C A N C E R I M M U N OT H E R A P Y
`
`Oncolytic viruses: a new class
`of immunotherapy drugs
`
`Howard L. Kaufman, Frederick J. Kohlhapp and Andrew Zloza
`
`Abstract | Oncolytic viruses represent a new class of therapeutic agents that promote
`anti-tumour responses through a dual mechanism of action that is dependent on selective
`tumour cell killing and the induction of systemic anti-tumour immunity. The molecular and
`cellular mechanisms of action are not fully elucidated but are likely to depend on viral
`replication within transformed cells, induction of primary cell death, interaction with tumour
`cell antiviral elements and initiation of innate and adaptive anti-tumour immunity. A variety
`of native and genetically modified viruses have been developed as oncolytic agents, and
`the approval of the first oncolytic virus by the US Food and Drug Administration (FDA) is
`anticipated in the near future. This Review provides a comprehensive overview of the basic
`biology supporting oncolytic viruses as cancer therapeutic agents, describes oncolytic
`viruses in advanced clinical trials and discusses the unique challenges in the development
`of oncolytic viruses as a new class of drugs for the treatment of cancer.
`
`Tumour-associated antigen
`A protein derived from tumour
`cells that can be recognized
`by the immune system.
`An immune response may be
`triggered because a T cell
`has survived negative thymic
`selection for the cognate
`antigen, or more probably
`because the normal host
`protein has been mutated
`within the cancer cell.
`
`Danger signals
`Nuclear and cytosolic proteins
`that are released by cells
`during tissue injury or necrosis,
`and stimulate the innate and
`adaptive immune system.
`
`Rutgers Cancer Institute
`of New Jersey, 195 Little
`Albany Street, Room 2004,
`New Brunswick,
`New Jersey 08901, USA.
`Correspondence to H.L.K. 
`e‑mail: howard.kaufman@
`rutgers.edu
`doi:10.1038/nrd4663
`Corrected online
`18 January 2016
`
`Oncolytic virus immunotherapy is a therapeutic approach
`to cancer treatment that utilizes native or genetically
`modified viruses that selectively replicate within tumour
`cells. The ability of viruses to kill cancer cells has been
`recognized for nearly a century, but only over the past
`decade have clinical trials documented a therapeutic
`benefit in patients with cancer1–3. Interest in oncolytic
`viruses has been increasing, based on a better under‑
`standing of viral biology, tumour immunology and
`molecular genetics. Furthermore, a recent randomized
`Phase III clinical trial demonstrated an improved durable
`response rate for patients with advanced melanoma
`who were treated with a modified herpes simplex virus
`type 1 (HSV‑1), encoding granulocyte–macrophage 
`colony‑stimulating factor (GM‑CSF) 3. This virus,
`termed talimogene laherparepvec (T‑VEC; Amgen),
`is widely anticipated to be the first oncolytic virus
`immuno therapy to be approved by the US Food and
`Drug Administration (FDA) for the treatment of can‑
`cer. The success of T‑VEC is likely to promote further
`drug development within this new class of cancer
`therapeutics.
`Of the nearly 1 million vertebrate viruses, approxi‑
`mately 320,000 are thought to infect mammalian cells4.
`Viruses have several shared properties; these include a
`genetic element composed of single‑ or double‑stranded
`DNA or RNA and the ability to infect host cells and
`replicate under permissive conditions (TABLES 1,2).
`
`The outcome of viral infections can be highly variable
`depending on the pathogenic nature of the virally
`encoded genes, interactions between the virus and the
`host immune system and the ability of the virus to repli‑
`cate and/or induce latency following infection. Insights
`into the mechanisms of viral entry, replication, induc‑
`tion and/or suppression of immune responses and lytic
`versus latent infections have led to an intense interest
`in utilizing viruses for the treatment of human diseases
`and have been used to select oncolytic vectors for the
`treatment of specific types of cancers. In contrast to
`standard viral‑based ‘vaccines’, oncolytic viruses directly
`infect and lyse tumour cells in situ. They do not nec‑
`essarily require defined antigens to be included in the
`vector as tumour-associated antigens may be released by
`dying tumour cells. Oncolytic viruses can also provide
`additional danger signals that can promote an efficient 
`antitumour immune response.
