INTRODUCTION T cell-based immunotherapy in combination with checkpoint blockade has shown some promise in the generation of durable responses in glioblastoma (GBM); however, this disease remains ...nearly universally fatal. In murine GBM, we have previously demonstrated the existence of a unique CD3+ CD8+ T-cell phenotype on tumor infiltrating lymphocytes (TILs) consisting of CD39+ and PD1+ that appears to be tumor-specific in an interferon gamma (IFNg)-YFP reporter model (data unpublished, presented at CNS 2019). METHODS We implanted 104 KR158b.luc murine glioma cells into the right caudate nucleus of IFNg-YFP reporter mice. At 28 days, tumors were harvested, and TILs were sorted into two separate groups (CD39+ PD1+ YFP+ triple positive - TP and CD39− PD1− YFP− triple negative -TN) using a BD FACS Aria II. Sorted cells were were co-cultured with expansion CD3/28 beads, IL-2, and +/- aPD1 for 1 week. TILs were then put through an in vitro restimulation assay for co-culture to evaluate response to specific and non-specific tumor types with KR158b.luc, B16F0 (melanoma) and alone. IFNg quantification in supernatant was done via ProQuantum Immunoassay. RESULTS Sorted TP cells that were co-cultured without aPD1 and sorted TN cells that were co-cultured with or without aPD1 demonstrated minimal to no IFNg secretion (< 3.0 pg/ml). Sorted TP cells that were co-cultured with aPD1 demonstrated an increase above baseline in response to KR158b.luc (12.47 pg/ml: p < 0.1), B16F0 (7.19 pg/ml: p = NS), and alone (36.93 pg/ml: P < .0001) CONCLUSION We have previously identified a population of TILs in murine glioma that we believe are tumor specific. Sorted functional analysis demonstrates that these cells are in a state of exhaustion when attempts are made to restimulate them against tumor targets. The addition of aPD1 recovers exhaustion from these TILs in this functional assay. These data have important implications for the future development of TIL based therapy in human GBM.
Abstract
INTRODUCTION
Adoptive cell therapy has been demonstrably promising in the treatment against cancer, but two major barriers have prevented its application to brain stem gliomas (BSG). First ...is the penetrance of T cells into BSGs, and the second is that the heterogeneity of brain tumors inevitably results in antigen loss variants when targeting single tumor antigens. We address both limitations with a novel approach of concomitant transfer of hematopoietic stem and progenitor cells (HSC) with adoptive cell therapy in two preclinical models of BSG.
METHODS
We use syngeneic models of brain stem glioma, one with wildtype H3.3 and another that harbors the H3.3K27M mutation. Tumor cells were implanted into the brain stems of mice. HSCs were isolated from bone marrow. Tumor-reactive T cells were generated ex vivo against each tumor line by employing dendritic cells pulsed with tumor-RNA. Tumor-reactive T cells were adoptively transferred into tumor bearing mice with or without HSC co-transfer.
RESULTS
The efficacy of adoptive cell therapy was significantly increased in cohorts that received HSC co-transfer against both brain stem glioma models. Interestingly, T-cell infiltration into BSGs was only detected in cohorts that received HSC co-transfer. In addition, our observations provide evidence that HSCs co-transferred with IFNγ-secreting anti-tumor T cells differentiate into professional antigen presenting cells as characterized by CD11c, MHC-II, and a lack of MDSC markers. These cells then mount the capacity to capture and cross-prime tumor-derived antigens to both adoptively transferred T cells and host T cells, addressing the diversity of antigens within brain tumors.
Abstract
Glioblastoma (GBM) remains a deadly disease with an overall survival of 18 months. Despite advances in cancer immunotherapy, especially in the context of solid tumors derived outside of the ...central nervous system, GBM remains difficult to treat. This may be due, in part, to reduced T cell function and expansion of suppressive myeloid cells within the periphery. Here we sought to determine if GBM impacts the phenotype and gene expression of progenitor populations within the bone marrow where hematopoietic stem and progenitor cells (HSPCs) originate.
Using RNAseq, we found HSPCs derived from intracranial glioma-bearing mice possess altered gene expression relative to HSPCs derived from non-tumor-bearing mice. In addition, we found glioma-bearing mice possess an expansion of myeloid-derived suppressor cells (MDSCs) within their bone marrow and are significantly more suppressive on T cell proliferation and T cell-mediated tumor cell killing than MDSCs isolated from non-tumor-bearing mice. We also determined HSPCs derived from glioma-bearing mice are more likely to differentiate into MDSCs than HSPCs derived from non-tumor-bearing mice.
