D Taquet
email: dtaquet@hakodate.kosen-ac.jp

M Ono

M Krause-Ono

M Kuriyama

Developing Cross-Cultural Awareness in Provincial Hokkaido by Connecting Exchange Students and Kosen Japanese Students

Keywords: international exchange, intercultural communication, global studies in provincial areas, collaborative learning between Japanese and foreign students

Japan has been able to greatly increase the number exchange students in higher education, as a way to improve its global ranking and competitiveness on the international scene. As of May 2018, there were 298,980 international students in Japanese higher education and language institutes. Through various coordinated efforts, the government has so far been successful in reaching a steady increase of 12% each year since 2015, based on the JASSO (Japan Services Student Organization) survey. However, not all regions or prefectures are equal in this effort. Indeed, data show a great discrepancy between urban areas, and provincial Japan, the Kanto region, and unsurprisingly the greater Tokyo metropolitan area, concentrating close to half the total population of international students. As teachers in the Hokkaido area, that only hosts 1.7% of those students, our goal with this practical research is to provide more opportunities for local students to interact with their international counterparts. As cross-cultural understanding, and development of autonomous communication methods, English is a tool, not a finality. The three-step approach, spread over 3 years, consists first in 2-day seminars, then written communication using Moodle chatrooms, and finally group videolink discussions with Skype or GInet. In this presentation, we will focus on the rationale behind the project, the challenges we have encountered and how the use of technologies have provided some answers for the development of a better, and more effective framework. The authors of this study believe it will provide valuable data and a solid framework that could be applied in other technical institutes all through Japan.

Introduction

Globalization of technical education and crosscultural communication have been bustling concepts in the technical programs all over the world for the past few years. However, in order to develop such skills, and ambitions, Japanese students must first be accustomed to communicating with foreigners, to apply their classroom knowledge to practical communicative situations. This opportunity is readily available in large Japanese cities, or large universities which have a large foreign exchange student body, but what can be done for smaller cities and universities? The goals of the present educational research were to provide Japanese students with opportunities to get engaged into multicultural, multiracial, and multi-lingual environment. Furthermore, for the purpose of this study English is not considered an objective, but a tool among others to successfully achieve cross-cultural understanding. Finally, in a globalized, ultra-connected world, the constant flow of information and news makes it hard, and sometimes impossible to identify, and retain important world events, and crosscultural affairs. This project also put the emphasis on urbanism, environmental issues, and disaster preventions in several countries.

Objectives

This research group was formed through a clear objective: analyse, encourage, and develop best practices for cross-cultural communication through a variety of methods. We based our methods and efforts on an approach that was previously conducted by [START_REF] Ishikawa | International Video Conferencing Classes with Communication-Based Activities (Japanese)[END_REF] to ensure access to direct intercultural exchange for students located in regional areas.

The area we are focusing on in this research is located between the southern and central parts of Hokkaido, where are based the three higher education institutions our team members work at: NIT Hakodate College, Muroran Institute of Technology, and NIT Tomakomai College (Figure 1). Hokkaido, the northernmost island of Japan, and by far the largest prefecture, is only home to 5.4 million people, for a density of 64.5 people per inhabitable km2. As point of reference, Tokyo's density is 6168, Osaka is 4639.

With only 1.7% of Japan's total number of exchange students, and a density just 1% percent of that of Tokyo, we can understand that the chances of interactions are few and far between. Even then, most of Hokkaido exchange students are centralized at Hokkaido University in Sapporo, which gathers 2349 of the 3614 foreign students in the prefecture.

As such, we can see the importance of developing such opportunities for communication in rural areas such as ours. The timeframe of this research was divided into 3 distinct parts.

Educational Practices Step 1: On-Site Seminars

First, and what we felt what the most important, were the camp training lessons. There, Japanese students were invited along their international counterparts to two onsite educational gatherings.

The first seminar was held in the sounthernmost city of the group, Hakodate.

The seventeen participants, who had been recruited from each of the institutes, represented five countries: China, Cambodia, Japan, Malysia, and Mongolia. The engineering field were also varied: chemistry, production systems, mechanical, civil, electrical, and information science. (Figure 2).

