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Prostate cancer is one of the most prevalent cancers in men. Replication-competent oncolytic herpes simplex virus (oHSV) vectors are a powerful anti-tumor therapy that can exert at least two effects: direct cytocidal activity that selectively kills cancer cells and induction of anti-tumor immunity. In addition, oHSV vectors can also function as a platform to deliver transgenes of interest. In these studies, we have examined the expression of a xenogeneic homologue of the prostate cancer antigen, prostatic acid phosphatase (PAP), with the goal of enhancing virotherapy against PAP-expressing tumors. PAP has already been used for cancer vaccination in prostate cancer patients. Here we show that treatment with oHSV bPΔ6 expressing xenogeneic human PAP (hPAP) significantly reduces tumor growth and increases survival of C57/BL6 mice bearing mouse TRAMP-C2 prostate tumors, whereas expression of syngeneic mouse PAP (mPAP) from the same oHSV vector did not enhance anti-tumor activity. Treatment of mice bearing metastatic TRAMP-C2 lung tumors with oHSV expressing hPAP resulted in fewer tumor nodules. To our knowledge, this is the first report of oncolytic viruses being used to express xenoantigens. This data lends support to the concept of combining oncolytic and immunogenic therapies as a way to improve therapy of metastatic prostate cancer.
Prostate cancer is the most common cancer amongst men in developed countries and in the United States alone over 180,000 new cases were diagnosed in 2008 1. Treatment options for prostate cancer include surgery with radical prostatectomy, hormone therapy, chemotherapy 2 and radiation 3. However, severe secondary effects are common amongst patients and there is no curative treatment once the primary tumor metastasizes 4. The introduction of genetically-engineered oncolytic viruses as a new therapeutic avenue in the battle against cancer began with the use of oncolytic herpes simplex viruses (oHSV) 5. Typically, oHSV carry mutations in the viral genome that enable them to replicate in and kill cancer cells, without harming normal tissue 6. Amongst the benefits of oHSV vectors are that they are easily manipulated, can carry transgene inserts 7 and have already been safely used in human subjects with a variety of cancers 8, 9. Another advantage of oHSV is that it can interact synergistically with other therapeutic modalities, including chemotherapeutic agents 10, 11 and radiotherapy 12, thereby promoting better therapeutic outcomes by targeting more then one aspect of cancer biology.
Previous work from our laboratory and others has shown that oHSVs can be used as an in situ vaccine to generate a tumor-specific host immune response 13-16. Prostatic acid phosphatase (PAP) is a prostate-specific antigen that is expressed in both prostate cells and prostate cancer cells 17 as well as other adenocarcinomas 18. However, as it is a self-antigen it is not immunogenic due presumably to tolerance. It has previously been shown that immunization of rats with human PAP (hPAP) generates a CTL response leading to tissue-specific prostatitis 19. Furthermore, immunization of metastatic prostate cancer patients with recombinant mouse PAP (mPAP) loaded dendritic cells has been shown to result in an anti-tumor immune response and clinical stabilization of the disease, as indicated by a decreased rise of serum PSA levels 18. This suggests that vaccination with a xenogeneic homolog can break tolerance to a self-antigen, human PAP (hPAP), inducing a Th1-type cytokine response to both mouse and human PAP antigens. Other studies have confirmed this concept of using a xenogenic form of an antigen to break anti-tumor tolerance to other proteins, such as: prostate stem cell antigen, where DNA vaccination induced anti-tumor immune responses against TRAMP-C1 tumors 19, prostate-specific membrane antigen (PSMA) 20, gp100 in melanoma 21, HER2 expressed from a replication-deficient adenovirus vector in breast cancer 22, and EGFR 23. We therefore hypothesized that an oHSV expressing a xenogenic PAP could be used to treat prostate cancer.
In the present study we have combined the ability of oHSV to specifically replicate in and kill cancer cells with the immunogenic effects of in situ vaccination with a xenogeneic PAP protein to create a more powerful therapeutic strategy for prostate cancer. Our results show that this combined approach achieves improved inhibition of established tumor growth and animal survival compared to oHSV alone or when expressing syngeneic mPAP. These results represent an encouraging step towards the development of a viable therapy for prostate cancer and describe a novel approach that should be applicable to other oncolytic viruses.
