Direct Intratumoral Administration Research Articles

Nonclinical pharmacodynamics of boron neutron capture therapy using direct intratumoral administration of a folate receptor targeting novel boron carrier.

Boron neutron capture therapy (BNCT) is a precise particle radiation therapy known for its unique cellular targeting ability. The development of innovative boron carriers is crucial for the advancement of BNCT technologies. Our previous study demonstrated the potential of PBC-IP administered via convection-enhanced delivery (CED) in an F98 rat glioma model. This approach significantly extended rat survival in neutron irradiation experiments, with half achieving long-term survival, akin to a cure, in a rat brain tumor model. Our commitment to clinical applicability has spurred additional nonclinical pharmacodynamic research, including an investigation into the effects of cannula position and the time elapsed post-CED administration. In comprehensive in vivo experiments conducted on an F98 rat brain tumor model, we meticulously examined the boron distribution and neutron irradiation experiments at various sites and multiple time intervals following CED administration. The PBC-IP showed substantial efficacy for BNCT, revealing minimal differences in tumor boron concentration between central and peripheral CED administration, although a gradual decline in intratumoral boron concentration post-administration was observed. Therapeutic efficacy remained robust, particularly when employing cannula insertion at the tumor margin, compared to central injections. Even delayed neutron irradiation showed notable effectiveness, albeit with a slightly reduced survival period. These findings underscore the robust clinical potential of CED-administered PBC-IP in the treatment of malignant gliomas, offering adaptability across an array of treatment protocols. This study represents a significant leap forward in the quest to enhance BNCT for the management of malignant gliomas, opening promising avenues for clinical translation.

Usefulness of direct intratumoral administration of doxorubicin hydrochloride with an electro-osmosis–assisted pump

Patients receiving chemotherapy by intravenous (i.v.) or oral administration of anticancer drugs often experience side effects. In this study, an electro-osmotic flow (EO) pump was used for the direct administration of an anticancer drug with minimum side effects. Doxorubicin hydrochloride (DXR) was used as an anticancer drug, and its antitumor effect and toxicity were evaluated in comparison with i.v. administration. Balb/c female mice were subcutaneously transplanted with a breast cancer cell line (4T1/Luc) stably expressing luciferase, and 20 μL of DXR solution (1.0 mg/mL) was administered intratumorally (i.t.) at a slow rate (0.6 µL/min) using an EO pump or rapidly using a syringe. For comparison, 100 μL of DXR solution was injected through the tail vein at the same dose and a 5-times higher dose. A tumor growth inhibitory effect without significant weight loss was observed with direct i.t. administration of DXR using an EO pump. On the other hand, no suppressive tumor growth effect was observed with i.v. administration of DXR at the same dose. Although there was no significant difference in the suppression effect on tumor growth between i.t. administration with EO pump and syringe, the peripheral skin concentration of DXR were decreased after slow administration with EO pump compared with that after rapidly administration with a syringe. These results indicated that direct i.t. administration of DXR with lower dosing using an EO pump at slower administration rate may be useful for exhibiting antitumor effects and suppressing systemic side effects. In addition, the blood concentration and the peripheral skin concentration of DXR after administration at lower rate with EO pump were decreased compared with those after the rapidly administration with a syringe.

Circular mRNAs: More stable RNAs lead to more persistent protein expression

Yang et al. established a novel type of circular mRNA called cmRNA. cmRNA contains an echovirus 29-derived internal ribosomal entry point element to promote ribosome binding and a newly designed spacer to enhance translation. Their findings suggest that this type of circular mRNA can mediate strong and persistent expression of various types of proteins. Furthermore, in addition to delivery via lipid nanoparticle (LNP), cmRNAs were discovered suitable for direct intratumoral administration. By giving mice cmRNAs encoding mixed cytokines directly intratumorally, they achieved successful modulation of intratumoral and systemic antitumor immune responses and enhanced anti-programmed cell death protein 1 (PD-1) antibody-induced tumor suppression.

