Deliver anti-PD-L1 into brain by p-hydroxybenzoic acid to enhance immunotherapeutic effect for glioblastoma
Graphical abstract
Introduction
Glioblastoma (GBM) is the most common and deadly primary malignant intracranial tumor. Due to its poor prognosis, the current standard care, which usually combines surgical resection and chemoradiation, rarely effectively improves the median overall survival and 5-year survival rate [1,2].
Since the lymphatic drainage system has been proven to exist in brain [3,4], immunotherapy becomes a promising approach for brain tumor treatment, especially for glioma with typical cancer-associated immunosuppression [[5], [6], [7], [8]]. In GBM, signal transducer and activator of transcription 3 (STAT3) pathway upregulates expression of interleukin-10 (IL-10), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β), but limits the capacity of dendritic cell (DC) of tumor associated antigen presentation, thus inducing recruitment of regulatory phenotype T cells (Treg) and suppressing T effector cells. Furthermore, under the impact of suppressing cytokines such as TGF-β and IFN-γ, programmed cell death 1 ligand 1 (PD-L1) is overexpressed in glioblastoma, activating programmed death 1 receptor (PD-1) and inducing the apoptosis and anergy of T effector cells. Besides, immunosuppression is not confined to tumor microenvironment [9], which might also be related to PD-L1 over expression on tumor-derived extracellular vesicles [10].
So far, extensively immunotherapeutic approaches [9] have been investigated, including immune checkpoint inhibitors [11,12], vaccines [13], chimeric antigen receptor T-cells [14] and oncolytic viruses [15], etc. Immune checkpoint blockade, by antibodies against PD-1, cytotoxic T lymphocyte antigen 4 (CTLA-4) or PD-L1, has been proven effective for many solid tumors [[16], [17], [18]]. However, in clinical trials for GBM [[19], [20], [21]], combination of nivolumab and ipilimumab ended with severe immune-related adverse effects [22,23], and avelumab (anti-PD-L1 antibody) made only negligible improvements on the progression-free survival [24], though higher PD-L1 expression is usually related with effective immunotherapy in the peripheral solid tumors [25]. In contrast, checkpoint inhibitors were partially effective for the treatment of brain metastases clinically [26,27]. Although it remains unclear for exact underlying mechanisms of such variation in clinical response. Disrupted BBB induced by metastatic lesions might be one of reasonable explanation [28]. Indeed, the major obstacle for immunotherapy for glioma is the presence of BBB that restricts the entry of checkpoint inhibitors into the brain [28,29]. So effective delivery of antibody across BBB into brain is crucial for enhancing its efficacy.
Benzamide analogues have been proven to be able to cross the BBB mediated by D1 and D2 dopamine receptors [30] that prominently exist in most parts of the central nervous system. p-Hydroxybenzoic acid (pHA), one of benzamide analogues, has been successfully applied as the brain-targeting ligand for delivery systems [31,32].
In the present study, the conjugate of anti-PDL1 antibody and pHA was designed for immunotherapy of glioma (Fig. 1), which was expected to have affinity for CD274 protein (PD-L1), and capability of BBB-crossing, to provide new possibility of macromolecules for treatment of brain disease.
Section snippets
Materials, mice and cell lines
The anti-PDL1 antibody (αPDL1) was purchased from Biolegend (Clone: 10F.9G2, USA). p-Hydroxybenzoic acid (pHA) was from Sigma-Aldrich (St.Louis, Mo). 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC) was provided by Meilunbio (China). Spin Desalting Columns (7 K MWCO) were purchased by Thermo Fisher (USA). CD274 protein was purchased from Sino Biological (China). Dopamine (DA) was purchased from Meilunbio (China). Murine IFN-gamma was
Preparation and characterization of pHA-αPDL1 conjugate
The pHA-αPDL1 conjugate was synthesized via covalently conjugation of the activated amino group in αPDL1 with the sulfhydryl group introduced in pHA via the maleimide linker (Fig. 2A). The sulfhydryl group of pHA was attached with exposed maleimide groups of the activated antibody. MALDI-TOF (Fig. 2B) showed that the conjugation was successful with one monoclonal αPDL1 modified with an average of six pHA molecules. Furthermore, the increase in the molecular weight displayed by SDS-PAGE (Fig. 2
Conclusion
pHA-αPDL1 was developed to deliver anti-PD-L1 antibody into brain for treatment of glioma. pHA decoration on αPDL1 did not impair its binding affinity with PD-L1 protein and its capability of blocking immune checkpoints. Addition of brain-targeting properties assisted αPDL1 to cross BBB, consequently increasing its distribution into the brain as well as the glioma. Furthermore, intravenous pHA-αPDL1 administration prolonged the survival time of glioma-bearing mice by alleviating
Acknowledgements
This work was supported by National Natural Science Foundation of China (No.81690263), Shanghai Education Commission Major Project (2017-01-07-00-07-E00052) and National Basic Research Program of China (973 Program, No.2013CB932500).
References (53)
- et al.
How immunotherapies are targeting the glioblastoma immune environment
J. Clin. Neurosci.
(2018) - et al.
Ipilimumab in patients with melanoma and brain metastases: an open-label, phase 2 trial
Lancet Oncol.
(2012) - et al.
Pembrolizumab for patients with melanoma or non-small-cell lung cancer and untreated brain metastases: early analysis of a non-randomised, open-label, phase 2 trial
Lancet Oncol.
(2016) - et al.
Towards immunotherapy for Pediatric brain Tumors
Trends Immunol.
(2019) - et al.
Design of Y-shaped targeting material for liposome-based multifunctional glioblastoma-targeted drug delivery
J. Control. Release
(2017) - et al.
Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses
Immunity
(2007) - et al.
Nanodisk-based glioma-targeted drug delivery enabled by a stable glycopeptide
J. Control. Release
(2018) - et al.
Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: distribution, dose-response, and mechanisms of delivery
J. Control. Release
(2018) - et al.
A facile approach to functionalizing cell membrane-coated nanoparticles with neurotoxin-derived peptide for brain-targeted drug delivery
J. Control. Release
(2017) - et al.
Immune checkpoint inhibitor toxicities
Mayo Clin. Proc.
(2019)