Immune Competent Murine Model Research Articles

An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells

Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer.

P08.46 Synergistic Therapeutic Efficacy via Immunomodulatory Platelet Rich Fibrin Patch (PRF-P) in Combination with Oncolytic Adenovirus for the Treatment of Glioma

Abstract Introduction: Oncolytic virotherapy is an interesting approach for the treatment of cancer, including glioblastoma. Recent studies in this area suggest a synergistic approach to combine oncolytic virotherapy with immunomodulatory approaches. In this study, we have examined the role of platelet-rich fibrin patch (PRF-P) to enhance anti-tumoral immune response in immune-competent murine model C57/BL when used in conjunction with tumor specific oncolytic Ad5RGD-CMV-E1Δ24. This PRF-P is derived from blood, enriched with lymphocytes, platelets, macrophages (M1), and pro-inflammatory cytokines. Application of PRF-P has been shown to enhance immune cell migration and proliferation. HYPOTHESIS: We hypothesize that PRF-P provides local immunomodulation by supplying pro-inflammatory chemokines, as well as recruiting and enhancing proliferation of cytotoxic T cells, which may lead to an enhanced anti-tumoral immune response when combined with oncolytic virotherapy. METHODS: Blood from donor mice bearing GL261-OVA, a murine glioma cell line expressing ovalbumin (OVA), as a model antigen was collected into non-anticoagulant tubes and processed according to platelet-rich fibrin protocol. Five days after tumor implantation, a stereotactic intra-tumor injection of Ad5RGD-CMVΔ24 viral vector was performed. 48 hours post viral vector administration, a craniotomy was performed and the autologous PRF-P was applied over the tumor. For immunological flow and immunohistochemical analyses, mice were sacrificed five days post treatment. Results: We first performed a survival analysis in immuno-competent murine model. Mice injected with oncolytic virus alone had a significantly prolonged survival compared to PBS treated group (P<0.001). In addition, when used in combination with immunomodulatory PRF-P, this prolonged survival benefit was extended (P<0.001). Also, immunohistochemical analysis showed a decreased implanted tumor size in both virus alone and combinatory ­treatment groups. Most interestingly, we observed an increased number of tumor specific CD8+ cells and antigen cross-presenting and lymph node tropic dendritic cells (CD11b-/CD11C+/ CD103+) from therapeutic treatment groups. CONCLUSION: Our observation collectively represents a novel anti-glioma oncotherapy and immunomodulatory approach and provides a platform for the evaluation and translation of this promising anti-glioma strategy. We will further discuss additional immunomodulatory application: oncolytic virus expressing checkpoint inhibitor PD-1 along with the PRF-P treatment.

In vivo toxicological assessment of electrochemically engineered anodic alumina nanotubes: a study of biodistribution, subcutaneous implantation and intravenous injection

Electrochemically engineered anodic alumina nanotubes (AANTs) have recently shown good in vitro biocompatibility. However, in vivo toxicological and pathological studies are required to clarify the bio-safety of this novel nanomaterial. Herein, we present a pioneering pilot toxicity study on AANTs in immune-competent murine models (Balb/c mice, 8 weeks). AANTs were administered by intravenous (IV) injection and subcutaneous (SC) implantation routes considering the future toxicological implications associated with this nanomaterial for potential biomedical applications. AANTs, 736 nm long and 90 nm in outer diameter, were chosen as a nanomaterial model. We demonstrate that IV injected AANTs do not have any effect on the mortality or body weight of these animal models within 28 days at three different doses (20, 50, 100 mg kg-1). The biodistribution of AANTs characterized by fluorescence imaging and inductively coupled plasma revealed the accumulation of AANTs in the liver and spleen after IV injection. When AANTs were injected intravenously, the highest dose of 100 mg kg-1 caused moderate hepatotoxicity, identified by histopathological analysis. The implantation of AANTs subcutaneously and directly under the skin leads to an inflammatory response, which is a typical foreign body reaction. Taken together, this work provides new insights into the toxicity patterns of new nanomaterials such as AANTs and establishes a rationale for the design of functional AANTs for future biomedical applications.