Intratumoral delivered novel circular mRNA encoding cytokines for immune modulation and cancer therapy

The success of the two mRNA vaccines developed by Moderna and BioNTech during the COVID-19 pandemic increased research interest into the application of mRNA technologies. Compared with the canonical linear mRNA used in these vaccines, circular mRNA has been found to mediate more potent and durable protein expression and demands a simpler manufacturing procedure. However, the application of circular mRNA is still at the initiation stage, and proof of concept for its use as a future medicine or vaccine is required. In the current study, we established a novel type of circular mRNA, termed cmRNA, based on the echovirus 29-derived internal ribosome entry site element and newly designed homology arms and RNA spacers. Our results demonstrated that this type of circular mRNA could mediate strong and durable expression of various types of proteins, compared with typical linear mRNA. Moreover, for the first time, our study demonstrated that direct intratumoral administration of cmRNA encoding a mixture of cytokines achieved successful modulation of intratumoral and systematic anti-tumor immune responses and enhanced anti-programmed cell death protein 1 (PD-1) antibody-induced tumor repression in a syngeneic mouse model. This novel circular mRNA platform is thereby suitable for direct intratumoral administration for cancer therapy.

LOCL-07 LOCO-REGIONAL INFUSION OF GB-13 (IL13.E13K-PE4E) AS A POTENTIALLY PROMISING TREATMENT FOR RECURRENT HIGH-GRADE GLIOMA

INTRODUCTIONHigh grade gliomas (HGG) are devastating diseases with largely unchanged survival outcomes despite decades of research. Recent studies suggest the interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is selectively upregulated in up to 80% of HGG, including glioblastoma (GBM) and diffuse midline gliomas (DMG) harboring H3K27 alterations. Immunotoxins targeting IL-13Rα2 have been demonstrated as safe and have shown some benefit for patients with HGG in previous phase I/II and III clinical trials. We hypothesized that by using GB-13 (IL13.E13K-PE4E), a novel peptide-toxin that binds IL-13Rα2 with high specificity and possesses a Pseudomonas exotoxin moiety, we would enhance the anti-tumor effects of this immunotherapy for HGG in vitro and in vivo while decreasing off-target toxicity. METHODSWe examined the pharmacological effects of GB-13 in multiple patient-derived cell lines and rodent models of HGG. GBM and DMG lines were used to confirm IL-13Rα2 expression and sensitivity towards GB-13. Tumor naïve rats were evaluated for toxicity, and orthotopic PDX mice were used to monitor tumor size and survival following loco-regional infusion of GB-13. RESULTSGB-13 induced a potent cytotoxic response strongly predicated on IL-13Rα2 expression in vitro. No treatment-related adverse effects were noted after 7-day continuous intracranial infusion of GB-13 in tumor naïve rats. Further, in IL-13Rα2-upregulated orthotopic PDX mice, direct intratumoral administration of GB-13 via convection-enhanced delivery abrogated tumor growth and prolonged survival. CONCLUSIONSGiven these promising results as well as the critical need for novel therapies in CNS malignancies, we are progressing to human trials using GB-13 targeting recurrent HGG. Ongoing safety studies in tumor-bearing animals will be able to define dose levels for the initial adult study-arm and the following pediatric study-arm. In this Phase 1 clinical trial, we hypothesize that loco-regional infusion of GB-13 will safely enhance tumor clearance by causing selective killing of IL-13Rα2-upregulated HGG cells.

IL-13Rα2 Status Predicts GB-13 (IL13.E13K-PE4E) Efficacy in High-Grade Glioma.

