Cytokine Gene Therapy Research Articles

The Use of Targeted Cytokines as Cancer Therapeutics in Glioblastoma.

Cytokines play an important role in regulating the immune response. Although there is great interest in exploiting cytokines for cancer immunotherapy, their clinical potential is limited by their pleiotropic properties and instability. A variety of cancer cell-intrinsic and extrinsic characteristics pose a barrier to effective treatments including cytokines. Recent studies using gene and cell therapy offer new opportunities for targeting cytokines or their receptors, demonstrating that they are actionable targets. Current efforts such as virotherapy, systemic cytokine therapy, and cellular and gene therapy have provided novel strategies that incorporate cytokines as potential therapeutic strategies for glioblastoma. Ongoing research on characterizing the tumor microenvironment will be informative for prioritization and combinatorial strategies of cytokines for future clinical trials. Unique therapeutic opportunities exist at the convergence of cytokines that play a dual role in tumorigenesis and immune modulation. Here, we discuss the underlying strategies in pre- and clinical trials aiming to enhance treatment outcomes in glioblastoma patients.

Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model.

This study evaluates a short therapy schedule for bladder cancer using BCG Tokyo. BCG Tokyo was evaluated in vitro using bone marrow derived dendritic cells, neutrophils, RAW macrophages and the murine bladder cancer cell line, MB49PSA, and compared to other BCG strains. BCG Tokyo > BCG TICE at inducing cytokine production. In vivo, high dose (1 × 107 colony forming units (cfu)) and low dose (1 × 106 cfu) BCG Tokyo with and without cytokine genes (GMCSF + IFNα) were evaluated in C57BL/6J mice (n = 12–16 per group) with orthotopically implanted MB49PSA cells. Mice were treated with four instillations of cytokine gene therapy and BCG therapy. Both high dose BCG alone and low dose BCG combined with cytokine gene therapy were similarly effective. In the second part the responsive groups, mice (n = 27) were monitored by urinary PSA analysis for a further 7 weeks after therapy cessation. More mice were cured at day 84 than at day 42 confirming activation of the immune system. Cured mice resisted the re-challenge with subcutaneous tumors unlike naïve, age matched mice. Antigen specific T cells recognizing BCG, HY and PSA were identified. Thus, fewer intravesical instillations, with high dose BCG Tokyo or low dose BCG Tokyo with GMCSF + IFNα gene therapy, can induce effective systemic immunity.

Highlights of Immunomodulation in Salmonella-Based Cancer Therapy.

Bacteria-mediated cancer therapy (BMCT) is an emerging tool that may advance potential approaches in cancer immunotherapy, whereby tumors are eradicated by the hosts’ immune system upon recruitment and activation by bacteria such as Salmonella. This paper provides an emphasis on the immunomodulatory effects that encompasses both the innate and adaptive immune responses inherently triggered by Salmonella. Furthermore, modifications of Salmonella-based treatment in the attempt to improve tumor-specific immune responses including cytokine therapy, gene therapy, and DNA vaccine delivery are likewise discussed. The majority of the findings described herein incorporate cell-based experiments and murine model studies, and only a few accounts describe clinical trials. Salmonella-based cancer therapy is still under development; nonetheless, the pre-clinical research and early-phase clinical trials that have been completed so far have shown promising and convincing results. Certainly, the continuous development of, and innovation on, Salmonella-based therapy could pave the way for its eventual emergence as one of the mainstream therapeutic interventions addressing various types of cancer.

Genetically engineered mesenchymal stem cells: targeted delivery of immunomodulatory agents for tumor eradication.

