Oncolytic Viruses for the Treatment of Advanced Melanoma

Beverly E Barton, PhD

Melanoma

Overview
Melanoma cases make up nearly 5% of new cancers diagnosed in the United States, making it the fifth most common type of cancer in this country.¹ With the introduction of targeted therapy, melanoma treatment has undergone rapid changes in recent years, resulting in an overall 5-year survival rate of 91.3%.¹ Among these targeted treatments are BRAF inhibitors dabrafenib (Tafinlar) and vemurafenib (Zelboraf), and the MEK inhibitor trametinib (Mekinist).^2,3 These drugs are indicated for those who have a V600 BRAF mutation; approximately 50% of melanoma patients bear such a mutation, resulting in continual activation of the MAPK pathway.⁴ In patients whose tumors bear KIT mutations, the use of an appropriate inhibitor such as imatinib (Gleevec) may be warranted.⁵ A subset of patients with another mutation, NRAS, is particularly responsive to high doses of interleukin (IL)-2.⁶ One might suspect from these data that immunologic targeting should be a viable approach to advanced melanoma treatment; indeed, the use of the cytotoxic T-lymphocyte antigen-4 (CTLA-4)-inhibiting antibody ipilimumab (Ipi; Yervoy) has substantiated this supposition with a significant increase in overall survival.⁷ Despite these advances, the 5-year survival rate for advanced melanoma (having distant metastases) is only 16.1%,¹ underscoring the need to continue the search for even more effective treatments for this patient population. Cutting-edge therapy includes the use of oncolytic viruses designed to deliver immunostimulating cytokines while simultaneously killing tumor cells. This review will discuss the history and potential use of immunotherapeutic oncolytic viruses for the treatment of advanced melanoma, with particular attention to talimogene laherparepvec (TVEC), one such oncolytic virus currently in phase 3 trials.

Oncolytic Viruses in Cancer Therapy
As early as 1949, Alice Moore, MD, showed that infection with the Russian Far East encephalitis virus or with the influenza virus could induce regression of sarcomas in mouse models.^8,9 In 1999, Csatary and Bakács¹⁰ reported the successful treatment of a patient with glioblastoma multiforme using a Newcastle disease virus (NDV) vaccine. After receiving the NDV vaccine, the patient’s tumor shrank, and objective neurologic responses improved. Indeed, cancer virotherapy dates back to older observations that unexplained tumor regressions sometimes accompanied virus infections; the earliest such report is infection with NDV in a patient with gastric cancer.¹¹ Aggressive tumor cells have impaired antiviral responses, such as elaboration of interferons (IFNs), and at the same time show higher permissiveness for virus replication.¹² The development of modern molecular biology created the necessary technology to modify viruses according to clinical needs.¹³ Thus, viruses can easily be engineered to express transgenes of choice, such as immune-enhancing cytokines or cytolytic proteins. Table 1 briefly summarizes oncolytic viruses in clinical trials. Viruses from several families are in trials for their therapeutic potential as vaccines against a variety of solid tumors. Several viruses are modifications or variants of veterinary pathogens (eg, vaccinia, Seneca Valley virus [SVV], parvovirus, NDV, and vesicular stomatitis virus [VSV]). Some have been engineered to express cytokines or chemokines, notably IFN-?, and granulocyte-macrophage colony-­stimulating factor (GM-CSF).