MP83-08 LOCAL T LYMPHOCYTE SUB-POPULATION RESPONSES TO INTRAVESICAL BCG AND CHEMOTHERAPY IN AN IMMUNE COMPETENT MURINE MODEL OF BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-08 LOCAL T LYMPHOCYTE SUB-POPULATION RESPONSES TO INTRAVESICAL BCG AND CHEMOTHERAPY IN AN IMMUNE COMPETENT MURINE MODEL OF BLADDER CANCER Max Kates, Nikolai Sopko, Hotaka Matsui, Xiaopu Liu, Leonardo Reis, Noah Hahn, Alex Baras, Christina Kochel, Charles Drake, and Trinity Bivalacqua Max KatesMax Kates More articles by this author , Nikolai SopkoNikolai Sopko More articles by this author , Hotaka MatsuiHotaka Matsui More articles by this author , Xiaopu LiuXiaopu Liu More articles by this author , Leonardo ReisLeonardo Reis More articles by this author , Noah HahnNoah Hahn More articles by this author , Alex BarasAlex Baras More articles by this author , Christina KochelChristina Kochel More articles by this author , Charles DrakeCharles Drake More articles by this author , and Trinity BivalacquaTrinity Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2190AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has previously been shown that CD4 and CD8 cells are necessary to achieve BCG mediated antitumor reponses. Prior studies have also shown increases in the peripheral blood of pro-inflammatory CD4 cells and cytokines. However, it is currently unknown how infiltrating lymphocyte populations in bladder tumors change with intravesical therapy. The objective of the present study was to understand the effect of intravesical BCG and platinum-based chemotherapy on T cell subpopulations in an immune competent murine model of non-muscle invasive bladder cancer. METHODS Fischer 344 rats aged 7 weeks received 1.5mg/kg N-Nitroso-N-methylurea (MNU) every other week for 6 weeks (4 doses). Dysplasia begins by week 8 and by week 16 the majority of rats have a NMIBC phenotype with a mix of CIS, high grade Ta, and T1 disease. Beginning week 8 following the first MNU dose, rats were intravesically administered 0.3ml of BCG (n=5,1 vial Tice® suspended in 50ml saline), cisplatin (n=5, 1mg/ml), or saline (n=5) weekly for 6 total doses. All animals were sacrificed at week 16, and bladders (including tumors) were digested into single cell suspensions for flow cytometry. T lymphocyte subpopulations were then compared between the 3 groups using one-way ANOVA tests. RESULTS After whole bladder digestion, samples yielded 1-2 million cells per bladder. Rats treated with BCG had a 42% rise in CD4+ cells compared to saline controls (51.4% vs 34.8%, p<0.001). No significant differences in FoxP3+ CD4+ cells (20.6% vs 16.4%, p=0.12) or CD8+ cells (19.3% vs 16.4%, p=0.28) were observed between animals receiving BCG and saline control. Animals receiving intravesical cisplatin had no significant differences in CD4+ (34.8% in cisplatin vs 29.5% in controls, p=0.15), Foxp3+ CD4+ cells (13.9% vs 16.4%, p=0.25), or CD8+ cells (16.4% vs 16.9%, p=0.85) compared to animals receiving saline controls. CONCLUSIONS In an immune competent murine model of bladder cancer, our analysis of lymphocytes in the bladder wall suggests that BCG induces a large increase in CD4+ effector T cells, without a significant change in Foxp3+ regulatory T cells or CD8+ T cells. This large Th1 anticancer immunosurveillance response is not seen with intravesical platinum based chemotherapy. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1083-e1084 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Max Kates More articles by this author Nikolai Sopko More articles by this author Hotaka Matsui More articles by this author Xiaopu Liu More articles by this author Leonardo Reis More articles by this author Noah Hahn More articles by this author Alex Baras More articles by this author Christina Kochel More articles by this author Charles Drake More articles by this author Trinity Bivalacqua More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP83-09 TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND (TRAIL) POTENTIATES THE EFFECT OF BACILLUS CALMETTE-GUÉRIN (BCG) IN UROTHELIAL CARCINOMA MODEL

