A novel A33 promoter-based conditionally replicative adenovirus suppresses tumor growth and eradicates hepatic metastases in human colon cancer models

Purpose: A33 antigen is a membrane-bound protein expressed in intestinal epithelium that is overexpressed in 95% of primary and metastatic colorectal carcinomas but is absent in most epithelial tissues and tumor types. We hypothesized that A33 promoter might be useful in the design of a conditionally replicative adenovirus for the treatment of colorectal cancer (CRC). Experimental Design: We cloned an A33 promoter fragment (A33Pr) that extends from -105 to +307 bp. Using luciferase activity as a reporter gene, we showed that A33Pr was active inCRC cell lines. We next constructed a conditionally replicative adenovirus named AV22EL where E1A was placed under the control of A33Pr. The tumor-specific oncolytic effect of AV22EL was investigated both in vitro and in vivo. Results: AV22EL induced specific in vitro lysis of human CRC cell lines that expressed A33 and have negligible lytic capacity on cells that lacked or hadminimal A33 expression, including normal human colonic cells. In vivo, a marked reduction of tumor growth and increased long-term survival rates were observed in nude mice xenografted with s.c. CRC tumors. Combination with 5-fluorouracil induced an additive effect in vitro with no toxic effects in vivo. Remarkably, AV22EL completely eliminated established hepatic metastases in >90% of mice and restored hepatic function according to biochemical parameters. Its systemic administration induced E1A expression only in the hepatic metastasis but not in normal organs. Conclusions: These data show that AV22EL is a stringently regulated and potent oncolytic agent for the treatment of CRC.Facultad de Ciencias Veterinaria

Effective tumor immunotherapy: start the engine, release the brakes, step on the gas pedal,...and get ready to face autoimmunity

Cellular immune responses can destroy cancer cells, achieving the cure of experimental malignancies. An expanding wealth of knowledge on the molecular basis of how to prime and amplify a T cell response has fueled a number of strategies successful at treating established tumors (rather than merely preventing tumor grafting). The most efficacious approaches operate at different stages, including: 1) priming the immune response using tumor antigen-expressing dendritic cells or tumor cells transfected with genes that render them immunogenic, 2) sustaining and amplifying immunity using agonistic monoclonal antibodies against costimulatory molecules or immune-potentiating cytokines, and 3) eliminating mechanisms that self-regulate the strength of the immune response, such as inhibitory receptors or regulatory T cells. A rational combination of such approaches holds great hope for cumulative and synergistic effects, but there is also evidence that they can open the flood-gates for unwanted inflammatory reactions. The next decade can be envisioned as the time when the first reproducibly efficacious combination regimes for cancer immunotherapy will become available and widely used in the clinic, as clinicians learn the best strategies and try to harness their potentially damaging effects

Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12

Interleukin-12 (IL-12) has been shown to possess potent immunoregulatory and antitumoral effects. We have evaluated the anti-oncogenic potential and the mechanisms of the antitumoral effect of in vivo adenovirus-mediated transfer of IL-12 gene in a murine model of colon cancer. AdCMVIL-12 was constructed to permit coordinated production of p40 and p35 subunits of IL-12 gene to obtain the maximum IL-12 bioactivity. Infection of murine colon cancer CT-26 cells in vitro with AdCMVIL-12 resulted in the production of high levels of IL-12. In vivo gene therapy of colon cancer nodules by intratumoral injection of AdCMVIL-12 induced a local increase in IL-12 and interferon-gamma levels and a complete regression of the tumor in 26 of 34 (76%) mice. Tumor disappeared between days 7 and 10 after vector administration. The antitumoral effect was mediated by CD8+ T cells and was associated with the generation of cytotoxic T lymphocytes against colon cancer cells. Animals that eliminated the tumor were protected against a second administration of neoplastic cells. Treatment with AdCMVIL-12 of one tumor nodule also caused regression of established tumors at distant sites. These data demonstrate that AdCMVIL-12 is a useful therapeutic tool for established colon cancer in mice and should be considered for application in humans

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12

Gene therapy for liver diseases: recent strategies for treatment of viral hepatitis and liver malignancies

Gene therapy has emerged as a powerful and very plastic tool to regulate biological functions in diseased tissues with application in virtually all medical fields. An increasing number of experimental and clinical studies underline the importance of genes as curative agents in the future. However, intense research is needed to evaluate the potential of gene therapy to improve efficacy and minimise the toxicity of the procedure

Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells

OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura

Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12

The use of gene therapy to enhance antitumor immunity has emerged as a promising procedure to fight cancer. In this study we have tested the ability of an adenovirus carrying interleukin 12 (IL-12) gene (AdCMVIL-12) to eliminate tumoral lesions in 3 animal models of orthotopic hepatocellular carcinoma (HCC). Intratumoral injection of AdCMVIL-12 in animals with a single big tumor nodule implanted in the liver resulted in significant inhibition of tumor growth in a dose-dependent manner. Fifty percent of animals that received a dose of 5 x 10(9) plaque-forming units, showed complete regression of the tumor 2 weeks after treatment. In animals with 2 independent tumor nodules in the left liver lobe, injection in only one of them of 5 x 10(9) pfu AdCMVIL-12 induced, 15 days after therapy, complete regression of 50% of treated tumors and also of 50% of untreated lesions, with 60% long-term survival. Rats that were tumor free after therapy with AdCMVIL-12 showed protection against tumor rechallenge. A group of rats received the carcinogen diethylnitrosamine and developed multiple hepatic dysplasic nodules of 1 to 5 mm in diameter. These animals were treated by intrahepatic artery injection of either AdCMVIL-12 (5 x 10(9) pfu) or control vector. In this model AdCMVIL-12 induced complete tumor regression in 20% of treated rats and inhibited tumor growth in 60% of cases with an increase in rat survival. Activation of natural killer (NK) cells and inhibition of angiogenesis were found to be antitumor mechanisms set in motion by AdCMVIL-12. Our data indicate that experimental HCC can be efficiently treated by intratumoral or intravascular injection of adenovirus expressing IL-12

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50-100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies

Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12

Despite the efficacy of IL-12 in cancer experimental models, clinical trials with systemic recombinant IL-12 showed unacceptable toxicity related to endogenous IFNgamma production. We report that systemic administration of a recombinant adenovirus encoding IL-12 (AdCMVmIL-12) has a dramatically different survival outcome in a number of mouse pure strains over a wide range of doses. For instance at 2.5 x 10(9) p.f.u., systemic AdCMVmIL-12 killed all C57BL/6 mice but spared all BALB/c mice. Much higher IFNgamma concentrations in serum samples of C57BL/6 than in those from identically treated BALB/c were found. Causes for heterogeneous toxicity can be traced to differences among murine strains in the levels of gene transduction achieved in the liver, as assessed with adenovirus coding for reporter genes. In accordance, IL-12 serum concentrations are higher in susceptible mice. In addition, sera from C57BL/6 mice treated with AdCMVmIL-12 showed higher levels of IL-18, a well-known IFNgamma inducer. Interestingly, lethal toxicity in C57BL/6 mice was abolished by administration of blocking anti-IFNgamma mAbs and also by simultaneous depletion of T cells, NK cells, and macrophages. These observations together with the great dispersion of IFNgamma produced by human PBMCs upon in vitro stimulation with IL-12, or infection with recombinant adenovirus encoding IL-12, suggest that patients might also show heterogeneous degrees of toxicity in response to IL-12 gene transfer

Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens

Intralesional administration of cultured dendritic cells (DCs) engineered to produce IL-12 by in vitro infection with recombinant adenovirus frequently displays eradicating efficacy against established subcutaneous tumors derived from the CT26 murine colon carcinoma cell line. The elicited response is mainly mediated by cytolytic T lymphocytes. In order to search for strategies that would enhance the efficacy of the therapeutic procedure against less immunogenic tumors, we moved onto malignancies derived from the inoculation of MC38 colon cancer cells that are less prone to undergo complete regression upon a single intratumoral injection of IL-12-secreting DCs. In this model, we found that repeated injections of such DCs, as opposed to a single injection, achieved better efficacy against both the injected and a distantly implanted tumor; that the use of semiallogeneic DCs that are mismatched in one MHC haplotype with the tumor host showed slightly better efficacy; and that the combination of this treatment with systemic injections of immunostimulatory anti-CD137 (4-1BB) monoclonal antibody achieved potent combined effects that correlated with the antitumor immune response measured in IFN-gamma ELISPOT assays. The elicited systemic immune response eradicates concomitant untreated lesions in most cases. Curative efficacy was also found against some tumors established for 2 weeks when these strategies were used in combination. These are preclinical pieces of evidence to be considered in order to enhance the therapeutic benefit of a strategy that is currently being tested in clinical trials. Supplementary Material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html