IFN-alpha Gene Therapy Research Articles

Abstract PR13: Development of chimeric forms of IFN-alpha for “on demand” in vivo cancer gene therapy

Abstract The immunosuppressive tumor microenvironment is a major hurdle for cancer therapy. Our lab previously developed a cell and gene therapy strategy for the tumor-targeted delivery of IFN-alpha based on the transduction of hematopoietic stem/progenitor cells (HSPC) with lentiviral vectors (LV) expressing the IFN-alpha gene under the control of the TIE2 enhancer/promoter. The specificity of this strategy is ensured by the restricted expression of IFN-alpha in a population of HSPC-derived tumor-infiltrating macrophages expressing the TIE2 receptor (TEM), which are recruited at the tumor site. TEM-mediated delivery of IFN-alpha has shown efficacy in different tumor models of both solid and hematopoietic origin, including a model of human glioma in immunodeficient mice. New data obtained in a more relevant immunocompetent mouse model of orthotopic glioma closely reproducing several features of the human disease show dramatic tumor inhibition in mice treated with IFN-alpha gene therapy. We are currently characterizing the contribution of IFN-induced antiangiogenic and modulatory activity on the innate and adaptive immune system underlying such response. To further improve the therapeutic index of our strategy, we are developing inducible strategies to superimpose a timer and a rheostatic switch on the amount of IFN-alpha secreted in the tumor microenvironment. Indeed, whereas a sustained cytokine output could ensure efficacy and long-term protection from tumor recurrence, it may raise concerns for long-term effects, especially in case of cancer eradication. By fusing a destabilizing domain (DD) to a protein of interest (POI), the former can confer its instability to the latter. This destabilization can be rescued in a reversible and dose-dependent manner with the addition of a small molecule specifically binding to the DD. To apply this technology to our strategy, we have designed and in vitro tested different fusion proteins of IFN-alpha (DD-IFN-alpha) with or without the addition of flexible or cleavable linkers and selected them for their capacity to be stabilized in presence of their specific ligand in vitro. Through this approach, we have identified fusion proteins with low basal activity and high fold induction upon ligand treatment. These novel regulated forms of IFN-alpha are functional and their specific activities are comparable to the wild type. Based on preliminary evidence of in vivo ligand-regulated IFN release, we are now testing the safety and efficacy of our new platforms in inducing antitumor responses in melanoma, colon, and glioma models of cancer. This abstract is also being presented as Poster B31. Citation Format: Filippo Birocchi, Melania Cusimano, Anna Ranghetti, Tiziano Di Tomaso, Barbara Costa, Peter Angel, Nadia Coltella, Luigi Naldini. Development of chimeric forms of IFN-alpha for “on demand” in vivo cancer gene therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR13.

Local interferon‐α gene therapy elicits systemic immunity in a syngeneic pancreatic cancer model in hamster

The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN-alpha did not show cross-species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN-alpha effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN-alpha augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN-alpha gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN-alpha adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM-1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell- and natural killer cell-mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM-1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity.

Gene therapy of ovarian cancer with IFN-α-producing fibroblasts: comparison of constitutive and inducible vectors

Ovarian cancer represents a malignancy suitable for cell and gene therapy approaches owing to its containment within the peritoneal cavity, even at advanced tumor stages. As regulation of transgene expression would be preferable for conducting clinical trials for reasons of safety, we investigated whether intraperitoneal (i.p.) administration of retroviral vector-transduced fibroblasts encoding murine interferon-alpha (IFN-alpha) could have therapeutic activity, and compared its effect with the antitumor effects of fibroblasts producing IFN-alpha under a rapamycin analogue (AP21967)-inducible promoter. Human and murine fibroblasts were recruited into the solid component of transplantable ovarian cancer-grown i.p. in severe combined immunodeficiency mice. Multiple administrations of fibroblasts producing IFN-alpha in a constitutive manner showed therapeutic efficacy, leading to significant prolongation of survival in the majority of animals, associated with inhibition of tumor angiogenesis. Compared to cells transduced by the constitutive vector, fibroblasts transduced by the inducible vector released twofold higher IFN-alpha levels in vitro, following induction by AP21967, and production of the cytokine was under pharmacologic control both in vitro and in vivo. However, these cells elicited only modest therapeutic effects in vivo. Overall, these findings indicate that intracavitary IFN-alpha gene therapy using engineered fibroblasts requires sustained production of IFN-alpha to achieve durable antitumor effects.