Held during students' free-time in early December 2017, the main theme of the seminar was the introduction to cross-cultural communication, along with with a lecture comparing urbanism and city development between the three research cities (Hakodate, Muroran, and Tomakomai). This lecture was given in Japanese by M. Kuriyama, professor of civil engineering in the deprtament for engineering innovation at NIT Tomakomai. The students, divided into groups, also had an opportuinity to reflect upon stereotypes, and how they influence communication.

Figure 2. Students presenting some aspects of their cultural background in Hakodate.

The second on-site seminar took place during a 3-day weekend in October 2018 in Otaki seminar, Date city, west of Tomakomai. The theme of that seminar was one that echoed with all students-Japanese and foreign-Disasters; their prevention, human causes, and effect they have on city planning and environment. 19 students from the following areas were represented: India, Indonesia, Japan, Malaysia, Mongolia, Thailand, Vietnam, each group being led by a PhD student from that country, who were studying at MIT. Although group discussions were conducted in Japanese and English, students had to focus on creating a presentation in English. The educational practice was two-fold: visiting a location that had been hit by a disaster near Toya Lake, and for students to use their engineering skills to discuss and reflect upon disaster prevention methods in the four countries (Figure 3). The discussions had all the more impact that it took place a few weeks after the large 6.6 magnitude hokkaido earthquake on September 6th 2018. Most students, both Japanese and international, were in Hokkaido during the disaster and were able to share their impressions, memories, and fears about the earthquake.

Students were also taken to lake Toya, famous for its beautiful scenery but also for bearing the marks of a recent volcanic erruption. Indeed, Mount Usu errupted in 2000 spewing burning gases and mud. It was the mud slides that had the most impact on local towns, destroying whole roads and buildings on their path (Nakano). Although no one died, the damage incurred was estimated to be 10.3 billion yen (approx, 92 million US dollars), and 4700 inhabitant of nearby municipalities had to be evacuated (Figure 4). Students participating in the research groups were able to see first-hand the immense power and potential for destruction that such disaster could bring to nearby locations. It also motivated them to create posters describing environmental issues in the 4 countries of the PhD students, who led each group (Figure 5).

Educational Practices Step 2: Moodle Communication

Once the on-site camps have been completed, our next task was set up a moodle-like textchat system for students to participate. Indeed, creating a balance between teacher control and student autonomy, a moodle-like system will not only help them improve student's comprehensive language ability, but also their autonomy, a key-component in learners' motivation, through peer communication [START_REF] Ma | The Application of Moodle-Based Platform in College English Learning[END_REF]. Unfortunately, and quite ironically, while setting the communication modules was systematic, the authors did not anticipate that motivating the foreign students to join, and participate would be much more challenging a task. Some reasons include, but may not be limited to: school/university calendar differences, different approach to digital literacy, lack of motivation, different culture.

Future Practices

At the time of this paper's submission, the authors are planning another on-site training that will also include digital literacy reinforcement. Indeed, a group of Italian students from a technical high-School coming to NIT Hakodate College in early September of 2019, will participate in a seminar in Otaki.

Furthermore, the final stage in our three-step objective will be to create video-links using BlueJeans or Skype, giving more autonomy to students, reinforcing their motivation, and thus creating a long-lasting communication line between the Japanese and foreign students. As Information and Communication Technologies (ICT) are used and developed differently in Italy and Japan, putting these protocols in place will require precise communication, and dedication between the teachers and students of the various schools and countries.

We are hoping to gather empirical data on students' motivations through questionnaire, thus leaving the possibility for further improvement in a follow-up research.

Conclusions

The objectives of this research were quite ambitious to begin with. We wanted to offer Japanese students from provincial areas an opportunity, a gateway to crosscultural understanding, while expanding their horizons concerning issues that future technicians and engineers will need to focus on and solve: environmental changes, and disaster preventions. The first part of the project was succesful, but a lack of coordination between group members, and difference in school calendars made participation to digital communication harder to set up and enact. Further reflection is needed to analyze, and overcome the logistical challenges.

Figure 1 .

 1 Figure 1. Location of the research team members.

Figure 3 .

 3 Figure 3. One of the groups brainstorming ideas for their poster session. 3 countries were represented in that group (Japan, Thailand, and Mongolia).

Figure 4 .

 4 Figure 4. A residential building, swept by a mudslide near Toya Lake.

Figure 5 .