To construct oHSVs expressing human PAP (bPΔ6-hPAP) or the mouse homolog (bPΔ6-mPAP), we used the Flip-Flop HSV-BAC technology 7. A third construct, lacking a transgene (bPΔ6-empty), was also generated to be used as a control (Supplementary Fig. 1). The backbone for these vectors was HSV-1 strain Patton, from which PΔ6-BAC was constructed by inserting the BAC cassette into the ICP6 (UL39) gene, as previously described 7. The genomic structure of the vectors was confirmed by restriction endonuclease digestion and gel electrophoresis and the recombinant vectors all express β-galactosidase, which is detectable after X-gal histochemistry (data not shown). While mouse prostate cancer cells are much less susceptible to oHSV replication than human prostate cancer cells (compare 24 and 25) or Vero cells, they still replicate and spread in mouse TRAMP-C2 and RM-1 prostate cancer cells (Fig 1A). The transgene expressing viruses replicate less well than the parental wild-type strain Patton (WT, Fig 1B), likely due to the ICP6 mutation, however, there is no difference between bPΔ6-hPAP and bPΔ6- empty (Fig 1B). A dose response curve for killing TRAMP-C2 cells is illustrated in Fig 1C. Again there is no difference between bPΔ6-hPAP and bPΔ6-empty, demonstrating that expression of hPAP does not alter the in vitro replication or cytoxicity of bPΔ6.
Next we confirmed that the viruses express the inserted transgenes. The CMV IE promoter was used to drive transgene expression, as we have described before 26, 27, because it provides high level expression. Vero cells were infected with either bPΔ6-hPAP, bPΔ6-mPAP or bPΔ6-empty and RT-PCR analysis was carried out for mPAP, using mouse transgene specific primers, and a band corresponding to the expected mPAP RNA was only seen in bPΔ6-mPAP infected Vero cells (Fig 2A). Expression of the hPAP transgene in mouse tumor cell lines was detected with human-specific anti-PAP antibody. Western blot analysis reveals a band corresponding to hPAP in all lanes where cells were infected with bPΔ6-hPAP (+, Fig 2B). As a control, we show the expression of endogenous hPAP in human LNCaP prostate cells but not in 293 cells (Fig 2B, right). In order to test the immunogenic effects of the oHSV PAP vectors it was necessary that the mouse prostate tumor model expressed mPAP. RT-PCR analysis confirmed the presence of mPAP RNA in both TRAMP-C2 and RM-1 prostate cancer cells (Fig. 2C). Ultimately, TRAMP-C2 was selected as our experimental tumor cell line since previous data from our lab has shown that these cells are responsive to oHSV treatment in both primary and metastatic tumor models 25, 28. Finally, in order to verify that bPΔ6-hPAP expresses human PAP protein in vivo, we implanted TRAMP-C2 cells subcutaneously into C57/BL6 immunocompetent mice. Once tumors were established, the mice were injected intra-neoplastically with bPΔ6-hPAP or bPΔ6-empty. Forty-eight hours post-injection, hPAP was detected in tumors treated with bPΔ6-hPAP, but not bPΔ6-empty (Fig 2D).
TRAMP-C2 cells were implanted subcutaneously in syngeneic C57/BL6 mice. Once tumors were established, mice were randomized into four treatment groups: bPΔ6-hPAP, bPΔ6-mPAP, bPΔ6-empty, and PBS only. Each mouse was treated four times intra-neoplastically with 1×107 PFU / injection over a period of 10 days and then monitored for tumor growth and survival. Mice treated with oHSV lacking transgene (bPΔ6-empty) or oHSV expressing a non-xenogenic form of PAP (bPΔ6-mPAP) showed a slower rate of tumor growth, with no difference between mPAP and empty (p=0.7) and not significantly different from the PBS-treated mice (PBS vs mPAP p=0.09, PBS vs empty p=0.08). However, mice treated with the xenogenic hPAP-expressing oHSV (bPΔ6-hPAP) showed a significant inhibition in tumor growth when compared to bPΔ6-mPAP (p=0.03), bPΔ6-empty (p=0.04) or PBS (p=0.002) groups (Fig 3A). This inhibition translated into a significant increase in survival (Fig. 3B) when compared to mice treated with bPΔ6-mPAP (p=0.01), bPΔ6-empty (p=0.01), or PBS only (p=0.0008), with the median survival increasing from 29 days for PBS to 39 for hPAP. In a separate experiment, 3 intra-tumoral injections of bPΔ6-hPAP resulted in significantly extended survival compared to bPΔ6-empty or PBS (data not shown). This indicates that expression of xenogenic hPAP is enhancing anti-tumor activity.
