be thoroughly evaluated prior to the initiation of the trial to ensure that the facilities are sufficient for storing and administering talimogene laherparepvec, as well as having the appropriate facilities for the collection and storage of human specimens. Additionally, all clinical site personnel involved in the handling or administration of talimogene laherparepvec will be trained according to the study protocol, and all supportive documentation, including the IPIM. A thorough study-specific training will occur prior to the initiation of the study via a formal investigator meeting and/or on-site study initiation visit.

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4. Size of the Site The size of each site will vary but it is important to note that contamination of the site at which the administration is performed is expected to be minimal, when suitable precautions are adhered to.

5. Method(s) to be Used for the Release Talimogene laherparepvec is intended for intrahepatic injection into hepatocellular carcinoma (HCC) and metastatic liver tumors (non-HCC) by a trained medical professional in a medical study site facility. Injections will be performed using the coaxial injection technique under ultrasound or CT guidance. A needle of larger diameter than the talimogene laherparepvec dosing syringe needle (introducer needle) will first be inserted into the lesion. Talimogene laherparepvec will be administered via a filled dosing syringe through the introducer needle.

6. Quantities of GMOs to be Released A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Vials containing 1.15 mL of talimogene laherparepvec will be supplied to study site pharmacies in two strengths; 106 PFU/mL or 108 PFU/mL.

The maximum volume of talimogene laherparepvec administered at any dose is 4.0 mL or 8.0 mL for any individual lesion. The maximum dose in any one treatment is 8.0 mL.

7. Disturbance on the Site (Type and Method of Cultivation, Mining, Irrigation, or Other Activities) There will be no disturbance at the sites of dose preparation/administration.

8. Worker Protection Measures Taken During the Release It is expected that workers will comply with the instructions included in the IPIM during the preparation and administration of talimogene laherparepvec. This includes completion of specific training by individuals who will be administering talimogene laherparepvec.

Key handling practices and containment requirements for both Biosafety Level 1 (BSL-1) and Biosafety Level 2 (BSL-2) organisms obtained from globally available biosafety guidelines are presented in Table 3 and Table 4.

Universal biosafety practices are similar amongst the guidelines and are typically followed by medical facilities when handling injectable medicinal products and medical waste, such as:

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 Restricted access  Safe storage  Training of personnel  Availability and use of Personal Protective Equipment (PPE; laboratory coats, gowns, gloves and safety glasses)  Established routine practices for dealing with potentially biohazardous materials such as patient samples/fluids and medical waste (autoclaves, sharps bins, incinerators, disinfectants and appropriate cleanable surfaces).

Precautions for use are provided in the latest versions of the IB and Safety Data Sheet (SDS), issued to each study site by Amgen.

Additional Information Related to Exposure to Talimogene Laherparepvec A needle stick injury, spill, or splash back during administration may result in accidental exposure of Healthcare Professionals to talimogene laherparepvec. The ICP34.5 gene deletion is intended to allow only tumor selective replication and limited or no viral replication in normal tissues. However, talimogene laherparepvec injection can result in signs or symptoms of primary infection at the site of exposure. A few reports of accidental exposure in study personnel have been received. In one of the cases, the exposed physician developed clinical signs/symptoms of a herpetic whitlow-like lesion at the site of the accidental needle stick injury that resolved without sequelae. An initial antibody assay was positive for an HSV-type virus. A confirmatory PCR assay was conducted 10 days after the accidental exposure and was positive for a virus with the ICP47 deletion, indicating that the virus was most likely talimogene laherparepvec. None of the other exposed individuals reported signs or symptoms of infection. In some cases oral acyclovir or valacyclovir was administered.

Accidental Exposure to Talimogene Laherparepvec Accidental exposure to talimogene laherparepvec may lead to signs or symptoms suggestive of herpetic infection. Healthcare personnel may be exposed to talimogene laherparepvec during preparation or administration. In the event of an accidental occupational exposure through a splash to the eyes or mucous membranes, flush with clean water for at least 15 minutes. In the event of exposure to broken skin or needle stick, clean the site thoroughly with soap and water and/or disinfectant. Healthcare personnel with open skin wounds or who are immunosuppressed should not administer talimogene laherparepvec or should not come into direct contact with the talimogene laherparepvec injection site(s). Healthcare personnel preparing or administering intralesional injections and applying protective dressing to injected lesions must observe safety precautions (eg, wear protective gown or laboratory coat, safety glasses, and

Technical and Scientific Information on the GMO EudraCT: 2014-005386-67 Talimogene laherparepvec Page 49 gloves) to avoid direct contact with talimogene laherparepvec. Close contacts may be exposed to talimogene laherparepvec via direct contact with injected lesions, protective dressings, or physical contact with body fluids (eg, blood and urine) of treated subjects. Individuals with open skin lesions should not come into direct contact with the talimogene laherparepvec injection site(s). Individuals who are immunosuppressed should avoid direct contact with treated subjects. Close contacts assisting subjects in applying protective dressing to injected lesions must wear protective gloves and must observe safety precautions to avoid direct contact with talimogene laherparepvec. In the event of a secondary exposure (eg, leakage through occlusive dressing to subject or contacts) to talimogene laherparepvec, advise exposed individuals to clean the site thoroughly with soap and water and/or a disinfectant. Exposed individuals should contact healthcare provider for signs of systemic (fever, aches, nausea, and malaise) or local (for example, pain, redness and swelling) infection.

