Oncolytic Immunotherapy: A Local and Systemic Antitumor Approach

Oncolytic immunotherapy Oncolytic immunotherapy the use of a genetically modified virus to attack tumors and induce a systemic immune response to cancer is a rapidly evolving area of therapy, but the basic idea behind it is not new. The ability of viruses to affect cancer was first observed in 1904, and for more than a century potential virus-induced remissions have been observed in a limited number of patients. However, the effect with naturally occurring viruses is limited and short-lived, with the risk of serious infection and death. More recently, there have been attempts to genetically modify these viruses for a more potent, selective, and permanent anticancer effect. 1 Oncolytic immunotherapy uses a modified oncolytic virus that selectively replicates in tumor cells for an antitumor effect through a proposed dual mode of action 2 : Oncolytic: Direct cytotoxic activity due to the replication of the virus and cell lysis Immunotherapy: Indirect induction of a systemic antitumor immune response 3

Many potential genetic modifications to oncolytic viruses have been identified for enhanced effect Many viruses have been studied as potential vectors for oncolytic immunotherapy. This includes the insertion, deletion, and/or inactivation of various genes to achieve desired effects. 3 Modifications allow the virus to selectively replicate in and destroy tumor cells and release progeny viruses. The viruses infect, replicate selectively in, and lyse other tumor cells for a potent oncolytic effect. 4,5 Additional genetic modifications enhance oncolytic cell death and express cytokine genes to activate a systemic immune response. 3 Effect Virus Possible gene modifications Adenovirus E1B-55kd and/or E1A CR2 deletion Use of tissue- or tumor-specific promoters Selectivity 3,6 HSV-1 ICP34.5 gene or ICP6 gene inactivation/deletion TK gene deletion Use of tissue- or tumor-specific promoters Vaccinia virus TK gene deletion SPI-1 and SPI-2 gene deletion Enhanced oncolytic cell death 3,4 Poxvirus HSV-1 Adenovirus TK gene disruption ICP47 deletion and earlier expression of US11 E3 or FMG gene expression E1B-19kd gene deletion Cytokine expression 3 Various IL-4, IL-12, GM-CSF, MCP-1, and IFN gene insertion FMG = fusogenic membrane glycoprotein, GM-CSF = granulocyte-macrophage colony-stimulating factor, HSV-1 = herpes simplex virus 1, IFN = interferon, IL = interleukin, MCP = monocyte chemotactic protein, SPI = spleen focus forming virus proviral integration oncogene, TK = thymidine kinase. 5

Genetic modifications reduce the ability of viruses to replicate in healthy cells Figure A Figure B Selectivity may be increased by deleting or inactivating genes in the virus that are critical for replication in healthy cells but are not necessary for replication in tumor cells. 3 HSV-1 relies on the neurovirulence factor ICP34.5 to overcome the host cell s normal response to infection, which includes production of IFNα. IFNα signaling inhibits viral replication. By inactivating the gene in the virus that encodes the neurovirulence factor, the virus will no longer be able to replicate in healthy cells. However, these viruses are able to replicate in tumor cells even without neurovirulence factors. 7,8 The chart shows the results of a preclinical model of healthy human cells. Cells treated with HSV-1 modified by the deletion of the neurovirulence factor ICP34.5 and cultured in the presence of IFNα resulted in a 62,000-fold reduction of viral replication (Figure A). 9 The perpetuated cycle of tumor-selective replication and oncolysis after release of newly synthesized viruses can be visualized in a mouse model of a colorectal carcinoma infected with an oncolytic virus containing the LacZ gene. After staining, LacZ expression was detected as early as day 1, with maximum expression seen on days 2 to 3 and continuing through day 7 (Figure B). 10 Virus yield (pfu/ml) 10 7 Preclinical model of virus replication 10 6 10 5 10 4 10 3 10 2 10 0.0 HSV Adapted from Mulvey M, et al. J Virol. 2004. HSV with ICP34.5 deletion - IFNα + IFNα DAY 1 DAY 3 DAY 7 Viral infiltration (blue) of murine colorectal carcinoma cells Reprinted from Surgery 141, Malhotra S, et al. Use of an oncolytic virus secreting GM-CSF as combined oncolytic and immunotherapy for treatment of colorectal and hepatic adenocarcinomas, 520-529, Copyright 2007, with permission from Elsevier. 7

