Summary
The use of viruses to treat human malignancy is not a new concept but has only recently evolved into a clinically viable therapy. Spurred by advances in molecular biology that have allowed rela- tively easy manipulation of the viral genome, a number of different viruses have been evaluated and shown to have promise as anticancer agents. Of these, herpes simplex virus (HSV) has been perhaps the most intensively investigated. Several strains of replication competent oncolytic HSV have been developed, some of which have been used in clinical trials. Continuing research efforts are aimed at manipulating the viral genome to more specifically target tumor cells, to further enhance efficacy while maintaining safety, and to assess the role of oncolytic HSV in combination with chemotherapy and radiation therapy.
Key Words: Herpes simplex virus (HSV); oncolytic viral therapy.
1. INTRODUCTION
More than a century has passed since the possibility of using viral pathogens to treat cancer was first recognized. In 1893, remission of leukemia following natural viral infections was reported. Shortly thereafter, Pasteur observed regression of cervical can- cer in a patient after vaccination with an attenuated rabies virus (1). Anecdotes of tumor growth inhibition following bouts of severe viral illnesses, such as measles or mumps, were also reported. Initial, sporadic attempts to treat cancer with viruses yielded encouraging results (2); however, the viral treatment for cancer was not pursued further because of fear of uncontrolled viral infection and lack of effective antiviral agents.
From: Cancer Drug Discovery and Development: Gene Therapy for Cancer Edited by: K. K. Hunt, S. A. Vorburger, and S. G. Swisher © Humana Press Inc., Totowa, NJ
213
13 Therapy in Preclinical and Clinical Trials
Richard H. Pin, MD , Maura Reinblatt, MD , Yuman Fong, MD , and William R. Jarnagin, MD
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ONCLUSIONSherpes simplex viruses (HSV) were developed and are now under investigation as potential cancer therapies (3). The ability to modify the viral genome to selectively target tumor cells for lysis represented a major advance and has allowed oncolytic viral therapy to emerge as a viable and potentially important cancer therapy. A major focus of investigation in this regard has been the creation of viral strains with mutations or deletions of certain key genes, the protein products of which are expressed at higher levels in tumor cells compared with normal cells, thereby attenuating toxicity to normal tissues. Other efforts have involved the creation of tumor-specific promoters that con- trol viral growth genes and modification of genes encoding viral envelope proteins to restrict viral infection to tumor cells that express specific receptors. Over the past 10 yr, oncolytic HSV have moved from the laboratory bench to patient care. Today, genetically engineered viruses are the most commonly used gene delivery vehicles in clinical trials, with three mutated HSV being tested in phase I studies (4–6).
2. LYTIC CYCLE
HSV are comprised of an outer envelope, tegument, and inner capsid containing the viral genomic DNA (see Fig. 1). The virus first attaches to the cell through glyco- proteins on the viral envelope that bind to receptors on the cell membrane. Once bound, the viral envelope delivers the tegument and capsid into the cytoplasm where the tegu- ment transfers the capsid to the nucleus. Within the nucleus, the capsid releases the double-stranded viral DNA for replication and transcription (see Fig. 2). The viral tegu- ment protein, VP16, initiates the cascade of gene expression. The immediate-early genes are the first set of genes transcribed and serve to promote the early genes that encode proteins required for viral replication. Once viral replication is initiated, the late genes necessary for viral assembly are activated. In this fashion, herpesviruses subvert the cellular machinery for their own replication. The new viral progeny that are pro- duced ultimately lyse the cell and infect and lyse neighboring cells to repeat the replica- tive cycle. An attractive feature of replication competent HSV and other oncolytic viruses is that a large number of progeny virus arise from relatively few initially infected cells so that injection of a large viral load can be avoided, a feature now being exploited specifically to treat malignant disease.