 5 Figure 5. Group picture with students holding their posters.

Acknowledgements

This research has been funded through the JSPS KAKENHI program, Grant Number JP17K02921


Céline Chevaleyre

Anthony Novell

Nicolas Tournier

Ambre Dauba

Steven Dubois

Dimitri Kereselidze

Erwan Selingue

Benoit Jego

Bernard Maillère

Benoit Larrat

Hervé Nozach

Charles Truillet
email: charlestruillet@universite-paris-saclay.fr

Efficient PD-L1

Keywords: ImmunoPET, therapeutic ultrasound, Fc receptor, neonatal, PD-L1, Immune Checkpoint Inhibitors, Glioblastoma

INTRODUCTION

Patients diagnosed with glioblastoma (GBM) have a harrowing overall survival of 13 to 16 months following the standard-of-care treatments [START_REF] Mooney | Current Approaches and Challenges in the Molecular Therapeutic Targeting of Glioblastoma[END_REF]. GBM is the most common primary brain tumor and is highly aggressive. Immunotherapies have the possibility to be a keystone in eradicating GBM as their microenvironment is immunosuppressive. Ex vivo staining of biopsies have shown that the immune checkpoint programmed death ligand 1 (PD-L1) is expressed by most GBM neoplastic tissues, which suggests a promising role for immune checkpoints inhibitors (ICI) such as anti-PD(L)1 antibodies [START_REF] Berghoff | Programmed death ligand 1 expression and tumorinfiltrating lymphocytes in glioblastoma[END_REF].

However, the efficacy of ICI in treating brain malignancies such as GBM has been proven insufficient to improve patient overall survival [START_REF]An Investigational Immuno-therapy Study of Temozolomide Plus Radiation Therapy With Nivolumab or Placebo, for Newly Diagnosed Patients With Glioblastoma (GBM, a Malignant Brain Cancer) -Study Results -ClinicalTrials[END_REF][START_REF] Reardon | Effect of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: The CheckMate 143 Phase 3 Randomized Clinical Trial[END_REF][START_REF]An Investigational Immuno-therapy Study of Nivolumab Compared to Temozolomide, Each Given With Radiation Therapy, for Newly-diagnosed Patients With Glioblastoma (GBM, a Malignant Brain Cancer) -Study Results -ClinicalTrials[END_REF]. Durations of response were nevertheless greater in patients treated with the anti-PD1 antibody nivolumab compared to those treated with the standard-of-care in the Checkmate 143 trial (NCT02017717) [START_REF] Reardon | Effect of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: The CheckMate 143 Phase 3 Randomized Clinical Trial[END_REF]. Clinical trials notably informed on the need for an effective minimally-invasive method to select patients who will benefit from ICI and monitor the immune response at the central nervous system (CNS) level. PD-L1 expression by neoplastic tissues or its microenvironment is the most predictive biomarker of anti-PD(L)1 therapy response in most tumors [START_REF] Taube | Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti-PD-1 Therapy[END_REF][START_REF] Topalian | Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer[END_REF]. It has not been investigated in GBM as PD-L1 expression is usually evaluated by immunochemistry on tumor biopsies. Moreover, PD-L1 expression is known to be spatially heterogeneous and to change over time with treatments [START_REF] Nishino | Monitoring immune-checkpoint blockade: response evaluation and biomarker development[END_REF]. Repeated biopsies over the therapy course are neither always feasible nor acceptable, particularly for CNS localizations. In this regard, non-invasive detection of biomarkers by immunoPET has emerged as a powerful tool to monitor response to ICI therapy. ImmunoPET imaging using radiolabeled antibodies targeting PD-(L)1 allows quantification and the in vivo assessment of the inter-and intratumoral heterogeneity of the biomarker expression [START_REF] Niemeijer | Whole body PD-1 and PD-L1 positron emission tomography in patients with non-small-cell lung cancer[END_REF]. However, immunoPET imaging within the CNS remains challenging due to the inability of antibodies to cross the blood-brain barrier (BBB). Niemeijer et al. and Nienhuis and al. obtained uneven accumulation between lesions of the anti-PD1 antibody 89 Zr-nivolumab or 18 F-adnectins targeting PD-L1 in brain metastases of patients [START_REF] Niemeijer | Whole body PD-1 and PD-L1 positron emission tomography in patients with non-small-cell lung cancer[END_REF][START_REF] Nienhuis | F-BMS986192 PET Imaging of PD-L1 in Metastatic Melanoma Patients with Brain Metastases Treated with Immune Checkpoint Inhibitors: A Pilot Study[END_REF]. This uptake in some brain metastases but not all within a patient probably reflects the tumor-induced loss in the integrity of the BBB rather than the local expression of targeted biomarkers. It is therefore essential to propose strategies to overcome the BBB and improve the brain kinetics of radiolabeled antibodies to enable quantitative estimation of immune biomarkers such as PD-L1 in infiltrative brain tumors like GBM using immunoPET [START_REF] Van Tellingen | Overcoming the blood-brain tumor barrier for effective glioblastoma treatment[END_REF].

