Abstract There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic ...effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.
While immune-cell infiltrated tumors, such as human papillomavirus positive (HPV+) ororpharyngeal squamous cell carcinomas (OPSCC) have been associated with an improved clinical prognosis, there is ...evidence to suggest that OPSCCs are also subjected to increased immunoregulatory influence. The objective of this study was to assess whether patients with clinically aggressive OPSCC have a distinct immunosuppressive immune signature in the primary tumor.
This retrospective case-control study analyzed 37 pre-treatment tissue samples from HPV+ and HPV-negative OPSCC patients treated at a single institution. The cases were patients with known disease recurrence and the controls were patients without disease recurrence. An mRNA-expression immune-pathway profiling was performed, and correlated to clinical outcomes. The TCGA head and neck cancer database was utilized to make comparisons with the institutional cohort.
In our cohort, HPV-negative and HPV+ patients with known disease recurrence both had significantly increased suppressive monoctyte/macrophage and granulocyte cell-expression-profile enrichment. Similar findings were found in the TCGA cohort when comparing HPV-negative to positive patients.
our study demonstrates that patients with recurrent HPV+ OPSCC had suppressive monocyte/macrophage and granulocyte immune-cell enrichment, similar to those seen in the more aggressive HPV-negative OPSCC.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
BackgroundDiffuse midline glioma (DMG) is a universal fatal glial brain cancer in children. We tested our novel multilamellar mRNA lipid particle aggregate vaccine (RNA-LPA, IND19304—Sayour),1 a ...tumor-agnostic treatment platform that encapsulates tumor specific RNA and delivers the payload in a highly immunogenic fashion, as an approach to treating this currently incurable cancer.MethodsUsing the K2 DMG model,2 we implant H3K27M-expressing DMG cells into the 4th ventricle of P1-P3 neonatal C57BL/6 mice. RNA-LPA generated from predicated human H3K27M epitopes or total-tumor mRNA are administered intravenously beginning at day 35. We performed multiparameter 3D geospatial fluorescent microscopy to characterize mRNA transduction. Immunologic responses to treatment were evaluated by multiparameter flow cytometry, microscopy, and cytokine profiling.ResultsMice developed clinical neurological signs of disease by day 30–35. RNA-LPAs targeting human H3K27M epitopes were found to be immunogenic in wild-type mice. Intriguingly, nonspecific enhanced green fluorescent protein (eGFP)-RNA-LPAs resulted in statistically significant survival benefits compared to mice treated with empty LPs. However, tumor-specific RNA-LPAs (either H3K27M-specific or total tumor mRNA-derived) also enhanced survival and additionally resulted in a subset of mice with long-term survival. This survival benefit was observed despite the development of clinical hydrocephalus in mice treated with RNA-LPAs. 3D microscopy established that tumors demonstrated invasive disease and microvascular erosion in mice. We found that mRNA transduces fibroblastic reticular cells (FRCs) in the spleen and lymph nodes, prompting widespread immune activation. Treatment with RNA-LPA led to massive increases in production inflammatory cytokines (i.e. TNF-α) and chemokines (i.e. CCL2), which led to recruitment of the majority of circulating monocytes and lymphocytes to secondary lymphoid organs.ConclusionsRNA-LPAs extend survival in our highly aggressive DMG model, including curative outcomes in cohorts treated with either total tumor or H3K27M RNA-LPs. These data suggest that RNA-LPs are capable of stimulating host adaptive immune responses against established DIPG tumors. Signs of hydrocephalus in treated mice may indicate pseudoprogression due to immunologic response, yet mice were frequently able to survive this development. Future studies will further characterize the immunologic response in these mice and support expansion of our existing IND for a multi-institutional phase I clinical trial for children with DMG, who currently have no curative options.AcknowledgementsWe appreciate funding from the ChadTough Defeat DIPG Foundation and the DIPG/DMG Research Funding Alliance. John Ligon and Elias Sayour contributed equally and are co-senior authors.ReferencesMendez-Gomez H, DeVries A, Castillo P, Stover B, Qdaisat S, Von Roemling C, Ogando-Rivas E, Weidert F, McGuiness J, Zhang D, Chung MC, Li D, Zhao C, Marconi C, Campaneria Y, Chardon-Robles J, Grippin A, Karachi A, Thomas N, Huang J, Milner R, Sahay B, Sawyer WG, Ligon JA, Silver N, Simon E, Cleaver B, Wynne K, Hodik M, Molinaro A, Guan J, Kellish P, Doty A, Lee J-H, Carrera-Justiz S, Rahman M, Gatica S, Mueller S, Prados M, Ghiaseddin A, Mitchell DA, Sayour EJ. mRNA aggregates harness danger response for potent cancer immunotherapy. medRxiv. 