3. ATTENUATION AND SAFETY
A number of different mutations in the genome of replication-competent viruses attenuate their toxicity to normal cells. The gene L
134.5 exists as two copies in the HSV genome and is largely responsible for neurotoxicity. The L
134.5 gene product prevents the infected host cell from shutting off its cellular machinery, a normal host response to viral infection, and allows ongoing viral replication. A deletion in the L
134.5 gene there- fore hinders viral replication, and has been shown to limit viral spread within the cen- tral nervous system (7). Another important gene for replication, UL39, encodes the enzyme ribonucleotide reductase (RR). This enzyme is essential for the reduction of ribonucleotides to deoxyribonucleotides, which form the DNA substrate pool (8).
Ribonucleotide reductase therefore plays a critical role in viral DNA synthesis, and
deletions in RR limit viral replication. Eukaryotic cells also produce a cellular variant
of RR during cell division. This cellular RR has significant sequence homology to that of viral RR and can be used to promote viral replication in dividing cells (9). Therefore, HSV with deletions in UL39 can only replicate in rapidly dividing cells, such as some cancer cells, which produce sufficiently high levels of exogenous, cellular RR (10).
Additional mutations in the viral genome allow the host to respond more vigorously to viral infection. The 47 gene normally functions to down-regulate MHC class I pep- tides on the surface of virally infected cells. This cloaks an infected cell from the host’s immune system. A mutation in the 47 gene enables major histocompatability com- plex (MHC) class I peptide expression, allowing antigen presentation to CD8
+cells.
This interaction improves the hosts ability to identify and destroy infected cells and limits the ability of virus to generate new progeny (Table 1) (11).
Viruses have also been constructed with safety features that prevent undesired infection.
A temperature-sensitive mutation in the immediate-early F4 gene leads to inhibition of viral replication at temperatures greater than 39.5°C, thereby offering some protection against a potentially serious viral-mediated illness. Furthermore, an intact thymidine kinase gene confers sensitivity to the antiherpetic drugs acyclovir and gancyclovir. These addi- tional safeguards make attenuated HSV an attractive vector for cancer gene therapy.
4. VIRAL ONCOLYTIC THERAPY
Herpesviruses are effective against a broad spectrum of human tumors. G207 is a mutant HSV with deletions in both copies of L
134.5, and a mutation in UL39 (Table 1).
This attenuated virus was first described in the treatment of malignant gliomas, and fur- ther research has demonstrated its efficacy in treating a wide range of tumors types, including breast, bladder, colon, gallbladder, stomach, liver, pancreas, and the oro- digestive tract (10,12–17). In these studies, G207 has been able to effectively kill can- cer cells in vitro and reduce experimental animal tumor burdens in vivo. Furthermore, G207 has demonstrated preclinical safety in BALB/c mice and Aotus monkeys. In BALB/c mice, doses of 1 × 10
7plaque-forming units (pfu) of G207 injected intrace- rebrally did not produce any adverse effects (18). Likewise, a dose of 1 × 10
9pfu in Aotus monkeys did not result in any pathology (19). Clinical safety has been further confirmed in a phase I clinical trial of patients with malignant gliomas resistant to stan- dard therapies subjected to direct intratumoral injections of G207. None of the patients in this study developed serious adverse effects, and eight patients had radiographic reduction in tumor volume (Table 2) (5).
Fig. 1. HSV structure. (A) The viral envelope contains membrane-bound glycoproteins that aid in docking the virus and binding to receptors on the tumor cell membrane. (B) The proteinaceous tegu- ment is released in the cytoplasm and guides the inner icosahedral capsid to the nucleus. (C) The capsid delivers the 152-kb double-stranded DNA viral genome to the nucleus.