Cancer immunotherapy emerged as a novel therapeutic option that employs enhanced or amended native immune system to create a robust response against malignant cells. The systemic therapies with immune-stimulating cytokines have resulted in substantial dose-limiting toxicities. Targeted cytokine immunotherapy is being explored to overcome the heterogeneity of malignant cells and tumor cell defense with a remarkable reduction of systemic side effects. Cell-based strategies, such as dendritic cells (DCs), fibroblasts or mesenchymal stem cells (MSCs) seek to minimize the numerous toxic side effects of systemic administration of cytokines for extended periods of time. The usual toxicities comprised of a vascular leak, hypotension, and respiratory insufficiency. Natural and strong tropism of MSCs toward malignant cells made them an ideal systemic delivery vehicle to direct the proposed therapeutic genes to the vicinity of a tumor where their expression could evoke an immune reaction against the tumor. Compared with other methods, the delivery of cytokines via engineered MSCs is safer and renders a more practical, and promising strategy. Large numbers of genes code for cytokines have been utilized to reengineer MSCs as therapeutic cells. This review highlights the recent findings on the cytokine gene therapy for human malignancies by focusing on MSCs application in cancer immunotherapy.

Gmcsf and Ifnα gene therapy improves the response to BCG immunotherapy in a murine model of bladder cancer.

Aim: To develop a strategy to improve response to bacillus Calmette-Gueri (BCG) using cytokine gene therapy (Gmcsf + Ifnα). Materials & methods: MB49-PSA tumor-bearing C57BL/6N mice were assigned into four groups: control; Gmcsf + Ifnα therapy; BCG therapy or combined therapy (Gmcsf + Ifnα and BCG). In schedule 1, cytokine gene therapy was delivered before BCG therapy (eight instillations). In schedule 2, cytokine gene and BCG therapy were instilled alternatively (eight instillations). Tumors were analyzed by immunohistochemistry and mRNA analysis and urinary immune cells by flow cytometry. Results: Combined therapy in schedule 2 reduced tumor growth, increased immune cell recruitment and was associated with reduced inflammation when compared with BCG therapy. Conclusion: Alternating cytokine gene delivery with BCG therapy modulates the tumor environment increasing receptivity to BCG.

Biologic and mechanical aspects of tendon fibrosis after injury and repair

ABSTRACTTendon injuries of the hand that require surgical repair often heal with excess scarring and adhesions to adjacent tissues. This can compromise the natural gliding mechanics of the flexor tendons in particular, which operate within a fibro-osseous tunnel system similar to a set of pulleys. Even combining the finest suture repair techniques with optimal hand therapy protocols cannot ensure predictable restoration of hand function in these cases. To date, the majority of research regarding tendon injuries has revolved around the mechanical aspects of the surgical repair (i.e. suture techniques) and postoperative rehabilitation. The central principles of treatment gleaned from this literature include using a combination of core and epitendinous sutures during repair and initiating motion early on in hand therapy to improve tensile strength and limit adhesion formation. However, it is likely that the best clinical solution will utilize optimal biological modulation of the healing response in addition to these core strategies and, recently, the research in this area has expanded considerably. While there are no proven additive biological agents that can be used in clinical practice currently, in this review, we analyze the recent literature surrounding cytokine modulation, gene and cell-based therapies, and tissue engineering, which may ultimately lead to improved clinical outcomes following tendon injury in the future.

Combination of Suicide and Cytokine Gene Therapies as Surgery Adjuvant for Canine Mammary Carcinoma.

The incidence of canine mammary carcinoma varies with age, breed, and spay status, being among the main tumors appearing in intact female dogs. Thirty-six canine mammary carcinoma patients received injections of canine interferon-β (cIFN-β) and HSV-thymidine kinase/ganciclovir (HSV-tk/GCV) carrying lipoplexes, into the tumor bed, immediately after surgery. Next, they started periodic subcutaneous injections of lipoplexes carrying a human granulocyte-macrophage colony stimulating factor and interleukin-2 mixed with allogeneic mammary carcinoma extracts. This combined strategy was safe and well tolerated. In addition, only two out of 26 patients treated with complete surgery developed a local relapse, and 0 out of 29 stage II and III patients displayed distant metastases, suggesting both local and systemic antitumor activities. The most encouraging result was the long survival times: 22 > 1 year (where 13 > 2 and 4 > 3 years), while maintaining a good quality of life. The preliminary results in five patients presenting with local disease, an additional HSV-tk/GCV plus cIFN-β gene treatment induced local antitumor activity, evidenced by four objective responses (one complete, three partial) and one stable disease. This successful outcome supports further studies to validate this approach not only for canine veterinary patients, but also for translation to human patients.