Talimogene laherparepvec is sensitive to acyclovir or any anti-viral drug that is activated by the viral thymidine kinase gene and may be administered, if clinically indicated.

Accidental Spills Talimogene laherparepvec spills should be treated with a virucidal agent and absorbent materials. All materials contaminated with Talimogene laherparepvec must be disposed of in compliance with local institutional guidelines.

Given the attenuated nature of the modified organism, the fact that the parent organism is not known to spread through aerosols, and the minimal manipulations required in drawing the dose from a vial into a syringe (with unlikely potential aerosol from the dead space of the needle) it is not considered necessary to perform the preparation procedure in a biosafety cabinet. Similarly, no further precautions are required when administering the product by i.l. injection.

The worker protection measures recommended for the preparation and administration of talimogene laherparepvec are therefore generally in accordance with those recommended globally for the handling of BSL-1/BSL-2 organisms in a research setting. The main difference between the handling recommendations for talimogene laherparepvec and those recommended for wild-type HSV-1 (globally classified as BSL-2; see Table 1) is the use of a biosafety cabinet, which is not required for talimogene laherparepvec due to the limited potential for the production of aerosols during dose preparation and administration.

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Ultimately, the actual measures applied will be in accordance with local (site-specific) practices.

Transportation The following transportation classification is recommended:

IATA – UN Number: 3245 Proper shipping Name – Genetically Modified Micro-Organism

Talimogene laherparepvec is not considered infectious per the IATA definition. IATA defines infectious substances as “pathogens which can cause disease in humans or animals” but specifies that substances in which “pathogens have been neutralized or inactivated such that they no longer pose a health risk” or are “unlikely to cause disease in humans or animals” are exempt from the infectious substances definition. Talimogene laherparepvec meets both of these exemption criteria. First, talimogene laherparepvec is an attenuated version of HSV-1, modified so that replication occurs selectively in tumor cells. Because the virus is attenuated in normal cells, no adverse effects would be expected for healthy individuals or animals that inadvertently come in contact with it. Second, data from animals and humans support that talimogene laherparepvec is unlikely to cause disease, therefore exempting it from the IATA definition of infectious.

Talimogene laherparepvec is a Genetically Modified Micro-Organisms (GMMO) since it is HSV-1 that has been genetically altered in a way that does not occur in nature. Talimogene laherparepvec was generated from HSV-1 strain JS1 by deletion of ICP34.5 (required for neurovirulence and efficient replication in non-tumor cells) and ICP47 (blocks antigen presentation in HSV-1 infected cells), and insertion of the gene for GM-CSF. Deletion of ICP34.5 from HSV-1 is known to render the virus avirulent. GMMOs which do not meet the definition of toxic or infectious substances must be assigned to UN 3245. The Talimogene Laherparepvec Safety Data Sheet (Amgen, 2015), which includes instructions for handling spills, accompanies all shipments.

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Table 3. Summary of Recommended Containment Precautions for BSL-1 Infectious Agents

Personal Protective Secondary Barriers Region Practices Equipment Primary Barriers (Facilities) WHO Good microbiological Laboratory coats and No primary barriers required Laboratory bench and sink Laboratory Biosafety Manual technique gloves; eye, face required (3rd Ed., 2004). Pages 2-3 and protection, as needed 9-15. EU Good microbiological Suitable protective No primary barriers required Bench - Surfaces resistant Directive 2009/41/EC as laboratory practices clothing to water, acids, alkalis, amended. Annex IV; Table IA solvents, disinfectants, decontamination agents and easy to clean Autoclave on site US Standard microbiological Laboratory coats and No primary barriers required Laboratory bench and sink Biosafety in Microbiological and practices gloves; eye, face required Biomedical Laboratories (5th Ed., protection, as needed 2009). Page 59. Canada Good microbiological Laboratory coats and No primary barriers required Laboratory bench and sink Canadian Biosafety Guidelines laboratory practices gloves; eye, face required 3rd Ed., 2004. Page 19-22 protection, as needed Autoclave available Australia/ New Zealand Limited access Laboratory coats and No primary barriers required Laboratory bench and sink AS/NZS 2243.3:2010 Safety in Biohazard warning signs gloves; eye, face required protection, as needed laboratories - Microbiological Staff shall be trained in the safety and containment clean-up of microbiological Pages 38-40 spills

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Table 4. Summary of Recommended Containment Precautions for BSL-2 Infectious Agents