Parvovirus mainly causes disease in dogs and other animals, although parvovirus B19 is the causative agent of fifth disease (erythema infectiosum) in humans. The H-1 strain is a nonpathogenic virus, and its natural hosts are the rat and other rodent species. Unlike many other parvoviruses, it replicates in human cells while displaying no immunosuppressive effects. It is now in a trial for treatment of primary or recurrent glioblastoma.¹⁴ Details regarding transgene expression by the viral vaccine have not yet been published. Vaccinia is known to many as the cowpox virus, formerly used to immunize against smallpox. An engineered variant, JX-594, was created to destroy cancer cells through replication-dependent lysis. In addition, it activates immune responses through expression of its GM-CSF transgene and shows marked cancer cell selectivity through mechanisms driving its replication: activation of epidermal growth factor receptor signaling in tumor cells and inactivation of the type I IFN response pathway.¹⁵ Herpes simplex virus-1 (HSV-1), the causative agent of most cold sores, is also familiar. Although humans have levels of immunity to this common pathogen, research has shown that such immunity does not seem to interfere with antitumor effects.¹⁶ An oncolytic HSV-1 vaccine bearing a GM-CSF transgene will be discussed in detail below. Adenovirus, the common cold virus, is the basis for several vaccines. The H101 variant, the first commercialized oncolytic vaccine, was approved by China’s regulatory agency in 2005 for the treatment of nasopharyngeal carcinoma when used in conjunction with cisplatin.¹⁷ VSV is normally an animal pathogen. Humans generally lack immunity to VSV (natural infections are for the most part asymptomatic); thus, VSV is an attractive candidate for oncolytic virotherapy.¹⁸ Reovirus, normally an enteric pathogen, has a preference for replication in KRAS-mutated cells. This attribute makes it a good choice for use in colorectal cancer, since 40% to 50% of patients with this cancer bear activating KRAS mutations in their tumors.¹⁹ Other solid tumors, such as head and neck squamous cell carcinoma and prostate cancer, may be treated by reovirus vaccines in the future. Measles virus derived from an attenuated strain shows broad antitumor activity. Preliminary data indicate that the measles virus interacts with CD46 when it is overexpressed on tumor cells (this is true for nearly all tumor types), causing its cytopathic effects of syncytia formation and cell lysis. Normal cells expressing lower amounts of surface CD46 appear to be protected from the deleterious effects of the measles vaccine.²⁰ SVV normally infects swine. In humans, it displays a tropism for neuroendocrine tumors. An advantage of SVV over several other viruses is the ability to generate high titers following administration, which was observed to aid in eradication of tumors in model systems.²¹ Coxsackie virus is an enterovirus; the A16 strain is the most common cause of hand, foot, and mouth disease in the United States.²² The A21 strain has oncolytic activity against a variety of solid tumors, including prostate, melanoma, and breast. In combination with doxorubicin, Coxsackie A21 induced greater tumor regression than either agent as monotherapy in a murine breast cancer model.²³

Table 1

Results from the clinic indicate that oncolytic viral vaccines are generally well-tolerated, even at the highest doses.²⁴ Risk of disease transmission is lowered by using replication-defective vectors, or attenuated viruses, or those not normally pathogenic to humans. In addition, using viral vectors that do not integrate into the human genome lessens the potential for human gene mutations.²⁴

Mechanisms of Action of Oncolytic Viruses
Oncolytic viral vaccines work in several ways. The specific cellular tropism of the viral vector is exploited to determine the type of cancer to be treated.²⁴ As mentioned above, some, such as measles-based vaccines, induce desired cytopathic effects, including syncytia formation and tumor cell lysis, by binding to cell surface receptors overexpressed on tumor cells.²⁰ Oncolytic viral vaccines induce cell death through apoptosis, necrosis, or autophagy.²⁵ Direct killing may be a function of infection efficiency,²⁶ which is why obtaining high viral titers is desirable, as with SVV-derived vaccines.²¹ Successful viral vaccines induce cross-priming of naive CD8+ T cells in lymph nodes; this would imply that there be antigenic determinants expressed by the viral vectors.²⁶ It is certainly possible to engineer viral vaccines to express transgenes that enhance antigen presentation to dendritic cells or otherwise stimulate immune responses. Heat-shock proteins are used in such a vaccine approved in Russia, although the vaccine failed to confer significant recurrence-free survival in patients with renal cell carcinoma following nephrectomy.²⁷ An additional feature of viral vaccines is the ability to sensitize tumor cells to the effects of conventional therapeutic modalities, such as chemotherapy or radiotherapy.²⁸

