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Background: Since the preponderance of pediatric gliomas are mutationally 'bland,' immune checkpoint inhibitors are unlikely to mediate therapeutic benefit. Alternately, immunologic response can be induced de novo against pediatric gliomas with mRNA cancer vaccines. Messenger RNA represents a paradigm shift in vaccinology (i.e. COVID-19) given its flexibility, commercialization, and propensity to confer rapid protection with only a single vaccine. Objective: We sought to develop a new mRNA platform with an optimized backbone for insertion of both personalized and/or “off the shelf” (i.e. H3K27M) transcripts for rapid induction of anti-tumor activity against pediatric gliomas. Approach: We synthesized an mRNA backbone with optimized 5' and 3' UTRs for delivery of gene transcripts pertinent to pediatric brain tumors using a lipid-nanoparticle (NP) delivery vehicle. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” or multi-lamellar package. Results: We demonstrate immunogenicity of RNA-NPs delivering either personalized glioma mRNA or H3K27M mRNA. RNA-NPs localize to myeloid cells in murine KR158b brain tumors and activate dendritic cells that supplant regulatory intratumoral myeloid populations inducing antigenrecall response with long-term survivor benefit. Our optimized mRNA backbone yielded significantly improved anti-tumor efficacy compared with commercial backbones. We have shown this approach can be refined for co-delivery of immunomodulatory RNAs (i.e. GM-CSF) and/or delivery of siRNAs targeting immunoregulatory axes (PD-L1) in murine brain tumors (GL261). We have since established safety of RNA-NPs in acute/chronic murine GLP toxicity studies without cross-reactivity to normal-brain, and launched a large-animal canine brain tumor trial which demonstrated RNANPs to be feasible, safe and immunologically active. Conclusion: RNA-NPs reprogram the brain tumor microenvironment while inducing a gliomaspecific immune response. We have since received FDA-IND approval for first-in-human trials.

INTRODUCTION Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. How the different tumor cells interact with immune cells and how this metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. OBJECTIVES The objectives are to unravel the various molecular signals and metabolic link that underlie the interaction of SCCs with the GBM microenvironment, in particular with the suppressive immune compartment, and to effectively target these interactions for better therapeutics. METHODS Multiple murine glioma cell lines were used to establish metabolic heterogeneity and communications, while various genetic and pharmacological approaches were applied to assess the effect of disrupting the metabolic interplay between SCCs and the immune system. RESULTS We determined that SCCs exhibit distinct metabolic dependencies, involving preferential lipid metabolism supported by enhanced fatty acid uptake. We also found that tumor progression is regulated by the interaction of SCCs with the immune system and established that SCCs recruit immune suppressive M2-like macrophages to the tumor microenvironment, which in turn work against tumor immune rejection by inhibiting T cell anti-tumor activity. The immune microenvironment shaped by SCCs is marked by specific metabolic features enhancing lipid exchange capacities that are exploited by SCCs to support their survival and functions. Importantly, disrupting lipid metabolic exchange sensitized tumors to chemotherapy. CONCLUSION Our results reveal that metabolic interactions between SCCs and tumor-associated macrophages within the GBM microenvironment play a critical role in the development of drug and immune resistant tumors. This study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities.

BACKGROUND Brain tumors are notoriously difficult to treat in part due to their isolation behind the blood brain barrier and their power to suppress antitumor immune responses. We have previously reported cationic liposome formulations capable of delivering immune modulatory nucleic acids to immune cells in various peripheral organs, but there is currently no reliable method to deliver nucleic acids into brain tumors without direct injection into the tumor site. OBJECTIVE Here, we report the development of a customized lipid nanoparticle to deliver immune modulatory nucleic acids to immune cells in brain tumors. APPROACH Cationic liposomes composed of varying lipid combinations were evaluated for their ability to deliver functional mRNA and siRNA to innate immune cells in vitro and in intracranial tumor models. Nucleic acids were labelled with Cy3 to monitor particle distribution in vivo. RESULTS Lipids composed of DOTAP and cholesterol selectively delivered mRNA and siRNA to intracranial GL261 and KR158b tumors. Interestingly, these particles selectively delivered these nucleic acids to CD45+ white blood cells with minimal delivery to CD45- tumor cells or normal brain tissue. Encapsulation of siRNA blocking programmed death ligand 1 (PDL1) significantly reduced PDL1 expression on innate immune cells in brain tumors, with the greatest effects on tumor-associated macrophages. Co-administration of systemic checkpoint blockade with intravenous administration of these lipid nanoparticles bearing PDL1 siRNA enabled systemic and intratumoral checkpoint blockade, leading to 37% long term survivorship in an otherwise fatal intracranial tumor model. CONCLUSIONS Our customized lipid nanoparticles enable potent intratumoral immune modulation via delivery of nucleic acids to immune cells in brain tumors.