`Although incompletely understood, oncolytic viruses
`are thought to mediate antitumour activity through two
`distinct mechanisms of action: selective replication
`within neoplastic cells, resulting in a direct lytic effect
`on tumour cells; and induction of systemic antitumour
`immunity. The relative contribution of these mechanisms
`may differ depending on the nature and type of cancer
`cell, the characteristics of the viral vector, and the inter‑
`action between the virus, tumour microenvironment and
`host immune system.
`
`642 | SEPTEMBER 2015 | VOLUME 14
`
`www.nature.com/reviews/drugdisc
`
`© 2016 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
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`
`

`

`Table 1 | Properties of select DNA viruses
`Adenovirus
`
`Vaccinia virus
`
`Herpesvirus
`
`Parvovirus H1
`
`35 kb
`
`190 kb
`
`154 kb
`
`5 kb
`
`70–90 nm
`
`70–100 nm
`
`200 nm
`
`18–28 nm
`
`Group I: dsDNA
`
`Adenoviridae
`
`Naked
`
`Icosahedral
`
`Group I: dsDNA
`
`Poxviridae
`
`Complex coats
`
`Complex
`
`Group I: dsDNA
`
`Herpesviridae
`
`Enveloped
`
`Icosahedral
`
`Group II: ssDNA
`
`Parvoviridae
`
`Naked
`
`Icosahedral
`
`Nucleus and cytoplasm
`
`Cytoplasm
`
`Nucleus and cytoplasm
`
`Nucleus and cytoplasm
`
`Baltimore classification
`Family
`Virion
`Capsid symmetry
`Replication site
`Cell receptor*
`Nuclear integration
`Transgene capacity
`Wild-type virus infects
`non-replicating cells
`Virulence of wild-type virus‡
`Antivirals
`Immunogenicity¶
`Haemagglutination
`Blood–brain barrier
`penetration
`Achievable titre (PFU per ml)
`5 × 108
`1010
`109
`1012
`CAR, coxsackie-adenovirus receptor; dsDNA, double-stranded DNA; HVEM, herpesvirus entry mediator; MTD, maximum tolerated dose; N/A, not applicable;
`PFU, plaque forming unit; ssDNA, single-stranded DNA. *Only well-validated receptors included, others may have been reported. ‡In humans. ¶Upon re-exposure.
`
`CAR
`
`+
`
`++
`
`–
`
`+/–
`
`+
`
`–
`
`+/–
`
`–
`
`Unknown
`
`HVEM, nectin 1, nectin 2
`
`Sialic acid residues
`
`–
`
`+++
`
`–
`
`+/–
`
`+
`
`–
`
`–
`
`–
`
`+
`
`+++
`
`–
`
`–
`
`+
`
`–
`
`–
`
`–
`
`+
`
`N/A
`
`+
`
`+
`
`–
`
`+
`
`+
`
`+
`
`Certain viruses have the ability to enter cancer cells
`and selectively replicate within such cells. Although
`oncolytic viruses can enter both normal and cancer cells,
`the inherent abnormalities in the cancer cell response to
`stress, cell signalling and homeostasis provide a selective
`advantage for viral replication5. The normal host cell anti‑
`viral machinery, which is responsible for the detection and
`clearance of viruses, may also be abnormal in cancer cells.
`For example, the protein kinase R (PKR) is a critical factor
`that helps in clearing intracellular viral infections. PKR
`may be absent in some cancer cells, allowing increased
`viral replication, whereas it may be active in other cancer
`cells, such as low‑grade tumours, and these differences
`can influence the therapeutic activity of an oncolytic virus.
`The immune response to oncolytic viruses appears to
`be an important component of the antitumour effect, but
`it can be a double‑edged sword. On the one hand, viruses
`can help to promote an immune response against the
`tumour cells by allowing tumour antigen presentation
`in the context of an active viral infection. On the other
`hand, neutralizing antiviral responses may block virus
`replication and ongoing infection of tumour cells. The
`therapeutic outcome depends on a complex interplay
`
`between these opposing forces. When systemic immunity
`is fully engaged, however, therapeutic responses may be
`seen in both locally injected tumours and at distant sites
`of un‑infected tumour growth.
`Many viruses have been proposed as vectors for
`oncolytic virus immunotherapy, and considerable work
`has been done to optimize viral vectors by attenuating
`pathogenicity and enhancing immunogenicity6. To date,
`adenoviruses, poxviruses, HSV‑1, coxsackieviruses,
`poliovirus, measles virus, Newcastle disease virus
`(NDV), reovirus, and others, have entered into early‑
`phase clinical trials. Two viruses, T‑VEC and H101
`(Shanghai Sunway Biotech) have now achieved regula‑
`tory review. H101 is a genetically modified oncolytic
`adenovirus that, in combination with chemotherapy,
`was approved for the treatment of nasopharyngeal
`carcinoma in China in November 2005 (REFS 3,7).