The expression of cytokine transgenes aids the anti­tumor effects of viral vectors in several ways, depending on the cytokine. IFN-? induces tumor cell apoptosis (through induction of caspase-7 and downstream activation of deoxyribonuclease-?), tumor cell cycle arrest, activation of immune responses, and induction of cytokine and chemokine secretion such as tumor necrosis factor-?.²⁹ IFN-? also enhances immune response by cross-priming antitumor cytolytic CD8+ T-cell responses; in addition, local production of IFN abrogates many of the adverse responses seen with systemic IFN-? administration.²⁶ Early vaccine prototypes using IL-2 transgenes on NDV vectors had unacceptable toxicity levels due to IL-2. Therefore, another T cell–stimulating cytokine, IL-15, is under investigation. IL-15 has less toxicity than IL-2 but similarly strong capability to stimulate CD8+ T cells.³⁰ GM-CSF is one of the most potent immunostimulatory cytokines to be used as a viral vaccines transgene. GM-CSF enhances tumor antigen presentation, recruits dendritic cells to receive presented tumor cell antigen, and aids in the coordinated activation of CD4+ and CD8+ T cells.³¹ Animal models reveal that tumor cells infected with viruses having GM-CSF transgenes have dense infiltrates comprising professional antigen-presenting cells (eg, dendritic cells and macrophages). In addition, numerous natural killer cells were noted.³¹ These results imply that GM-CSF activates antitumor mechanisms through antigen-specific and antigen-nonspecific pathways.³¹

Talimogene Laherparepvec: Engineering HSV-1 for Melanoma Therapy
HSV-1 is a member of the human herpes virus family, among whose other members are varicella zoster (causative agent of chickenpox and shingles), Epstein-Barr virus, human cytomegalovirus, and Kaposi sarcoma virus. It is a large, enveloped, linear double-stranded DNA virus, 152 kilobases in length, coding for about 85 gene products. It does not integrate into host genomes. The HSV-1 genome is thus well-suited to engineer for construction of viral vaccines.³² Early versions of HSV-1 viral vaccines expressed so-called suicide genes, such as thymidine kinase and cytosine deaminase.³³ However, vectors expressing immunostimulatory transgenes such as GM-CSF have progressed the furthest.

To create TVEC, the virus was modified so that it replicates within tumors by deleting ICP34.⁵ (in wild-type HSV-1, this gene enables the virus to replicate in healthy neural tissue and helps overcome the effects of host-secreted IFN-?).³⁴ In addition, ICP47 was deleted to enhance tumor-selective replication (ICP47 inhibits human transporter proteins and blocks antigen presentation). Finally, TVEC expresses GM-CSF as a transgene.³⁵ In preclinical glioma models, HSV-1 having deletions of either ICP34.5 or ICP47 significantly reduced tumor volumes; when both genes were deleted and GM-CSF was expressed, the antitumor effect was the most significant.³⁶ Early human trials comparing GM-CSF with Flt3 ligand transgenes revealed that GM-CSF was superior for recruiting dendritic cells, the melanoma lesions of volunteers having dense mature dendritic cell infiltrates.³⁷ Furthermore, there were increased numbers of CD8+ T cells found in lesions, as well as systemically, after vaccination with an active HSV-1 vector compared with vaccination with inactivated virus.³⁸

To date, TVEC is registered in 3 trials^39-41; results are available for 2 of them. In these trials, TVEC was administered intratumorally.²⁶ The OPTiM trial39 finished accruing, and preliminary results were recently released. This trial compared TVEC with GM-CSF (sargramostim) in patients with stage IIIB-IV unresectable melanoma. Analysis of data from 436 patients enrolled in the trial revealed that the primary end point, durable response rate, was met. The US Food and Drug Administration under a special protocol assessment⁴² accepted this primary end point. A summary of the results, presented in Table 2, shows that in the patients who received TVEC, overall survival increased by 4.4 months. This improvement approached clinical significance at 23.3% for the TVEC arm versus 18.9% for the sargramostim arm (P=.51; hazard ratio 0.787). Moreover, the durable response rate was 16% for those who received TVEC versus 2% for those who received sargramostim.⁴³ Final data analysis has not yet been reported. Subset analyses of the patterns of durable responses revealed that of 48 patients having a durable response, 83% remained in remission at the median follow-up more than 18.4 months later. Twenty-three patients had progressive disease prior to responding, a common observation with immunotherapies in general, reinforcing protocols that indicate treatment should continue through progression until responses are achieved (Table 2).⁴⁴ Phases 1 and 2 of this trial revealed no virus shedding or elevated serum GM-CSF. Induction of tumor antigen–specific immune responses was observed. T cells found in tumor infiltrates often displayed memory T-cell phenotype; in addition, decreased numbers of monocyte-derived suppressor cells were observed.^35,45,46 The most common adverse events seen in the phase 3 trial were fatigue, chills, and pyrexia. Fewer than 3% of patients in either arm experienced adverse events of grade 3 or greater.⁴³