High-grade gliomas (HGG) are devastating diseases in children and adults. In the pediatric population, diffuse midline gliomas (DMG) harboring H3K27 alterations are the most aggressive primary malignant brain tumors. With no effective therapies available, children typically succumb to disease within one year of diagnosis. In adults, glioblastoma (GBM) remains largely intractable, with a median survival of approximately 14 months despite standard clinical care of radiation and temozolomide. Therefore, effective therapies for these tumors remain one of the most urgent and unmet needs in modern medicine. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface transmembrane protein upregulated in many HGGs, including DMG and adult GBM, posing a potentially promising therapeutic target for these tumors. In this study, we investigated the pharmacological effects of GB-13 (also known as IL13.E13K-PE4E), a novel peptide–toxin conjugate that contains a targeting moiety designed to bind IL-13Rα2 with high specificity and a point-mutant cytotoxic domain derived from Pseudomonas exotoxin A. Glioma cell lines demonstrated a spectrum of IL-13Rα2 expression at both the transcript and protein level. Anti-tumor effects of GB-13 strongly correlated with IL-13Rα2 expression and were reflected in apoptosis induction and decreased cell proliferation in vitro. Direct intratumoral administration of GB-13 via convection-enhanced delivery (CED) significantly decreased tumor burden and resulted in prolonged survival in IL-13Rα2-upregulated orthotopic xenograft models of HGG. In summary, administration of GB-13 demonstrated a promising pharmacological response in HGG models both in vitro and in vivo in a manner strongly associated with IL-13Rα2 expression, underscoring the potential of this IL-13Rα2-targeted therapy in a subset of HGG with increased IL-13Rα2 levels.

Intratumoral cancer immunotherapy exploiting anti-viral immunity.

After a long period of endeavor, immunotherapy has become the mainstream of cancer therapies. This success is mostly ascribed to immune checkpoint blockade, chimeric antigen receptor-transduced T cell therapies, and bispecific antibodies. However, these methods have been effective or applicable to only a limited proportion of patients so far. Thus, further development of broadly applicable and effective immunotherapies is eagerly anticipated. Given that innate immunity is key to the induction of robust adaptive immunity and that the immunosuppressive tumor microenvironment is a major hurdle to overcome, intratumoral immunotherapy in which delivery of immunostimulatory microbial agents to the tumor site triggers innate immunity in situ is a rational strategy. There has been a plethora of preclinical and clinical trials conducted involving the delivery of either mimetics of viral nucleic acids or oncolytic viruses intratumorally to trigger innate immunity via various nucleic acid sensors in the tumor site. Many of these have shown significant antitumor effects in mice, particularly in combination with immune checkpoint blockade. Oncolytic herpes simplex virus type 1 has been approved for the treatment of advanced melanoma in the United States and Europe and of glioblastoma in Japan. Whereas direct intratumoral administration has mainly been chosen as a delivery route, several promising compounds amenable to systemic administration have been developed. Intratumoral delivery of immunostimulatory agents will become an important option for cancer immunotherapy as an off-the-shelf, broadly applicable, and rational strategy that exploits the physiology of immunity, namely anti-microbial immunity.

The impact of immunotherapy on a glioma immune interaction model

Glioblastoma, a highly aggressive in primary brain tumor and has exceptionally poor prognosis, is refractory to conventional treatments, such as chemotherapy, surgery and radiation. This study aims at aiding in the design of more successful glioma therapy. To understand the dynamics under what circumstances the glioma cells can be eradicated, we propose and analyze a mechanistic model for malignant gliomas and immune system interplays that may ensue upon direct intra-tumoral administration of immunotherapeutic agent T11 target structure. Our mathematical model encompasses considerations of the interactive dynamics of glioma cells, glioma-specific CD8+T cells, macrophages, immuno-suppressive cytokine TGF-β and immuno-stimulatory cytokine IFN-γ. The proposed mathematical model successfully retrieved clinical response to the immunotherapeutic drug T11 target structure for glioma cells. Our model enables us to identify the treatment regimen for a determined time window, in order to obtain an admissible concentration of glioma cell population. To mathematically model the dynamics of malignant gliomas development, before and after administration of immunotherapeutic treatment strategy, we derived the local asymptotic stability for the biologically feasible equilibrium points and the local relative controllability conditions for this coupled system of nonlinear ordinary differential equations. The system undergoes sensitivity analysis to identify the most sensitive parameters with respect to glioma cells. Numerical simulations were conducted for model verification and for retrieving putative treatment scenarios. The model simulations suggest that the immunotherapy has an impact in reducing the growth of glioma cell population and also an impact in enhancing the cell count of macrophages and CD8+T cell populations.