Another widely tested mutant herpes virus is NV1020. Initially failing as a vaccine against HSV-1 and HSV-2, NV1020 subsequently demonstrated potent antitumor pro- perties. This virus differs from G207 in that it contains a deletion in only one copy of L
134.5, as well as deletions in the viral genes UL24 and UL56 (Table 1) (20). NV1020 has effectively treated a variety of human tumor xenografts including those of the colon, prostate, pancreas, and head and neck. Moreover, NV1020 is being evaluated in a phase I clinical trial for liver metastases from colorectal cancer (4). To date, 9 patients with hepatic metastases have received a maximum dose of 1.3 × 10
7pfu of NV1020 delivered through a percutaneous hepatic arterial catheter. No patients suffered severe reactions related to viral inoculation and all patients demonstrated radiographically stable dis- ease and reduced carcinoembryonic antigen (CEA) levels during the, admittedly, very short 28-d observation period. The adverse effects were mild and consisted of fever (7 of 9), nausea (3 of 9), and headache (2 of 9) (Table 2) (4).
Another virus, 1716, has deletions in both copies of the L
134.5 gene. This virus has been successful in the treatment of a variety of tumors in animal models and has recently completed clinical trials assessing safety (Table 2). In two phase I clinical tri- als in patients with recurrent gliomas, a maximum dose of 1 × 10
5pfu was admini- stered through direct intratumoral injection. No patients suffered adverse effects and viral replication could be demonstrated in resected tumor specimens (6,21). In a third phase I clinical trial involving patients with stage 4 melanoma, 1 × 10
3pfu of 1716 was injected into a single nodule. Following treatment, histopathologic necrosis and HSV antigen could be documented in tumor cells (22).
NV1066 is a unique mutant HSV that contains a transgene encoding an enhanced
green fluorescent protein (GFP). It is attenuated through a deletion in one copy of
Fig. 2. HSV lytic cycle. (A) The herpes virion attaches to the tumor cell membrane. (B) After enve- lope fusion with the cell membrane, the large genome enters the nucleus where viral DNA is repli- cated and transcribed. A cascade of gene expression ensues which culminates in the production and assembly of new viral particles. (C) With viral replication and egress, the tumor cell is lysed and the viral effect amplified. Progeny virions can then infect neighboring tumor cells. The herpes replicative cycle often is completed within 24 h and is the basis of herpes oncolytic viral therapy.L
134.5, as well as deletions in the F0 and F4 genes. The F0 and F4 gene products pre- vent cellular apoptosis and stimulate other viral genes necessary for replication (Table 1).
Viral infection can be observed using excitation and emission filters that visualize GFP.
In this respect, NV1066 has the potential for effective detection and treatment of can- cer. In a murine esophageal cancer model, intraperitoneal metastases less than 2 mm in diameter could be differentiated from normal tissues by fluorescent filtered laparoscopy.
Furthermore, tumor burdens were reduced 73% in NV1066-treated animals compared with controls, and 4 of 8 mice treated with NV1066 had no evidence of gross peritoneal disease 4 wkafter infection (23).
5. GENE DELIVERY
Herpesviruses are effective gene transfer agents, and several strains have been devel- oped that contain immunostimulatory or other genes while maintaining replication com- petence and oncolytic activity. Two such strains are NV1034 and NV1042, which contain the transgenes granulocyte-macrophage colony-stimulating factor (GM-CSF) and inter- leukin-12 (IL-12), respectively. These viruses are particularly attractive because of their combined immunostimulatory and oncolytic properties, a treatment approach that appears to be more effective than either approach alone. NV1034 and NV1042 have demonstrated efficacy in squamous cell xenograft tumors, with NV1042 specifically eliciting “memory” tumor immunity (Table 1) (24). In addition, a mutant HSV contain- ing a GM-CSF gene insertion has recently entered phase I clinical trial in the United Kingdom. This GM-CSF expressing virus, OncoVEX
GM-CSF, contains deletions in the L
134.5 and F4 genes to attenuate its virulence. OncoVEX
GM-CSFis being tested by direct injection into several tumor types including melanoma, breast, head and neck, and
Table 1
Virus Mutations Transgene Comments
G207 UL39 LacZ Safety confirmed in phase I trial
L134.5 (both copies) Effective against a broad array of tumors 1716 L134.5 (both copies) None Safety confirmed in phase I trial NV1020 Joint region HSV-2 Originally developed as an HSV vaccine
(Lg134.5 one copy, segment Ongoing phase I clinical trial UL24, UL56)
G47) UL39 Lac Z Enhanced antitumor immune response
L134.5 (both copies) F47
NV1066 L134.5 (one copy) GFP Useful in detecting small tumor deposits
F0, F4, TK Inhibits esophageal tumor xenografts
NV1034 UL56 GM-CSF Efficacy demonstrated in SCC xenografts
F47 LacZ
NV1042 UL56 IL-12 Inhibits SCC xenograft growth
F47 LacZ Elicits memory tumor immunity
HSV, herpes simplex virus; TK, thymidine kinase; GFP, green fluorescent protein; SSC, squamous cell carcinoma.