Abstract 1819: Reprogramming the tumor microenvironment by targeting endothelial DNA repair

Abstract The tumor microenvironment (TME) plays a critical role in orchestrating immune infiltration, tumor progression and response to therapeutics. Therefore, strategies to manipulate the TME including cytokine gene therapy, antibodies to reverse macrophage polarization etc are under active investigation. We have identified an alternative approach to target the TME by disrupting DNA repair in the tumor endothelial cells (ECs). We discovered a seven-microRNA (miR) signature induced in ECs in in vitro and in vivo by oxidative stress and DNA damage. The top miR candidate in this signature, miR-103 altered the TME by inducing DNA damage in ECs, eliciting type I interferons, upregulating immune costimulatory receptors and decreasing PD-L1 expressing tumor associated macrophages and granulocytes. Moreover, miR-103 treatment had a paracrine effect on the tumors and upregulated Fas and TRAIL receptors. Mechanistically, these functions of miR-103 were largely due to its downregulation of the three prime exonuclease TREX1. Local, systemic or vascular targeted delivery of miR-103 decreased both angiogenesis and tumor burden in multiple mouse tumor models. Complementary to the role of miR-103, two additional miRs in the miR signature, miRs 494 and 99b each induced senescence in the vasculature by downregulating the Mre11a-Rad50-NBN (MRN) complex. Ectopic expression of miRs 494 or 99b decreased telomerase activity, increase p21 levels with a concomitant decrease in pRb levels. Vascular targeted delivery of miR-494 decreased angiogenesis in vivo whereas systemic delivery decreased tumor growth. Interestingly, both miR mimics and the MRN knockdowns induced the transcription of a number of senescence associated genes including CD44. Taken together these data suggest that miRs 494 and 99b targeting of the MRN complex induces senescence. The MRN complex interacts with the ATM kinase, histone H2AX and TREX1 suggesting that the miRs we identified disrupt critical nodes of a DNA Damage Response (DDR) network. Our findings reveal a complex, miR mediated cross talk between EC DNA damage pathways, the TME and tumor cells. These interactions can be exploited for designing therapies that synergize with tumor cell killing to enhance host anti-tumor responses. Citation Format: Cristina Espinosa-Diez, RaeAnna Wilson, Rebecca Ruhl, Nathan Kanner, Namita Chatterjee, Clay Hudson, Sudarshan Anand, Shushan Rana. Reprogramming the tumor microenvironment by targeting endothelial DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1819. doi:10.1158/1538-7445.AM2017-1819

Reprogrammed chondrocytes engineered to produce IL-12 provide novel ex vivo immune-gene therapy for cancer.

The somatic cell reprogramming technology was applied to a novel and promising ex vivo immune-gene therapy strategy for cancer. To establish a novel ex vivo cytokine gene therapy of cancer using the somatic cell reprogramming procedures. Mouse fibroblasts were converted into chondrocytes and subsequently transduced with IL-12 gene. The resultant IL-12 induced chondrogenic cells were irradiated with x-ray and inoculated into mice bearing CT26 colon cancer. The irradiation at 20 Gy or higher totally eliminated the proliferative potential of the cells, while less significantly influencing the IL-12 production from the cells. An inoculation of the irradiated IL-12 induced chondrogenic cells significantly suppressed tumor by inducing tumor-specific cytotoxic T lymphocytes, enhancing natural killer tumoricidal activity and inhibiting tumor neoangiogenesis in the mice. The somatic cell reprogramming procedures may provide a novel and effective means to treat malignancies.