The engineering of antibodies is currently investigated to improve their brain delivery [START_REF] Kouhi | Brain Disposition of Antibody-Based Therapeutics: Dogma, Approaches and Perspectives[END_REF]. This includes modulation of neonatal Fc receptor (FcRn) mediated transcytosis. Reducing the affinity of an antibody for the FcRn has notably been proposed to optimize the peripheral and brain kinetics of radiolabeled antibodies for immunoPET [START_REF] Kenanova | Tailoring the Pharmacokinetics and Positron Emission Tomography Imaging Properties of Anti-Carcinoembryonic Antigen Single-Chain Fv-Fc Antibody Fragments[END_REF]. According to the tissue considered, FcRn is responsible for the recycling and transcytosis of the Fc-containing proteins [START_REF] Pyzik | The Neonatal Fc Receptor (FcRn): A Misnomer? Front Immunol[END_REF]. FcRn's physiological function is to bind endogenous immunoglobulin G (IgG) and albumin at acidic pH to protect them from lysosomal degradation, maintaining their serum homeostasis [START_REF] Roopenian | FcRn: the neonatal Fc receptor comes of age[END_REF]. The loss of affinity for the FcRn obtained by substituting two key amino acids of the Fc-domain of an IgG (H310A and H435Q) decreases the plasma half-life of antibodies [START_REF] Kim | Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn[END_REF][START_REF] Bouleau | Optimizing immunoPET imaging of tumor PD-L1 expression: pharmacokinetics, biodistribution and dosimetric comparisons of 89Zr-labeled anti-PD-L1 antibody formats[END_REF]. Besides the advantages for PET imaging, the affinity loss for the FcRn could impact IgG brain distribution. FcRn is highly expressed by the CNS endothelium [START_REF] Schlachetzki | Expression of the neonatal Fc receptor (FcRn) at the bloodbrain barrier[END_REF]. Although there has yet to be a consensus regarding the role of FcRn in controlling IgG transport across brain endothelial cells. Some studies on the brain distribution of IgG in FcRn knock-out mice led to the conclusion of an absence of a FcRn mediated transcytosis at the BBB [START_REF] Chen | The effect of the neonatal Fc receptor on human IgG biodistribution in mice[END_REF][START_REF] Abuqayyas | Investigation of the role of FcγR and FcRn in mAb distribution to the brain[END_REF]. However, those studies are subject to discussion due to potential compensatory mechanisms associated with FcRn depletion. Moreover, some evidence supports a FcRn-mediated efflux of IgG from the brain to the circulation. After intracranial injection, brain clearance of IgG with improved FcRn affinity was faster compared with IgG with reduced FcRn affinity [START_REF] Cooper | Efflux of monoclonal antibodies from rat brain by neonatal Fc receptor, FcRn[END_REF]. This property was used effectively to promote IgG-mediated amyloid plaque removal in a mouse model of Alzheimer's disease [START_REF] Deane | IgG-Assisted Age-Dependent Clearance of Alzheimer's Amyloid β Peptide by the Blood-Brain Barrier Neonatal Fc Receptor[END_REF]. To our knowledge, the particular role of FcRn at the blood-tumoral barrier (BTB) has never been investigated.

