Impact of blood-brain barrier permeabilization induced by ultrasound associated to microbubbles on the brain delivery and kinetics of cetuximab: An immunoPET study using ⁸⁹Zr-cetuximab

Highlights

• FUS and MB significantly increase the delivery of CTX across the BBB.

• PET is a powerful method to assess the PK of the passage of mAb through the BBB.

• ⁸⁹Zr-DFO-CTX was used to follow quantitatively enhanced brain exposure to CTX.

• ImmunoPET-combined FUS is a clinically relevant strategy for GBM innovative therapy.

Abstract

Epidermal growth factor receptor (EGFR), involved in cell proliferation and migration, is overexpressed in ~50% of glioblastomas. Anti-EGFR based strategies using monoclonal antibodies (mAb) such as cetuximab (CTX) have been proposed for central nervous system (CNS) cancer therapy. However, the blood-brain barrier (BBB) drastically restricts their brain penetration which limits their efficacy for the treatment of glioblastomas. Herein, a longitudinal PET imaging study was performed to assess the relevance and the impact of focused ultrasound (FUS)-mediated BBB permeabilization on the brain exposure to the anti-EGFR mAb CTX over time. For this purpose, FUS permeabilization process with microbubbles was applied on intact BBB mouse brain before the injection of ⁸⁹Zr-labeled CTX for longitudinal imaging monitoring. FUS induced a dramatic increase in mAb penetration to the brain, 2 times higher compared to the intact BBB. The transfer of ⁸⁹Zr-CTX from blood to the brain was rendered significant by FUS (k_uptake = 1.3 ± 0.23 min⁻¹ with FUS versus k_uptake = 0 ± 0.006 min⁻¹ without FUS). FUS allowed significant and prolonged exposure to mAb in the brain parenchyma. This study confirms the potential of FUS as a target delivery method for mAb in CNS.

Introduction

Epidermal growth factor receptor (EGFR) plays a key role in essential cellular functions that include proliferation and migration. EGFR signaling mediates oncogenic progression and metastasis in several human malignancies including peripheral and CNS cancers such as glioma [1] [2]. EGFR vIII (deletions of exons 2–7) is the most prevalent mutation observed for EGFR. EGFR vIII is expressed in ~50% of glioblastomas which amplifies the expression of the wild-type protein [3]. Overexpression of EGFR is associated with poor prognosis in glioma patients [4]. The EGFR pathway is therefore regarded as a promising therapeutic target against CNS malignancies. In peripheral cancer, anti-EGFR monoclonal antibodies (mAb) were shown to inhibit cell proliferation, enhance apoptosis, and reduce angiogenesis, invasiveness and metastasis [5]. Anti-EGFR mAbs such as cetuximab (CTX) are active against EGFR vIII and prevent the binding of endogenous ligand [6]. Although anti-EGFR mAbs show favorable effects in colorectal, head and neck and non-small cell lung cancers, clinical trials using CTX in patients with glioblastoma did not show significant improvement over standard of care regimens [7,8]. The blood-brain barrier (BBB) is the main obstacle limiting the passage of therapeutics into the brain [9]. Insufficient exposure of CNS tumors to CTX across the BBB is widely assumed to explain the limited therapeutic efficacy.

For glioma, the abnormal tumor vasculature issued from the overproduction of proangiogenic factors leads to a compromised blood-tumor barrier (BTB) which is assumed to allow the extravasation of small and large molecules [10]. However, in reality, the penetration of drugs into the compromised BTB is very low, leading to inefficient tumor treatment. In early stages of glioblastoma (GBM), tumor own vasculature is not yet very well developed and the BTB is apparently not disrupted. At theses stages, BTB resembles the BBB and prevents efficient passage of cancer therapeutics, including small molecules and antibodies [11]. Moreover as glioma progresses, BTB presents some degree of heterogeneity i.e. all glioma have clinically significant regions of tumor with an intact BBB limiting the diffusion of treatment [12,13]. As mAb can be blocked from reaching the areas where the BBB remained intact, there is a clear need for strategies that can deliver immunotherapeutic agents into tumors.

The reigning paradigm to overcome this blockage shifted to the establishment of a safe and effective method to improve the brain delivery of mAb. Recent preclinical and clinical studies based on osmotic BBB disruption using intra-arterial mannitol infusion provided encouraging results in terms of improved drug delivery and safety, nevertheless these methods do not allow for controlled and localized BBB permeabilization [14]. The combination of injected microbubbles with low intensity focused ultrasound (FUS) is the most advanced method for controlled, minimally-invasive and transient BBB disruption in vivo [[15], [16], [17]]. Low acoustic pressures, produced by FUS, induce microbubble oscillations (alternation of expansion and shrinkage of microbubbles) near the vessel wall that can result in tight junction loosening due to local stress via push-pull mechanisms or microstreaming [18]. In preclinical model, FUS was recently shown to lead to a 3.5 fold increase in the brain concentrations of mAb [19]. However, current methods used to quantify drug concentrations in the brain are invasive or destructive (mass spectrometry, Elisa assay, HPLC). Non-invasive imaging technique such as magnetic resonance imaging (MRI) or positron emission tomography (PET) have therefore been developed to address the brain uptake of labeled mAbs, thus paving the way for the translational study of the neuropharmacokinetics of biologics [20,21]. However, to our knowledge, no in vivo imaging was proposed to study brain mAb concentrations over a prolonged time to provide an in-depth understanding of the neuropharmacokinetics and overall brain exposure to mAbs in vivo.

In the present study, the immunoPET paradigm was used to evaluate over time (up to 1 week) the impact of FUS-induced BBB opening on the delivery of CTX to the whole brain in mice with intact BBB. We used the long half-life (t_1/2 = 78.4 h) radionuclide zirconium 89 (⁸⁹Zr) which is relevant to the biological half-life of mAbs. In this work, kinetic modeling of the brain delivery of ⁸⁹Zr-CTX was performed to describe the initial transfer rate of CTX across the BBB and to address its retention by the brain over time.

We have found that FUS can increase significantly the uptake and the transfer rate of labeled mAb. Permeabilized ⁸⁹Zr-DFO-CTX stayed at the proximity of the FUS field for up to 72 h which further consolidate the rationale of using FUS as a controlled delivery method and ⁸⁹Zr PET imaging as a suitable monitoring technique.