Interestingly, we found components of immunotherapy are capable of redirecting cell fate differentiation of glioma-bearing HSPCs. We determined HSPCs cultured in T cell supernatants are capable redirecting their differentiation from suppressive MDSCs towards stimulatory DCs. Finally, using two murine glioma models, we found adoptive cellular therapy using HSPCs derived from glioma-bearing mice is capable of providing a similar survival benefit as adoptive cellular therapy using HSPCs derived from non-tumor-bearing mice.
Immunotherapy has been demonstrably effective against multiple cancers, yet tumor escape is common. It remains unclear how brain tumors escape immunotherapy and how to overcome this immune escape.
We ...studied KR158B-luc glioma-bearing mice during treatment with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested the immunogenicity of primary and escaped tumors using T-cell restimulation assays. We used flow cytometry and RNA profiling of whole tumors to further define escape mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T cells through the use of escape variant total tumor RNA and administered these cells as ACT. In addition, programmed cell death protein-1 (PD-1) checkpoint blockade was studied in combination with ACT.
Escape mechanisms included a shift in immunogenic tumor antigens, downregulation of MHC class I, and upregulation of checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater recognition of escaped tumors than primary tumors. When administered as ACT, these T cells prolonged median survival of escape variant-bearing mice by 60%. The rational combination of ACT with PD-1 blockade prolonged median survival of escape variant glioma-bearing mice by 110% and was dependent upon natural killer cells and T cells.
These findings suggest that the immune landscape of brain tumors are markedly different postimmunotherapy yet can still be targeted with immunotherapy.
Abstract
BACKGROUND
Glioma-induced immune disfunction has been described in a limited number of studies, and here we further demonstrate that gliomas also interrupt the cellular differentiation ...programming and outcomes of hematopoietic stem cells (HSC) in the bone marrow. HSCs from glioma-bearing mice are re-programmed and driven towards expansion of myeloid lineage precursors myeloid-derived suppressor cells (MDSC) in secondary lymphoid organs. However, we found this is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of CNS malignancies, and here we describe how glioma-induced disfunction is reversed by this immunotherapeutic platform.
METHODS
The impact of orthotopic KR158B glioma on HSCs was evaluated in an unbiased fashion using single cell RNAseq of lineage- cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor-specific dendritic cells, tumor-reactive T cells, and HSCs isolated from glioma-bearing or non-tumor-bearing mice was used to evaluate cell fate differentiation and survival.
RESULTS
We observed differential gene expression of HSCs in tumor-bearing versus healthy mice coupled with an expansion of myeloid lineage subsets in glioma-bearing mice. Interestingly, MDSCs from glioma-bearing mice demonstrated hyper suppressive capacity as compared to MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame the suppressive properties of glioma-bearing HSCs. When HSCs from tumor-bearing mice were transferred with ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.
CONCLUSIONS
These findings demonstrate the suppressive disposition in HSCs from glioma-bearing hosts can be redirected using ACT for significant survival benefit. Collectively, these data demonstrate gliomas impact the hematopoietic compartment at the progenitor level to promote expansion of suppressive myeloid cells and their progenitors. However, ACT can overcome this bias and provide lasting anti-tumor effects.
Abstract
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.
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Background: Despite aggressive clinical interventions, glioblastoma (GBM) remains almost universally fatal. In a pilot, randomized, and blinded clinical trial, we recently ...demonstrated that administration of RNA-loaded dendritic cell (DC) vaccines was associated with significantly improved progression-free and overall survival in patients with GBM (Mitchell et al, Nature 2015). Furthermore, clinical outcomes correlated with migration of Indium-111-labeled DCs to vaccine-site draining lymph nodes (LNs) measured by SPECT/CT imaging. While these studies demonstrated that DC migration may be an important clinical biomarker for response to DC vaccination, the complexity and regulatory requirements associated with nuclear labeling to track DC migration limits widespread application of this technique. We have therefore developed RNA-loaded magnetic nanoparticles (RNA-NPs) to enhance DC migration to LNs and track that migration with a widely available imaging modality (i.e. MRI). Methods: Cationic liposomes were loaded with iron oxide nanoparticles with or without cholesterol modification. The resulting nanoparticles were complexed with RNA and used to transfect DCs ex vivo. RNA-NP-loaded DsRed+ DCs were then injected intradermally into mice and tracked noninvasively with T
2
-weighted 11T MRI before excision and quantification with flow cytometry. Results: In vitro experiments demonstrate that iron oxide loading does not reduce RNA-NP-mediated transfection of DCs. Additionally, replacement of cationic lipids with cholesterol increased RNA-NP transfection of the DC2.4 cell line and enhanced the T cell stimulatory capacity of treated bone marrow-derived dendritic cells (BMDCs). Compared to electroporation, RNA-NPs enhanced DC migration to lymph nodes and reduced T
2
MRI intensity in DC-bearing lymph nodes. Conclusions: This data suggests that iron oxide-loaded RNA-NPs enable noninvasive cell tracking with MRI and enhance DC migration to lymph nodes. We have further shown that inclusion of cholesterol in RNA-NPs augments the stimulatory capacity of transfected DCs. Future work will consider effects of RNA-NPs on antitumor immune responses and the utility of MRI-detected DC migration as a biomarker of vaccine efficacy.