Transcranial application of low-intensity focused ultrasound (FUS) with the injection of microbubbles (MB) was shown to enable the brain delivery of drugs by reversibly disrupting the BBB. Local mechanic and shear stress induced by the oscillations of MB (alternation of expansion and compression of the gaseous core of the MB) loosen the tight junctions and increase the pinocytic activity of endothelial cells [START_REF] Dauba | Recent Advances on Ultrasound Contrast Agents for Blood-Brain Barrier Opening with Focused Ultrasound[END_REF][START_REF] Deprez | Opening doors with ultrasound and microbubbles: Beating biological barriers to promote drug delivery[END_REF]. FUS are investigated in preclinical models and clinical trials to treat numerous CNS diseases, notably GBM [START_REF] Sun | Closed-loop control of targeted ultrasound drug delivery across the blood-brain/tumor barriers in a rat glioma model[END_REF][START_REF] Chen | Neuronavigation-guided focused ultrasound for transcranial blood-brain barrier opening and immunostimulation in brain tumors[END_REF][START_REF] Mainprize | Blood-Brain Barrier Opening in Primary Brain Tumors with Non-invasive MR-Guided Focused Ultrasound: A Clinical Safety and Feasibility Study[END_REF]. FUS-aided immunoPET was useful in demonstrating the increased brain delivery of radiolabeled antibodies from a therapeutic perspective [START_REF] Tran | 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[END_REF][START_REF] Sheybani | ImmunoPET-informed sequence for focused ultrasoundtargeted mCD47 blockade controls glioma[END_REF]. Meng et al. reported enhanced delivery of trastuzumab across the BBB with magnetic resonance-guided FUS in patients with HER2-positive breast cancer and brain metastases. In this study, the HER2-positive status of the brain metastases was determined on surgical specimens previously collected at different temporality before treatments [START_REF] Meng | MR-guided focused ultrasound enhances delivery of trastuzumab to Her2-positive brain metastases[END_REF]. The NCT05879120 clinical trial aims to estimate the median overall survival of patients with recurrent GBM with FUS BBB opening and neo-adjuvant pembrolizumab [START_REF] Anderson Cancer | Randomized Study of Neo-adjuvant and Adjuvant Pembrolizumab With and Without Targeted Blood Brain Barrier Opening Using Exablate MRI-guided Focused Ultrasound (Exablate MRgFUS) for Recurrent Glioblastoma[END_REF]. The inclusion criteria of patients in this study do not include the determination of the brain tumors' PD(L)1 status. FUS-aided immunoPET could provide a companion theranostic approach to assess the likelihood of response of brain tumors to immunotherapy.

Here, we aimed to investigate the potential of FUS-aided immunoPET to provide quantitative imaging of PD-L1 expression by glioblastoma tumors within the brain. The human recombinant IgG1, C4, targeting human and murine PD-L1 and its engineered low FcRn affinity (H310A/H435Q) mutant (C4 Fc-MUT ) were used to perform FUS-aided immunoPET in a syngeneic glioblastoma murine model (Scheme 1). Kinetic modeling was performed to interpret brain PET data, estimate PD-L1 expression, and elucidate the importance of FcRn function on the transport of the radiolabeled antibody across the BBB/BTB.

METHODS

Production of the C4 ligands. C4 is a human recombinant IgG1 cross-reacting with human and murine PD-L1 [START_REF] Truillet | Imaging PD-L1 Expression with ImmunoPET[END_REF]. The C4 ligands production has been performed as already described in Bouleau et al. [START_REF] Bouleau | Optimizing immunoPET imaging of tumor PD-L1 expression: pharmacokinetics, biodistribution and dosimetric comparisons of 89Zr-labeled anti-PD-L1 antibody formats[END_REF].

Briefly, they were obtained by transient transfection of HEK293 FreestyleTM cells (Thermo-Fisher) with AbVec2.0-IGHG1 and AbVec1.1-IGLC plasmids corresponding to the IgG C4 heavy and light chains, respectively. After transient cell transfection, the supernatant was collected and purified using Lambda FabSelect columns (GE Healthcare).

FcRn binding assay. IgG/human FcRn and IgG/murine FcRn affinity were measured using the Lumit™ FcRn Binding Immunoassay kit (Promega) following the protocol described by Nath et al. [START_REF] Nath | Deciphering the Interaction between Neonatal Fc Receptor and Antibodies Using a Homogeneous Bioluminescent Immunoassay[END_REF]. To measure IgG/mFcRn interaction, hFcRn was substituted by recombinant mFcRn with terminus biotin (ACROBiosystems). The recombinant mFcRn was introduced at a concentration of 0.25 mg/mL. Experiments were run in triplicate. Normalized luminescence data were generated by assigning 100% to the maximum bioluminescent signal obtained in the absence of IgG.