The effects of gene therapy with granulocyte-macrophage colony-stimulating factor in the regression of tumor masses in fibrosarcoma mouse model.

Cytokine gene therapy is one of the cancer treatment strategies. Recently, granulocyte-monocyte colony-stimulating factor (GM-CSF), as an important cytokine in activating dendritic cells and boosting the anti-tumor immune responses, has been utilized as an immunotherapeutic agent in cancer gene therapy. The purpose of the present investigation was to study the GM-CSF gene therapy effects in regression of tumor masses in fibrosarcoma mouse model. To investigate the therapeutic efficacy of GM-CSF, WEHI-164 tumor cells were transfected with murine GM-CSF plasmid. For cytokine production by transfected cells, enzyme-linked immunosorbent assay test was used. Fibrosarcoma mouse model established with transfected cells which were injected subcutaneously into Balb/c mice. Tumor sizes were measured by caliper. Mice were sacrificed and the tumors were extracted. The expression of GM-CSF was studied by real-time polymerase chain reaction (PCR) and immunoblotting. The expression of Ki-67 (a tumor proliferative marker) in tumor masses was studied by immunohistochemical staining. The group treated with GM-CSF indicated a decrease in tumor mass volume (P = 0.001). The results of western blotting and real-time PCR showed that GM-CSF expression increased in the group treated with GM-CSF (with a relative expression of 1.36). Immunohistochemical staining showed that Ki-67 expression has reduced in the group treated with GM-CSF. Monotherapy with GM-CSF has therapeutic effects on the regression of tumor masses in the fibrosarcoma mouse model.

Cytokine gene therapy for treatment of ischemia and hypoxia stress induced gastrointestinal injury

Cytokine gene therapy for treatment of ischemia and hypoxia stress induced gastrointestinal injury

Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination.

Bubble-assisted ultrasound is a versatile technology with great potential in immunotherapy and vaccination. This technology involves the exposure of immune cells (i.e., dendritic cells, lymphocytes) in-vitro or diseased tissues (i.e., brain, tumor) in-vivo to ultrasound treatment with gas bubbles. Bubble destruction leads to physical forces that induce the direct delivery of weakly permeant immuno-stimulatory molecules either into the cytoplasm of immune cells, or through the endothelial barrier of diseased tissues. Hence, therapeutic antibodies (i.e., antibody-based immunotherapy) and cytokine-encoding nucleic acids (i.e., cytokine gene therapy) can be successfully delivered into diseased tissues, thus improving immune responses. In addition, protein antigens, as well as antigen-encoding nucleic acids (pDNA, mRNA), can be delivered into dendritic cells (i.e., dendritic cell-based vaccines), thus leading to a long-lasting prophylactic or therapeutic immunization. This chapter focuses on the state-of-the-art of bubble-assisted ultrasound in the field of immunotherapy and vaccination.

Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer.

BackgroundGene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system.MethodsWe studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte–macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models.ResultsWe showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan.ConclusionsWe demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier – a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0433-0) contains supplementary material, which is available to authorized users.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

Vaccine immunotherapy for prostate cancer: from mice to men.

Preclinical studies demonstrated the ability of an adenovirus/PSA (Ad/PSA) vaccine to induce strong anti-PSA immune responses, and these responses were capable of destroying prostate-specific antigen (PSA)-secreting mouse prostate tumors. A series of preclinical studies have demonstrated the superiority of the Ad/PSA vaccine to other PSA vaccines for the induction of anti-PSA immune responses, the ability of Ad/PSA vaccination combined with cytokine gene therapy and the TLR9 agonist CpG to enhance the anti-prostate tumor immunotherapy, and the reduction of negative regulatory elements when the vaccine was combined with 5-fluoruracil administration. A phase I clinical trial of the Ad/PSA vaccine in men with metastatic castrate-resistant prostate cancer demonstrated the safety of the vaccine even at the highest single dose permitted by the FDA. Currently, a phase II trial of the Ad/PSA vaccine is underway treating patients in two protocols. Thus far 81 patients have been enrolled and vaccinated. Early results from the patients evaluated to date demonstrated the induction of anti-PSA T cell responses, and the majority of patients evaluated at this time had demonstrated an increase in PSA doubling times.