Table 2

A phase 1b/2 trial in naive patients with unresectable stage IIIB-IV melanoma examines the safety and efficacy of TVEC in combination with ipilimumab (Ipi).⁴⁰ Ipi is a monoclonal antibody blocking CTLA-4, thereby preventing the interaction between costimulatory molecules, CD80 or CD86, and CTLA-4. This short-circuits the normal ebbing of T-cell–mediated responses and thereby enhances antitumor immunity.⁴⁷ Ipi has been approved for melanoma therapy in the United States since 2011 due to its positive effects on survival in melanoma patients.⁷ In the TVEC + Ipi trial, 17 patients have been evaluated thus far. The objective response rate of patients who received TVEC + Ipi was 41%. Of these, 24% achieved a complete response and 18% had a partial response. The response rates of patients on either therapy alone were not stated.⁴⁸ Further analysis by flow cytometry revealed that patients who received TVEC + Ipi had significantly higher numbers of CD8+ T cells at the time of evaluation compared with baseline (Table 2).⁴⁸ Phenotyping and additional analyses continue.

Conclusion
Oncolytic viruses are both old and cutting-edge as treatment for solid tumors. Using genetic engineering, viruses can be modified to enhance tumor-specific replication, abrogate unwanted tissue tropisms, and carry antitumor payloads. Among these payloads, immunostimulatory cytokines such as GM-CSF are proving to be effective in the clinic, although other novel molecules will surely follow.²⁵ This is a promising area of investigation, and its potential is only beginning to be realized.