Adenovirus-mediated interferon α gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer

We previously demonstrated a characteristically high sensitivity of pancreatic cancer cells to interferon alpha (IFN-α) gene transfer, which induced a more prominent growth suppression and cell death in pancreatic cancer cells than in other types of cancers and normal cells. The IFN-α protein can exhibit both direct cytotoxicity and indirect immunological antitumour activity. Here, we dissected and examined the two mechanisms, taking advantage of the fact that IFN-α did not show any cross-species activity in its in vivo effect. When a human IFN-α adenovirus was injected into subcutaneous xenografts of human pancreatic cancer cells in nude mice, tumour growth was significantly suppressed due to cell death in an adenoviral dose-dependent manner. The IFN-α protein concentration was markedly increased in the injected subcutaneous tumour, but leakage of the potent cytokine into the systemic blood circulation was minimal. When a mouse IFN-α adenovirus was injected into the same subcutaneous tumour system, all mice showed significant tumour inhibition, an effect that was dependent on the indirect antitumour activities of IFN-α, notably a stimulation of natural killer cells. Moreover, in this case, tumour regression was observed not only for the injected subcutaneous tumours but also for the untreated tumours at distant sites. This study suggested that a local IFN-α gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its dual mechanisms of antitumour activities and lack of significant toxicity.

Interferon-α Gene Therapy by Lentiviral Vectors Contrasts Ovarian Cancer Growth Through Angiogenesis Inhibition

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.

Interferon-α gene therapy prevents aflatoxin and carbon tetrachloride promoted hepatic carcinogenesis in rats

Retrovirus-mediated interferon alpha (IFN-alpha) gene transfer was evaluated with regard to its possible protective effects against aflatoxin B1 (AFB1)-initiated and carbon tetrachloride (CCl4)-promoted hepatic carcinogenesis in rats. To our knowledge, this is the first time an experimental in vivo gene therapy trial was conducted in Egypt. Two genes were examined in liver tissue by RT-PCR: the first was glutathione-S-transferase placental (GST-P) isoenzyme, as an early marker to detect hepatic malignancy; the second was IFN-alpha gene expression to detect the efficiency of gene uptake and its persistence after transduction. Forty male rats, divided equally into 4 groups, were included in the study: the first group was the control; the second group received CCl4 0.2 ml subcutaneously twice weekly for 12 weeks and AFB1 0.25 mg/kg body wt intraperitoneally twice weekly for 6 weeks; the third group received IFN-alpha (10(8) pfu) intravenously in the tail vein prior to the start of CCl4 and AFB1 injections; and the fourth group received IFN-alpha (10(8) pfu) by intrahepatic injection under ultrasonography guide after termination of the CCl4 and AFB1 injection schedule. The results showed that IFN-alpha has a marked and significant protective effect against hepatic fibrogenesis as well as hepatic carcinogenesis. Pathological examination of liver tissue proved that IFN-alpha minimized both fibrotic and cirrhotic processes. The amount of fibrosis was less in both groups receiving IFN-alpha, with more protection in the group that received IFN-alpha intravenously prior to CCl4 and AFB1. The results of RT-PCR showed that the IFN-alpha gene was significantly expressed in both groups receiving IFN-alpha, with a more intense expression in the group that received IFN-alpha by intrahepatic injection after termination of CCl4 and AFB1 injections. The IFN-alpha gene was detected after three months of gene transduction in rats receiving IFN-alpha intravenously prior to CCl4 and AFB1 and after one month of gene transduction in the post CCl4 and AFB1 rats. IFN-alpha gene was not expressed in the two groups that did not undergo gene transfer. Histopathological signs of premalignant macronodules were evident in the group receiving CCl4 and AFB1, but not IFN-alpha as well as in the group that received IFN-alpha at the end of the experiment. GST-P gene expression was also detected in these two groups, confirming early malignant transformation. In conclusion, IFN-alpha exerts significant protective effects, but more so when the gene is administered before fibrogenic and carcinogenic induction in hepatic tissues. IFN-alpha gene therapy may be justified in clinical trials for high-risk candidates with hepatic carcinogenesis.

Interferon-alpha and interleukin-12 gene therapy of cancer: interferon-alpha induces tumor-specific immune responses while interleukin-12 stimulates non-specific killing.

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-alpha and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-alpha and IL-12. Transduced MC38 cell lines expressing IFN-alpha or IL-12 (MC38-IFNalpha or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-alpha or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNalpha or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNalpha-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8(+) cells in wild-type tumors of mice inoculated with IFN-alpha-expressing cells. Leukocyte-depletion experiments implicated CD8(+) T cells in tumor rejection induced by IFN-alpha-transduction; both CD8(+) T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNalpha cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNalpha and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-alpha induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-alpha and IL-12 gene therapy might warrant clinical trials.