Personal Protective Secondary Barriers Region Practices Equipment Primary Barriers (Facilities) WHO BSL-1 plus: As BSL-1 BSCs for procedures with a BSL-1 plus: Laboratory Biosafety Manual Limited access high potential for producing Autoclave or other means of rd aerosols (3 Ed., 2004). Pages 2-3 and Biohazard warning signs decontamination should be 9-19. available in appropriate proximity EU BSL-1 plus: As BSL-1 plus: Minimise aerosol BSL-1 plus: Directive 2009/41/EC as Limited access Gloves (optional) dissemination Autoclave in the building amended. Annex IV; Table IA Biohazard warning signs US BSL-1 plus: As BSL-1 BSCs or other physical BSL-1 plus: Biosafety in Microbiological and Limited access containment devices used Autoclave available th for all manipulations of Biomedical Laboratories (5 Ed., Biohazard warning signs December 2009). Page 59 agents that cause splashes “Sharps” precautions or aerosols of infectious Biosafety manual defining materials any needed waste decontamination or medical surveillance policies Page 1 of 2

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Table 4. Summary of Recommended Containment Precautions for BSL-2 Infectious Agents

Personal Protective Secondary Barriers Region Practices Equipment Primary Barriers (Facilities) Canada BSL-1 plus: As BSL-1 BSCs must be used for As BSL-1 Canadian Biosafety Guidelines Limited access procedures that may rd produce infectious aerosols 3 Ed., 2004. Page 22-23 Biohazard warning signs and that involve high Training and/or supervision concentrations or large of staff commensurate with volumes of biohazardous their anticipated activities in material. the containment area Written procedures for emergencies (eg. spills/ clean up) must be available Australia/ New Zealand BSL-1 plus: As BSL-1 If the work produces a BSL-1 plus: AS/NZS 2243.3:2010 Safety in Instruction and training in significant risk from the Autoclave available laboratories - Microbiological handling infectious production of infectious safety and containment. microorganisms shall be aerosols, a biological safety Pages 40-44 provided to laboratory cabinet shall be used. personnel with regular updates Page 2 of 2

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9. Post-release Treatment of the Site Treatment of the medical facility after use will not be necessary, provided the precautions outlined above are adhered to when administering the product or when dealing with accidental spillages and breakages. However, work surfaces shall be decontaminated using a chemical disinfectant capable of virucidal activity following preparation and dosing of talimogene laherparepvec.

10. Techniques Foreseen for Elimination or Inactivation of the GMOs at the End of the Experiment Following administration of talimogene laherparepvec at the study site, materials used during injection (eg, gloves, needles, gauze) will be disposed of in accordance with local/regional and institutional requirements for biohazardous waste, as stated in the latest version of the IB.

In the medical facility, this will involve temporary containment in sharps bins or clearly marked bags (eg, biohazard, medical waste) prior to autoclaving and/or incineration either on or off site as per local institutional guidelines for handling potentially infectious materials.

Commonly in a medical setting, non-disposable equipment and other materials used during the procedure will be cleaned using a chemical disinfectant capable of virucidal activity for the required duration of contact, or sterilised by autoclaving consistent with local institutional guidelines for handling potentially infectious materials.

Typically, standard operating procedures for disposal within medical facilities (where the potential for contamination from other agents is potentially much more hazardous than that presented by talimogene laherparepvec) will be consistent with the guidance given in the WHO Laboratory Biosafety Manual, 3rd Ed (2004) as outlined below:

Contaminated (infectious) “sharps”: Hypodermic needles, scalpels, knives and broken glass; should always be collected in puncture-proof containers fitted with covers and treated as infectious.

After use, hypodermic needles should not be recapped, clipped or removed from disposable syringes. The complete assembly should be placed in a sharps disposal container. Disposable syringes, used alone or with needles, should be placed in sharps disposal containers and incinerated, with prior autoclaving if required. Sharps disposal containers must be puncture-proof/-resistant and must not be filled to capacity. When they are three-quarters full they should be placed in “infectious waste” containers and

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Contaminated (potentially infectious) materials for autoclaving and reuse: No pre-cleaning should be attempted of any contaminated (potentially infectious) materials to be autoclaved and reused. Any necessary cleaning or repair must be done only after autoclaving or disinfection.

Contaminated (potentially infectious) materials for disposal: Apart from sharps, which are dealt with above, all contaminated (potentially infectious) materials should be autoclaved in leak-proof containers (eg. autoclavable, color-coded plastic bags), before disposal. After autoclaving, the material may be placed in transfer containers for transport to the incinerator. If possible, materials deriving from healthcare activities should not be discarded in landfills even after decontamination. If an incinerator is available on the laboratory site, autoclaving may be omitted; the contaminated waste should be placed in designated containers (eg. color-coded bags) and transported directly to the incinerator. Reusable transfer containers should be leakproof and have tight-fitting covers. They should be disinfected and cleaned before they are returned to the laboratory for further use.

After administration of talimogene laherparepvec, the injection site will be covered with an occlusive dressing that must be kept in place for 7 days. Patients will be given extra dressings to take home and a sealable bag. If the dressings need to be changed while at home, patients must put the used dressing in the provided bag and seal properly. This must be given to a research team member for waste disposal.

11. Information on, and Results of, Previous Releases of the GMOs, Especially at Different Scales and in Different Ecosystems Detailed information on previous releases is provided in Section II.C.2 (h).

