Ann Thorac Surg 1999;68:1756-1760
© 1999 The Society of Thoracic Surgeons
Original Articles
Oncolytic therapy using a mutant type-1 herpes simplex virus and the role of the immune system
Eric S. Lambright, MDa,
David J. Caparrelli, MDa,
Abbas E. Abbas, MDa,
Takane Toyoizumi, MDa,
George Coukos, MDa,
Katherine L. Molnar-Kimber, PhDa,
Larry R. Kaiser, MDa
a Thoracic Oncology Laboratory, Harrison Department of Surgical Research, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA
Address reprint requests to Dr Kaiser, General Thoracic Surgery, Hospital of the University of Pennsylvania, 6 Silverstein Pavilion, Philadelphia, PA 19104
e-mail: kaiser{at}mail.med.upenn.edu
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
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Abstract
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Background. Herpes simplex virus (HSV)-1716, a replication-restricted herpes simplex virus type 1, has shown efficacy as an oncolytic treatment for central nervous system tumors, breast cancer, ovarian cancer, and malignant mesothelioma. We evaluated the efficacy of HSV-1716 in a murine lung cancer model, Lewis lung carcinoma.
Methods. Lewis lung carcinoma cells were infected with HSV-1716 and implanted in the flanks of mice at varying ratios of infected to uninfected cells. Tumor burden was assessed by measurement of the weight of the tumor nodule. The role of the immune system was examined by performing experiments in both immunocompetent and SCID mice. Tumors were implanted in the opposite flank to evaluate the vaccine effect.
Results. In immunocompetent and SCID animals, ratio of 1:10 (infected-to-uninfected) cells completely prevented tumor formation and ratio of 1:100 suppressed tumor growth. Established tumors at a distant site in the groups receiving HSV-1716 infected cells showed no difference in size versus control, suggesting absence of a vaccine effect.
Conclusions. We conclude that HSV-1716 may provide a oncolytic therapy for lung cancer even in the absence of immune system induction and a "carrier" cell could potentially deliver this vector.
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Introduction
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In recent years, one of the major thrusts of viral gene therapy for malignant disease has been delivery of suicide genes, such as thymidine kinase, or tumor suppressor genes, such as p53, with replication defective viruses [1, 2]. Although initial results have been encouraging, this method has potential drawbacks. First, these vectors are replication defective and it is unclear whether these viruses will be able to penetrate more than a few tumor cell layers [3]. Second, introduction of these adenoviral vectors has resulted in a strong antiadenoviral immune response in humans [4] and in various animal models [5–9]. This preexisting immunity has significantly decreased transgene expression in animal models [6, 7]. It is these drawbacks that have led to the development of replication competent or replication selective vectors for use in cancer gene therapy [10]. One such group of viruses is the replication-competent herpes simplex virus (HSV) type 1, which has a deletion in the RL1 region of the genome that contains the gamma 34.5 gene. This gene encodes for the ICP34.5 protein, which is a major determinant of herpes neurovirulence [11, 12]. Viruses with mutations in this region show drastically reduced neurovirulence and do not produce encephalitis when inoculated either intracerebrally or peripherally [11–13]. Moreover, these mutants replicate as well as their wild-type parental strain in a variety of dividing cells lines, but replicate poorly in cells not undergoing mitosis [14, 15]. These characteristics potentially make HSV-1716 an attractive vector for cancer gene therapy.
Previous studies have demonstrated that RL1 mutant herpes viruses, such as HSV-1716, replicate well in established dividing human glioma, melanoma, malignant mesothelioma, and ovarian cancer cell lines [16–20]. Infection of these cells by HSV-1716 results in cell death in the majority of patients. Several groups of researchers have shown efficacy in vivo in both immunocompromised and murine tumor models [17, 21–23]. Furthermore, Randazzo and colleagues [14] have shown that HSV antigen staining is restricted to the tumor mass with no spread to adjacent normal human skin or murine tissue in vivo.
Although viral gene therapy for malignant disease has shown promise, there are several problems that must be addressed if this therapy is to become a clinically viable treatment option. The cost associated with manufacturing large quantities of clinical grade viruses is extremely high and may limit the widespread use of a potentially effective therapy. Also, vectors have been ineffective in evading the host immune response. In the context of cancer gene therapy, however, it is still unclear whether the immune system will inhibit vector-mediated tumor lysis or aid in tumor destruction through the induction of a tumor-specific immune response. Regardless, the role of the immune response in viral cancer gene therapy must be investigated further. Therefore, in an effort to address some of these potential problems and increase the efficacy of replication-restricted vectors, such as HSV-1716, we have developed a method whereby cells that are infected with the mutant herpes virus would serve as "carrier cells" for this oncolytic vector.