Intramuscular electroporation delivery of IFN-alpha gene therapy for inhibition of tumor growth located at a distant site.

Although electroporation has been shown in recent years to be a powerful method for delivering genes to muscle, no gene therapy via electro-injection has been studied for the treatment of tumors. In an immunocompetent tumor-bearing murine model, we have found that delivery of a low dose of reporter gene DNA (10 microg) to muscle via electroporation under specific pulse conditions (two 25-ms pulses of 375 V/cm) increased the level of gene expression by two logs of magnitude. Moreover, administration of 10 microg of interferon (IFN)-alpha DNA plasmid using these parameters once a week for 3 weeks increased the survival time and reduced squamous cell carcinoma (SCC) growth at a distant site in the C3H/HeJ-immunocompetent mouse. IFN-alpha gene therapy delivered to muscle using electroporation demonstrated statistically significant (P < 0.05) therapeutic efficacy for treating SCC located at a distant site, compared with interleukin (IL)-2 or endostatin gene, also delivered by electro-injection. The increased therapeutic efficacy was associated with a high level and extended duration of IFN-alpha expression in muscle and serum. We also discovered that the high level of IFN-alpha expression correlated with increased expression levels of the antiangiogenic genes IP-10 and Mig in local tumor tissue, which may have led to the reduction of blood vessels observed at the local tumor site. Delivery of increasing doses (10-100 microg) of IFN-alpha plasmid DNA by injection alone did not increase antitumor activity, whereas electroporation delivery of increasing doses (10-40 microg) of IFN-alpha plasmid DNA did increase the survival time. Our data clearly demonstrate the potential utility of electroporation for delivery of gene therapy to muscle for the treatment of residual or disseminated tumors.

IFN-alpha-expressing tumor cells enhance generation and promote survival of tumor-specific CTLs.

IFN-alpha gene therapy has been successfully applied in several tumor models. Our studies involving the murine colorectal adenocarcinoma cell line MC38 confirm that IFN-alpha transduction of a poorly immunogenic tumor cell reduces tumorigenicity and leads to long-lasting tumor immunity. To investigate the effect of IFN-alpha transduction on the development of antitumor immune responses, we restimulated splenocytes from MC38-immune mice in vitro. Detection of MC38-specific cytotoxicity was markedly enhanced when murine IFN-alpha2-transduced MC38 (MC38-IFNalpha) or CD80-transduced MC38 (MC38-CD80) was used for restimulation compared with wild type (MC38-WT) or neomycin resistance gene-transduced MC38 (MC38-Neo) cells. MC38-specific CD8+ CTL line and clone were established from splenocytes of mouse immunized with MC38-IFNalpha. Stimulation with MC38-IFNalpha as well as MC38-CD80 enhanced the proliferation of MC38-specific CTLs in vitro much more effectively than stimulation with WT or MC38-Neo (p < 0.05). Coincubation of MC38-specific CTLs with MC38-IFNalpha or MC38-CD80 resulted in significantly less DNA fragmentation (8.0% and 12.8%, respectively) compared with coincubation of the CTLs with MC38-WT (43.5%; p < 0.001) or MC38-Neo cells (38.1%; p < 0.003). These results suggest that prevention of apoptotic cell death in tumor-specific CTLs may be one mechanism by which IFN-alpha-expressing tumor cells can promote the generation of antitumor immunity. The effect of IFN-alpha on CTLs appears to be similar to that of CD80, which also prevents apoptotic cell death after stimulation of T lymphocytes.

Interferon-alpha gene therapy for cancer: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells are both effective strategies for gene delivery in murine tumor models.

Stable transfection of tumor cells with IFN-alpha genes has been shown to result in abrogation of tumor establishment and induction of antitumor immunity. However, strategies suitable for the clinical application of IFN-alpha gene therapy for cancer have not been reported. In this study, we investigated two gene delivery systems capable of mediating the local paracrine production of high levels of biologically active IFN-alpha in murine tumor models: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells. In spite of the antiproliferative effects of IFN-alpha, it was possible to obtain stable retroviral producer cell lines and transduce a variety of murine tumor cells including syngeneic fibroblasts to stably secrete 2000-5000 U (40-100 ng) murine IFN-alpha/10(6) cells/24 h. IFN-alpha transduction of tumor cells abrogated tumorigenicity in establishment models and induced antitumor immunity in several murine tumor model systems. Importantly, IFN-alpha gene delivery using retrovirally transduced syngeneic fibroblasts was capable of suppressing the establishment of the poorly immunogenic TS/A mouse mammary adenocarcinoma and induced antitumor immunity. Particle mediated transient transfection of tumor cells using the gene gun led to the production of up to 20,000 U IFN-alpha/10(6) cells during the first 24 h and proved to be equally effective in suppressing establishment of TS/A adenocarcinoma and inducing antitumor immunity. These results suggest that retroviral transduction of autologous fibroblasts can serve as an effective gene delivery method for IFN-alpha gene therapy of cancer. Particle-mediated transfection of freshly isolated tumor cells may represent a clinically attractive alternative approach for nonviral gene delivery. Both strategies circumvent the difficulties in routinely establishing primary tumor cell lines from the vast majority of human cancers.