Dysplasia begins by week 8 and by week 16 the majority of rats have a NMIBC phenotype with a mix of CIS, high grade Ta, and T1 disease. Beginning week 8 following the first MNU dose, rats were intravesically administered 0.3ml of BCG (n1⁄45,1 vial Tice suspended in 50ml saline), cisplatin (n1⁄45, 1mg/ml), or saline (n1⁄45) weekly for 6 total doses. All animals were sacrificed at week 16, and bladders (including tumors) were digested into single cell suspensions for flow cytometry. T lymphocyte subpopulations were then compared between the 3 groups using one-way ANOVA tests. RESULTS: After whole bladder digestion, samples yielded 1-2 million cells per bladder. Rats treated with BCG had a 42% rise in CD4+ cells compared to saline controls (51.4% vs 34.8%, p<0.001). No significant differences in FoxP3+ CD4+ cells (20.6% vs 16.4%, p1⁄40.12) or CD8+ cells (19.3% vs 16.4%, p1⁄40.28) were observed between animals receiving BCG and saline control. Animals receiving intravesical cisplatin had no significant differences in CD4+ (34.8% in cisplatin vs 29.5% in controls, p1⁄40.15), Foxp3+ CD4+ cells (13.9% vs 16.4%, p1⁄40.25), or CD8+ cells (16.4% vs 16.9%, p1⁄40.85) compared to animals receiving saline controls. CONCLUSIONS: In an immune competent murine model of bladder cancer, our analysis of lymphocytes in the bladder wall suggests that BCG induces a large increase in CD4+ effector T cells, without a significant change in Foxp3+ regulatory T cells or CD8+ T cells. This large Th1 anticancer immunosurveillance response is not seen with intravesical platinum based chemotherapy.

Mitigation of hypertrophic scar contraction via anti-TNFalpha conjugated hyaluronic acid gels and poly(l-lactide-co-epsilon-caprolactone) electrospun scaffolds

Event Abstract Back to Event Mitigation of hypertrophic scar contraction via anti-TNFalpha conjugated hyaluronic acid gels and poly(l-lactide-co-epsilon-caprolactone) electrospun scaffolds Elizabeth Lorden1, Emily E. Friedrich2, Mohamed M. Ibrahim3, Latif Bashirov3, Howard Levinson3, 4, Newell R. Washburn2, 5, 6 and Kam W. Leong1, 7 1 Duke University, Biomedical Engineering, United States 2 Carnegie Mellon University, Biomedical Engineering, United States 3 Duke University Medical Center, Plastic and Reconstructive Surgery, United States 4 Duke University Medical Center, Pathology, United States 5 Carnegie Mellon University, Chemistry, United States 6 University of Pittsburgh, McGowan Institute for Regenerative Medicine, United States 7 Columbia University, Biomedical Engineering, United States Introduction: Hypertrophic scar (HSc) occurs in 40-70% of patients treated for third degree burn injuries. Clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Early mediation of the inflammatory phase of the immune response during wound healing is likely a key factor in rescuing thermally injured tissue from progressive wound remodeling and contraction. During the acute inflammation phase of wound healing, hyaluronic acid (HA) becomes highly prevalent in the wound bed. Conjugation of an anti-TNFα monoclonal antibody to high molecular weight HA has been shown to lead to decreased inflammatory response in an incisional wound model[1]. We explore the impact of (HA+anti-TNFα) hydrogels on HSc contraction using an immune-competent murine model[2]. We compare the efficacy of HA+anti-TNFα gel treatment with our previously described poly(l-lactide-co-ε-caprolactone) (PLCL) scaffold[3]. The long-term goal of this study is to develop an anti-scarring scaffold for treatment of large-area burn injuries. Materials and Methods: Hyaluronic acid sodium salt was covalently conjugated to anti-TNFα monoclonal antibody (anti-TNFα+HA) as previously described.[1] Following covalent conjugation, anti-TNFα mAB activity was analyzed via ELISA. Electrospinning was used to generate random fibrous PLCL scaffolds with an average fiber diameter of 6µm. Bovine type-I collagen was covalently attached to PLCL (ccPLCL). ccPLCL scaffolds were surgically inserted beneath skin grafts in a validated immune-competent murine HSc contraction model for 14 days, with comparison to skin graft alone or treatment with (anti-TNFα+HA). (anti-TNFα+HA) treatment groups contained 5% HA gel with approximately 400µg/mL anti-TNFα mAB. The gel was applied beneath skin graft at the time of surgery (single application group) and topically at 48h and 96h post-surgery (triple application group). Wounds were photographed daily and total wound area computed. Results: ELISA demonstrated that the activity of anti-TNFα mAB was not significantly affected by conjugation to HA. All data presented from animal studies are described in terms of percentage of original wound size, where 100% is the wound size at d0, and a fully contracted wound is 0% of its original size. All treatments were applied beneath a split-thickness skin graft. Murine wounds treated with skin grafts alone contracted to 50±9.5% at d14, wounds treated with a single application of anti-TNFα+HA contracted to 56±11%, while wounds treated with triple application of anti-TNFα+HA contracted to 64±8.0%. In contrast, wounds treated with ccPLCL scaffolds showed significantly decreased contraction of 82±1.2% at d14 (Fig 1). Discussion: At early time points, HSc contraction was significantly decreased in animals treated with either triple application of anti-TNFα+HA or ccPLCL scaffolds as compared to those treated with skin graft alone or single administration of anti-TNFα+HA. However, ccPLCL scaffolds showed a more effective inhibition of scar contraction at d14 as compared to all other groups. Conclusion: Conjugation of anti-TNFα+HA to PLCL may improve the inhibition of HSc contraction by combining the biochemical and mechanical cues to regulate the acute and chronic inflammatory events, respectively. Studies are ongoing to assess the synergistic effect of anti-TNFα+HA coating of PLCL scaffolds in vitro and in vivo.