Table 2 Oncolytic virusTumor typePatientsRoute of deliveryMaximum dose (pfu)Findings G207Recurrent glioma21Direct injection3×109G207 well-tolerated and safe 1 to 5 injectionsNo dose-limiting toxicity reached 1716Recurrent glioma9Direct injection1×105No adverse clinical symptoms No viral shedding or reacti latent HSV 4 of 9 patients alive Recurrent glioma12Direct injection1×105No toxicity observ Resection 4 to 9 d laterHSV DNA detected in 10 of 12 tumors Metastatic melanoma5Subcutaneous injection4×103Viral replication conf 1 to 4 injectionsNo viral toxicity reported Tumor necrosis with NV1020Hepatic colorectal 9 OngoingRegional (via 1.3×107No dose-limiting toxicities observ cancer metastasespercutaneous hepaticNV1020 confined to tumor cells artery catheter)All patients had a decrease in CEA levels 28 d after sur OncoVEXMelanoma8Direct injection1×107Ongoing trial (GM-CSF)BreastOngoingDoses well-tolerated thus f GastrointestinalNecrosis and viral replication Head and neckdetected in tumors Abbr:HSV,herpes simplex virus; PFU,viral plaque forming units; HAI pump,hepatic arterial infusion pump; CEA,carcinoembryonic antigen
218
various gastrointestinal cancers. Thus far, 8 patients have received a maximum dose of 1 × 10
7pfu, which was well-tolerated (Table 2) (25).
Herpesviruses may also be used purely as gene delivery vehicles, stripped of their ability to replicate. These replication-incompetent HSV, also known as HSV ampli- cons, possess the identical envelope, tegument, and capsid as replication-competent viruses and can therefore infect a wide array of tumor types (26). HSV amplicons can infect both dividing and nondividing cells and their genome can accept DNA inserts greater than 100 kilobases (kb) (27). Although they contain less than 2% of the viral genome and lack the ability to replicate, HSV amplicons are capable of utilizing the host cellular machinery to promote specific transgene expression. These replication- incompetent viruses have recently been manufactured using bacterial artificial chromo- some technology. With this production method, contamination of amplicon stocks by replication-competent viruses has been nearly eliminated (28).
An HSV amplicon expressing GM-CSF has been used in several studies, one of which reported improved survival of mice with subcutaneous human gliomas from 10% to 60% at 80 d (29). Another GM-CSF-expressing amplicon has been used to treat subcutaneous murine melanomas. The level of tumor inhibition proved to be dose- dependent, and, interestingly, tumoral injection of HSV amplicon reduced tumor growth in both the injected tumors and the noninoculated contralateral tumors (30). In another immunomodulatory approach, HSV amplicons were created to express T-cell costimu- lating factors, such as ligand B7.1. They were able to cause growth inhibition of esta- blished murine lymphomas. Again, direct HSV amplicon injection resulted in the regression of the injected tumor as well as the noninoculated contralateral tumor.
Furthermore, mice that demonstrated tumor regression were resistant to further tumor cell injections (31). An HSV amplicon expressing intercellular adhesion molecule-1 (ICAM-1) has been tested in rat hepatocellular and human colorectal cancer cell lines.