`This Review provides a comprehensive overview
`of critical issues in the development of oncolytic virus
`immunotherapy. We discuss preclinical and clinical data
`that support a role for oncolytic viruses in cancer therapy
`and detail some of the unique challenges in oncolytic
`viruse drug development.
`
`NATURE REVIEWS | DRUG DISCOVERY
`
` VOLUME 14 | SEPTEMBER 2015 | 643
`
`© 2016 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
`
`Replimune Limited Ex. 2014 - Page 2
`Transgene and Bioinvent International AB v. Replimune Limited
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`
`

`

`Table 2 | Properties of select RNA viruses
`Reovirus
`Coxsackievirus
`
`Seneca
`Valley Virus
`
`Poliovirus
`
`Measles virus
`
`Newcastle
`disease virus
`
`Vesicular
`stomatitis
`virus
`
`23 kb
`
`28 kb
`
`7 kb
`
`7.5 kb
`
`16 kb
`
`15 kb
`
`11 kb
`
`75 nm
`
`28 nm
`
`25–30 nm
`
`30 nm
`
`100–200 nm
`
`100–500 nm
`
`80 nm
`
`Baltimore
`classification
`
`Group III:
`dsRNA
`
`Group IV: ssRNA
`
`Group IV: ss(+)
`RNA
`
`Group IV: ss(+)
`RNA
`
`Group V: ss(–)
`RNA
`
`Group V: ss(–)
`RNA
`
`Group V ss(–)
`RNA
`
`Family
`Virion
`Capsid symmetry
`Replication site
`Cell receptor*
`
`Reoviridae
`
`Picornaviridae
`
`Picornaviridae
`
`Picornaviridae
`
`Paramyxoviridae Paramyxoviridae
`
`Rhabdoviridae
`
`Naked
`
`Naked
`
`Naked
`
`Naked
`
`Enveloped
`
`Enveloped
`
`Enveloped
`
`Icosahedral
`
`Icosahedral
`
`Icosahedral
`
`Icosahedral
`
`Icosahedral
`
`Helical
`
`Helical
`
`Cytoplasm Cytoplasm
`
`Cytoplasm
`
`Cytoplasm
`
`Cytoplasm
`
`Cytoplasm
`
`Cytoplasm
`
`Unknown
`
`Unknown
`
`CD155
`
`SLAM and CD46 Unknown
`
`LDLR
`
`–
`
`–
`
`CAR/ICAM-1/
`DAF
`–
`
`N/A
`
`–
`
`+/–
`
`–
`
`–
`
`+
`
`–
`
`–
`
`N/A
`
`+
`
`+
`
`–
`
`+
`
`+
`
`+
`
`+
`
`–
`
`109
`
`109
`
`N/A
`
`Nuclear integration –
`Transgene capacity N/A
`Wild-type virus
`+
`infects non-
`replicating cells
`Virulence of wild-
`type virus‡
`Antivirals
`Immunogenicity¶
`–
`Haemagglutination +
`Blood–brain barrier
`+
`penetration
`Achievable titre
`(PFU per ml)
`CAR, coxsackie-adenovirus receptor; DAF, decay accelerating factor; dsRNA, double-stranded RNA; ICAM-1, intercellular adhesion molecule 1; LDLR, low-density
`lipoprotein receptor; MTD, maximum tolerated dose; N/A, not applicable; PFU, plaque forming unit; SLAM, signalling lymphocytic activation molecule; ss(+)RNA,
`positive single-stranded RNA; ss(–)RNA, negative single-stranded RNA; VP, viral particle. *Only well-validated receptors included, others may have been reported.
`‡To humans. ¶Upon re-exposure.
`
`–
`
`N/A
`
`–
`
`–
`
`–
`
`+/–
`
`+
`
`+
`
`108
`
`–
`
`+
`
`–
`
`–
`
`–
`
`–
`
`–
`
`–
`
`+
`
`–
`
`+
`
`–
`
`–
`
`–
`
`+
`
`+
`
`+
`
`+
`
`–
`
`–
`
`–
`
`–
`
`1011
`
`108
`
`2 × 1010
`
`Cell antiviral response
`elements
`Intracellular and extracellular
`components of the inherent
`cellular response to viral
`infection, including detection
`(via protein kinase R (PKR),
`Toll-like receptors (TLRs),
`retinoic acid-inducible gene 1
`(RIG-1), and others) and
`initiation of the immune
`response through the release
`of cytokines (such as type I
`interferons), danger-associated
`molecular pattern signals
`(DAMPs) (such as high mobility
`group box 1(HMGB1),
`heat shock proteins (HSPs),
`and others), and pathogen-
`associated molecular patterns
`(PAMPs; viral products
`recognized by TLRs).