Biostable electrospun microfibrous scaffolds mitigate hypertrophic scar contraction in an immune-competent murine model

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30–90% will develop hypertrophic scars (HSc). In this study, we evaluate the impact of an elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In vitro, fibroblast-seeded PU scaffolds contracted significantly less and demonstrated fewer αSMA+ myofibroblasts compared to fibroblast-seeded collagen lattices. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. Our data suggest that electrospun ccPU scaffolds meet the requirements to reduce HSc contraction including reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, our data suggest that a more long-term scaffold may possess merit in reducing HSc. Statement of SignificanceIn severe burns treated with skin grafting, between 30% and 90% of patients develop hypertrophic scars (HSc). There are no therapies to prevent HSc, and treatments are marginally effective. This work is the first example we are aware of which studies the impact of a permanent electrospun elastomer on HSc contraction in a murine model that mimics the human condition. Collagen coated polyurethane scaffolds decrease αSMA+ myofibroblast formation in vitro, prevent stiffening of scar tissue, and mitigate HSc contraction. Unlike current standards of care, electrospun, polyurethane scaffolds do not lose architecture over time. We propose that the future bioengineering strategy of mitigating HSc contraction should consider a long-term elastomeric matrix which persists within the wound bed throughout the remodeling phase of repair.

IMPS-09COMBINATION OF DELTA-24-RGDOX AND ANTI-PDL1 ANTIBODIES INDUCES ANTI-TUMOR EFFECT AND ANTI-GLIOMA IMMUNE MEMORY

Immune checkpoint modulators are being tested as anti-glioma therapy in clinical trials. In this work, we combined an oncolytic adenovirus expressing the OX40L, called Delta-24-RGDOX, with the intracranial injection of antibodies against PDL-1. We first observed that infection of murine and human glioma cells with oncolytic viruses triggered immunogenic cell death by assessing secretion of ATP, and the cell death–associated release of the HMGB1 by infected cells using ELISA and Western blot. Cells infected with Delta-24-RGDOX expressed OX40L in in vitro and in vivo conditions, as assessed by flow cytometry. Direct injection of Delta-24-RGDOX into murine gliomas increased animal survival in an immune competent murine model (p < 0.0001), but not in immune deficient mice (p = 1.0). Flow cytomeric analyses of brain infiltrating lymphocytes in the immune competent model showed an overrepresentation of CD4+ (p < 0.0001) and CD8+ cells (p< 0.0001). In addition, histologic examination of the brains revealed tumor necrosis in the immune competent mice treated with Delta-24-RGDOX, but not in the immune deficient model. Importantly, expression of OX40L within the tumor did not result in brain or systemic toxicity. Rechallenging of survivors with implantation of gliomas jeopardized the growth of tumors in the majority of mice. Spleens from mice treated with Delta-24-RGDOX harbored cells that recognized untreated glioma cells. Treating these co-cultures with antibodies against OX40L prevented this immune response. Using OVA as a model antigen we demonstrated that after Delta-24-RGDOX infection, CD8 cells specifically recognized cellular antigens. Importantly, combination of treatment of Delta-24-RGDOX and anti-PDL1 antibodies resulted in 100% survival of glioma-bearing immune competent mice. Our results indicate that is feasible to combine the inflammatory and lytic properties of oncolytic viruses with the modulation of immune checkpoints. Combination of several immune checkpoints modulators and oncolytic viruses may be required to obtain an effective anti-glioma therapy in a majority of patients.

