Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyteassociated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross blood-brain barrier (BBB). Here we describe targeted nanoscale immunoconjugates (NICs) on natural biopolymer scaffold, poly(β-L-malic acid), with covalently attached a-CTLA-4 or a-PD-1 for systemic delivery across the BBB and activation of local brain anti-tumor immune response. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) results in an increase of CD8+ T cells, NK cells and macrophages with a decrease of regulatory T cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with NIC combination is significantly longer compared to animals treated with single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of tumor-targeted polymer-conjugated checkpoint inhibitors as an effective GBM treatment via activation of both systemic and local privileged brain tumor immune response.
One
of the major problems facing the treatment of neurological
disorders is the poor delivery of therapeutic agents into the brain.
Our goal is to develop a multifunctional and biodegradable nanodrug
delivery system that crosses the blood–brain barrier (BBB)
to access brain tissues affected by neurological disease. In this
study, we synthesized a biodegradable nontoxic β-poly(l-malic acid) (PMLA or P) as a scaffold to chemically bind the BBB
crossing peptides Angiopep-2 (AP2), MiniAp-4 (M4), and the transferrin
receptor ligands cTfRL and B6. In addition, a trileucine endosome
escape unit (LLL) and a fluorescent marker (rhodamine or rh) were
attached to the PMLA backbone. The pharmacokinetics, BBB penetration,
and biodistribution of nanoconjugates were studied in different brain
regions and at multiple time points via optical imaging.
The optimal nanoconjugate, P/LLL/AP2/rh, produced significant fluorescence
in the parenchyma of cortical layers II/III, the midbrain colliculi,
and the hippocampal CA1-3 cellular layers 30 min after a single intravenous
injection; clearance was observed after 4 h. The nanoconjugate variant
P/LLL/rh lacking AP2, or the variant P/AP2/rh lacking LLL, showed
significantly less BBB penetration. The LLL moiety appeared to stabilize
the nanoconjugate, while AP2 enhanced BBB penetration. Finally, nanoconjugates
containing the peptides M4, cTfRL, and B6 displayed comparably little
and/or inconsistent infiltration of brain parenchyma, likely due to
reduced trans-BBB movement. P/LLL/AP2/rh can now be functionalized
with intra-brain targeting and drug treatment moieties that are aimed
at molecular pathways implicated in neurological disorders.
There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (a4b1g1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 a4 and b1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma. Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.
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