Radiolabeling. The anti-PD-L1 C4 ligands were radiolabeled according to a previously published protocol [START_REF] Bouleau | Optimizing immunoPET imaging of tumor PD-L1 expression: pharmacokinetics, biodistribution and dosimetric comparisons of 89Zr-labeled anti-PD-L1 antibody formats[END_REF]. First, the p-isothiocyanatobenzyldesferrioxamine (p-NCS-Bz-DFO, Macrocyclics) was conjugated to the C4, and then the radiolabeling with 89 Zr-oxalate (PerkinElmer) was performed. After the purification of the DFO-anti-PD-L1 ligands with a PD-10 column (GE Healthcare), DFO-anti-PD-L1 ligands were incubated with [ 89 Zr]Zr-oxalate for 1 h at 37°C. The 89 Zr-labeled DFO-anti-PD-L1 ligand conjugates were then purified with a PD-10 column and buffer exchanged in HEPES solution (Gibco) with a Vivaspin centrifugal concentrator (Sartorius). Radiochemical purity was assessed by instant thin-layer chromatography (iTLC) and high-performance liquid chromatography (HPLC) analyses (Figure S1).

Cell culture. Murine glioma's cell line GL261 transfected to produce Green Fluorescent Protein (GL261-GFP) were obtained from the Institute of Neurophysiopathology, Aix-Marseille University. Cells were cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (Gibco) at 37 °C in a humidified atmosphere containing 95% air and 5% carbon dioxide.

Animals.

Animal experiments were performed on six weeks old female C57BL/6 NRj mice (Janvier Labs).

Animal experiments were performed according to the European Directive 2010/63/EU and its transposition into French law (Decree No. 2013-118). The research project was conducted at the CEA-SHFJ imaging platform (authorization D91-471-105) and was approved by a local ethics committee (CETEA-CEA DSV IdF). Mice were housed in standard conditions (microisolator polycarbonate cages, aspen wood as bedding material, 5 mice in each cage, room temperature 22 °C, humidity 40%) under a regular 12-h dark/light cycle. Food and water were available ad libitum. GL261 orthotopic model. 24 mice were orthotopically implanted with the syngeneic cell line GL261-GFP, 5.10 4 cells in 1 µL PBS into the striatum. With bregma as origin, implantations coordinates were X= 0mm, Y=+2mm, Z=-3mm. Mice were anesthetized with isoflurane (3% for induction and 2% for maintenance) in 100% O 2 . 0.05 mg.kg -1 of buprenorphine was subcutaneously administered at the end of the intervention to prolong analgesia. MRI. 14 days after GL261 implementation, anatomical T2-weighted and T1-weighted contrast-enhanced MRI were acquired with a 7T/90mm bore hole MRI scanner (Pharmascan scanner, Bruker). A Gadoliniumbased contrast agent (Dotarem ® , 1nm diameter, 100 µL by animal) was intravenously injected via a catheter. T1-weighted images were then acquired (MSME sequence, TE/TR = 8/340 ms, matrix = 256 × 256 × 64, resolution = 0.15 × 0.15 × 0.60 mm 3 , 10 averages, acquisitions time = 6 min). T2-weigthed images were acquired through a RARE sequence (TE/TR = 5/1800 ms, RARE factor = 16, matrix = 256 × 256 × 64, resolution = 0.12 × 0.12 × 0.12 mm 3 ).

Blood-brain barrier disruption.