Abstract 1360: Combination therapy of reovirus and PD-1 blockade effectively establishes tumor control via innate and adaptive immune responses

Abstract We have developed the use of reovirus as a systemically delivered oncolytic agent in both pre-clinical models and in early Phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, partly because of disruption of the PKR-mediated anti-viral response in malignant cells. In addition however, we have shown that anti-tumor therapy is directly associated with immune activation by virus replication in tumors. The immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, as well as the generation of adaptive anti tumor immune responses as a result of in vivo priming against tumor associated antigens released during that killing. Therefore, to exploit the immune components of reovirus anti-tumor therapy, we hypothesized that the combination of reovirus therapy with systemic checkpoint inhibition would augment therapeutic efficacy. To test the hypothesis, we used C57Bl/6 mice, an immune-competent murine model, with established subcutaneous (s.c.) B16 melanomas. In this model, intra-tumoral injection of reovirus into s.c. tumors generated moderate therapy. Provision of systemic anti-PD-1 antibody along with i.t. reovirus, significantly enhanced survival compared to i.t. reovirus alone (p&amp;lt;0.01) and led to &amp;gt;40% of mice being cured long term. Immune analysis suggested that the enhanced therapeutic benefit of reovirus plus checkpoint inhibition is contributed by at least two factors. First, blockade of PD-1 significantly enhanced the ability of NK cells to recognize (TNF-α secretion), and kill, reovirus-infected target tumor cells. Second, anti PD-1 antibody led to a significant reduction in Treg activity in reovirus-treated mice, with the overall effect of increasing the adaptive CD8+ anti-tumor T cell response. Furthermore, in vivo depletion studies demonstrated that NK cells had a dramatic effect in reducing the therapeutic efficacy of reovirus plus anti-PD-1 therapy. Overall, the results indicate that combination therapy of reovirus with PD-1 blockade confers significant survival benefit, by augmenting tumor-specific NK responses and specifically attenuating tumor-specific immunosuppression. These data also suggest that combination of PD-1 inhibition therapy with reovirus oncolytic/immunotherapy represents a readily translatable method to enhance the therapeutic efficacy. Citation Format: Karishma Rajani, Christopher Parrish, Kevin Shim, Liz Ilett, Fiona Errington-Mais, Jill Thompson, Tim Kottke, Rosa Maria-Diaz, Peter Selby, Hardev Pandha, Kevin Harrington, Alan Melcher, Matt Coffey, Shane Zaidi, Richard Vile. Combination therapy of reovirus and PD-1 blockade effectively establishes tumor control via innate and adaptive immune responses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1360. doi:10.1158/1538-7445.AM2015-1360

244. Factor VIII Immunogenicity Is a Barrier to AAV Gene Therapy for Hemophilia A

Liver-directed adeno-associated virus (AAV) gene therapy utilizing a bioengineered, high-expression coagulation factor VIII (FVIII) transgene (ET3) has been demonstrated by our laboratory to produce therapeutic FVIII levels in a naive, immune-competent murine model of hemophilia A (HA). Recently, other groups have shown that similar liver directed gene therapy approaches also have the potential to reverse pre-existing immunity, including inhibitors, to coagulation factor IX (FIX) in murine models of hemophilia B (HB). In the current study, we sought to determine if, similarly, ET3-AAV8 could reverse pre-existing FVIII immunity in HA mice. Cohorts of mice were immunized with either recombinant ET3 or human FVIII (hFVIII). Robust anti-FVIII immune responses were elicited as expected and characterized by ELISA (total IgG) and Bethesda (inhibitor) titers ranging from 6000-15,400 arbitrary units (AU) and 22-286 BU, respectively, for ET3 immunized mice, while hFVIII immunized mice showed respective ELISA and Bethesda titers ranging from 460-1480 AU and 23-63 BU. Subsequently, the mice were administered ET3-AAV8 at a dose (4e12 vp/kg) previously shown to produce complete correction of the FVIII deficiency (>50% normal human FVIII activity) and monitored for 16 weeks. No significant change was observed in either ELISA or Bethesda titers for either cohort indicating that the pre-existing immunity was not modulated substantively by FVIII-AAV8 (p>0.5). As Arruda and colleagues previously showed that liver-directed FVIII-AAV8 can eradicate pre-existing anti-FVIII antibodies in a canine model of HA harboring low Bethesda titers (£12 BU), we hypothesized that the capacity for ET3-AAV8 to eliminate pre-existing antibodies may exist under a certain immune threshold quantitatively linked to the inhibitor titer. To test this hypothesis, cohorts of HA mice were established with pre-existing Bethesda titers ranging from 0-25 BU (n=10). Following administration of ET3-AAV8, no significant changes in titers have been observed to date. A corollary question then arose regarding the immune modulation potential of FVIII-AAV8 in naive HA mice, which has been studied extensively for FIX-AAV in HB mice. Specifically, we tested if a mid (4e12vp/kg) or high (2e13vp/kg) dose of ET3-AAV8 could tolerize naive HA mice to recombinant FVIII infusions. FVIII production was dose dependent with the mid cohort averaging 70% normal human levels (0.7 IU/ml) and the high dose cohort averaging 200% normal levels (2 IU/ml) over 5 months. Mice were then challenged with weekly intravenous injections of ET3. All mice treated with the mid AAV dose developed anti-FVIII ELISA and Bethesda titers similar to challenged controls with a concomitant loss of detectable plasma FVIII activity. However, mice in the high dose cohort maintained FVIII activity indicating a possible antigen production requirement for immune modulation consistent with current models of active tolerance. Collectively, these preclinical data indicate that current clinical FIX-AAV gene therapy data may not predict clinical outcomes for similar FVIII-AAV products, especially as trials progress into previously untreated and inhibitor populations.