The transduced tumor cells produced high-level human ICAM-1 surface expression, increased lymphocyte infiltration in vivo, and decreased tumorigenicity (32).
HSV amplicons and replication-competent HSV have also been used in combina- tion. In murine colorectal cancer and rat hepatocellular carcinoma models, low dose regional vascular delivery of G207 along with HSV amplicon expressing the lympho- cyte stimulator, IL-2, enhanced reduction of tumor burden in the liver compared with G207 therapy alone was observed. This enhanced antitumor efficacy was abolished when CD4
+and CD8
+lymphocytes were depleted, suggesting that the improved anti- tumor response was immune-mediated (33).
6. TARGETING
A fundamental problem in anticancer treatment in general, as well as in antitumor
gene therapy, is the lack of an agent that specifically targets tumor cells. To overcome
this obstacle gene therapy approaches are exploiting certain innate tumor characteris-
tics. For instance, hypoxia is a prevalent condition of many solid tumors. Low tumor
oxygen tensions have been correlated with an increased metastatic potential and resist-
ance to standard chemoradiation therapy (34,35). This tumor hypoxia can be utilized to
distinguish malignant from normal tissues and may enable the selective targeting of
cancer cells in gene therapy (36). Recently, a modified UL39 gene driven by a hypoxia-
responsive enhancer was inserted in an oncolytic herpesvirus to treat murine colorectal
cancer cells. This hypoxia-inducible UL39 virus showed improved viral susceptibility
of hypoxic cancer cells compared with cells transfected with G207, a virus deficient in
baseline reduction of tumor weight and nodule count by 65% (37).
Improved viral targeting may also be achieved by exploiting genes that are expressed at high levels in tumors, such as CEA, F-fetoprotein (AFP), or prostate specific antigen (PSA). These genes are not significantly activated in normal, differentiated tissue but they are found to be overexpressed in a variety of different malignancies (CEA, colorectal, pan- creatic, gastric cancer; AFP, hepatocellular carcinoma, testicular cancer; PSA, prostate cancer) (38,39). This tumor-specific expression can be used to target cancer cells by expres- sion of critical viral growth genes from tumor-specific promoters (40–42). Viral prolifera- tion can be thus restricted to cells with high level expression of the tumor-associated gene, an approach that has already been shown effective in HSV and other viral systems (40,41).
The effective entry of viral particles into a cell depends on a sequence of inter- actions between viral glycoproteins and cell surface receptors. Hence, the modifica- tions of viral envelope glycoproteins is another strategy that may allow for selective targeting of tumors without attenuating viral oncolytic activity. Unlike other mutations which compromise the ability of the virus to replicate or to utilize cellular components, these glycoprotein mutations do not attenuate the viral lytic cycle once the cell is infected. R5111 is a genetically engineered HSV containing IL-13 insertional muta- tions in key envelope glycoproteins. R5111 has been shown to bind to the IL13RF2 receptor found on the surface of malignant gliomas only. This particular strain was found to specifically infect and replicate within cells bearing the IL13RF2 receptor, whereas cells lacking the receptor were not infected. Furthermore, R5111 replication was as robust as wild-type HSV in IL12RF2 receptor-positive cells (43).
7. CONCLUSIONS
The concept to use herpes virus to kill cancer led to its application that is now advancing from the laboratory the bench to the bedside. In a relatively short period of time, herpesviruses have been characterized, genetically modified, redirected to treat malignant disease, and utilized in numerous preclinical studies. Furthermore, several oncolytic viral mutants have been used in phase I trials that have demonstrated their safety in humans, with sporadic reports of antitumor efficacy. In addition, both replication- competent and replication-incompetent herpesviruses have been utilized for therapeutic transgene expression in the treatment of cancer. Future work must be focused on ways to enhance viral targeting without sacrificing oncolytic activity and to determine how best to combine oncolytic agents with conventional cancer treatments.
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