Efficacy of oncolytic adenovirus expressing suicide genes and interleukin-12 in preclinical model of prostate cancer

Oncolytic adenovirus-mediated suicide gene therapy has been shown to improve local tumor control in preclinical tumor models and in the clinic. Although local tumor control is important, for most human cancers, new therapies must also target metastatic disease if they are to have an impact on survival. Here, we test the hypothesis that adding cytokine gene therapy to our multimodal platform improves both local and metastatic tumor control in a preclinical model of prostate cancer. An oncolytic adenovirus (Ad5-yCD/mutTKSR39rep-mIL12) expressing two suicide genes and mouse interleukin-12 (IL-12) was generated. Relative to an adenovirus lacking IL-12 (Ad5-yCD/mutTKSR39rep), Ad5-yCD/mutTKSR39rep-mIL12 improved local and metastatic tumor control in the TRAMP-C2 prostate adenocarcinoma model, resulting in a significant increase in survival. Ad5-yCD/mutTKSR39rep-mIL12 resulted in high levels of IL-12 and interferon gamma in serum and tumor, increased natural killer (NK) and cytotoxic T-lymphocyte lytic activities, and the development of tumor-specific antitumor immunity. Immune cell depletion studies indicated that both the innate and adaptive arms of immunity were required for maximal Ad5-yCD/mutTKSR39rep-mIL12 activity. The results demonstrate that the addition of IL-12 significantly improves the efficacy of oncolytic adenovirus-mediated suicide gene therapy and provide the scientific basis for future trials targeting locally aggressive cancers.

Intricacies for posttranslational tumor-targeted cytokine gene therapy.

The safest and most effective cytokine therapies require the favorable accumulation of the cytokine in the tumor environment. While direct treatment into the neoplasm is ideal, systemic tumor-targeted therapies will be more feasible. Electroporation-mediated transfection of cytokine plasmid DNA including a tumor-targeting peptide-encoding sequence is one method for obtaining a tumor-targeted cytokine produced by the tumor-bearing patient's tissues. Here, the impact on efficacy of the location of targeting peptide, choice of targeting peptide, tumor histotype, and cytokine utilization are studied in multiple syngeneic murine tumor models. Within the same tumor model, the location of the targeting peptide could either improve or reduce the antitumor effect of interleukin (IL)12 gene treatments, yet in other tumor models the tumor-targeted IL12 plasmid DNAs were equally effective regardless of the peptide location. Similarly, the same targeting peptide that enhances IL12 therapies in one model fails to improve the effect of either IL15 or PF4 for inhibiting tumor growth in the same model. These interesting and sometimes contrasting results highlight both the efficacy and personalization of tumor-targeted cytokine gene therapies while exposing important aspects of these same therapies which must be considered before progressing into approved treatment options.

Abstract 4412: Combining cytokine gene therapy with conventional cancer chemotherapyfor treatment of metastatic renal cell carcinoma