Detailed information relating to the results of monitoring biodistribution, shedding and accidental exposure to talimogene laherparepvec is provided in Section II.C.2 (i).3.

No evidence of herpes infection attributable to talimogene laherparepvec outside of the tumour has been documented. In some studies a number of individuals (patients or medical personnel) had reports of oral lesions but where these where tested by culture of the virus and PCR analysis the causative agent was wild-type HSV (reactivating endogenous HSV) and not talimogene laherparepvec. It should be noted that reactivation of wild-type HSV-1 would not be unexpected in the population as a whole.

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B. INFORMATION ON THE ENVIRONMENT (BOTH ON THE SITE AND IN THE WIDER ENVIRONMENT) 1. Geographical Location and Grid Reference of the Site(S) (In Case of Notifications Under Part C the Site(S) of Release Will be the Foreseen Areas of Use of the Product) A list of the participating study sites in this clinical trial are listed in Appendix 2.

2. Physical or Biological Proximity to Humans and Other Significant Biota Talimogene laherparepvec will be administered to the subject by a trained medical professional at an approved study site. The product will be stored prior to administration in a secure, temperature monitored freezer at -70C or below in the pharmacy or other appropriate secure location.

Following the injection procedure, the injection sites will be covered by an occlusive dressing before subjects leave the clinical trial facility.

Given the nature of the product administration directly into the subject’s tumours, the use of an occlusive dressing to provide a physical barrier to virus leakage, and the absence/ low levels of shedding observed, the risk of exposure to talimogene laherparepvec to the study site personnel involved in the subject’s care and the subject’s family on returning home from the medical facility is minimal.

3. Proximity to Significant Biotopes, Protected Areas, or Drinking Water Supplies Given the nature of the product administration directly into the subject’s tumours, and procedures for waste treatment, the exposure to significant biotopes, protected areas and drinking water supplies is expected to be minimal. Humans are the only natural host for wild-type HSV-1 infection with no known vector, proximity to other biotopes is not a concern. The genetic modifications made to the parental virus in the construction of talimogene laherparepvec do not affect its selectivity to the host species.

The stability of talimogene laherparepvec in the environment is also unchanged from that of wild-type HSV-1, and will rapidly lose viability outside the target species (see Section II.A.10 (c)).

4. Climatic Characteristics of the Region(s) Likely to be Affected The risk of release of talimogene laherparepvec in to the environment is unrelated to climatic characteristics.

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The stability of talimogene laherparepvec in the environment is unchanged from that of wild-type HSV-1, and will rapidly lose viability outside the target species (see Section II.A.10 (c)).

5. Geographical, Geological and Pedological Characteristics The risk of release of talimogene laherparepvec in to the environment is unrelated to geographical, geological and pedological characteristics. The stability of talimogene laherparepvec in the environment is unchanged from that of wild type HSV-1, and will rapidly lose viability outside the target species (see Section II.A.10 (c)).

6. Flora and Fauna, Including Crops, Livestock and Migratory Species Since humans are the only natural host for wild-type HSV-1 infection with no known vector, proximity to other flora and fauna is not a concern.

7. Description of Target and Non-target Ecosystems Likely to be Affected No ecosystems are targeted in the use of talimogene laherparepvec. Ecosystems are not expected to be affected, since humans are the only natural host of the parental organism which is rapidly inactivated outside the host species (see Section II.A.10 (c)).

8. A Comparison of the Natural Habitat of the Recipient Organism with the Proposed Site(s) of Release The natural habitat of wild type HSV-1 is human beings. Talimogene laherparepvec will also be administered to human beings, though the genetic construct contains functional deletions of the neurovirulence factor (ICP34.5) rendering it unable to replicate in non-tumour cells (see Section II.C.2 (i) 1). (ie, replication is designed to be self-limiting to tumour cells through the functional deletion of the neurovirulence factor ICP34.5).

The potential for excessive population increase of talimogene laherparepvec in the environment is low, as described in Section IV.B.8.

9. Any Known Planned Developments or Changes in Land Use in the Region which Could Influence the Environmental Impact of the Release None known.

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IV. INFORMATION RELATING TO THE INTERACTIONS BETWEEN THE GMOs AND THE ENVIRONMENT A. Characteristics affecting survival, multiplication and dissemination 1. Biological Features Which Affect Survival, Multiplication and Dispersal Talimogene laherparepvec is an attenuatedversion of HSV-1, modified so that replication occurs selectively in tumour cells in the target human population and is therefore self-limiting. Considerable literature shows that HSV-1 deleted for ICP34.5 has been extensively utilised without incident in animals and humans (see Section II.C. (i) 1).

Its mode of transmission (dispersal) is through direct contact and is unchanged from that of wild type HSV-1 (see Section II.A.10 (d)).

Outside of the host, wild type HSV-1 is an enveloped virus which is sensitive to and rapidly inactivated by both physical inactivation (dehydration, heat, low pH) and disinfectants (lipid solvents and mild detergents); see Section II.A.10 (c)). The genetic modifications made during the construction of talimogene laherparepvec from wild type HSV-1 do not affect survivability outside the host organism.