2023:2023.03.12.23287108. doi: 10.1101/2023.03.12.23287108.Misuraca KL, Cordero FJ, Becher OJ. Pre-Clinical Models of Diffuse Intrinsic Pontine Glioma. Front Oncol. 2015;5:172. doi: 10.3389/fonc.2015.00172. PubMed PMID: 26258075; PMCID: PMC4513210.Ethics ApprovalWork approved under UF IACUC 202200000375
BackgroundPatients with relapsed and metastatic osteosarcoma have a 5 year overall survival <25%. Our group tested our novel multilamellar RNA lipid nanoparticle aggregate vaccine (RNA-LPA)1 as an ...approach to reprogram the immunosuppressive tumor microenvironment (TME)2 present in osteosarcoma, where immunotherapy has not yet been effective.MethodsTotal-tumor mRNA was amplified from tumor cell lines in mice or tumor biopsy in canines before complexation in lipid nanocarriers/cationic lipids, generating RNA-LPA for systemic administration. Preclinical murine models were generated using K7M2, KHOS or 143B osteosarcoma cells in either C57Bl/6, BALB/c or BALB/c SCID mice inoculated by tail vein injection to mimic minimal residual metastatic disease from pulmonary osteosarcoma. We treated client-owned canine patients (pet-dogs) with osteosarcoma through a comparative oncology clinical trial with the UF College of Veterinary Medicine (UF IACUC#202111376, PI: Milner). Using liquid-like solid (LLS) 3D tumoroid culture system,3 we established ex vivo models of murine, canine, and human osteosarcoma.ResultsTumor-specific RNA-LPAs elicited anti-tumor efficacy in the murine K7M2 model with long term survival (7/8 mice), with some survival benefit even with irrelevant pp65 RNA-LPAs (p<0.05); these findings correlated with an increase in intratumoral central memory T cells. Both tumor-specific and irrelevant RNA-LPAs reprogramed the innate immune microenvironment (decreased tumor associated macrophages and myeloid derived suppressor cells, p<0.01), but tumor-specific RNA-LPAs additionally resulted in activation of adaptive immunity (dendritic cells and T-cells). Five pet-dogs with osteosarcoma were safely treated with total-tumor RNA-LPAs, which were immunologically active, demonstrating changes in complete blood counts and serum cytokines within 6 hours of vaccine administration. One pet dog received 4 total RNA-LPAs every 2 weeks with initial objective radiographic resolution of pulmonary metastases, though this subject relapsed with new metastases. Osteosarcoma tumors from mice, canines, and human patients were used to successfully establish 3D tumoroid tissue culture for at least 32 days ex vivo.ConclusionsBoth tumor-specific and nonspecific ‘off the shelf’ RNA-LPA vaccines redirected immunosuppressive myeloid cells, a hallmark of the osteosarcoma TME.2 This agent, which is FDA-IND approved (BB-19304, Sayour) and in human clinical trials for patients with brain tumors (NCT04573140) will enter clinical trials for patients with osteosarcoma in the coming year (NCT04837547, not yet recruiting). Our 3D tumoroid system offers the opportunity to study the immune-modulating effect of RNA-LPAs and may be a meaningful correlate to both canine and human subjects enrolled on clinical trials.AcknowledgementsWe appreciate funding from the National Cancer Institute, the V Foundation, MIB Agents, Hyundai Hope on Wheels, and the Pediatric Cancer Research Foundation.Trial RegistrationNCT04573140ReferencesMendez-Gomez H, DeVries A, Castillo P, Stover B, Qdaisat S, Von Roemling C, Ogando-Rivas E, Weidert F, McGuiness J, Zhang D, Chung MC, Li D, Zhao C, Marconi C, Campaneria Y, Chardon-Robles J, Grippin A, Karachi A, Thomas N, Huang J, Milner R, Sahay B, Sawyer WG, Ligon JA, Silver N, Simon E, Cleaver B, Wynne K, Hodik M, Molinaro A, Guan J, Kellish P, Doty A, Lee J-H, Carrera-Justiz S, Rahman M, Gatica S, Mueller S, Prados M, Ghiaseddin A, Mitchell DA, Sayour EJ. mRNA aggregates harness danger response for potent cancer immunotherapy. medRxiv. 2023:2023.03.12.23287108. doi: 10.1101/2023.03.12.23287108.Ligon JA, Choi W, Cojocaru G, Fu W, Hsiue EH, Oke TF, Siegel N, Fong MH, Ladle B, Pratilas CA, Morris CD, Levin A, Rhee DS, Meyer CF, Tam AJ, Blosser R, Thompson ED, Suru A, McConkey D, Housseau F, Anders R, Pardoll DM, Llosa N. Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes. Journal for immunotherapy of cancer. 2021;9(5). doi: 10.1136/jitc-2020–001772. PubMed PMID: 34021032; PMCID: PMC8144029.Bhattacharjee T, Gil CJ, Marshall SL, Urueña JM, O’Bryan CS, Carstens M, Keselowsky B, Palmer GD, Ghivizzani S, Gibbs CP, Sawyer WG, Angelini TE. Liquid-like Solids Support Cells in 3D. ACS Biomater Sci Eng. 2016;2(10):1787–95. Epub 20160620. doi: 10.1021/acsbiomaterials.6b00218. PubMed PMID: 33440476.Ethics ApprovalStudies approved by UF IACUC 202111376 and 202009941, and UF IRB202001062