This delivery system has several major benefits. First, rapidly dividing cell lines allow these viruses to replicate very efficiently, producing as many as 6,000 plaque-forming units per infected cell. Second, administering these carrier cells could theoretically protect, at least initially, the viral vector from host neutralizing antibodies. Such carrier cell lines also could be engineered to enhance tumor killing and induce a tumor-specific immune response. Finally, the presence of these cells alone may have a positive effect on tumor killing through the induction of an inflammatory immune response.
Lewis lung carcinoma (LLC) is a spontaneous, nonimmunogenic murine lung cancer derived from C57BL/6 mice [24]. This line has been shown to grow very well in the flanks of immunocompetent C57BL/6 mice cells after subcutaneous injection [25]. The aggressive nature of this cell line and their nonimmunogenic character make it an excellent cell line to test our HSV-1716 vector in a syngeneic model of lung cancer.
The goals of this study were threefold. First, we sought to develop an immunocompetent animal model for lung cancer in which to study the oncolytic effect of this vector. Second, by demonstrating that only a minority of tumor cells need to be infected to allow viral spread and tumor lysis, we hoped to prove that our viral carrier cell delivery system could be an effective method of viral delivery. Last, by examining both immunocompetent and immunodeficient animals we wanted to assess the role of the immune system in HSV-1716-mediated tumor lysis and determine whether a tumor-specific immune response could be induced or evidence of a systemic response could be seen.
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Material and methods
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HSV-1716
HSV-1716 was originally isolated in the laboratory of S.M. Brown (Glasgow, Scotland) and passaged for use as previously described [16]. This mutant contains a 759-bp deletion in each copy of the RL1 region of the genome that is responsible for encoding the ICP34.5. Thus, no ICP34.5 protein expression occurs in HSV-1716-infected cells.
Cell lines and culture
Lewis lung carcinoma cells originated spontaneously as a carcinoma of the lung of a C57BL/6 mouse [24]. They were purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in RPMI-1640 medium with 10% fetal calf serum (FCS), 200 U/mL penicillin, 200 U streptomycin, and 200 mmol/L L-glutamine (Mediatech, Washington, DC). Cells were harvested during exponential growth of the cell culture.
In vitro studies of HSV-1716 on Lewis lung carcinoma
Cell viability assays were performed on LLC cells in 96-well plates at a density appropriate for the duration of the assay (1,000 to 2,000 cells/well). Twenty-four hours later, media was removed, cells were washed, and subsequently infected with HSV-1716 (diluted in no FCS RPMI) at multiplicity of infection (MOI) of 0.1, 1, 3, and 10 for 1 hour. Six wells were infected at each MOI. After 1 hour, media containing 10% heat-inactivated FCS was added and plates were incubated at 37°C. A sufficient number of plates were used to allow for viability assays at 48, 72, and 96 hours after infection. Viable cell number was assessed by colorimetric assay (CellTiter 96 Aqueous Nonradioactive MTS Cell Proliferation Assay; Promega, Madison, WI) that measures viable cell dehydrogenase activity by absorbency at 490 nm. The percentage of control growth is defined as the ratio of the mean absorbency at 490 nm of six treated wells minus background to the mean absorbency of six matched controls minus background. Background is defined as the mean absorbency of six wells containing media alone.
One-step viral growth curves
To construct one-step viral growth curves, LLC cells were plated on six-well plates at a density appropriate to maintain the cells in logarithmic growth for the duration of the experiment and infected 24 hours later with HSV-1716 at a MOI of 0.01. A sample was harvested at time points of 0, 6, 19, 24, and 48 hours by cell scraping and collection of the cell lysate. The samples were frozen and stored at -80°C. The cell lysates were then thawed and titered by plaque assay on baby hamster kidney cell monolayers, as previously described [16].
In vivo flank studies
A flank tumor model using LLC cells in C57BL/6 and SCID mice was used for all in vivo experimentation. The Animal Use Committees of the Wistar Institute and the University of Pennsylvania in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985) approved all animal protocols. Female C57BL/6 and SCID mice (aged, 5 to 8 weeks) were obtained from Jackson Laboratories (Bar Harbor, ME) and housed at the animal facilities of the Wistar Institute.