Abstract Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies and 85-90% of kidney cancer. Treatment was generally aggressive involving nephrectomy and excision of the metastatic lesion. The current study was designed to evaluate a new strategy for treatment of RCC, using combination of cytokine gene therapy with conventional cancer chemotherapy. The study established tumor metastasis in Balb/c mice using Renca cells as a model1. Treatment was performed on tumor bearing mice with hydrodynamic gene delivery of plasmids containing either mIL-2 or mIL-12 gene, separately, or in combination with or without two weekly injection of 5-flououracil (5-FU) (20 mg/kg, ip). The results showed that the lung, liver and kidney were the primary sites of tumor growth. 5-FU is capable of inhibiting tumor growth in the liver and kidney and effective in slowing down tumor growth in the lung when treatment started early and with high frequency. mIL-2 gene therapy inhibited tumor growth in the liver and kidney. The effect became more obvious if treatment started early. However, IL-2 alone is unable to eliminate all liver tumors. mIL-2 gene therapy had almost no effect on tumor growth in lung. mIL-12 gene therapy significantly blocked cancer-induced weight loss. mIL-12 inhibited tumor growth in the liver and kidney, but IL-12 alone is unable to eliminate all liver and kidney tumors. In addition, IL-12 inhibited lung swelling induced by tumor, especially when treatment was started early. Cytokine gene therapy combined with chemotherapy is effective in blocking tumor-induced weight loss, and tumor growth in the liver, lung and kidney. These results provide direct evidence in support that combined therapy of cytokine gene therapy with traditional chemotherapy is effective in treating RCC. Key words: Renal cell carcinoma, gene therapy, IL-12, IL-2, 5- flououracil 1 Jianfeng Li, Qian Yao and Dexi Liu (2011) Hydrodynamic cell delivery for simultaneous establishment of tumor growth in mouse lung, liver and kidney. Cancer Biol Ther 12:737-741. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4412. doi:1538-7445.AM2012-4412

Abstract 1546: Combined direct intratumoral adenoviral delivery and production of Rheoswitch®-regulated IL-12 and IFNα enhances antitumor activity in lung and breast cancer models

Abstract Current cancer immunotherapies have provided limited success in the clinic and innovative strategies are required to further enhance the effectiveness of an anti-tumor immune response.This study assessed anti-tumor activity utilizing intratumoral (i.t.) administration of adenovirus (Ad) with the novel Rheoswitch Therapeutic System®(RTS®), an inducible promoter system, for regulated expression of murine IL-12 or IFNα. Oral administration of a small molecule activator ligand (AL), INXN-1001, regulates the expression of IL-12 in Ad-RTS-mIL-12 and IFNα in Ad-RTS-mIFNα. The regulated expression and therapeutic benefit of IL-12 and IFNα from i.t. administered Ad-RTS-mIL-12 and Ad-RTS-mIFNα, either alone or in combination, was examined in syngeneic Lewis lung carcinoma (LLC) and syngeneic mammary carcinoma (4T1) models. In the LLC model, daily treatment with oral AL alone (administered in feed at 1000 ppm, representing a daily dose of ∼225 mg/kg/day) or i.t. cytokine gene therapy in the absence of AL did not result in significant inhibition of tumor growth compared to control, untreated tumors. In contrast, i.t. injection with 1010 vp Ad-RTS-mIL-12 or Ad-RTS-mIFNα and daily oral AL led to significant tumor growth inhibition by day 25 (72 and 71%, respectively; p<0.05). Notably, combined treatment of the LLC tumors with Ad-RTS-mIL-12 and Ad-RTS-mIFNα with oral AL resulted in significant anti-tumor effect compared to either treatment alone (97% growth inhibition; p<0.05) without overt toxicity as assessed by no change in body weight. In the 4T1 model, i.t. treatment with 1010 vp of Ad-RTS-mIL-12 plus AL or Ad-RTS-mIFNα plus AL led to 58 and 53% inhibition of tumor growth compared to control untreated tumors by day 34 (p<0.05). Notably, concomitant treatment with both Ad-RTS-mIL-12 plus AL and Ad-RTS-mIFNα plus AL resulted in enhanced anti-tumor activity with 80% growth inhibition. These data indicate that the combined treatment strategy using RTS-regulated IL-12 and IFNα in Ad vectors concomitant with AL induces effective therapeutic activity against aggressive murine tumors. Future studies will investigate the mechanism by which both IL-12 and IFNα exert their anti-tumor effect in the above tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1546. doi:1538-7445.AM2012-1546

Vaccines Targeting Tumor Blood Vessel Antigens Promote CD8+ T Cell-Dependent Tumor Eradication or Dormancy in HLA-A2 Transgenic Mice

We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.