Background Since the preponderance of pediatric gliomas are mutationally ‘bland,’ immune checkpoint inhibitors are unlikely to mediate therapeutic benefit. Alternately, immunologic response can be induced de novo against pediatric gliomas with mRNA cancer vaccines. Messenger RNA represents a paradigm shift in vaccinology (i.e. COVID-19) given its flexibility, commercialization, and propensity to confer rapid protection with only a single vaccine. Objective We sought to develop a new mRNA platform with an optimized backbone for insertion of both personalized and/or “off the shelf’ (i.e. H3K27M) transcripts for rapid induction of anti-tumor activity against pediatric gliomas. Approach We synthesized an mRNA backbone with optimized 5’ and 3’ UTRs for delivery of gene transcripts pertinent to pediatric brain tumors using a lipid-nanoparticle (NP) delivery vehicle. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” or multi-lamellar package. Results We demonstrate immunogenicity of RNA-NPs delivering either personalized glioma mRNA or H3K27M mRNA. RNA-NPs localize to myeloid cells in murine KR158b brain tumors and activate dendritic cells that supplant regulatory intratumoral myeloid populations inducing antigen-recall response with long-term survivor benefit. Our optimized mRNA backbone yielded significantly improved anti-tumor efficacy compared with commercial backbones. We have shown this approach can be refined for co-delivery of immunomodulatory RNAs (i.e. GM-CSF) and/or delivery of siRNAs targeting immunoregulatory axes (PD-L1) in murine brain tumors (GL261). We have since established safety of RNA-NPs in acute/chronic murine GLP toxicity studies without cross-reactivity to normal-brain, and launched a large-animal canine brain tumor trial which demonstrated RNA-NPs to be feasible, safe and immunologically active. Conclusion RNA-NPs reprogram the brain tumor microenvironment while inducing a glioma-specific immune response. We have since received FDA-IND approval for first-in-human trials (IND#BB-19304) in pediatric patients with high-grade gliomas (PNOC020 study, NCT04573140).

BACKGROUND While dendritic cell (DC) vaccine therapy has shown considerable promise for glioblastoma (GBM) patients (Mitchell et al. Nature, 2015), their advancement into human clinical trials has been fraught with challenges in the development, manufacturing, and marketing of successful cancer immunotherapies. To circumvent the challenges associated with cell therapy, we have developed a new platform technology consisting of tumor derived mRNA complexed into lipid-nanoparticles (RNA-NPs) for systemic delivery to DCs in vivo and induction of antigen specific T cell immunity against GBM. OBJECTIVES/ METHODS We sought to assess if surface and charge modifications to our custom lipid-NP could facilitate its localization to lymphoid organs and the brain tumor microenvironment. RESULTS We demonstrate that intravenous administration of our unmodified custom RNA-NPs mediate systemic activation of DCs; these include activation of CD11c+ cells in the brains of animals with intact blood brain-barriers (BBBs). RNA-NPs mediate antigen specific T cell immunity and anti-tumor efficacy with increased tumor infiltrating lymphocytes against a NF-1/p53 mutant glioma that recapitulates features of human GBM in immunocompetent mice. Modification of surface charge could direct these RNA-NPs to lymphoid organs (e.g. spleen, lymph nodes) while modification of the lipid backbone (with cholesterol) enhances localization to innate immune cells in NF-1/p53 mutant and GL261 gliomas. We therefore assessed if this customizable lipid-NP could be leveraged for delivery of immune checkpoint inhibitors (ICIs) (i.e. PD-L1 siRNA) to the brain tumor microenvironment. Compared with scrambled siRNA-NPs in combination with ICIs, surface modified siRNA-NPs (antagonizing PD-L1) in combination with ICIs mediated significant antitumor efficacy with 37% long term survivors in an otherwise fatal brain tumor model. CONCLUSION We designed multifunctional RNA-NPs with a simple, scalable synthesis method that enables delivery of nucleic acids to innate immune cells in lymphoid organs and brain tumors.