2. Known or Predicted Environmental Conditions which may Affect Survival, Multiplication and Dissemination (Wind, Water, Soil, Temperature, pH, etc.) Environmental conditions which may affect survival of talimogene laherparepvec outside the host are temperature, pH and environmental humidity, as for wild type HSV-1 (see Section II.A.10 (c)).

3. Sensitivity to Specific Agents The genetic modifications made during the construction of talimogene laherparepvec from wild type HSV-1 do not affect its sensitivity to physical and chemical inactivation.

Physical Inactivation Wild-type HSV virus is easily inactivated outside the host by exposure to pH <4, temperatures >56C for 30 min, pasteurization (60C for 10 h), and microwave heating for 4 min (Croughan & Behbehani, 1988; Jerome & Morrow, 2007).

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Chemical Inactivation Wild-type HSV virus is easily inactivated by lipid solvents (Jerome & Morrow, 2007). It can be inactivated by 0.5% Lysol in 5 min; by Listerine (1:1 mixtures) in 5 min; by 2,000 ppm (2,000 µL/L) of bleach in 10 min; by 70% isopropyl alcohol (1:1 mixtures) (Croughan & Behbehani, 1988). HSV is also susceptible to quaternary ammonium compounds (Wood and Payne, 1998). Most herpes viruses are also susceptible to 30% ethanol and isopropanol, 0.12% orthophenyl phenol, and 0.04% glutaraldehyde (Prince & Prince, 2001).

Susceptibility to Anti-viral Agents Antiviral medicinal products like acyclovir, valacyclovir and famciclovir can be used to inhibit wild-type HSV-1 replication (Drew, 2004; Usitane and Tinitigan, 2010) and are expected to be effective against talimogene laherparepvec. The in vitro sensitivity of talimogene laherparepvec to acyclovir has been demonstrated (see Section II.C.2 (i) 2).

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B. INTERACTIONS WITH THE ENVIRONMENT 1. Predicted Habitat of the GMOs The predicted habitat of talimogene laherparepvec is humans. Replication is designed to be self-limiting to tumour cells through the functional deletion of the neurovirulence factor ICP34.5. The use of talimogene laherparepvec will be limited to medical facilities and cancer care clinics, where controls are in place to limit use and spread beyond the defined patient treatment regimen.

2. Studies of the Behaviour and Characteristics of the GMOs and Their Ecological Impact Carried Out in Simulated Natural Environments, Such as Microcosms, Growth Rooms, Greenhouses Talimogene laherparepvec is an attenuated version of wild type HSV-1. The genetic modifications do not affect its natural host range, which is restricted to humans.

No specific studies have been conducted regarding transmission of talimogene laherparepvec between humans as such studies would be unethical.

It is not possible to model human transmission between treated and untreated animals since transmission of wild type HSV-1 between animals is not known to occur in nature, and the genetic modifications made to wild type HSV-1 resulting in talimogene laherparepvec attenuate the virus, further reducing the likelihood of transmission.

Biodistribution and virus shedding has however been monitored in both humans and animals following administration of talimogene laherparepvec (see Section II.C.2 (i) 3).

3. Genetic Transfer Capability Humans are the only natural host of the parental organism, wild type HSV-1, Talimogene laherparepvec is further attenuated by the functional deletion of the neurovirulence factor, ICP34.5 such that it replicates selectively in tumour cells. No transfer into other organisms is expected.

The transfer of genetic material is therefore limited to the virally mediated (non-integrative) transfer of the viral DNA between humans and theoretically, genetic exchange by homologous recombination with wild-type HSV-1 which could only occur if human cells were simultaneously infected with both wild type HSV-1 and talimogene laherparepvec. The likelihood of this occurring and the potential genetic variants which may be produced are discussed in Section II.C.2(c).

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4. Likelihood of Postrelease Selection Leading to the Expression of Unexpected and/or Undesirable Traits in the Modified Organism The selective pressure on talimogene laherparepvec will be towards reversion to wild type, since both gene deletions (ICP34.5 and ICP47) in talimogene laherparepvec limit its replication and survival in its host species.

The likelihood of this reversion to wild-type is still considered low, since it would require genetic exchange by homologous recombination with wild type HSV-1 which could only occur if human cells were simultaneously infected with both wild type HSV-1 and talimogene laherparepvec.

5. Measures Employed to Ensure and to Verify Genetic Stability. Description of Genetic Traits Which May Prevent or Minimise Dispersal of Genetic Material. Methods to Verify Genetic Stability A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

The functional deletions in ICP34.5 and ICP47 render the GMO unable to replicate efficiently in non-tumour cells, and less able to evade MHC mediated immunity. Therefore infection leading to replication of the GMO (and therefore potential for dispersal) is minimised to targeted tumour bearing humans.

A spontaneously occurring genetic variant of talimogene laherparepvec would require an initial recombination event(s) leading to the creation of the genetic variant itself. It is unlikely that a wild type virus would be in the same tissue as talimogene laherparepvec in subjects since the latter is directly injected into tumour cells and cannot spread effectively into normal tissue, while the pre-existing HSV-1 would be in the mucosal tissues or neuronal ganglia of the patient. The possibility of the creation of stable genetic variants with unintended characteristics is also minimised by the design of the talimogene laherparepvec genetic construct. See Section II.C.2 (c) for a full discussion of this topic.