Programmed cell death-1 blockade in combination with stereotactic radiation in an orthotopic mouse model of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) develops in an environment of chronic inflammation. Curative treatments are plagued by frequent recurrences locally near the treated tumor, regionally within the inflamed liver, and systemically, and yet no adjuvant therapies are known to prevent recurrence. Here the efficacy of immunotherapy with stereotactic radiation (SBRT) in an immune-competent murine model is evaluated. A syngeneic HCC cell line (Hep 55.1c) is injected into the liver of C57BL/6 mice using ultrasound guidance. The tumors are measured and treated using computed tomography and a single dose of intravenous contrast that is taken up by resident liver macrophages allowing visualization of tumors for at least 30 days. Stereotactic radiation is delivered using a Small Animal Radiation Research Platform. Three doses of 250 ug anti-Programmed Cell Death-1 antibody (αPD-1) are delivered concurrently with 30 Gy SBRT in 3 fractions. This combination reduces the growth rate of tumors and improves survival (p < 0.05). Addition of αPD-1 to SBRT is associated with increases in CD8+ cytotoxic T cells based on flow cytometry (Figure ​(Figure1a)1a) and CD3+ T cells based on six-color immunofluorescence, and this augmentation of immune response is associated with increased ICOS expression on T cells within tumor and spleen. Importantly, the tumors grow out rapidly after αPD-1 is discontinued. The percentage of regulatory T cells (CD4+CD25+FOXP3+) also increases significantly after αPD-1 alone and in combination with SBRT (p < 0.001), suggesting a potential mechanism by which cytotoxic T cells could be rendered anergic. In addition, the combination of αPD-1 plus SBRT induced programmed cell death-1 ligand expression on suppressive macrophages based on flow cytometry (Figure ​(Figure1b)1b) and immunofluorescence, suggesting another potential mechanism of resistance to long term immune control of the tumors. Conclusion: Tumor response to SBRT can be augmented by concurrent administration of αPD-1 antibody. The efficacy of this combination therapy is transient, however. The environment of chronic immune suppression in most patients affected by HCC adds to the rationale of this immune-radiotherapy combination, but the mechanisms of immune resistance will need to be overcome for this combination to generate lasting immunity. Figure 1

Changes in BAI1 and nestin expression are prognostic indicators for survival and metastases in breast cancer and provide opportunities for dual targeted therapies.

The 2-year survival rate of patients with breast cancer brain metastases is less than 2%. Treatment options for breast cancer brain metastases are limited, and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 (BAI1) is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify that BAI1 expression is significantly reduced in breast cancer and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is upregulated in several cancers. We found that higher Nestin expression significantly correlated with breast cancer lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high Nestin-expressing cells and deliver Vstat120 (extracellular fragment of BAI1). Finally, we created two orthotopic immune-competent murine models of breast cancer brain metastases and demonstrated 34.5ENVE extended the survival of immune-competent mice bearing intracranial breast cancer tumors.