Abstract
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.
Abstract
BACKGROUND
The blood-brain barrier (BBB) remains a potent obstacle for development of new therapies against glioblastoma (GBM). While activated T cells can cross the BBB, immunotherapy has ...yet to be fully unlocked for malignant brain tumors due their heterogeneity and immunosuppressive microenvironments. To overcome these challenges, our group has developed a novel treatment platform, which leverages the use of a clinically translatable nanoparticles (NPs) combined with personalized tumor derived mRNA to peripherally activate T cells against a heterogenous source of tumor antigens and reprogram the intratumoral milieu into an immune activated state.
OBJECTIVE
We sought to assess if RNA-NPs could activate systemic/intratumoral dendritic cells (DCs) and mediate a peripheral T cell response that could penetrate the GBM microenvironment.
RESULTS
We uncovered that RNA-NPs elicit potent innate immunomodulating effects through release of interferon-α (IFN-α) from plasmacytoid DCs (pDCs). After only a single RNA-NP vaccine, the bulk of systemic and intratumoral DCs in mice display an activated phenotype; DCs, harvested from intracranial tumors, elicit expansion of antigen specific T cell immunity. Tumor-specific RNA-NPs elicited enhanced survival outcomes in immunocompetent animals bearing NF-1/p53 mutant gliomas with increased intratumoral memory CD8+T-cells. Unlike immune checkpoint blockade (anti-PD-L1 mAbs), we found that RNA-NPs increase LFA-1 on peripheral CD8 splenocytes, which is necessary for activated T cell passage across the BBB. RNA-NPs also increased CCR2 on peripheral CD8 cells, which was dependent on IFN-α/β, as the percentage of CCR2+CD8+ splenocytes and anti-tumor activity (mediated by RNA-NPs) was abrogated in animals receiving concomitant type I IFN receptor (IFNAR1) mAbs.
CONCLUSION
Since LFA-1 is important for T cell trafficking across the BBB and CCR2 may promote chemotaxis to the brain (as GBMs are known to secrete CCL2), RNA-NPs may offer a new treatment modality and immunologic mechanism for unlocking peripheral T cell immunity against malignant gliomas.
To compare the efficacy and safety of 2 nonvalved glaucoma drainage devices (GDDs): Ahmed ClearPath (ACP) vs. Baerveldt glaucoma implant (BGI).
Single-center, retrospective, comparative study.
...Consecutive patients who underwent ACP or BGI surgery for glaucoma (250 mm
or 350 mm
models), had ≥ 6 months of follow-up, and no prior GDD implantation.
Chart review of ACP or BGI surgery in patients with glaucoma at Wills Eye Hospital (2020-2023).
The primary outcome measure was surgical failure at the end of follow-up, defined as intraocular pressure (IOP) > 21 or < 6 mmHg at 2 consecutive visits, progression to no light perception (NLP) vision, glaucoma reoperation, or implant removal. Secondary outcome measures included the rate of postoperative complications and changes in best corrected visual acuity (BCVA), IOP, and glaucoma medications.
A total of 128 eyes of 113 patients (63 ACP, 65 BGI) with similar baseline characteristics and a mean follow-up duration of 19.6 ± 10.8 (median 20.5) months were included. Surgical failure occurred in 12 eyes (9.4%) with no significant difference between ACP and BGI eyes (9.5% vs. 9.2%, respectively; P = 0.810). Reasons for failure included IOP > 21 mmHg (3/12, 25.0%), glaucoma reoperation (5/12, 41.7%), and tube removal (4/12, 33.3%). No eyes progressed to NLP vision. Kaplan-Meier survival analysis showed similar cumulative rate of surgical failure in both groups (P = 0.871). Both groups achieved significant IOP and medication reduction compared to their baseline. Final IOP, BCVA, and complication rates were similar in both groups, but medication number was significantly lower in the ACP group (P = 0.012). Both the 250 mm
and 350 mm
models had similar outcomes, but diplopia was significantly associated with the 350 mm
model of either implant (P = 0.012). Univariate logistic regression analysis did not identify either tube type or plate size as predictors of surgical failure.
This study compares the recently approved ACP vs. BGI. Both implants had similar surgical failures and complication rates. Final IOP was similar in both groups, but ACP achieved lower medication number. Diplopia was significantly associated with the use of 350 mm
model of either implant. Neither tube type nor plate size were significant predictors of surgical failure.
Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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