BACKGROUND The lack of appropriate preclinical murine glioblastoma models limits comprehensive toxicity/efficacy evaluation of investigational agents. To overcome this challenge, we evaluated the safety and activity of a new immunotherapeutic technology that we have pioneered (composed of tumor mRNA complexed into a custom lipid-nanoparticle formulation) in client-owned canines (pet dogs) diagnosed with malignant gliomas. OBJECTIVE/ METHODS Canine malignant gliomas were biopsied for generation of personalized tumor mRNA loaded into our custom lipid-nanoparticle (NP) vector. The patients received RNA-NPs intravenously beginning two weeks after their biopsy once weekly (x3) and no other anti-tumor therapeutic interventions. RESULTS Within a few hours after administration, tumor specific RNA-NPs elicited margination of peripheral blood mononuclear cells, which increased in the subsequent days/weeks post-treatment; suggesting that RNA-NPs mediate lymphoid honing of immune cell populations before egress. RNA-NPs also elicited increased: 1) serum interferon-α that spiked at 2 hours; 2) CD80 and MHCII on CD11c+ cells (demonstrating activation of peripheral DCs); and 3) interferon-γ + T-cells (i.e. activated T-cells). After receiving weekly RNA-NPs (×3), the canines had a steady course. Aside from low-grade fevers on the vaccination days, personalized tumor RNA-NPs (1x) were well tolerated with stable blood counts, chemistries, and renal/liver function tests. All patients assessed developed immunologic response with pseudoprogression or stable/smaller tumors by MRI. Although we have treated a small cohort, we have observed improvement in median/overall survival in all canine patients with terminal gliomas receiving RNA-NPs (compared with historical controls). CONCLUSION RNA-NPs were feasible, safe and immunologically active in client-owned canines with terminal gliomas. We have not appreciated significant toxicities in canines that would preclude investigation in humans at 1x dosing. Although these results need to be validated in larger canine data sets, these results suggest safety and activity of tumor specific RNA-NPs in canines with terminal gliomas.

BACKGROUND: Pediatric brain tumors are poorly immunogenic preventing current immunotherapies (i.e. checkpoint blockers) from having significant activity. Unlocking immunotherapy against these malignancies is predicated on reprogramming their immunogenicity. OBJECTIVE/METHODS: We developed a novel treatment platform, which leverages the use of clinically translatable lipid-nanoparticles (NPs) combined with tumor derived mRNA that simultaneously function as both vaccines and immunomodulating agents to reprogram the systemic/local microenvironments of pediatric brain cancers. RESULTS: Systemic tumor mRNA-NPs localize to lymphoid organs (i.e. liver, spleen lymph nodes) and to the intratumoral milieu, activating the immunologic microenvironment therein through release of type I interferon from plasmacytoid dendritic cells. This culminates in a CD4 and CD8 central memory T cell response against H3.1K27M murine gliomas. In immunologically resistant murine tumor models, RNA-NPs induce robust anti-tumor efficacy and mediate synergistic activity in settings where immune checkpoint inhibitors (i.e. anti-PD-L1 therapy) do not confer therapeutic benefit. We demonstrate safety of RNA-NPs in acute/chronic murine toxicity studies and in a client-owned canine (pet dog) diagnosed with a malignant glioma. In our first canine subject, RNA-NPs elicited activation of dendritic cells, release of type I interferon and activation of CD8+ T cells. CONCLUSION: RNA-NPs bypass MHC restriction and can be made readily available for all patients (and not only HLA specific haplotypes); they also provide a renewable antigen resource that can be used to continuously vaccinate patients for months/years after diagnosis. Through partnership with the Pacific Pediatric Neuro-Oncology Consortium (PNOC), we are in the final stages of FDA-IND submission before first-in-human trials.

BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) remains uniformly recalcitrant and thus necessitates development of novel targeted therapies. The histone mutation in H3K27M is conserved in the preponderance of DIPG patients and may be exploited as a neoepitope for cancer immunotherapy. We have developed a novel treatment platform, which leverages the use of clinically translatable nanoparticles (NPs) combined with H3K27M mRNA neoantigens for in vivo activation of dendritic cells and generation of DIPG specific T cells. OBJECTIVE/METHODS: Since neoantigens have been shown to mediate immunologic response through MHC class II, we sought to identify MHCII restricted epitopes spanning the H3.1K27M junction for development of RNA-NP vaccines against DIPG. RESULTS: We identified MHCII restricted H3.1K27M epitopes not present in the wild-type sequence. We constructed a 25-mer sequence (75 amino acids) spanning the range of epitopes, before cloning it into our custom pGEM-4z plasmid containing a poly A tail and 5’ UTR with a T7 promoter for amplification of MHC-II restricted H3.1K27M mRNA. We complexed this RNA with our custom NP formulation and investigated its immunogenicity in C57Bl/6 mice. Unlike MHC-I restricted epitopes (i.e. OVAlbumin’s SINFEKL epitope), MHC-II restricted H3K27M epitopes elicited significant increases in activated CD4 and CD8 central memory T cells. We demonstrate that this central memory phenotype correlates with anti-tumor efficacy and long-term survival outcomes in murine tumor models. CONCLUSION: RNA-NPs can be made readily available for all DIPG patients (and not only HLA specific haplotypes) providing a renewable antigen resource that can be used to continuously vaccinate patients for months/years after diagnosis. Herein, we identify MHCII restricted H3.1K27M epitopes for activation of immunologic central memory necessary for long-lived anti-tumor efficacy.