Highlights of This Issue

Tumor-targeting monoclonal antibodies represent a major advance in oncology therapy; however, due to their limited brain permeability, mAbs are not generally effective against brain tumors. Conjugation of an anti-HER2 mAb with Angiopep-2, a peptide recognized by LRP1 (low-density lipoprotein receptor-related protein 1), results in ANG4043, which enters brain physiologically, targets HER2+ tumors, and increases survival in mice bearing intracranial tumors. The Angiopep conjugation to mAbs, as well as to previously reported small molecules, is applicable in general to create brain-penetrant therapeutics for brain malignancies and other CNS disorders.Activation of the Hedgehog pathway is a feature of many different types of cancer, including highly lethal brain tumors. Accordingly, small molecules that can antagonize Hedgehog signaling are being developed as new anticancer drugs. Larsen and colleagues demonstrate that mebendazole, a widely used antiparasitic, can inhibit Hedgehog pathway activation by preventing the formation of the primary cilium, a tubulin-based cellular organelle that functions as a signaling hub. The off-label use of mebendazole as a Hedgehog inhibitor provides a promising new option for the management of brain tumors and other tumor types in which Hedgehog activation is prevalent.HSP90 inhibitors simultaneously affect multiple cancer-related proteins; therefore, they have potential use in advanced cancer treatment. However, because HSP90 also functions in normal cells, it is essential to balance efficacy and toxicity to maximize the antitumor potential of HSP90 inhibitors. Ohkubo and colleagues report on the biological activity of TAS-116, a novel, orally active HSP90α and β selective inhibitor. TAS-116 showed antitumor activity without detectable ocular toxicities in a rat model, due to greater TAS-116 distribution in target tumor tissues than in non-target eye tissues. The favorable therapeutic index of TAS-116 thus highlights its therapeutic potential.Treatment options for breast cancer brain metastases are limited and new therapies are needed. Here, Meisen and colleagues discovered the relationship of multifunctional brain angiogenesis inhibitor 1 (BAI1) and nestin expression with breast cancer brain metastases. Higher BAI1 expression is associated with better patient survival and higher nestin expression is significantly correlated with breast cancer lung and brain metastases. The application of 34.5ENVE, a Nestin-targeted oncolytic virus expressing a therapeutic extracellular fragment of BAI1 destroyed breast cancer brain metastases in two different immune-competent murine models of the disease. The results of these studies warrant the clinical investigation of 34.5ENVE for breast cancer brain metastases.

Liposome promotion of tumor growth is associated with angiogenesis and inhibition of antitumor immune responses

Liposomes have tremendous potential as drug carriers in the treatment of cancer. However, despite enhanced tumor drug delivery and decreased toxicity, patient survival rates have not improved significantly compared to corresponding free drug treatments. Importantly, we found that a liposomal nanoparticle currently used as a drug carrier in cancer patients enhanced tumor growth in an immune competent murine model of cancer. This was associated with increased tumor angiogenesis and suppression of antitumor immune responses as indicated by decreased cytokine production by tumor macrophages and cytotoxic T cells, diminished tumor infiltration of tumor-specific T cells, and decreased number of dendritic cells in tumor draining lymph nodes. These results suggest that carrier-induced immunosuppression and angiogenesis have the potential to reduce the antitumor effects of drugs loaded within. These findings may have significant implications for the current use and future development of anticancer nanoparticles and further investigations are urgently needed. From the Clinical EditorThis study discusses important implications of nanoliposome-based drug delivery systems in cancer therapy, and demonstrates that nanoliposomes may have immunosuppressive and angiogenetic properties, directly counterbalancing their anti-cancer activity, which may also have important clinical implications related to more widespread applications of such systems.

Efficacy and Long Term Survival of Genetically Modified T Cells Targeting CD19 in a Syngeneic Immune Competent Transgenic Murine Tumor Model Is Dependent on Prior Lymphodepleting Chemotherapy.