`
`Mechanisms of oncolytic virus immunotherapy
`The mechanisms through which oncolytic viruses mediate
`tumour rejection are incompletely understood. Most
`oncolytic viruses directly kill host tumour cells. This
`activity is influenced by the efficiency of cell receptor
`targeting, viral replication and host cell antiviral response
`elements8,9. The lytic potential of oncolytic viruses also
`depends on the type of virus, dose, natural and induced
`viral tropism, and the susceptibility of the cancer cell
`to the different forms of cell death (apoptosis, necrosis,
`pyroptosis and autophagy).
`In normal cells, a variety of signalling pathways oper‑
`ate to detect and clear pathogenic viral particles (FIG. 1).
`These pathways can be stimulated by local interferon
`(IFN) release or through intracellular Toll‑like receptors
`(TLRs), which are activated by viral elements. TLRs are cell
`surface and intracellular pattern recognition receptors that
`are activated in response to repeated sequences, so‑called
`pathogen‑associated molecular patterns (PAMPs), which
`
`are common to pathogenic bacteria and viruses. PAMPs
`may include elements of viral capsids, DNA, RNA and
`viral protein products. TLR signalling activates host
`cell antiviral responses and systemic innate immunity.
`Several downstream host cell factors involved in onco‑
`lytic virus clearance have been identified, including TNF‑
`associated factor 3 (TRAF3), IFN‑related factor 3 (IRF3),
`IRF7 and retinoic acid‑inducible gene 1 (RIG‑1). These
`factors activate the JAK–STAT (Janus kinase–signal
`transducer and activator of transcription) pathway, which
`coordinates the antiviral machinery in infected cells. The
`antiviral machinery reinforces local IFN release, which
`activates PKR activity. PKR is an intracellular protein
`kinase that recognizes double‑stranded RNA and other
`viral elements10,11. When activated by viral elements, PKR
`terminates cell protein synthesis and promotes rapid cell
`death and viral clearance. In cancer cells, IFN pathway
`signalling and PKR activity may be abnormal, and viral
`clearance is thwarted12.
`
`644 | SEPTEMBER 2015 | VOLUME 14
`
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`
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`
`R E V I E W S
`
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`
`

`

`a Healthy cell
`
`b Cancer cell
`
`IFN
`
`IFNR
`
`IFN
`
`IFNR
`
`Viral TLR
`ligands
`
`JAK
`
`Cytoplasm
`
`TLR
`
`Viral TLR
`ligands
`
`TLR
`
`࢑
`
`JAK
`
`PKR
`
`(cid:127) Reovirus
`(cid:127) Herpes
`(cid:127) Adenovirus
`(cid:127) Vaccinia
`(cid:127) Influenza
`
`Translation
`
`dsRNA
`
`MYD88
`
`TRIF
`
`࢑
`
`RIG-1
`NDV
`
`TRAF6
`
`TRAF3
`
`dsRNA
`
`MYD88
`
`TRIF
`
`RIG-1
`NDV
`
`TRAF6
`
`TRAF3
`
`NF-κB
`
`IRF7
`NDV
`
`IRF3
`NDV
`
`STAT
`
`IRF9
`
`(cid:127) VSV
`(cid:127) NDV
`(cid:127) Measles
`(cid:127) Vaccinia
`
`Apoptosis
`
`NF-κB
`
`Nucleus
`
`࢑
`IRF7
`NDV
`
`࢑
`
`IRF3
`NDV
`
`࢑
`
`PKR
`
`(cid:127) Reovirus
`(cid:127) Herpes
`(cid:127) Adenovirus
`(cid:127) Vaccinia
`(cid:127) Influenza
`
`STAT
`
`࢑
`IRF9
`
`(cid:127) VSV
`(cid:127) NDV
`(cid:127) Measles
`(cid:127) Vaccinia
`
`Translation
`
`Apoptosis
`
`Pro-
`inflammatory
`cytokines
`
`NF-κB
`
`Type I
`IFNs
`
`IRF7
`
`IRF3
`
`(cid:127) IRF7
`(cid:127) PKR
`
`STAT
`
`IRF9
`
`Pro-
`inflammatory
`cytokines
`
`NF-κB
`
`Type I
`IFNs
`
`IRF7
`
`IRF3
`
`(cid:127) IRF7
`(cid:127) PKR
`
`STAT
`
`IRF9
`
`Figure 1 | Oncolytic viruses can exploit cancer immune evasion pathways. a | Following viral infection, most
`normal cells activate an antiviral pathway that allows to contain viral infections. The antiviral machinery can be
`triggered by viral pathogen-associated molecular patterns (PAMPs) that activate Toll-like receptors (TLRs) or through
`the detection of viral nucleic acids by retinoic acid-inducible gene 1 (RIG-1). Once a virus is detected, a signalling
`Nature Reviews | Drug Discovery
`cascade through several type I interferon (IFN) elements (Janus kinase (JAK), signal transducer and activator of
`transcription (STAT), and interferon regulatory factor 9 (IRF9)) results in a programmed transcriptional pathway that