•Townes humanized knock-in mouse model recapitulates human globin gene switching.•Silencing of γ-globin is completed during postnatal development checkpoints 2 to 4 after birth of Townes mice.•Normal ...murine developmental erythropoiesis coincides with human globin gene switching in the Townes mouse model.•Hydroxyurea suppresses erythropoiesis in Townes mice to create an early persistent F-cell phenotype without re-activating γ-globin transcription.•Value of Townes mouse model as pre-clinical model for pharmacologic fetal hemoglobin inducers is dependent on specific mechanism of action of the drug.
Mechanisms that control the fetal-to-adult hemoglobin switch are attractive therapeutic targets in sickle cell disease. In this study, we investigated developmental γ-globin silencing in the Townes humanized knock-in mouse model, which harbors a construct containing the human γ-, βA-, and βS-globin genes, and examined the utility of this model in evaluation of pharmacologic induction of fetal hemoglobin (HbF). We studied mouse pups on the day of delivery (P0) to 28 days after birth (P28). Regardless of the hemoglobin genotype (SS, AS, or AA), the proportion of F cells in peripheral blood was 100% at P0, declined sharply to 20% at P2, and was virtually undetectable at P14. Developmental γ-globin silencing in Townes mice was complete at P4 in association with significantly increased BCL11A expression in the primary erythropoietic organs of the mouse. Hydroxyurea given at P2 significantly sustained elevated percentages of F cells in mice at P14. However, the percentage of F cells declined at P14 for treatment begun at P4. A lack of augmentation of γ-globin mRNA in erythroid tissues suggests that the apparent increase in HbF in red cells caused by hydroxyurea was not due to sustained or re-activation of γ-globin transcription, but was instead a function of erythropoiesis suppression. Thus, we provide new details of the hemoglobin switch in the Townes murine model that recapitulates postnatal γ- to β-globin switch in humans and identify the myelosuppressive toxicity of hydroxyurea as a superseding factor in interpreting pharmacologic induction of HbF.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
RNA vaccines have shown great promise as activators of immune responses against viral pathogens but their efficacy in cancer is unclear. Here, we describe a versatile, personalized ...mRNA-nanoparticle (RNA-NP) platform that can be customized to produce powerful anti-tumor immune activation, reprogram the brain tumor microenvironment, and predict vaccine efficacy just two days after treatment. Lipid mixtures (i.e. DOTAP, Cholesterol) with or without iron oxide nanoparticle-cores were complexed with mRNA encoding tumor antigens. Intravenous administration of RNA-NPs induced robust activation of innate immune cells resulting in prolonged survival in murine models of subcutaneous and intracranial melanoma. Inclusion of cholesterol in the lipid backbone enabled delivery of nucleic acids across the blood brain barrier and into tumor-associated myeloid cells in intracranial GL261 and KR158b tumors. These cholesterol-bearing liposomes not only activated innate immune cells in the tumor microenvironment (i.e. increased expression of CD80 and MHCII), but also enabled further manipulation of this compartment. Use of RNA-NPs to deliver siRNA targeting PD-L1 resulted in significant reduction in PD-L1 expression among tumor associated myeloid cells, leading to 37% long term survivorship in combination with systemic checkpoint blockade in an otherwise fatal model of GL261. We have also shown that IONPs incorporated into the cores of these particles enable non-invasive tracking of dendritic cells by MRI, enabling creation of a theranostic approach to: 1) enhance immunologic effects; 2) facilitate translocation across the blood-brain barrier; and 3) enable non-invasive imaging to predict response to RNA-NPs.