T cells may be genetically modified ex vivo to target specific antigens by retroviral transduction of genes encoding chimeric antigen receptors (CARs). We have previously constructed a CAR, termed 19z1, specific for the CD19 antigen expressed on most B cell malignancies. Human T cells modified to express the 19z1 CAR specifically eradicate systemic human CD19+ tumors in SCID-Beige mice. However, these models are limited by the xenogeneic nature of the human T cells and tumor cells and the immune compromised state of the host. Here, we studied the biology of adoptively transferred 19z1+ T cells in a syngeneic immune competent murine model designed to better mimic the clinical setting of patients with B cell malignancies. We utilized transgenic C57BL6 mice which lack expression of mouse CD19 (mCD19−/−) and have a single copy of the human CD19 (hCD19+/−) gene (C57BL6(mCD19−/− hCD19+/−)) kindly provided by Dr. T. Tedder, Duke University. These mice are functionally immune-competent with hCD19 expression restricted to the B cell population. To assess whether syngeneic 19z1+ T cells were capable of eradicating normal hCD19+ B cells, we infused C57BL6(mCD19−/− hCD19+/−) mice with either 19z1+ or control prostate specific membrane antigen-targeted (Pz1+) T cells. As assessed by flow cytometric analysis of peripheral blood, we neither found evidence of hCD19+ B cell aplasias in 19z1+ T cell treated mice nor were able to demonstrate the persistence of infused CAR+ T cells. To investigate whether the lack of 19z1+ T cell efficacy and persistence was due to an absence of homeostatic drive, we next lymphodepleted C57BL6(mCD19−/− hCD19+/−) mice with cyclophosphamide prior to T cell infusion. Mice lymphodepleted prior to 19z1+ T cell infusion demonstrated marked and sustained B cell aplasias when compared to lymphodepleted Pz1+ T cell and non-lymphodepleted T cell treated controls. Furthermore, while no CAR+ T cells were identifiable in the Pz1 and non-lymphodepleted control groups, 19z1+ T cells were consistently present in the peripheral blood of the cyclophosphamide pre-treated, 19z1+ T cell treated mice (3–5% of white blood cells). To assess the anti-tumor efficacy of the 19z1+ T cells, we next established a systemic tumor model utilizing mouse EL4 thymoma cells retrovirally modified to express hCD19 (EL4(hCD19)). C57BL6(mCD19−/− hCD19+/−) mice pre-treated with cyclophosphamide, subsequently infused systemically with EL4(hCD19) tumor, followed by systemic 19z1+ T cell infusion, had a significant survival advantage (80% survival at >120 days) over untreated controls or controls treated with Pz1+ T cells or 19z1+ T cells in the absence of lymphodepletion (0% survival). In conclusion, we have developed a syngeneic immune competent tumor model of hCD19 disease that is highly relevant to the clinical setting. Using this model, we demonstrate the significance of lymphodepletion on the prolonged in vivo persistence and anti-tumor efficacy of 19z1+ T cells. Data derived from this model will be correlated to findings obtained from a recently initiated clinical trial for patients with chronic lymphocytic leukemia, and will significantly impact the design of subsequent trials in the future.

Sequential plasmid vaccinations targeting normal melanocytes protects against challenge with melanoma in a syngenic murine model

2570 Background: Malignant melanoma is one of a handful of tumors responsive to immunotherapy. Tumor regression correlates with an expanded epitope response and induction of vitiligo. In fact, expansion of the immune response to include multiple melanoma differentiation antigen (MDA) targets correlates with clinical response in newer vaccine approaches. We hypothesized that a vaccine designed to induce epitope spreading against melanocytic antigens may be useful in melanoma. By directly targeting normal melanocytes in situ, we hoped to induce both autoimmunity and a broad anti-melanocytic response. To accomplish this, we utilized sequential plasmid vaccinations in an immune competent murine model for melanoma. Methods: C57/b6 mice were vaccinated with plasmid DNA encoding either the gene for chicken ovalbumin (cOVA) or empty vector intramuscularly (IM) weekly for two weeks. Mice (n=9) were then vaccinated intradermally (ID) with either cOVA or a control gene under the control of the tyrosinase promoter weekly for three weeks. Mice were challenged with subcutaneous injections of 105 B16 melanoma tumor cells or B16 containing the transgene cOVA (B16cOVA). Tumor size and survival were followed. Results: Vaccination with the cOVA gene IM resulted in 90% to 100% protection against B16cOVA tumor cells compared to 0% to 10% of control plasmid injected mice. Challenge with B16 resulted in equally poor survival between the two groups with 100% of mice being sacrificed by day 26. Surprisingly, if the mice were vaccinated with the cOVA gene ID under the control of the tyrosinase promoter after the initial IM vaccination with cOVA and subsequent tumor challenge, B16 growth was delayed compared to controls. Delay was not observed without the initial IM cOVA vaccination. Conclusions: Ectopic expression of antigens for which a strong pre-existing cellular immune response exists may allow targeting of an immune response to normally tolerant antigens. This system provides a relatively simple approach in understanding the requirements of both autoimmunity and cancer immunotherapy. No significant financial relationships to disclose.