`limits viral spread and can target infected cells for apoptosis or necrosis. Local IFN production induced by the innate
`immune response to viral infections may also promote antiviral activity through the IFN receptor (IFNR). TLRs signal via
`the myeloid differentiation primary response protein MYD88, TIR-domain-containing adapter-inducing IFNβ (TRIF),
`IRF7, IRF3 and nuclear factor-κB (NF-κB), inducing the production of pro-inflammatory cytokines and type I IFNs.
`The type I IFNs signal through the JAK–STAT signalling pathway, resulting in the upregulation of cell cycle regulators,
`such as protein kinase R (PKR) and IRF7, which limit viral spread by binding to viral particles and triggering type I IFN
`transcriptional pathways, promoting abortive apoptosis of infected cells and the production of cytokines that alert the
`immune system to the presence of a viral infection. b | In cancer cells, however, this process is disrupted. Cancer cells
`may downregulate key signalling components within the innate signalling pathway, including RIG-1, IRF7, and IRF3
`(REF. 1). This limits detection of viral particles by TLR and RIG-1, making cancer cells more susceptible to viral
`replication. Furthermore, cancer cells may downregulate key components of the type I IFN signalling pathway2–7,
`thereby limiting the pro-apoptotic and cell cycle regulatory effects of type I IFNs. Although data are limited, the figure
`depicts individual viruses near the factors and/or pathways that are known to promote viral elimination in normal cells
`(part a) or that support viral replication owing to factor deficiency in cancer cells (part b). dsRNA, double-stranded
`RNA; NDV, Newcastle disease virus; TRAF, TNF-associated factor; VSV, vesicular stomatitis virus.
`
`Different viruses can also manipulate distinct aberrant
`signalling factors within tumour cells to block apoptosis,
`which allows more time for the virus to complete its life
`cycle. Following viral replication, most oncolytic viruses
`induce cell death, which can directly eliminate viable
`tumour cells but also sets the stage for initiating systemic
`immune responses. Induction of host immune responses
`can be greatly aided by both the type of cell death and the
`
`release of danger signals from virus‑infected cells. For
`example, necrosis or pyroptosis are more immunogenic
`forms of cell death than apoptosis.
`
`Induction of systemic anti-tumour immunity. The induction
`of systemic innate and tumour‑specific adaptive immune
`responses appears to be a critical element for tumour
`eradication with oncolytic viruses. Following oncolytic
`
`NATURE REVIEWS | DRUG DISCOVERY
`
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`
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`R E V I E W S
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`

`

`cell death, tumour cells release tumour‑associated
`antigens that can serve to promote an adaptive immune
`response that mediates tumour regression at distant
`tumour sites that are not exposed to virus. They also
`release viral PAMPS and additional cellular danger‑
`associated molecular pattern signals (DAMPs; for
`example, heat shock proteins, high mobility group box 1
`(HMGB1) protein, calreticulin, ATP, and uric acid) and
`cytokines (for example, type I IFNs, tumour necrosis
`factor‑α (TNFα), IFNγ, and interleukin‑12 (IL‑12)), which
`promote the maturation of antigen‑presenting cells (APCs)
`such as dendritic cells. These activate antigen‑specific CD4+
`and CD8+ T cell responses. Once activated, CD8+ T cells
`can expand into cytotoxic effector cells with the ability to
`traffic to sites of established tumour growth, where they
`mediate anti‑tumour immunity upon antigen recognition
`(FIG. 2). However, the natural ability of viruses to induce a
`host antiviral immune response may result in clearance of
`the virus through neutralizing antiviral antibodies and/or
`cytotoxic T‑cell‑mediated immune responses. The extent
`to which viral neutralization influences the induction of
`tumour immunity is complex and can be influenced by
`many variables, most notably the characteristics of the
`virus and the tumour microenvironment.
