Mazzolini, G. (Guillermo)
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- Alpha(v)beta(3) integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment(Nature Publishing Group, 2001) Mazzolini, G. (Guillermo); Duarte, M. (Marina); Qian, C. (Cheng); Bilbao, R. (Roberto); Melero, I. (Ignacio); Narvaiza, I. (Íñigo); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Bustos, M. (Matilde)We previously reported that systemic injection of recombinant adenovirus resulted in a rim of gene transduction around experimental liver tumor nodules. This zone of higher infection is dependent on the alpha(v)beta(3) integrin, acting as an adenovirus internalization receptor, which is overexpressed in tissues surrounding liver metastases. When a recombinant adenovirus encoding interleukin-12 (AdCMVIL-12) is given into a subcutaneous tumor nodule in mice also bearing concomitant liver tumors, a fraction of AdCMVIL-12 reaches the systemic circulation and infects liver tissue, especially at the malignant/healthy tissue interface. As a result of the expression at this location of the interleukin-12 transgenes, VCAM-1 is induced on vessel cells and mediates the recruitment of adoptively transferred anti-tumor cytolytic T-lymphocytes. These studies provide mechanistic explanations for the potent therapeutic synergy observed between interleukin-12 gene transfer and adoptive T-cell therapy.
- Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens(Wiley-Blackwell, 2004) Mazzolini, G. (Guillermo); Alfaro, C. (Carlos); Duarte, M. (Marina); Qian, C. (Cheng); Melero, I. (Ignacio); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Feijoo, E. (Esperanza); Chen, L. (Lieping); Arina, A. (Ainhoa)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.
- Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy(American Association of Immunologist, 2000) Mazzolini, G. (Guillermo); Duarte, M. (Marina); Qian, C. (Cheng); Zaratiegui, M. (Mikel); Barajas, M. (Miguel); Melero, I. (Ignacio); Narvaiza, I. (Íñigo); Prieto, J. (Jesús)We have constructed a recombinant defective adenovirus that expresses functional murine IFN-gamma-inducible protein-10 (IP-10) chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into s.c. tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line displayed some antitumor activity but it was not curative in most cases. Previous studies have shown that injection of similar s. c. CT26 tumor nodules with adenovirus-encoding IL-12 (AdCMVIL-12) induces tumor regression in nearly 70% of cases in association with generation of antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect of suboptimal doses of AdCMVIL-12, reaching 100% of tumor eradication not only against injected, but also against distant noninjected tumor nodules. Colocalization of both adenoviruses at the same tumor nodule was required for the local and distant therapeutic effects. Importantly, intratumoral gene transfer with IL-12 and IP-10 generated a powerful tumor-specific CTL response in a synergistic fashion, while both CD4 and CD8 T cells appeared in the infiltrate of regressing tumors. Moreover, the antitumor activity of IP-10 plus IL-12 combined gene therapy was greatly diminished by simultaneous in vivo depletion of CD4+ and CD8+ T cells but was largely unaffected by single depletion of each T cell subset. An important role for NK cells was also suggested by asialo GM1 depletion experiments. From a clinical point of view, the effects of IP-10 permit one to lower the required gene transfer level of IL-12, thus preventing dose-dependent IL-12-mediated toxicity while improving the therapeutic efficacy of the elicited antitumor response.
- Gene therapy of cancer with interleukin-12(Bentham Science Publishers, 2003) Mazzolini, G. (Guillermo); Melero, I. (Ignacio); Prieto, J. (Jesús)IL-12 has demonstrated remarkable antitumor activity when used directly as a recombinant protein or when different viral or non-viral vectors transfer its genes. At enhancing tumor immunity, IL-12 acts as a bridge between innate and adaptive immune responses due to its ability to induce proliferation and activation of NK, NKT, and T cells. In addition, IL-12 inhibits tumor angiogenesis mainly through IFN gamma-dependent production of the chemokine IP10. As a result, IL-12 can eliminate several types of tumors developed in rodents. Pre-clinical experience forecasted a quick and successful clinical translation, but the encouraging results observed in animals were not reproduced in patients. Moreover, unacceptable toxicity resulting from IFN gamma overproduction was observed in 2 renal carcinoma patients included in a phase II clinical trial that consisted in systemic administration of rIL-12. As a consequence, development of IL-12 as an antitumor agent was temporarily halted while the high expectations raised among clinicians faded away. Gene transfer methods are designed to confine IL-12 production in the tumor environment preventing systemic toxicity. Tumor cells, dendritic cells, or autologous fibroblasts have been transfected with recombinant adenoviruses or retroviruses to secrete IL-12 locally, showing good efficacy and safety profiles. IL-12 combination with other immunotherapy approaches synergizes to achieve even better results. Encouraging pilot clinical results have been recently obtained from the first phase I trial studying adenovirus mediated in vivo gene transfer of IL-12 into lesions of advanced cancer patients. Further improvements will follow from: i) increases in the efficacy of gene transduction; ii) development of tumor specific promoters; iii) development of regulatable and long-term expression vectors and iv) combination with other immunological and non-immunological anticancer therapies.