6. Routes of Biological Dispersal, Known or Potential Modes of Interaction with the Disseminating Agent, Including Inhalation, Ingestion, Surface Contact, Burrowing, etc Talimogene laherparepvec is an attenuated version of HSV-1, modified so that replication occurs selectively in tumour cells in the target human population and is therefore self-limiting. Humans are the only natural host forwild-type HSV-1 infection, and other species are known to be carriers or vectors under natural conditions.

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Considerable literature shows that HSV-1 deleted for ICP34.5 has been extensively utilised without incident in animals and humans (see Section II.C. (i) 1).

Its mode of transmission (dispersal) is through direct contact and is unchanged from that of wild-type HSV-1 (see Section II.A.10 (d)).

7. Description of Ecosystems to Which the GMOs Could be Disseminated Humans are the only natural host for wild-type HSV-1 infectionand the genetic modifications introduced in the construction of talimogene laherparepvec do not affect host range but do attenuate its replicative capacity in normal cells through the functional deletion of ICP34.5.

Dissemination could therefore only occur between human beings. However, due to the ICP34.5 gene deletion no replication would be expected in normal cells of treated individuals exposed or other humans unintentionally to the attenuated virus.

8. Potential for Excessive Population Increase in the Environment The potential for excessive population increase of talimogene laherparepvec in the environment is low, due to:

 Requirement for its host species (human) for replication and absence of other vector species (see Sections II.A.7 and II.A.8)  Attenuation of the virus rendering it highly restricted for replication in normal cells (see Section II.C.2 (a))  Administration directly into the tumours of eligible patients; replication in the patient will be self-limiting, dependant on tumour burden  Low incidence of shedding of infective virus from treated individuals (see Section II.C.2 (i) 3).  Low persistence and viability outside the host organism (see Section II.A.10 (c)); high sensitivity to physical and chemical agents (see Section IV.A.3).

9. Competitive Advantage of the GMOs in Relation to the Unmodified Recipient or Parental Organism(s) Talimogene laherparepvec is defective in the expression of two proteins which are present in all wild-type HSV-1 strains and are essential for neurovirulence and immunity evasion (see Section II.C.2 (a)). Talimogene laherparepvec is therefore at a competitive disadvantage when compared to its parent strain.

10. Identification and Description of the Target Organisms if Applicable The target organism is humans, specifically melanoma patients with unresected or metastatic disease.

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11. Anticipated Mechanism and Result of Interaction Between the Released GMOs and the Target Organism(s) if Applicable Talimogene laherparepvec is based on a newly isolated strain of HSV-1 that has been engineered to replicate selectively in tumours, killing tumour cells by viral lysis, followed by spread of talimogene laherparepvec within the tumour and further tumour cell lysis. Additionally, the oncolysis of tumour cells by talimogene laherparepvec also releases and exposes an array of antigens to initiate a systemic immune response, and this is augmented through the expression of an immune stimulatory protein, hGM-CSF from the virus (Study 4647-00041). Released tumour antigens are expected to be taken up by antigen presenting cells (APCs) which then traffic to lymph nodes and present to T cells, inducing an immune response. hGM-CSF increases the activity of APCs, enhancing the immune responses. This immune response is intended to provide a systemic anti- tumour effect, including the shrinkage of tumours which do not come into direct contact with talimogene laherparepvec, reduction of micrometastatic disease, and protection against future relapse.

12. Identification and Description of Non-target Organisms Which May be Adversely Affected by the Release of the GMO, and the Anticipated Mechanisms of any Identified Adverse Interaction Humans are the only natural host for wild type HSV. The modified organism talimogene laherparepvec is functionally deleted for ICP34.5 (neurovirulence factor) so that the virus replicates selectively in tumours and is attenuated in healthy tissue (see Section II.C.2 (i) 1). Talimogene laherparepvec will only be administered as an investigational medicinal product to subjects enrolled in the clinical trial.

The only non-target organisms which could be affected are unintended human recipients (healthcare workers and close contacts of the patient).

There have been no cases of transmission of talimogene laherparepvec from a patient to an unintended human recipient observed during development. In addition, it is not expected that transmission would lead to disease in healthy humans, due to the deletion of ICP34.5.

In the unlikely event that transmission to a healthy unintended human recipient occurs and manifests as an active infection which is shown to be a result of talimogene laherparepvec, it is likely that the safety profile in healthy subjects would be at worst similar to that observed in a HSV-1 wild type infection (see Section II.C.2 (i) 1). The subject may be treated with approved antiviral agents (eg. acyclovir) if required (see Section II.C.2 (i) 2).

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13. Likelihood of Postrelease Shifts in Biological Interactions or in Host Range A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

The likelihood of post-release shifts in biological interactions or host range is negligible. Even a potential recombination event between talimogene laherparepvec and wild type virus could only result in a reversion to wild-type like interactions in the host species (humans).