Cell mixing studies in immunocompetent and SCID mice
In this series of experiments, each animal (C57BL/6) was injected with a subcutaneous left flank infection of 3 x 105 LLC cells in 0.1 mL of cell culture media (no FCS) on day 0. On day 3, animals were similarly injected in the right flank with a total of 3 x 105 LLC in ratios of 1:10, 1:100, and 1:1,000 infected-to-uninfected cells (n = 10 per group). Control animals (n = 10) received 3 x 105 LLC cells (100% uninfected) in a similar manner. The LLC cells were infected with HSV-1716 (MOI 5) in cell culture media (no FCS) for 1 hour. Ten percent heat-inactivated FCS culture media was then added and the cells were incubated at 37°C for 3 hours before trypsinization, mixing, and injection. Animals were examined daily and tumors measured periodically. Animals were sacrificed by carbon dioxide asphyxiation when control tumors hindered the animals’ ability to ambulate. Tumor burden was assessed by measurement of weight of the excised tumor nodule. Similar experiments were carried out using SCID mice.
Statistical analysis
All of the in vitro cell viability data are expressed as the mean ± standard deviation of the mean. Tumor weights for all in vivo experiments are expressed as the mean ± standard error of the mean. Mean tumor weights between treatment groups were compared using analysis of variance. When significant differences were found (p < 0.05), Fisher’s test was used to test among groups.
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Results
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HSV-1716 exerts an oncolytic effect on Lewis lung carcinoma in vitro
To measure directly the cell killing efficiency of HSV-1716 on LLC, in vitro cell viability assays were performed. Cell viability was assessed at several doses of virus (ranging from MOI 0.1 to 10), as well as at varying time points after infection. HSV-1716 killed the target LLC cells in a time- and dose-related manner (Fig 1).
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Fig 1. In vitro cytolytic effect of HSV-1716 on Lewis lung carcinoma cells. The effect of HSV-1716 was assessed by colorimetric proliferation assay at multiple times points and variable multiplicities of infection. Values are expressed as a percentage of noninfected cells. Data is expressed in mean ± standard deviation.
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HSV-1716 infects and replicates in Lewis lung carcinoma
To assess the ability of HSV-1716 to replicate in LLC, one-step viral growth curves were performed. The LLC supported the growth of HSV-1716 as shown in Figure 2. As expected, recovery of active viral particles at t = 6 hours was decreased with respect to the initial viral inoculum as a result of viral uptake and disassembly before replication. However, by 24 hours, an increase over the initial viral inoculum was observed. The LLC did support the replication of HSV-1716 with viral burst size of approximately 20.
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Fig 2. HSV-1716 one-step viral growth curve on Lewis Lung Carcinoma cells. An inoculum of virus (0.01 multiplicity of infection) was given at time zero. Burst size was calculated to be 20.
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Infecting LLC with HSV-1716 decreases tumor growth in flank mixing studies
Because efficacy of this oncolytic vector relies on the ability of the virus to replicate, lyse cells, and spread within a tumor mass, we first performed mixing studies in the flanks of immunocompetent C57BL/6 mice and SCID mice. On the right, or treated flank, an infected-to-uninfected ratio of 1:10 completely prevented tumor formation in 10 animals (p = 0.0005). Significant growth inhibition was also seen at a ratio of 1:100. The mean tumor weight for uninfected controls was 0.092 ± 0.021 g, whereas tumors in the 1:100-treated group averaged 0.026 ± 0.011 g (p = 0.0095) (Fig 3). Left flank tumors in all groups showed no differential growth suggesting the absence of a measurable vaccine effect. These results demonstrate that HSV-1716 can effectively decrease tumor growth even when a percentage (as low as 1%) of cells are infected. However, we were not able to demonstrate a measurable vaccine or distant "bystander effect."
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Fig 3. Cell mixing study in immunocompetent C57BL/6 mice. Tumor weight on day 20. Statistical significance was found in the infected-to-uninfected groups at 1:10 (p = 0.0005) and 1:100 (p = 0.0095). Error bars reflect standard error of the mean. (LLC = Lewis lung carcinoma.)