`The release of tumour‑associated antigens, especially in
`combination with local cytokine and DAMP release, can
`be beneficial for inducing innate and adaptive immune
`responses against cancer cells (FIG. 2). This effect may be
`especially important for mediating tumour regression at
`distant tumour sites that are not injected or exposed to
`virus. Preclinical studies have demonstrated the impor‑
`tance of tumour‑specific CD8+ T cells in mediating tumour
`rejection with oncolytic viruses13.
`Type I IFNs and DAMPs can also directly activate
`natural killer (NK) cells, which are part of the innate
`immune response. NK cells can kill target cells with
`downregulated major histocompatibility complex (MHC)
`class I expression, which is a common occurrence in
`cancer cells14,15. The influence of NK cells may depend
`on both the host species and the characteristics of the
`virus16,17. Furthermore, NK cells may be detrimental to
`the effectiveness of oncolytic viruses by eliminating virally
`infected cells18. The factors that influence the balance
`between immune‑mediated viral clearance and induction
`of antitumour immunity are incompletely understood.
`
`Counteracting cancer-mediated immune evasion. Cancer
`cells have evolved sophisticated strategies for avoiding
`immune‑mediated destruction. For example, tumour
`cells and the microenvironment can express immune‑
`inhibitory surface receptors that inactivate effector
`immune cells, and secrete factors — such as IL‑10, trans‑
`forming growth factor‑β (TGFβ) and indoleamine‑2,3‑
`dioxygenase (IDO) — that facilitate the recruitment of
`immune‑suppressive cells, such as tumour‑associated
`macrophages19 and myeloid‑derived suppressor cells20,
`to sites of tumour growth21. Oncolytic viruses modify
`this suppressive microenvironment through a variety
`of mechanisms that alter the cytokine milieu and the
`type of immune cells within the tumour microenviron‑
`ment22,23. These changes promote immune‑mediated
`
`tumour cell recognition and eradication, and can trigger
`tumour‑associated antigen and epitope spreading24,25.
`In the presence of danger signals and TLR engagement,
`the levels of type I IFNs and other inflammatory media‑
`tors increase, further potentiating systemic immunity
`against the cancer.
`The killing of cancer cells can result in the release of
`novel cancer antigens (neo-antigens) that may have been
`previously hidden to the immune system because of
`restricted presentation (FIG. 2). This effect was recently
`reported following the treatment of cancer patients with
`immunotherapeutic T cell checkpoint inhibitors26,27. Such
`neo‑antigens may be taken up by local APCs in the context
`of a pro‑inflammatory environment, which can trigger an
`immune response against the neo‑antigen. If new T cell
`clones are generated, they may be able to circulate and kill
`antigen‑expressing cancer cells, including cancer cells that
`were not infected by the virus. The immune response has
`also been associated with a ‘immune‑associated’ bystander
`effect, in which local release of cytotoxic perforins and
`granzymes may result in the killing of nearby tumour cells,
`even in the absence of direct antigen expression28. This
`is distinct from the virus bystander effect (BOX 1), which
`relates to the replication of the virus inside cancer cells and
`its spread to previously un‑infected cancer cells.
`
`Oncolytic virus biodistribution. Physical barriers that
`reduce the spread of oncolytic viruses include necrosis,
`calcification, hypoxia, acidosis, increased proteolytic
`activity, and a high interstitial pressure29–31. Furthermore,
`tumours are dense with extracellular matrix and are
`poorly vascularized. The majority of clinical studies
`with oncolytic viruses (such as adenovirus, poxvirus,
`HSV‑1, measles, and reovirus) have used intratumoural
`injections to bypass the tumour architectural barriers.
`However, intratumoural injections are limited to tumours
`that are physically accessible through clinical palpation or
`direct imaging. As discussed above, injection of an onco‑
`lytic virus into one tumour lesion can induce a systemic
`anti‑tumour response that can overcome physical limita‑
`tions, as evidenced in the T‑VEC OPTIM Phase III clini‑
`cal trial3. Other viruses, such as Seneca Valley Virus, can
`be delivered intravenously because of natural resistance
`to haemagglutination, a process resulting in premature
`viral clearance and reduced delivery to the tumour site
`following intravenous delivery32.