Treatment of human hepatocellular carcinoma by fibroblast-mediated human interferon alpha gene therapy in combination with adoptive chemoimmunotherapy.

The therapeutic effect of the fibroblast-mediated human interferon (IFN alpha) gene therapy in combination with interleukin-2 (IL-2) activated killer cells (AK)/doxorubicin (i.e., adoptive chemoimmunotherapy) on nude mice bearing the human hepatocellular carcinoma (HCC) was investigated. A fibroblast cell clone (NIH3T3-IFN alpha+) secreting 1024 U/ml human IFN alpha was obtained from 14 positive clones by BMGNeo-IFN alpha DNA transfection, G418-resistant selection, limiting dilution and assay of IFN alpha activity. After i.p. implantation of NIH3T3-IFN alpha+ encapsulated into collagen, serum human IFN alpha activity could be detected from 12 h to day 15 with a peak at 72 h. AK were prepared from human peripheral mononuclear cells costimulated in vitro by IL-2 and inactivated human SMMC 7721 HCC cells. When the NIH3T3-IFN alpha+ cells were i.p. implanted into the HCC-bearing nude mice, the grown of HCC was inhibited and the survival time of the mice was extended. The growth of HCC was inhibited more obviously when AK was i.v. injected and IL-2 was i.p. injected after the NIH3T3-IFN alpha+ cells had been implanted. The best therapeutic effect was achieved when NIH3T3-IFN alpha+ cells were used in combination with IL-2/AK/doxorubicin. All these results suggested that the fibroblast-mediated human IFN alpha gene therapy could be used to treat the human hepatocellular carcinoma effectively and that when used in combination with IL-2-based adoptive chemoimmunotherapy, the therapeutic effect would be better.

In vitro modulation of the invasive and metastatic potentials of human renal cell carcinoma by interleukin-2 and/or interferon-alpha gene transfer.

Continuous local delivery of interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) via gene transfer appears to be more effective than systemic therapy in preventing the growth of human renal cell carcinoma (RCC) in vitro and in vivo. To understand further if cytokine-gene transfection of RCC could alter certain cellular properties that are associated with the invasive and metastatic potentials of tumor, the authors characterized six cell lines that produce IL-2 and/or IFN-alpha in their expression of intercellular adhesion molecule-1 (ICAM-1) and CD44; binding affinity to extracellular matrix (ECM) components (fibronectin, laminin, type IV collagen, and vitronectin); and preference in forming homotypic aggregation and mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), tumor transforming growth factor-beta (TGF-beta) and type IV collagenase. These six lines were compared with control vector transfected parental R11 line. The expression of ICAM-1 and CD44 was determined by fluorescence-activated cell sorter (FACS) analysis, the tumor cell binding affinity to ECM components was measured by cell attachment assay, the degree of homotypic aggregation was quantified by cell aggregation assay, and the mRNA levels of c-myc, EGF-R, TGF-beta, and collagenase were analyzed by a quantitative polymerase chain reaction analysis. Both IL-2-gene- and IFN-alpha-gene-modified R11 exhibited enhanced expression of ICAM-1, suppression of CD44, and decreased binding affinity to ECM components, when compared with the R11-control vector. All cytokine-producing tumor lines showed a decreased preference to form homotypic aggregation. Interferon-alpha gene transfer downregulated c-myc, EGF-R, and type IV collagenase mRNA expression, whereas only the higher producers of IL-2 downregulated TGF-beta mRNA expression. Exogenous IL-2 and/or IFN-alpha treatment of a IFN-alpha-resistant RCC enhanced both HLA class I antigen and ICAM-1 expression and suppressed CD44 expression, but had no effect on tumor growth rate. The local production of high concentrations of IL-2 and IFN-a at the tumor site may directly alter tumor properties associated with invasive and metastatic phenotypes of RCC. Interleukin-2 and/or IFN-alpha gene therapy may be an effective strategy for treatment of patients with advanced renal cancer.