References
1. Howlader N, Noone AM, Krapcho M, et al. SEER Cancer Statistics Review, 1975-­2011. National Cancer Institute. Bethesda, MD. http://seer.cancer.gov/csr/1975_2011/, based on November 2013 SEER data submission, posted to the SEER website, April 2014.
2. Ascierto PA, Grimaldi AM, Acquavella N, et al. Future perspectives in melanoma research. Meeting report from the “Melanoma Bridge. Napoli, December 2nd-4th 2012.” J Transl Med. 2013;11:137.
3. Saraceni MM, Khushalani NI, Jarkowski A III. Immunotherapy in melanoma: recent advances and promising new therapies [published online March 27, 2014]. ­J Pharm Pract.
4. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507-2516.
5. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Melanoma. Version 4.2014. www.nccn.org/professionals/ physician_gls/f_guidelines.asp. Accessed June 13, 2014.
6. Joseph RW, Sullivan RJ, Harrell R, et al. Correlation of NRAS mutations with clinical response to high-dose IL-2 in patients with advanced melanoma. J Immunother. 2012;35:66-72.
7. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711-723.
8. Moore AE. The destructive effect of the virus of Russian Far East encephalitis on the transplantable mouse sarcoma 180. Cancer. 1949;2:525-534.
9. Moore AE. Effect of inoculation of the viruses of influenza A and herpes simplex on the growth of transplantable tumors in mice. Cancer. 1949;2:516-524.
10. Csatary LK, Bakács T. Use of Newcastle disease virus vaccine (MTH-68/H) in a patient with high-grade glioblastoma. JAMA. 1999;281:1588-1589.
11. Csatary LK. Viruses in the treatment of cancer. Lancet. 1971;2:825.
12. Moehler M, Goepfert K, Heinrich B, et al. Oncolytic virotherapy as emerging immunotherapeutic modality: potential of parvovirus H-1. Front Oncol. 2014;4:92.
13. Vacchelli E, Eggermont AM, Sautès-Fridman C, et al. Trial watch: oncolytic viruses for cancer therapy. Oncoimmunology. 2013;2:e24612.
14. Geletneky K, Huesing J, Rommelaere J, et al. Phase I/IIa study of intratumoral/intracerebral or intravenous/intracerebral administration of Parvovirus H-1 (Parv­ Oryx) in patients with progressive primary or recurrent glioblastoma multiforme: ParvOryx01 protocol. BMC Cancer. 2012;12:99.
15. Parato KA, Breitbach CJ, Le Boeuf F, et al. The oncolytic poxvirus JX-594 selectively replicates in and destroys cancer cells driven by genetic pathways commonly activated in cancers. Mol Ther. 2012;20:749-758.
16. Carson J, Haddad D, Bressman M, et al. Oncolytic herpes simplex virus 1 (HSV-1) vectors: increasing treatment efficacy and range through strategic virus design. Drugs Future. 2010;35:183-195.
17. Liang M. Clinical development of oncolytic viruses in China. Current Pharm Biotechnol. 2012;13:1852-1857.
18. Muik A, Stubbert LJ, Jahedi RZ, et al. Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity and enhance oncolytic potency. Cancer Res. 2014;74:1-12.
19. Maitra R, Seetharam R, Tesfa L, et al. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan. Oncotarget. 2014;5:2807-2819.
20. Msaouel P, Iankov ID, Dispenzieri A, et al. Attenuated oncolytic measles virus strains as cancer therapeutics. Curr Pharm Biotechnol. 2012;13:1732-1741.
21. Rudin CM, Poirier JT, Senzer NN, et al. Phase I clinical study of Seneca Valley Virus (SVV-001), a replication-competent picornavirus, in advanced solid tumors with neuroendocrine features. Clin Cancer Res. 2011;17:888-895.
22. Centers for Disease Control and Prevention. Hand, Foot, and Mouth Disease (HMFD). Centers for Disease Control and Prevention website. www.cdc.gov/hand-foot-mouth/index.html. Updated May 22, 2013. Accessed May 21, 2014.
23. Skelding KA, Barry RD, Shafren DR. Enhanced oncolysis mediated by Coxsackievirus A21 in combination with doxorubicin hydrochloride. Invest New Drugs. 2012; 30:568-581.
24. Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol. 2012;30: 658-670.
25. Bartlett DL, Liu Z, Sathaiah M, et al. Oncolytic viruses as therapeutic cancer vaccines. Mol Cancer. 2013;12:103.
26. Elsedawy NB, Russell SJ. Oncolytic vaccines. Expert Rev Vaccines. 2013;12:1155-1172.
27. Wood C, Srivastava P, Bukowski R, et al. An adjuvant autologous therapeutic vaccine (HSPPC-96; vitespen) versus observation alone for patients at high risk of recurrence after nephrectomy for renal cell carcinoma: a multicentre, open-label, randomised phase III trial. Lancet. 2008;372:145-154.