Intratumoral Nanofluidic System for Enhancing Tumor Biodistribution of Agonist CD40 Antibody

AbstractTumor uptake and biodistribution of immunotherapy is associated with clinical response as well as toxicity. To augment immunotherapy bioavailability in the tumor, an intratumoral administration route via direct injection or local release technologies has emerged as an appealing approach. Here the biodistribution of an agonistic anti‐CD40 monocolonal antibody (CD40 mAb) when sustainably delivered via an intratumoral nanofluidic drug‐eluting seed (NDES) is evaluated in comparison to systemic or direct intratumoral administration. The NDES achieves sustained drug release through diffusion by leveraging electrostatic nanoconfinement within nanochannels, without requiring internal or external actuation. Using the 4T1 murine mammary carcinoma model, the biodistribution of Alexa Fluor‐700 conjugated CD40 mAb is tracked via fluorescence imaging analysis, comparing three routes of administration over 7 and 14 days. NDES‐treated cohort shows sustained high levels of intratumoral CD40 mAb without off‐target organ exposure, compared to the intraperitoneal and direct intratumoral administration. Moreover, radiation pre‐treatment of the 4T1 tumors augments tumor retention of CD40 mAb in the NDES group. Overall, sustained intratumoral release of CD40 mAb via the NDES improves local drug bioavailability without systemic dissemination, suggesting an enhanced approach for immunotherapy administration.

Abstract 944: exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen-presenting cells resulting in improved tumor antigen-specific adaptive immune response

Abstract Background: The Stimulator of Interferon Genes (STING) pathway is an attractive target in immuno-oncology. Selective activation of the STING pathway in antigen presenting cells (APCs) is essential for eliciting a potent and specific anti-tumor immune response. STING is ubiquitously expressed in normal cells including T cells and endothelial cells. Direct intra-tumoral administration of a free STING agonist results in activation of the STING pathway in all cells, resulting in loss of viability of immune cells, tissue damage, and systemic immune activation due to vascular leakage. Exosomes are an efficient natural messenger system that delivers macromolecules between cells. Leveraging exosome biology, we have developed a novel engineered exosome therapeutic, exoSTING, to selectively target the STING pathway in tumor resident APCs. Results: exoSTING is composed of exosomes engineered to express high levels of Protein X (PrX), a transmembrane glycoprotein naturally occurring on exosomes, and loaded with a STING agonist. Following intra-tumoral injection in checkpoint refractory B16F10 tumors, exoSTING selectively activates STING in APCs, leading to anti-tumor immunity with greater than 100-fold improvement in potency compared to free STING agonist. The activity of exoSTING is substantially diminished in the PrX knock out exosomes, highlighting the importance of this surface glycoprotein for preferential activation of APCs. In contrast to the free STING agonist, exoSTING is retained within the injected tumor, minimizing systemic exposure. Furthermore, exoSTING administration preserves the viability of T cells and APCs, reduces collateral tissue damage, and does not induce systemic cytokine production, resulting in a broader therapeutic window in contrast to free STING agonist. exoSTING produced an increased systemic tumor-specific T cell response as demonstrated by elimination of non-injected abscopal tumors. The specificity of exoSTING activity was demonstrated using a STING knockout mouse (Tmem173gt/J). T cells and myeloid cells play a critical role in exoSTING mediated anti-tumor immunity, in contrast to NK cells which are not required. exoSTING treatment results in significant induction of PD-L1 expression (P<0.005). In combination with PD1 checkpoint blockade, exoSTING shows enhanced efficacy over high-dose free STING agonist on abscopal tumors. Conclusion: exoSTING is an engineered exosome therapeutic that leverages exosome biology and specifically targets the STING pathway in APCs, resulting in greater potency with preserved viability of T cells and APCs, greater systemic tumor antigen specific immune response, reduced systemic cytokine production, and enhanced efficacy. Citation Format: Su Chul Jang, Raymond J. Moniz, Chang Ling Sia, Rane A. Harrison, Damian Houde, Nikki Ross, Ke Xu, Nuruddeen Lewis, Raymond Bourdeau, Christine McCoy, Tong Zi, Agata Villiger-Oberbek, Scott Estes, Jorge Sanchez-Salazar, Kevin Dooley, William K. Dahlberg, Sriram Sathyanarayanan, Kyriakos D. Economides. exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen-presenting cells resulting in improved tumor antigen-specific adaptive immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 944.