Abstract
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).
Abstract
BACKGROUND
High-grade gliomas are an aggressive subset of CNS tumors in need of novel treatment strategies. However, tumor heterogeneity, the immunosuppressive tumor microenvironment (TME) ...and the lack of immunogenic tumor specific targets have limited the translational power of many immunotherapies for high-grade gliomas. The promising application of mRNA vaccines has the potential to restore immunosurveillance through peripheral and intratumoral reprograming of the TME.
METHODS
Our lab has developed a lipid particle multilamellar aggregate (RNA-LPA) vaccine that encapsulates tumor specific RNA and delivers the payload to the immune system in a highly immunogenic fashion, similar to that of a virus. We sought to assess anti-tumor activity across preclinical murine models and investigate the mechanisms for response.
RESULTS
We demonstrated the immune activation potential of RNA-LPAs encoding for glioma associated antigens (i.e., pp65 and H3K27M) in preclinical murine models. We also showed activity of personalized RNA-LPA in canine and human studies (NCT04573140) for patients with high-grade gliomas. Exogenous RNA is recognized by pathogen recognition receptors (PRRs) which trigger a type I interferon (IFN) response and proinflammatory cytokine production. While most RNA based vaccines activate an innate immune response primarily through toll-like receptor 7 (TLR7), we reveal that RNA-LPAs activate the innate immune system through retinoic acid-inducible gene 1 protein (RIG-I). As a result of PRR signaling, the type I IFN and cytokine/chemokine response (IL-6, G-CSF, TNF-alpha, CXCL9, CXCL10, CCL2, CCL4) mediates massive recruitment of activated dendritic cell and activated T cell in lymphoreticular organs where antigen presenting cells and cytolytic T cell colocalize to initiate an adaptive immune response against glioma specific antigens.
CONCLUSION
RNA-LPAs are a novel platform immunotherapy that initiate anti-tumor immunity against malignant brain tumors through noncanonical activation of intracellular PRRs and widespread recruitment of peripheral blood mononuclear cells to sites of RNA-LPA localization.
Abstract
BACKGROUND
Glioblastoma (GBM) can be an effective teacher in the war on COVID-19, as an operative vaccine for either must elicit near-immediate protective responses that overcomes disease ...heterogeneity and immune suppression. Current prophylactic strategies against COVID-19 utilize mRNA vaccines targeting small fragments of the SARS-CoV-2 genome, but these may not induce robust T cell responses or elicit immunity quickly enough.
OBJECTIVE
We sought to adapt an FDA-IND approved mRNA vaccine in GBM against COVID-19 for: 1) activation of near immediate immune responses, 2) targeting of full-length SARS-CoV-2 structural proteins, and 3) induction of bidirectional (B and T cell) adaptive immunity.
METHODS
We utilized a novel engineering design that layers mRNA into a lipid-nanoparticle (NP) shell (much like an onion); this allows greater packaging of mRNA per particle to quickly boost innate/adaptive immune responses against full-length glioblastoma antigens or SARS-CoV-2 structural proteins.
RESULTS
In small and large animal models, RNA-NPs safely mimic viremia activating the quiescent immune system in only a few hours for induction of protective immunity against its mRNA payload. RNA-NPs activate dendritic cells (DCs), upregulate critical innate gene signatures, and induce antigen-specific cellular and humoral immunity. We found that mice receiving SARS-CoV-2 spike RNA-NPs had more effector T cells after vaccination with significant memory recall expansion after in vitro re-stimulation with overlapping SARS-CoV-2 spike peptide mix. We also found increased release of MIP-1-alpha (i.e. CCL3) previously shown by our group (Mitchell et al. Nature 2015) to be responsible for Th1 mediated memory recall to infectious vaccine antigens in GBM patients.
CONCLUSION
SARS-CoV-2 RNA-NPs elicit memory recall response after vaccination. We have obtained FDA-IND approval (BB-19304, Sayour) in GBM with SARS-CoV-2 specific amendment (BB-20871) underway to support first-in-human trials of RNA-NPs targeting both GBM and COVID-19.
PDF