`The blood–brain barrier may limit the ability of some
`viruses (and many other drugs) to reach primary brain
`tumours and brain metastases. This may be overcome
`by direct injection into central nervous system (CNS)
`tumours or through the use of external reservoirs that
`communicate with sites of brain tumours. Parvovirus
`naturally crosses the blood–brain barrier, allowing for the
`delivery of this oncolytic virus via the intravascular route.
`Parvovirus H‑1PV has been used in clinical trials of glio‑
`blastoma multiforme (GBM)33. However, there have been
`relatively few studies of oncolytic virus distribution in vivo
`to evaluate viral penetrance throughout the CNS33–35.
`Tumour size and heterogeneity can present another
`barrier to virus biodistribution. Moreover, growth‑
`arrested cancer cells in hypoxic environments are less
`
`Epitope spreading
`A process whereby tissue
`damage during an initial
`immune response against
`one antigen may lead to the
`priming of self-reactive T
`and/or B cells targeting other
`areas (or epitopes) within
`the initial antigen or against
`other antigens.
`
`Neo‑antigens
`New antigens, often derived
`from cell metabolic pathway
`proteins, and commonly
`expressed in tumour cells.
`Neo-antigens may appear
`as a consequence of epitope
`spreading following initial
`immune attack on a tumour
`cell triggered by an unrelated
`antigen.
`
`Checkpoint inhibitors
`Monoclonal antibodies that
`inhibit negative immune
`checkpoints. Immune
`checkpoints control important
`intracellular signalling
`pathways in the immune
`system that either activate
`or inhibit immune responses.
`Checkpoint inhibitors have
`shown significant promise in
`the treatment of cancer.
`Most notably, blockade of
`cytotoxic T lymphocyte
`antigen 4 (CTLA4) and
`interactions between
`programmed cell death 1
`(PD1) and programmed cell
`death 1 ligand 1 (PDL1) have
`had significant impact on
`the treatment of melanoma,
`lung cancer, and possibly
`many other cancers.
`
`646 | SEPTEMBER 2015 | VOLUME 14
`
`www.nature.com/reviews/drugdisc
`
`© 2016 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
`
`Replimune Limited Ex. 2014 - Page 5
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Oncolytic
`virus
`
`Infection
`
`Cancer cell
`
`(cid:127) Viral proteins
`(cid:127) Viral genome
`
`Release/
`secrete
`
`(cid:127) ER stress
`(cid:127) Genotoxic stress
`
`ROS
`
`Cytokine
`receptors
`
`NK cell
`
`Cytotoxicity
`
`CD8+ T cell
`
`Cytokine
`receptors
`
`Cytotoxicity
`
`IL-2R
`
`IL-2
`
`Viral
`oncolysis
`
`ROS
`
`Release
`
`Release
`
`CD28
`
`TCR
`
`CD4+ T cell
`
`Antigen
`uptake
`
`TLR
`
`MHC MHC
`
`CD40L
`
`PAMPs
`(cid:127) Viral capsids
`(cid:127) Viral DNA
`(cid:127) Viral dsRNA/ssRNA
`(cid:127) Viral proteins
`
`DAMPs
`(cid:127) HSPs
`(cid:127) HMGB1
`(cid:127) Calreticulin
`(cid:127) ATP
`(cid:127) Uric acid
`
`Cytokines
`(cid:127) Type I interferons
`(cid:127) TNFα
`(cid:127) IFNγ
`(cid:127) IL-12
`
`(cid:127) Type I IFNs
`(cid:127) DAMPs/PAMPs
`
`(cid:127) Type I IFNs
`(cid:127) Cytokines
`
`DAMPs/
`PAMPs
`
`(cid:127) Viral antigens
`(cid:127) TAAs/neoantigens
`
`(cid:127) CD80/CD86
`(cid:127) Chemokine receptors
`
`Activation
`
`CD40
`
`TCR
`
`MHC
`
`Antigen presenting cell
`
`Figure 2 | The induction of local and systemic anti-tumour immunity by oncolytic viruses. The therapeutic efficacy
`of oncolytic viruses is determined by a combination of direct cancer cell lysis and indirect activation of anti‑tumour
`immune responses. Upon infection with an oncolytic virus, cancer cells initiate an antiviral response that consists of
`Nature Reviews | Drug Discovery
`endoplasmic reticulum (ER) and genotoxic stress. This response leads to the upregulation of reactive oxygen species (ROS)
`and the initiation of antiviral cytokine production. ROS and cytokines, specifically type I interferons (IFNs), are released
`from the infected cancer cell and stimulate immune cells (antigen presenting cells, CD8+ T cells, and natural killer (NK)
`cells). Subsequently, the oncolytic virus causes oncolysis, which releases viral progeny, pathogen-associated molecular