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To begin to assess the role of the immune system in our model, we then performed a flank mixing study in immunodeficient SCID mice. As in the C57BL/6 experiments, the animals were first injected with 3 x 105 LLC cells on the left flank on day 0 and on day 3 the animals were injected with ratios of infected-to-uninfected LLC. Studies in SCID mice demonstrated that in the infected-to-uninfected group at 1:10 there was prevention of tumor formation (p < 0.0001). In addition, a significant difference (p < 0.0001) was found in the 1:100 treated group. The mean tumor weight for the control animals was 0.115 ± 0.013 g, whereas tumors in the 1:100 treated group averaged 0.004 ± 0.002 g (Fig 4). As expected, no vaccine effect was observed in the SCID mice.
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Fig 4. Cell mixing study in immunodeficient SCID mice. Tumor weight on day 20. A statistical difference was demonstrated in the 1:10 and the 1:100 versus control (p < 0.005 and 0.0095, respectively). Error bars reflect standard error of the mean. (LLC = Lewis lung carcinoma.)
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Comment
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In this study we sought to determine the efficacy of an attenuated HSV in treating a murine model of lung carcinoma and to attempt to delineate the role of the immune system in this model of cancer gene therapy. We demonstrated that a replication-restricted herpes simplex virus, HSV-1716, efficiently kills LLC cells in vitro, suggesting its utility in the treatment of adenocarcinoma of the lung. Our results indicated that there is a clear dose-related effect of the virus on LLC. However, because large-scale production of mutant HSV has significant technical difficulties, we have developed a method to amplify the viral load delivered. In vivo we have been able to show that as few as 1 in 100 LLC cells needs to be infected to significantly inhibit tumor growth in both immunocompetent and immunodeficient animals. This finding suggests that viral carrier cells may serve as a means of vector delivery. Using these cells as efficient viral factories may address several of the major concerns with the therapeutic modality of gene therapy, specifically the manufacturing cost of clinical grade virus and vector delivery.
The host immune response in malignant disease remains a complex issue. This is further complicated by the use of an immunogenic viral vector as a method of treatment. In this study, we show that only a percentage of LLC cells must be infected to decrease tumor growth in flank-mixing studies in both immunocompetent and immunodeficient animals. These in vivo data suggest that induction of an immune system response is not required for the oncolytic effects of HSV-1716. In addition, we sought to determine whether there was any effect of HSV-1716 therapy on tumor at a distant site suggestive of a tumor-specific immune response. We determined that there was no difference in remote site tumor size in those animals receiving viral therapy versus control. These results suggest that there is no induction of a tumor-specific immune response or distant vaccine effect.
In theory, the ideal oncolytic vector would not only lyse tumor cells efficiently but would also induce a specific antitumor immune response in the host. Carrier cells may play a major role initially by protecting the virus from this neutralizing immunity. However, induction of a strong immune response by manipulation of the carrier cells with cytokines or other immunomodulators could potentially augment its oncolytic effects. Several groups have looked at cytokines, including interleukins (IL) 2, 4, and 6, interferon-
, and tumor necrosis factor-
, with regard to their role in preventing tumor growth and metastasis [24–27]. In one study, the systemic administration of IL-4 decreased tumor growth and significantly increased survival [27]. In another study of IL-2 and IL-4, Ohe and colleagues [25] demonstrated that locally increased levels of IL-2 induced a tumor-directed immune response. Furthermore, IL-6-transfected LLC cells have also been shown to decrease tumorigenicity as well as reduce metastatic competence [26]. In addition, efficacy of another ICP34.5 mutant, R3616, was augmented in an experimental tumor model of glioma by coexpression of IL-4 [28]. The administration of an IL-12-expressing HSV-1 amplicon together with G207, which is an ICP34.5 and ribonucleotide reductase attenuated HSV-1 strain, decreased tumor growth in a colorectal carcinoma model and induced a local and systemic antitumor immune response [29]. Carrier cells could be engineered to express particular cytokines while still remaining permissive for viral replication, thus augmenting their oncolytic effects. A balance between efficient viral replication and induction of the host immune response will be essential for this therapy to be effective.
In conclusion, this study demonstrates that attenuated virus HSV-1716 efficiently kills LLC cells and it may provide a new oncolytic therapy for lung cancer even in the absence of a specific tumor-mediated immune response. In addition, we demonstrate that only a percentage of tumor cells must be infected to effectively suppress tumor growth in a murine model. This observation adds credence to the concept of carrier cells and the ability to manipulate the carrier cells to augment the immune response is clearly one of the major theoretic advantages to this therapeutic approach.
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Acknowledgments
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This study was supported by grants from the National Cancer Institute (PO-66726-S1) and the University of Pennsylvania Research Foundation.
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Abstract
Introduction