28. Goldufsky J, Sivendran S, Harcharik S, et al. Oncolytic virus therapy for cancer. Oncolytic Virotherapy. 2013;2:31-46.
29. Yoshida J, Mizuno M, Wakabayashi T. lnterferon-? gene therapy for cancer: basic research to clinical application. Cancer Sci. 2004;95:858-865.
30. Niu Z, Bai F, Sun T, et al. Recombinant Newcastle disease virus expressing IL15 demonstrates promising antitumor efficiency in melanoma model [published online March 22, 2014]. Technol Cancer Res Treat.
31. Dranoff G. GM-CSF-based cancer vaccines. Immunol Rev. 2002;188:147-154.
32. Goins WF, Huang S, Cohen JB, et al. Engineering HSV-1 vectors for gene therapy. Methods Mol Biol. 2014;1144:63-79.
33. Varghese S, Rabkin SD. Oncolytic herpes simplex virus vectors for cancer virotherapy. Cancer Gene Ther. 2002;9:967-978.
34. Melchjorsen J, Matikainen S, Paludan SR. Activation and evasion of innate antiviral immunity by herpes simplex virus. Viruses. 2009;1:737-759.
35. Senzer NN, Kaufman HL, Amatruda T, et al. Phase II clinical trial of a granulocyte-macrophage colony-stimulating factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma. J Clin Oncol. 2009; 27:5763-5771.
36. Liu BL, Robinson M, Han ZQ, et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther. 2003; 10:292-303.
37. Mach N, Gillessen S, Wilson SB, et al. Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand. Cancer Res. 2000;60:3239-3246.
38. Benencia F, Courrèges MC, Conejo-Garcia JR, et al. HSV oncolytic therapy upregulates interferon-inducible chemokines and recruits immune effector cells in ovarian cancer. Mol Ther. 2005;12:789-802.
39. National Institutes of Health. A randomized phase 3 clinical trial to evaluate the efficacy and safety of treatment with OncoVEXGM-CSF compared to subcutaneously administered GM-CSF in melanoma patients with unresectable stage IIIb, IIIc and IV disease. www.clinicaltrials.gov/ct2/show/NCT00769704. Updated June 2012. Accessed May 22, 2014.
40. National Institutes of Health. Phase 1b/2, multicenter, open-label trial to evaluate the safety and efficacy of talimogene laherparepvec and ipilimumab compared to ipilimumab alone in subjects with previously untreated, unresected, stage IIIb-IV melanoma. www.clinicaltrials.gov/ct2/show/study/NCT01740297. Updated May 21, 2014. Accessed May 22, 2014.
41. National Institutes of Health. A phase 2, multicenter, single-arm trial to evaluate the biodistribution and shedding of talimogene laherparepvec in subjects with unresected, stage IIIb to IVM1a melanoma. www.clinicaltrials.gov/ct2/show/NCT0201 4441. Updated May 22, 2014. Accessed May 22, 2014.
42. US Food and Drug Administration. Guidance for Industry. Special Protocol Assessment. www.fda.gov/cder/guidance/index.htm. Accessed May 22, 2014.
43. Kaufman HL, Andtbacka RHI, Collicho FA, et al. Primary overall survival (OS) from OPTiM, a randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous (SC) granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment (tx) of unresected stage IIIB/C and IV melanoma. J Clin Oncol. 2014;32(suppl). Abstract 9008a.
44. Ross MI, Andtbacka RHI, Puzanov I, et al. Patterns of durable response with intralesional talimogene laherparepvec (T-VEC): results from a phase III trial in patients with stage IIIb-IV melanoma. J Clin Oncol. 2014;32(suppl). Abstract 9026.
45. Kaufman HL, Bines SD. OPTIM trial: a phase III trial of an oncolytic herpes virus encoding GM-CSF for unresectable stage III or IV melanoma. Future Oncol. 2010;6: 941-949.
46. Hu JC, Coffin RS, Davis CJ, et al. A phase I study of OncoVEXGM-CSF, a second-generation oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor. Clin Cancer Res. 2006;12:6737-6747.
47. Ascierto PA, Marincola FM, Ribas A. Anti-CTLA4 monoclonal antibodies: the past and the future in clinical application. J Transl Med. 2011;9:196.
48. Puzanov I, Milhem MM, Andtbacka RHI, et al. Primary analysis of a phase 1b multicenter trial to evaluate safety and efficacy of talimogene laherparepvec (T-VEC) and ipilimumab (ipi) in previously untreated, unresected stage IIIB-IV melanoma. J Clin Oncol. 2014;32(suppl). Abstract 9029.
49. Kaufman HL, Andtbacka RHI, Collichio FA, et al. Primary overall survival (OS) from OPTiM, a randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous (SC) granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment (tx) of unresected stage IIIB/C and IV melanoma. J Clin Oncol. 2014;32:5s(suppl). Abstract 9008a.

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