Radioluminescent nanoparticles for radiation-controlled release of drugs

The present work demonstrates a novel concept for intratumoral chemo-radio combination therapy for locally advanced solid tumors. For some locally advanced tumors, chemoradiation is currently standard of care. This combination treatment can cause acute and long term toxicity that can limit its use in older patients or those with multiple medical comorbidities. Intratumoral chemotherapy has the potential to address the problem of systemic toxicity that conventional chemotherapy suffers, and may, in our view, be a better strategy for treating certain locally advanced tumors. The present study proposes how intratumoral chemoradiation can be best implemented. The enabling concept is the use of a new chemotherapeutic formulation in which chemotherapy drugs (e.g., paclitaxel (PTX)) are co-encapsulated with radioluminecsnt nanoparticles (e.g., CaWO4 (CWO) nanoparticles (NPs)) within protective capsules formed by biocompatible/biodegradable polymers (e.g., poly(ethylene glycol)-poly(lactic acid) or PEG-PLA). This drug-loaded polymer-encapsulated radioluminescent nanoparticle system can be locally injected in solution form into the patient's tumor before the patient receives normal radiotherapy (e.g., 30–40 fractions of 2–3 Gy daily X-ray dose delivered over several weeks for locally advanced head and neck tumors). Under X-ray irradiation, the radioluminescent nanoparticles produce UV-A light that has a radio-sensitizing effect. These co-encapsulated radioluminescent nanoparticles also enable radiation-triggered release of chemo drugs from the polymer coating layer. The non-toxic nature (absence of dark toxicity) of this drug-loaded polymer-encapsulated radioluminescent nanoparticle (“PEG-PLA/CWO/PTX”) formulation was confirmed by the MTT assay in cancer cell cultures. A clonogenic cell survival assay confirmed that these drug-loaded polymer-encapsulated radioluminescent nanoparticles significantly enhance the cancer cell killing effect of radiation therapy. In vivo study validated the efficacy of PEG-PLA/CWO/PTX-based intratumoral chemo-radio therapy in mouse tumor xenografts (in terms of tumor response and mouse survival). Results of a small-scale NP biodistribution (BD) study demonstrate that PEG-PLA/CWO/PTX NPs remained at the tumor sites for a long period of time (> 1 month) following direct intratumoral administration. A multi-compartmental pharmacokinetic model (with rate constants estimated from in vitro experiments) predicts that this radiation-controlled drug release technology enables significant improvements in the level and duration of drug availability within the tumor (throughout the typical length of radiation treatment, i.e., > 1 month) over conventional delivery systems (e.g., PEG-PLA micelles with no co-encapsulated CaWO4, or an organic liquid, e.g., a 50:50 mixture of Cremophor EL and ethanol, as in Taxol), while it is capable of maintaining the systemic level of the chemo drug far below the toxic threshold limit over the entire treatment period. This technology thus has the potential to offer a new therapeutic option that has not previously been available for patients excluded from conventional chemoradiation protocols.

Immunomodulation of the Tumor Microenvironment: Turn Foe Into Friend.

Immunotherapy, where the patient's own immune system is exploited to eliminate tumor cells, has become one of the most prominent new cancer treatment options in the last decade. The main hurdle for classical cancer vaccines is the need to identify tumor- and patient specific antigens to include in the vaccine. Therefore, in situ vaccination represents an alternative and promising approach. This type of immunotherapy involves the direct intratumoral administration of different immunomodulatory agents and uses the tumor itself as the source of antigen. The ultimate aim is to convert an immunodormant tumor microenvironment into an immunostimulatory one, enabling the immune system to eradicate all tumor lesions in the body. In this review we will give an overview of different strategies, which can be exploited for the immunomodulation of the tumor microenvironment and their emerging role in the treatment of cancer patients.