14. Known or Predicted Interactions with Non-target Organisms in the Environment, Including Competitors, Preys, Hosts, Symbionts, Predators, Parasites and Pathogens None known or predicted.

15. Known or Predicted Involvement in Biogeochemical Processes None known or predicted.

16. Other Potential Interactions with the Environment None known or predicted.

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V. INFORMATION ON MONITORING, CONTROL, WASTE TREATMENT AND EMERGENCY RESPONSE PLANS A. MONITORING TECHNIQUES 1. Methods For Tracing The GMOs, And For Monitoring Their Effects A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Monitoring of the direct and indirect effects of talimogene laherparepvec in subjects will be achieved by the clinical assessments defined in the clinical trial protocol. Study investigators will monitor subjects throughout treatment and will report adverse effects to Amgen Global Safety according to the requirements stipulated in the protocol.

Amgen will conduct a surveillance program to aid the assessment of any potential risks to third parties following treatment of subjects with talimogene laherparepvec.

2. Specificity (to identify the GMOs, and to distinguish them from the donor, recipient or, where appropriate the parental organisms), Sensitivity and Reliability of the Monitoring Techniques See Sections II.C.2(f) and II.C.2(g).

3. Techniques for Detecting Transfer of the Donated Genetic Material to Other Organisms The literature search revealed that non-human infection is rare, but identified that HSV-1 infection has been reported in a variety of species

4. Duration and Frequency of the Monitoring A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

Monitoring will occur throughout the subject’s participation in the study, including a period of safety follow-up, as defined in the study protocol, upon permanent discontinuation from the study.

Clinical assessments will be made according to the predefined schedule detailed in the study protocol. At each visit, the subject must be interviewed regarding their knowledge of any possible exposures or events that may have occurred in their close contacts.

The investigator is responsible for ensuring that all SAEs and AEs observed by the investigator or reported by the subject that occur after signing of the informed consent through to a predefined period after the last dose of study medication are recorded in the subject’s medical record and are submitted to Amgen. Any SAE must be submitted to Amgen within 24 hours following the investigator’s knowledge of the event.

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B. CONTROL OF THE RELEASE 1. Methods and Procedures to Avoid and/or Minimise the Spread of the GMOs Beyond the Site of Release or the Designated Area for Use Distribution and Supply Talimogene laherparepvec is an investigational medicinal product for use only in approved clinical trials by trained medical professionals at an authorised study site.

It will be supplied by Amgen directly to the study site and appropriate records/ traceability of shipments will be maintained in line with the requirements of Good Clinical Practice (GCP).

Talimogene laherparepvec is provided as a sterile frozen liquid in a single-use vials sealed with grey rubber stoppers, Flurotec-coated on the product side.

Storage Talimogene laherparepvec must be stored in a secure, temperature monitored freezer at -70C or below in the pharmacy or other appropriate secure location until planned use.

Dose Preparation and Administration Precautions to be adhered to during dose preparation and administration are outlined in Section III.A.8 of this document, and in the IPIM.

It is appropriate to dispense vials of drug such that product is then drawn up into syringes in the room used for product administration, although this may also optionally occur elsewhere, eg, in the pharmacy, although local institutional guidelines must also be followed.

The injection site should be swabbed with alcohol before and following injection, and then covered with a dry occlusive dressing before the patient leaves the study site facility. The occlusive dressing covers the injection site for up to 1 week. Given the nature of the product administration directly into the patient’s tumours, the use of an occlusive dressing provides a physical barrier to virus leakage. The absence/low levels of shedding observed during clinical development minimises the risk of exposure of the medical professionals involved in the patients’ care and the patients’ family on returning home from the study site facility to talimogene laherparepvec.

Spills: In the context of this clinical trial, spills are unlikely to constitute a volume greater than 4 mL (ie. 4 vials). Spills will be treated as outlined in Section III.A.10 of this document.

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Disposal: Talimogene laherparepvec is sensitive to inactivation by a variety of commonly available physical and chemical methods (see Section IV.A.3). Waste disposal is described in Section III.A.10.

2. Methods and Procedures to Protect the Site from Intrusion by Unauthorised Individuals Talimogene laherparepvec must be stored in a secure, temperature monitored freezer at -70C or below in the pharmacy or other appropriate secure location. Intrusion by unauthorised individuals is anticipated to be adequately controlled.

3. Methods and Procedures to Prevent Other Organisms from Entering the Site No other procedures are required to prevent other organisms from entering the site, since talimogene laherparepvec is an attenuated version of wild-type HSV-1, which itself has no known vectors for whichhumans are the only natural host. Within a medical facility, general pest control and cleaning procedures will be in place as dictated by site specific procedures for general hygiene.

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C. WASTE TREATMENT 1. Type of Waste Generated Waste generated from i.l. administration of talimogene laherparepvec will be limited to:

 Used vials and needles  Used swabs and items used to clean injected area  Used dressings applied to the injection sites  Personal Protective Equipment used at the point of administration and when replacing or removing the used dressing

2. Expected Amount of Waste Vials containing 1.15 mL of talimogene laherparepvec supplied to pharmacies will be provided in two strengths; 106 PFU/mL or 108 PFU/mL.