Oncolytic cell death using oncolytic viruses with target gene modifications can result in antitumor activity in preclinical models Figure A Figure B In a preclinical model of glioma, an HSV-1 oncolytic virus was modified with the deletion/inactivation of one or two different genes in order to generate tumor-selective replication and enhance tumor cell kill. These deletions led to significantly slowed or reduced tumor growth over time when injected into mice (Figure A). 5 In a preclinical model of prostate cancer, an oncolytic adenovirus with or without E3 gene expression was injected into mice. Six weeks after a single injection, these modifications resulted in an up to 4-fold reduction in tumor volume (Figure B). 11 Average tumor diameter (mm) 12 10 8 6 4 2 0 Glioma mouse model 5 0 7 14 21 28 35 Vehicle HSV with single gene deletion HSV with deletion of 2 genes P = 0.014 for difference Relative tumor volume (%) 400 350 300 250 200 150 100 50 0 Prostate cancer mouse model 11 Vehicle 0 1 2 3 4 5 6 7 Adenovirus without E3 gene Adenovirus with E3 gene Days Weeks Adapted from Liu BL, et al. Gene Ther. 2003. Adapted from Yu DC, et al. Cancer Res. 1999. 9

Oncolytic cell death and cytokine expression can initiate a systemic immune response Figure A Figure B In addition to releasing newly synthesized viruses, the oncolysis of tumor cells also releases and exposes an array of tumor antigens to initiate a systemic immune response (Figure A). The tumor antigens elicit an adaptive immune response that is patient- and tumor-specific, possibly enhancing efficacy. Further, the virus may be modified to express and release specific cytokines (eg, GM-CSF, IL-4, IL-12, MCP-1, or IFN). 3,12 GM-CSF secreting tumor cells can stimulate dendritic cell expansion and infiltration of tumor tissue. The effect of GM-CSF on dendritic cells and induction of antitumor immunity has been documented in preclinical models. Melanoma cells engineered to express GM-CSF but irradiated to prevent cell division were injected into mice, resulting in an accumulation of tumor-infiltrating dendritic cells (Figure B). 13 Illustrated rendering of the release of antigens, viruses, and cytokines following oncolytic cell death Local dendritic cell infiltration (brown stain) after cytokine expression from GM-CSF secreting cells in a mouse model of melanoma Reprinted from Cancer Research 60, Mach N, et al. Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocytemacrophage colony-stimulating factor or Flt3-ligand, 3239-3246, Copyright 2000, with permission from the American Association for Cancer Research. 11

Systemic effect: Antigen-presenting cells process and present an array of tumor antigens to activate T cells for a systemic immune response Figure A Figure B HSV-1 Control (inactivated virus) Dendritic cells capture tumor antigens after oncolysis and process them for presentation on MHC molecules. 14 By exposing dendritic cells to an array of tumor antigens, a robust and broad immune response may be achieved. 12,15 Mature dendritic cells can interact with CD4+ and CD8+ T cells to produce CD8+ effector T cells with cytotoxic potential (Figure A). 14,16 In a preclinical model of ovarian cancer, treatment with an oncolytic virus can induce tumor infiltration of CD8+ T cells (Figure B). 17 Cytotoxic T cells that recognize different tumor antigens on tumor cells can also disperse throughout the body to destroy tumor cells and lead to destruction of distant tumor cells. 18 Illustrated rendering of activated dendritic cells presenting tumor antigens to T cells CD8+ T cells (stained brown) after HSV-1 or inactivated virus Reprinted by permission from Macmillan Publishers Ltd: Molecular Therapy. Benencia F, et al. HSV oncolytic therapy upregulates interferon-inducible chemokines and recruits immune effector cells in ovarian cancer. 12:789-802. Copyright 2005. 13

Systemic effect: In preclinical models, a systemic immune response resulted in destruction of distant tumor cells Figure A Figure B Local injection may enhance immunotherapeutic response while reducing systemic exposure to healthy cells. 19 Preclinical studies demonstrate that a locally induced, tumor-specific immune response can result in the systemic destruction of distant tumor cells. A modified oncolytic HSV-1 expressing IL-12 injected into a murine colorectal adenocarcinoma tumor on days 0 and 7 resulted in significantly greater antitumor effects compared with a modified oncolytic HSV-1 that did not express the cytokine (Figure A). In addition, significant tumor growth reduction was also observed in a bilateral tumor that had not been injected, demonstrating a systemic immune response (Figure B). 6,20 Tumor volume (mm 3 ) 5000 4000 3000 2000 1000 Injected tumor 20 Vehicle Modified HSV-1 Modified HSV-1 expressing IL-12 5000 4000 3000 2000 1000 Noninjected tumor 20 0 0 5 10 15 20 0 0 5 10 15 20 Days post-inoculation Adapted from Toda M, et al. J Surg Oncol. 2011. 15

Oncolytic immunotherapy is an approach that utilizes an oncolytic virus to activate a tumor-specific immune response Genetic modifications of the virus result in selective replication in tumor cells while reducing the ability of the virus to replicate in healthy cells. 21 By exposing an array of tumor antigens through tumor cell lysis, oncolytic immunotherapy may induce a robust immune response in patients. 12,15 In addition, insertion of an immunomodulatory cytokine gene and local cytokine expression attracts and activates dendritic cells and T cells for a systemic immune response to tumor cells. 13 Induction of a systemic immune response can then result in death of distant tumor cells. 22 Illustrated rendering of T-cell expansion and tumor cell destruction 17

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