Abstract 1607: Durable efficacy and anti-cancer immunity following intratumoral administration of messenger RNAs encoding IL-36γ, IL-23 and OX40L

Abstract Immunologic responses to cancer involve complex interactions of cells within the tumor microenvironment (TME). These interactions are mediated in part through co-stimulatory and inhibitory transmembrane proteins, cytokines and chemokines. Direct intratumoral (ITu) administration of messenger RNA results in synthesis of proteins that can prime and enhance an anti-cancer immune response. Priming, expansion and migration of anti-cancer T-cell clones may also result in a systemic or ‘abscopal’ response in distal lesions. There is mounting preclinical evidence that OX40-OX40L signaling boosts anti-cancer immunity via co-stimulatory activity on T-cells. We generated mRNA constructs encoding OX40L with miR-122 binding sites that have been shown to suppress protein translation in hepatocytes yet not within tumor cells. A single ITu injection of OX40L mRNA, formulated in a lipid nanoparticle, resulted in transfection of up to 25% of live tumor cells, predominantly cancer and myeloid cells. OX40L protein persisted for 7 days after mRNA injection, and a resulting increase in tumor T-cells was observed. Repeat dosing of OX40L mRNA induced complete responses in a model that is sensitive to immune-meditated therapies, such as anti-PD1/PD-L1 blockade, yet was ineffective in a related model that might better represent an immunologically barren or immunosuppressive TME. The OX40L-resistant tumor model was also resistant to anti-PD1 treatment and was shown to contain fewer infiltrating immune cells. As this model may represent a subset of human cancers that do not respond well to checkpoint inhibitors, it was used to test various mRNA constructs designed to cooperatively harness components of both the innate and adaptive immune systems in igniting a productive anti-cancer response. Both IL-36γ and IL-23 have established roles in mediating immune responses in humans and have been implicated in driving various inflammatory diseases. IL-36γ activates innate immune cells and promotes Th1 responses, whereas IL-23 has been implicated in Th1/Th17 immunity as well as in the modulation of antigen presenting cells. Repeated weekly intratumoral co-administration of mRNAs encoding IL-23 and IL-36γ resulted in durable complete responses in both anti-PD1-sensitive and -resistant tumors in a synergistic fashion. A further modest improvement in outcomes was observed when the IL-23/36γ mRNA doublet was co-administered with OX40L mRNA. Further pharmacodynamic and mechanism of action studies with this combination are underway. ITu injection of mRNA encoding combinations of co-stimulatory proteins and cytokines represents a novel platform for cancer immunotherapy, and may be able to induce responses in patient tumors that are resistant to checkpoint inhibition. The co-formulated combination of mRNAs encoding human OX40L, IL-23 and IL-36γ is planned to enter clinical trials in 2017. Citation Format: Josh P. Frederick, Ailin Bai, Susannah Hewitt, Russ Karp, John Zielinski, Kana Ichikawa, Ameya Apte, Dyane Bailey, Kristen Arnold, Sam Farlow, Darren Potz, Bo Ying, Graham MacLean, Kerry Benenato, Maja Sedic, Steve M. Kelsey. Durable efficacy and anti-cancer immunity following intratumoral administration of messenger RNAs encoding IL-36γ, IL-23 and OX40L [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 1607. doi:10.1158/1538-7445.AM2017-1607

Abstract 5089: Antitumor activities elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1

Abstract Interleukin (IL)-28A/IFN-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect antitumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of antiproliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8+ T cells play a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8+ T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific antitumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ deficient mice, the antitumor activity of AdIL-28A was completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided an additive antitumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer gene therapy. Note: This abstract was not presented at the meeting. Citation Format: Muneo Numasaki, Hiroki Tsukamoto, Yoshihisa Tomioka. Antitumor activities elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1 [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 5089. doi:10.1158/1538-7445.AM2017-5089

Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

Abstract LB-096: Induction of systemic immunity through single-site intratumoral CD40 activation and checkpoint blockade eradicates melanoma in the brain