The maximum possible total volume for an individual patient is 4.0 mL or 8 mL ( 4 or 8 vials) per treatment. Based on a maximum talimogene laherparepvec treatment duration of 9 months, a total of 96 vials could be administered to each subject in this timeframe.

Each administration will result in the waste identified above.

The anticipated number of subjects to be administered talimogene laherparepvec as part of this clinical trial in the EU are provided in Section III.A.6.

3. Description of Treatment Envisaged Talimogene laherparepvec is sensitive to inactivation by a variety of commonly available physical and chemical methods (see Section IV.A.3).

Since talimogene laherparepvec will be administered in an approved study site facility, all associated waste will be disposed of in line with standard practice for medical waste (see Sections III.A.8 and III.A.10).

The information leaflet provided to each subject instructs that disposal of any soiled dressings should occur via the study site at their next scheduled visit. The subject is provided with additional dressings, disposable gloves and resealable bags, and specific instructions to be followed to minimise the risk of unintended exposure to the environment.

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D. EMERGENCY RESPONSE PLANS 1. Methods and Procedures for Controlling the GMOs in Case of Unexpected Spread The only organism which may act as a mechanism for the spread of talimogene laherparepvec are humans are the only natural host for wild-type HSV-1 infection and it is not known to be zoonotic or reversezoonotic under natural conditions.

Talimogene laherparepvec is an attenuatedversion of HSV-1, modified so that replication occurs selectively in tumour cells. Considerable literature shows that HSV-1 deleted for ICP34.5 has been extensively utilized without incidentin animals and humans (see Section II.C. (i) 1).

Because the virus is attenuated in normal cells, exposure is highly unlikely to lead to replication and shedding in those not intended to receive the treatment. As such, the likelihood of spread is very low (see Section IV.B.8).

In the unlikely event of the transmission of talimogene laherparepvec to an unintended human recipient, the affected patient can be treated with approved antiviral treatments such as acyclovir, if clinically indicated to alleviate any symptoms of primary infection and potential recurrence (if deemed necessary). Further spread from the individual can be mitigated by educational materials to increase awareness of the infection and preventative measures which can be taken to prevent transmission to close contacts.

Any spread of talimogene laherparepvec to unintended human recipients is likely to be isolated to single cases in discrete geographical locations. The risk of widespread infection is considered negligible.

2. Methods for Decontamination of the Areas Affected, for example Eradication of the GMOs Talimogene laherparepvec cannot persist outside its host organism for long periods and maintain viability. Since it is sensitive to even moderately harsh conditions, it is considered highly unlikely that spread would occur in the environment from fomites and talimogene laherparepvec would quickly be rendered non-viable by the prevailing conditions (see Section II.A.10 (c)).

Decontamination of areas in which a recently treated patient had frequented (their home and or treatment room) could be implemented by applying chemical disinfectants capable of virucidal activity (see Section IV.A.3) to areas of likely contact (for example, frequent touch-points such as handles, door knobs, hard surfaces, railings and hand-holds, washing facilities and lavatories).

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3. Methods for Disposal or Sanitation of Plants, Animals, Soils, etc. that were Exposed During or After the Spread Decontamination of plants, (non-human) animals and soils will not be required, since humans are the only natural host of the organism and it will rapidly become non-viable outside a human host (see Section II.A.10 (c)).

4. Methods for the Isolation of the Area Affected by the Spread There are no plans for isolation of an area should horizontal transfer occur between a patient receiving talimogene laherparepvec and an unintended human recipient. Measures outlined in Section V.D.1 may be implemented to reduce the risk of further spread.

5. Plans for Protecting Human Health and the Environment in Case of the Occurrence of an Undesirable Effect Talimogene laherparepvec will be administered as an investigational medical product in this clinical trial, which will be conducted in accordance with the EU Clinical Trials Directive and GCP.

Information is collected regarding all individual adverse events reported to Amgen by study investigators. Suspected unexepected serious adverse reactions (SUSARs) and annual safety reports are submitted by Amgen to the concerned National Competent Authorities and Ethics Committees. Procedures are in place at both the National Competent Authorities and Amgen to monitor, review and act on urgent safety information relating to investigational medicinal products so that human health is protected.

Information relating to specific monitoring activities in relation to the environment is provided in Section V.A.

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APPENDICES

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Appendix 1.

Responsible Scientists CVs and Signature Pages

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A full version of this section, including confidential information, is provided in the full version submitted to the Belgian Authorities.

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Appendix 2.

List of the Participating Study Sites (Section III.B.1)

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The participating sites in Beglium are as follows:

Site Name Address UCL Cliniques Universitaires Saint Luc Avenue Hippocrate 10, 1200 Bruxelles Universitair Ziekenhuis Gent De Pintelaan 185, 9000 Gent Universitair Ziekenhuis Leuven Campus Gasthuisberg Herestraat 49, 3000 Leuven Universitair Ziekenhuis Antwerpen Wilrijkstraat 10, 2650 Edegem