To investigate the therapeutic efficacy of bPΔ6-hPAP in a metastatic tumor model, we injected TRAMP-C2 cells intravenously via tail vein. TRAMP-C2 cells metastasize primarily to the lungs 28. Tumor bearing mice were systemically treated via tail vein injection with bPΔ6-hPAP, bPΔ6-mPAP, bPΔ6-empty, or PBS 4 times over a period of 10 days. Mice were sacrificed at day 60 and the lungs were stained with India ink to detect lung tumor nodules, which were measured and counted. Mice treated with bPΔ6-hPAP showed significantly fewer tumors per group then the bPΔ6-empty (p=0.015) or PBS (p= 0.017) groups and also a significant reduction in tumor burden when compared with bPΔ6-empty (p=0.034) (Table 1).
In an effort to improve the therapeutic potential of the oHSV vector system we combined oHSV with a xenoantigen transgene, designed to stimulate an auto-immune response against a prostate antigen, PAP. For these studies we developed a new oHSV vector using wild-type HSV-1 strain Patton as the backbone and Flip-Flop HSV-BAC technology 7 to insert the PAP transgenes into the ICP6 locus of the oHSV genome in a rapid fashion. Many oHSV vectors contain ICP6 mutations, which inactivates viral ribonucleotide reductase, impeding virus replication in normal quiescent cells except those lacking p16, such as many tumor cells 29. HrR3, with an insertion of E. coli LacZ inactivating ICP6 in the HSV-1 KOS backbone was the first such vector 30, 31. bPΔ6 has the same LacZ insertion into ICP6 as hrR3, but in a Patton backbone. Expression of PAP did not affect virus replication or tumor cell killing in vitro. To test the xenoantigen strategy, we needed a tumor model with endogenous mPAP expression that progressed slowly enough to allow the treated animals time to generate an immune response, and that was susceptible to oHSV. For these reasons we chose the TRAMP-C2 tumor cell line. Incorporating hPAP transgene into the oHSV vector bPΔ6, led to a reduction in tumor burden in subcutaneous and metastatic prostate tumor models that did not occur with mPAP. Because of limited oHSV replication in mouse prostate cancer cells, syngeneic mouse prostate tumors have been difficult to treat with oHSV, except with the addition of immune-modulatory transgenes, such as IL-12 25, 28.
Our data support the concept that better outcomes can be achieved by combining oncolytic viral therapy with immunotherapy than with oncolytic viral therapy alone. The ability of hPAP, but not mPAP, to enhance the anti-tumor activity of oHSV indicates that a specific immune response is at work, engendered by exposure to the xenogeneic PAP protein and resulting in a loss of tolerance to syngeneic PAP. Previous studies vaccinating rats with vaccinia virus or plasmid expressing xenogeneic human PAP transgenes demonstrated the induction of a MHC class I-dependent CTL-mediated anti-PAP auto-immune response, but not an antibody response 32, 33. In these studies the response differed to some extent between rat strains and the animals did not bear prostate tumors. To our knowledge, ours is the first report of an oncolytic virus expressing a xenoantigen for cancer vaccination. It is likely that the combination of hPAP with expression of other immunomodulatory molecules will further improve the efficacy of oHSV-mediated immunotherapy. As we have shown that oHSV expressing hPAP is efficacious in treating mouse prostate cancer, we hypothesize that oHSV expressing mPAP would be effective in treating human prostate cancer. This could be addressed in a clinical trial for the treatment of prostate cancer.
Supplementary Figure 1. Construction of PAP-expressing oHSV. Use of the Flip-Flop HSV-BAC system to construct recombinant oHSV vectors is illustrated in this cartoon and has been previously described 7, 26. Briefly, hPAP cDNA 38 was cloned into the shuttle vector pVec92 to generate pVec92-hPAP. pVec92-hPAP was inserted into bPΔ6-BAC via Cre/LoxP recombination. The bPΔ6-CMV-hPAP-shuttle plasmid was isolated and co-transfected into Vero cells with pCAGGS-Flp, a FLPe-expressing plasmid. Flp/Frt recombination removes the BAC, antibiotic resistance, GFP, and stuffer sequences so the viral genome can be efficiently packaged into virions, and the recombinant virus (bPΔ6-hPAP) harvested and plaque-purified.
The authors would like to thank Dr M Crew for providing the mPAP cDNA and Dr Timothy C. Thompson for providing RM-1 cells. This work was supported in part by grants to RLM from the DOD (W81XWH-04-1-0254) and the NIH (R01 CA102139).
CONFLICT OF INTEREST The authors declare no conflict of interest.