BACKGROUND: In a randomized phase III study for recurrent glioblastoma (GBM), combination therapy with immune checkpoint inhibitors (ICIs) nivolumab and ipilimumab failed to improve overall survival. Since ICIs appear to be dependent on host-myeloid (and not tumor-cell) expression of PD-L1 (Tang et al. JCI, 2018), we sought to assess if tumor-mRNA nanoparticles (NPs) could prime the systemic and intratumoral GBM milieu with activated PD-L1+ host-myeloid cells sensitizing immunologically ‘cold’ tumors to immunotherapy. METHODS: We systemically administered personalized tumor-derived mRNA encapsulated in translatable lipid-NPs with excess positive charge for enhanced delivery and transfection of peripheral/intratumoral antigen presenting cells (APCs) in murine and large animal canine malignant glioma models. RESULTS: RNA-NPs activate systemic immunity within twenty-four hours, inducing significant increases in the percentage of CD11c+ myeloid-cells expressing PD-L1 and CD86. After only a single RNA-NP vaccine, the bulk of APCs within the spleen, liver, lymph nodes, bone marrow and tumor display an activated phenotype. These PD-L1+ APCs (CD11c+MHC-II+CD86+PD-L1+ cells from intracranial tumors) did not suppress immunity, but rather, heightened interferon-(IFN)-γ reactivity. Addition of ICIs (to animals primed with RNA-NPs) augmented peripheral/intratumoral PD-1+CD8+ cells and mediated synergistic anti-tumor efficacy in settings where ICIs alone did not confer therapeutic benefit. These synergistic effects were mediated by type–I-interferon released from PD-L1+ plasmacytoid dendritic-cells. In translational studies, personalized mRNA-NPs (from whole tumor-transcriptome) were safe and active in a client-owned canine with a spontaneous malignant glioma. In this patient, RNA-NPs elicited robust immunologic activity with increased percentages of activated PD-L1+ APCs and IFNγ+CD8+ cells. CONCLUSION: RNA-NPs elicit widespread immune activation concomitant with inducible PD-L1 expression on intratumoral APCs that can be therapeutically exploited. Since RNA-NPs bypass cost/complexity of cellular therapeutics, are amenable to central distribution, and can be made within days of tumor resection, these formulations can be expeditiously translated as biomodulators of GBM immunogenicity.

While recurrent medulloblastoma (MB) remains almost uniformly fatal, our group has shown a trend (p=0.07) toward improved survival in patients receiving autologous tumor-RNA pulsed dendritic cells (DCs) coupled with ex vivo expanded total RNA activated T-cells. However, the advancement of autologous cellular therapeutics remains encumbered by significant cost, complexity and time to generation. To circumvent these challenges, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor-derived RNA for near-immediate induction of systemic immunity against MB. We identified a clinically translatable NP formulation for the delivery of RNA to antigen presenting cells for induction of anti-tumor T-cell immunity. When administered intravenously, RNA-NPs increased expression of co-stimulatory molecules on CD11c+ cells throughout reticuloendothelial organs and within the tumor microenvironment; this phenotype was dependent on type I interferon. Targeted inhibition of type I interferon signaling (via INFAR1-mAbs) abrogated anti-tumor immunity mediated by RNA-NPs. We enhanced the immunogenicity of this platform by simply combining mRNAs encoding for immunomodulatory molecules (i.e. HCV-PAMPs, GM-CSF) or by combining RNA-NPs with immune checkpoint inhibitors. Addition of checkpoint inhibitors to RNA-NPs mediated synergistic anti-tumor activity in settings where PD-1 or PD-L1 inhibition alone did not confer therapeutic benefit. In a pre-clinical cellular immunotherapy model targeting an anaplastic murine MB, RNA NPs superseded DCs in mediating anti-tumor activity. Based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of RNA-NPs targeting malignant brain tumors in canines (UF-IACUC#:201609430) before first in-human evaluation.