- MAGE antigens: therapeutic targets in hepatocellular carcinoma?(Elsevier, 2004) Mazzolini, G. (Guillermo); Alfaro, C. (Carlos); Melero, I. (Ignacio); Sarobe, P. (Pablo); Feijoo, E. (Esperanza)
- Intratumoural administration of dendritic cells: hostile environment and help by gene therapy(Informa Healthcare, 2005) Marin, V. (Verónica); Mazzolini, G. (Guillermo); Alfaro, C. (Carlos); Busto, V. (Victoria); Melero, I. (Ignacio); Huarte, E. (Eduardo); Vera, M. (María); Tirapu, I. (Íñigo); Palencia, B. (Belén); Arina, A. (Ainhoa)Like paratroopers in special operations, dendritic cells (DCs) can be deployed behind the enemy borders of malignant tissue to ignite an antitumour immune response. 'Cross-priming T cell responses' is the code name for their mission, which consists of taking up antigen from transformed cells or their debris, migrating to lymphoid tissue ferrying the antigenic cargo, and meeting specific T cells. This must be accomplished in such an immunogenic manner that specific T lymphocytes would mount a robust enough response as to fully reject the malignancy. To improve their immunostimulating activity, local gene therapy can be very beneficial, either by transfecting DCs with genes enhancing their performance, or by preparing tumour tissue with pro-inflammatory mediators. In addition, endogenous DCs from the tumour host can be attracted into the malignant tissue following transfection of certain chemokine genes into tumour cells. On their side, tumour stroma and malignant cells set up a hostile immunosuppressive environment for artificially released or attracted DCs. This milieu is usually rich in transforming growth factor-beta, vascular endothelial growth factor, and IL-10, -6 and -8, among other substances that diminish DC performance. Several molecular strategies are being devised to interfere with the immunosuppressive actions of these substances and to further enhance the level of anticancer immunity achieved after artificial release of DCs intratumourally.
- Anti-ICAM-2 monoclonal antibody synergizes with intratumor gene transfer of interleukin-12 inhibiting activation-induced T-cell death(American Association for Cancer Research, 2003) Mazzolini, G. (Guillermo); Alfaro, C. (Carlos); Gabari, I. (Izaskun); Baixeras, E. (Elena); Qian, C. (Cheng); Melero, I. (Ignacio); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Feijoo, E. (Esperanza); Arina, A. (Ainhoa)PURPOSE: Systemic treatment with an anti-ICAM-2 monoclonal antibody (mAb; EOL4G8) eradicates certain established mouse tumors through a mechanism dependent on the potentiation of a CTL-mediated response. However, well-established tumors derived from the MC38 colon carcinoma cell line were largely refractory to this treatment as well as to intratumor injection of a recombinant adenovirus encoding interleukin-12 (IL-12; AdCMVIL-12). We sought to design combined therapy strategies with AdCMVIL-12 plus anti-ICAM-2 mAbs and to identify their mechanism of action. EXPERIMENTAL DESIGN: Analysis of antitumor and toxic effects were performed with C57BL/6 mice bearing established MC38 tumors. Anti-ovalbumin T-cell receptor transgenic mice and tumors transfected with this antigen were used for in vitro and in vivo studies on activation-induced cell death (AICD) of CD8(+) T cells. RESULTS: Combined treatment with various systemic doses of EOL4G8 mAb plus intratumor injection of AdCMVIL-12 induced complete regression of MC38 tumors treated 7 days after implantation. Unfortunately, most of such mice succumbed to a systemic inflammatory syndrome that could be prevented if IFN-gamma activity were neutralized once tumors had been rejected. Importantly, dose reduction of EOL4G8 mAb opened a therapeutic window (complete cure of 9 of 18 cases without toxicity). We also show that ICAM-2 ligation by EOL4G8 mAb on activated CTLs prevents AICD, thus extending IFN-gamma production. CONCLUSIONS: Combination of intratumor gene transfer of IL-12and systemic anti-ICAM-2 mAb display synergistic therapeutic and toxic effects. CTL life extension resulting from AICD inhibition by anti-ICAM-2 mAbs is the plausible mechanism of action.
- An anti-ICAM-2 (CD102) monoclonal antibody induces immune-mediated regressions of transplanted ICAM-2-negative colon carcinomas(American Association for Cancer Research, 2002) Albar, J.P. (Juan P.); Mazzolini, G. (Guillermo); Gabari, I. (Izaskun); Melero, I. (Ignacio); Prieto, J. (Jesús); Tirapu, I. (Íñigo); Camafeita, E. (Emilio); Relloso, M. (Miguel); Schmitz, V. (Volker); Rodriguez-Calvillo, M. (Mercedes); Corbi, A.L. (Angel L.)Monoclonal antibodies (mAbs) can mediate antitumor effects by indirect mechanisms involving antiangiogenesis and up-regulation of the cellular immune response rather than by direct tumor cell destruction. From mAbs raised by immunization of rats with transformed murine endothelial cells, a mAb (EOL4G8) was selected for its ability to eradicate a fraction of established colon carcinomas that did not express the EOL4G8-recognized antigen. The antigen was found to be ICAM-2 (CD102). Antitumor effects of EOL4G8, which required a functional T-cell compartment, were abrogated by depletion of CD8(+) cells and correlated with antitumor CTL activity, whereas only a mild inhibition of angiogenesis was observed. Interestingly, we found that EOL4G8 acting on endothelial ICAM-2 markedly enhances leukotactic factor activity-1-independent adhesion of immature dendritic cells to endothelium-an effect that is at least in part mediated by DC-SIGN (CD209).
- Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells(Elsevier, 2001) Mazzolini, G. (Guillermo); Duarte, M. (Marina); Gabari, I. (Izaskun); Rifon, J. J. (Jose J.); Rocha, E. (Eduardo); Melero, I. (Ignacio); Prieto, J. (Jesús); Rodriguez-Calvillo, M. (Mercedes)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(Wiley Blackwell, 2001) Mazzolini, G. (Guillermo); Genove, G. (Guillem); Qian, C. (Cheng); Sangro, B. (Bruno); Barajas, M. (Miguel); Bilbao, R. (Roberto); Melero, I. (Ignacio); Narvaiza, I. (Íñigo); Prieto, J. (Jesús); Schmitz, V. (Volker)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.