Abstract Background: Melanoma brain metastases (MBM) are a rapidly growing clinical problem with up to 60% of melanoma patients developing MBM over the course of their disease. Locoregional treatment with surgery, radiotherapy, radiosurgery, and newer drug classes such as checkpoint inhibitors and targeted agents against BRAF-mutation melanoma have shown limited effectiveness against MBM (Lancet 2015; 16: e486-97). MBM patients continue to have poor clinical outcomes and life expectancies of only 3-7 months. Thus, there is a significant unmet need for effective therapies for MBM patients. Direct intratumoral administration of a non-replicating adenovirus encoding a CD40-targeting chimeric immunostimulatory protein (ISF35) leads to generation of potent melanoma-specific T cells. When combined with checkpoint inhibitors, ISF35 generates synergistic systemic anti-melanoma immunity that eradicates both injected and uninjected distant melanoma with 40% of mice cured using a B16 model. Based on this evidence of systemic activity, we hypothesized that ISF35/checkpoint inhibitors combination treatment may have systemic activity against MBM. Methods: A MBM mouse model was developed using luciferase-expressing B16 (B16-Luc). B16-Luc cells were subcutaneously (s.c.) implanted in the right flank of C57BL/6 mice 12 days prior to treatment. B16-Luc cells were then injected into the brain four days prior to treatment. Following tumor establishment at both sites, ISF35 was injected intratumorally in the right flank tumor on days 0, 4, 9, and 14. Simultaneously, anti-PD1 and anti-CTLA-4 antibodies were systemically administered. Results: Intratumoral administration of ISF35 in combination with anti-PD-1 and anti-CTLA-4 significantly increased (p<0.01) mouse survival compared to untreated mice. Median survival was not reached for the ISF35 plus checkpoint combination, was 10 days for untreated mice, 26 days for ISF35 monotherapy, and 17 days for anti-PD-1/anti-CTLA-4 combination therapy. ISF35/anti-PD-1/anti-CTLA-4 combination treatment of s.c. tumors resulted in complete and durable abscopal regression of brain tumors as assessed by bioluminescent imaging 30 days following initiation of treatment. In contrast, brain melanoma tumors continued to grow in the ISF35 monotherapy or anti-PD-1/anti-CTLA-4 treatment groups. The systemic anti-tumor activity of ISF35/anti-PD-1/anti-CTLA-4 was associated with greater production of melanoma-specific CD8 T cells with an activated phenotype, including upregulated PD-1 surface expression. Conclusions: These results suggest ISF35 may improve the effectiveness of checkpoint inhibitors therapy for metastatic melanoma, including in the brain. Citation Format: Manisha Singh, Christina Vianden, Adi Diab, Patrick Hwu, Willem W. Overwijk. Induction of systemic immunity through single-site intratumoral CD40 activation and checkpoint blockade eradicates melanoma in the brain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-096.

Design of reloadable in situ forming implants for delivery of doxorubicin

Design of reloadable in situ forming implants for delivery of doxorubicin

Modulated electro-hyperthermia enhances dendritic cell therapy through an abscopal effect in mice.

The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3+ and CD8+ cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3+ and CD8+ cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.

Comparing routes of delivery for nanoliposomal irinotecan shows superior anti-tumor activity of local administration in treating intracranial glioblastoma xenografts

Liposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients. We compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice. Our results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan. Our results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.

Mechanisms of action underlying the immunotherapeutic activity of Allovectin in advanced melanoma

Allovectin (velimogene aliplasmid) is a cancer immunotherapeutic currently completing a pivotal phase 3 study for metastatic melanoma. Consisting of a bicistronic plasmid encoding both major histocompatibility complex (MHC) class I heavy and light chains (HLA-B7 and β2-microglobulin, respectively) formulated with a cationic lipid-based system, it is designed for direct intratumoral administration. Following injection into a single lesion, the product is intended to induce anti-tumor immune responses against both treated and distal lesions. Both the plasmid and lipid components of Allovectin contribute to the biological activity of the drug product, and its therapeutic activity is hypothesized to derive from multiple mechanisms of actions (MOAs). These include the induction of both cytotoxic T-cell and innate immune responses directed against allogeneic as well as tumor-derived targets, consequences of both an increased MHC class I expression on tumor cells and the induction of a localized immune/inflammatory response. In this paper, we review Allovectin's proposed MOAs, placing their contributions in the context of anti-tumor immunity and highlighting both preclinical and clinical supporting data.