`patterns (PAMPs), danger-associated molecular pattern signals (DAMPs), and tumour associated antigens (TAAs)
`including neo-antigens. The release of viral progeny propagates the infection with the oncolytic virus. The PAMPs
`(consisting of viral particles) and DAMPs (comprising host cell proteins) stimulate the immune system by triggering
`activating receptors such as Toll-like receptors (TLRs). In the context of the resulting immune‑stimulatory environment,
`TAAs and neo-antigens are released and taken up by antigen presenting cells. Collectively, these events result in the
`generation of immune responses against virally infected cancer cells, as well as de novo immune responses against
`TAAs/neo-antigens displayed on un‑infected cancer cells. CD40L, CD40 ligand; dsRNA, double-stranded RNA;
`HMGB1, high mobility group box 1; HSP, heat shock protein; IL-2, interleukin-2; IL-2R, IL-2 receptor; MHC, major
`histocompatibility complex; ssRNA, single-stranded RNA; TCR, T cell receptor; TNFα, tumour necrosis factor-α.
`
`likely to be permissive to infection31,36,37. Stromal cells,
`such as cancer‑associated fibroblasts, may be infected
`by oncolytic viruses but are non‑permissive to viral
`replication. Thus, fibroblasts may act as a decoy reservoir
`for oncolytic viruses, reducing the delivery of infectious
`virions to cancer cells38. Another mechanism that may
`limit the overall effectiveness of oncolytic viruses is the
`susceptibility of cancer cells to apoptosis, which may
`be induced by viral infection or other factors39. If cells
`undergo apoptosis too rapidly, this will reduce the time
`
`for viral replication and propagation and decrease the
`amount of active virus in the tumour, ultimately limiting
`the active intratumoural dose.
`
`Development of oncolytic viruses as drugs
`As oncolytic viruses are live viral particles, the over‑
`all design of oncolytic virus strategies must consider
`approaches to tumour cell targeting and attenuating
`viral pathogenesis, as well as approaches to limit viral
`immunogenicity while promoting tumour cell killing
`
`NATURE REVIEWS | DRUG DISCOVERY
`
` VOLUME 14 | SEPTEMBER 2015 | 647
`
`© 2016 Macmillan Publishers Limited. All rights reserved
`
`R E V I E W S
`
`Replimune Limited Ex. 2014 - Page 6
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Box 1 | Virus bystander effect
`
`The optimal method for the administration of oncolytic viruses is not established but
`most clinical trials have utilized direct injection into established tumours, with some
`evaluating intravenous delivery. Because oncolytic viruses are capable of replication,
`only a limited number of tumour cells need to be infected. A significant bystander effect
`can be anticipated by the local replication of the virus and its release into the
`surrounding tumour where viral particles can then infect new tumour cells. If the host
`antiviral immune response does not neutralize the virus, infection may continue to
`propagate. The inclusion of suicide genes within the oncolytic virus can further enhance
`the bystander effect and achieve much greater cell killing. However, the contribution
`of the bystander effect to the overall therapeutic effectiveness of the oncolytic virus
`needs to be demonstrated in a randomized clinical trial to be more fully evaluated.
`
`and immunogenicity. The flexibility of recombinant
`engineering has allowed the exploration of a number of
`strategies to enhance the effectiveness of oncolytic viruses.
`
`Targeting oncolytic viruses to cancer cells. Many of the
`oncolytic viruses that are currently in the clinic have a
`natural tropism for cell surface proteins that are aber‑
`rantly expressed by cancer cells (FIG. 3). For example,
`HSV‑1 uses the herpesvirus entry mediator (HVEM) and
`selected nectins for cell entry. These surface receptors are
`overexpressed on some cancer cells, including melanoma
`and various carcinomas40. Measles virus, specifically the
`Edmonston strain, utilizes the surface receptor CD46
`for cell entry41. CD46 normally func