Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis.

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

Harnessing SLE Autoantibodies for Intracellular Delivery of Biologic Therapeutics.

Intracellular delivery of therapeutic antibodies is highly desirable but remains a challenge for biomedical research and the pharmaceutical industry. Approximately two-thirds of disease-associated targets are found inside the cell. Difficulty blocking these targets with available drugs creates a need for technology to deliver highly specific therapeutic antibodies intracellularly. Historically, antibodies have not been believed to traverse the cell membrane and neutralize intracellular targets. Emerging evidence has revealed that anti-DNA autoantibodies found in systemic lupus erythematosus (SLE) patients can penetrate inside the cell. Harnessing this technology has the potential to accelerate the development of drugs against intracellular targets. Here, we dissect the mechanisms of the intracellular localization of SLE antibodies and discuss how to apply these insights to engineer successful cell-penetrating antibody drugs.

Pro-inflammatory and proliferative microglia drive progression of glioblastoma.

Scant understanding of the glioblastoma microenvironment and molecular bases hampers development of efficient treatment strategies. Analyses of gene signatures of human gliomas demonstrate that the SETD2 mutation is correlated with poor prognosis of IDH1/2 wild-type (IDH-WT) adult glioblastoma patients. To better understand the crosstalk between SETD2 mutant (SETD2-mut) glioblastoma cells and the tumor microenvironment, we leverage single-cell transcriptomics to comprehensively map cellular populations in glioblastoma. In this study, we identify a specific subtype of high-grade glioma-associated microglia (HGG-AM). Further analysis shows that transforming growth factor (TGF)-ß1 derived from SETD2-mut/IDH-WT tumor cells activates HGG-AM, exhibiting pro-inflammation and proliferation signatures. Particularly, HGG-AM secretes interleukin (IL)-1ß via the apolipoprotein E (ApoE)-mediated NLRP1 inflammasome, thereby promoting tumor progression. HGG-AM present extensive proliferation and infiltration to supplement the activated microglia pool. Notably, TGF-ß1/TßRI depletion dramatically reduces HGG-AM density and suppresses tumor growth. Altogether, our studies identify a specific microglia subpopulation and establish the cellular basis of interactions between HGG-AM and glioblastoma cells.

EZH2 Phosphorylation Promotes Self-Renewal of Glioma Stem-Like Cells Through NF-κB Methylation.

Cancer stem-like cells (CSCs) is a cell population in glioma with capacity of self-renewal and is critical in glioma tumorigenesis. Parallels between CSCs and normal stem cells suggest that CSCs give rise to tumors. Oncogenic roles of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) have been reported to play a crucial role in glioma tumorigenesis. Herein, we focus on mechanistic contributions of downstream molecules to maintaining stemness of glioma stem-like cells (GSCs). Transcriptional factor, NF-κB, co-locates with MELK/EZH2 complex. Clinically, we observe that the proportion of MELK/EZH2/NF-κB complex is elevated in high-grade gliomas, which is associated with poor prognosis in patients and correlates negatively with survival. We describe the interaction between these three proteins. Specifically, MELK induces EZH2 phosphorylation, which subsequently binds to and methylates NF-κB, leading to tumor proliferation and persistence of stemness. Furthermore, the interaction between MELK/EZH2 complex and NF-κB preferentially occurs in GSCs compared with non-stem-like tumor cells. Conversely, loss of this signaling dramatically suppresses the self-renewal capability of GSCs. In conclusion, our findings suggest that the GSCs depend on EZH2 phosphorylation to maintain the immature status and promote self-proliferation through NF-κB methylation, and represent a novel therapeutic target in this difficult to treat malignancy.

Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma.

Purpose: The role of cholesterol biosynthesis in hedgehog pathway activity and progression of hedgehog pathway medulloblastoma (Hh-MB) were examined in vivo Statins, commonly used cholesterol-lowering agents, were utilized to validate cholesterol biosynthesis as a therapeutic target for Hh-MB.Experimental Design: Bioinformatic analysis was performed to evaluate the association between cholesterol biosynthesis with hedgehog group medulloblastoma in human biospecimens. Alterations in hedgehog signaling were evaluated in medulloblastoma cells after inhibition of cholesterol biosynthesis. The progression of endogenous medulloblastoma in mice was examined after genetic blockage of cholesterol biosynthesis in tumor cells. Statins alone, or in combination with vismodegib (an FDA-approved Smoothened antagonist), were utilized to inhibit medulloblastoma growth in vivoResults: Cholesterol biosynthesis was markedly enhanced in Hh-MB from both humans and mice. Inhibition of cholesterol biosynthesis dramatically decreased Hh pathway activity and reduced proliferation of medulloblastoma cells. Statins effectively inhibited medulloblastoma growth in vivo and functioned synergistically in combination with vismodegib.Conclusions: Cholesterol biosynthesis is required for Smoothened activity in the hedgehog pathway, and it is indispensable for the growth of Hh-MB. Targeting cholesterol biosynthesis represents a promising strategy for treatment of Hh-MB. Clin Cancer Res; 24(6); 1375-88. ©2018 AACR.

Restore the brake on tumor progression.

Sonic hedgehog (Shh) signaling plays a key role in regulation of normal development. The negative feedback mechanism mediated by the transcriptional factor, Gli3, acts to finely tune Shh signaling, providing tight control of normal developmental processes. Hyperactivation of Shh signaling often leads to many human malignancies, including basal cell carcinoma and medulloblastoma (MB). However, how tumor cells sustain the aberrant activation of Shh signaling is still not completely understood. We recently revealed that during MB formation, tumor cells express Nestin, a type VI intermediate filament protein, which maintains uncontrolled Shh signaling by abolishing negative feedback by Gli3. Therefore, Nestin expression is a necessary step for MB formation. These findings highlight the novel function of Nestin in regulating Shh signaling, as well as the important role of a disrupted negative feedback mechanism in MB tumorigenesis. Further, restoration of the intrinsic negative feedback by repressing Nestin expression represents a promising approach to treat MB as well as other Shh signaling associated malignancies.

Astrocytes Promote Medulloblastoma Progression through Hedgehog Secretion.

Astrocytes, the most abundant type of glial cells in the brain, play critical roles in supporting neuronal development and brain function. Although astrocytes have been frequently detected in brain tumors, including medulloblastoma (MB), their functions in tumorigenesis are not clear. Here, we demonstrate that astrocytes are essential components of the MB tumor microenvironment. Tumor-associated astrocytes (TAA) secrete the ligand sonic hedgehog (Shh), which is required for maintaining MB cell proliferation despite the absence of its primary receptor Patched-1 (Ptch1). Shh drives expression of Nestin in MB cells through a smoothened-dependent, but Gli1-independent mechanism. Ablation of TAA dramatically suppresses Nestin expression and blocks tumor growth. These findings demonstrate an indispensable role for astrocytes in MB tumorigenesis and reveal a novel Ptch1-independent Shh pathway involved in MB progression. Cancer Res; 77(23); 6692-703. ©2017 AACR.

Nestin Mediates Hedgehog Pathway Tumorigenesis.

The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here, we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation, resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Cancer Res; 76(18); 5573-83. ©2016 AACR.