A focused transducer (active diameter 25 mm, focal depth 20 mm, axial resolution 5 mm, lateral resolution 1 mm, Imasonic) centered at 1.5 MHz was used to disrupt the BBB. The transducer was connected to a single-channel programmable generator (Image Guided Therapy) and mounted on a motorized XYZ-axis stage. and positioned above the mouse head maintained under anesthesia with isoflurane (3% for induction and 1.5% for maintenance) in a 50:50 mixture of air-O 2 . The device was coupled to the mouse skull using a latex balloon (filled with deionized and degassed water) and centrifugated coupling gel. The distance between the transducer and the skull was adjusted by the displacement of the motorized axis (Z) and the filling of the balloon in order to get the center of the of the brain, at the focal distance (i.e., 20 mm). SonoVue® microbubbles (Bracco) were intravenously administrated in the tail vein via a bolus (50 µL) before the beginning of the FUS or sham sessions. The FUS sequence was similar to the one described in Felix et al. and already validated for efficient and safe BBB disruption on healthy mice [START_REF] Tran | 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[END_REF][START_REF] Felix | Ultrasound-Mediated Blood-Brain Barrier Opening Improves Whole Brain Gene Delivery in Mice[END_REF]. Reversibility of the BBB opening within 24h after FUS was confirmed in mice using [ 18 F]2-fluoro-2-deoxy-sorbitol, a PET marker of BBB integrity (Figure S2).

Briefly, the FUS sequence is composed of quasi-continuous ultrasonic waves transmitted with duty cycle of 69% at a peak negative pressure of 420 kPa (considering a transmission through mouse's skull of 80% at 1.5 MHz). A raster scan (XY-axis) of 6 mm x 6 mm was synchronized to the generator output in order to induce a whole brain BBB opening (Scheme S1). The sequence of 5.1s was repeated 25 times for a total exposure of 126.75 s.

microPET/CT imaging. On day 15 post GL261-GFP implementation, a 60-min dynamic PET scan was performed concurrently with radioligand injections of 89 Zr-DFO-C4 (3.5±0.3 MBq, 4.97 MBq/nmol, n=8) or 89 Zr-DFO-C4 Fc-MUT (3.5±0.5 MBq, 9.89 MBq/nmol, n=16) under camera after the FUS protocol. The radioligand injection was performed quickly after the end (1.7 ± 0.2 min) of the FUS BBB opening protocol. 20-min static PET scans were subsequently acquired at selected times post-injection (5 h, 22 h, 46 h, 70 h, and 7 days). A sham workflow (without emission of ultrasound waves) was applied on 6 tumorbearing animals before the injection of 89 Zr-DFO-C4 Fc-MUT and imaged accordingly.

PET emission scans were performed using an Inveon microPET scanner and an Inveon microPET/CT scanner (Siemens). After each PET scan, a transmission scan or a CT scan were performed for photon attenuation correction. PET images were reconstructed with the Inveon Acquisition Workspace software (2.1) using a three-dimensional ordinary Poisson ordered-subset expectation maximization followed by a maximum a posteriori algorithm (OP-OSEM3D-MAP). Normalization, as well as corrections for dead-time, scatter, decay and attenuation, were applied to all PET data. Dynamic PET acquisitions were reconstructed in 24 frames averaging signal on the period from 0.5 to 5 min resulting in a sequence of images of 3x30, 5x60, 5x120, 3x180, 3x240, 4x300, and 1x150 s.

Image analysis was performed with the PMOD software (v3.9). A volume of interest (VOI) was defined in the left cardiac ventricle to obtain blood radioligand concentration. MRI and brain PET acquisitions were all repositioned with the T2w MRI as reference. VOI were defined in selected brain areas, defining the T1w contrast-enhanced volume, the PET contrast-enhanced volume, and the contralateral hemisphere. Concentrations in VOI are expressed as percentage of injected dose (%ID/cc = activity (Bq/cc) / injected dose (Bq)) and time activity curves (TACs) were extracted (Table S1-4).

Immunofluorescence. After the last imaging session, mice were sacrificed, and their brains were collected, immersed in isopentane, and frozen in liquid nitrogen. A set of fixed frozen brain sections (10 µm) were incubated with a rat anti-mouse PD-L1 primary antibody (1:500, clone 10F.9G2, Biolegend). The slides were then incubated with an AF546-conjugated donkey anti-rat secondary antibody (1:1000, Jackson Laboratories). Adjacent brain sections were incubated with an AF546-conjugated goat anti-human secondary antibody (1:1000, Jackson Laboratories) to stain the injected C4. Adjacent slides were used for hematoxylin/eosin (H&E) staining. Slides were fixed in neutral buffer formalin 10%, then stained with Harris hematoxylin (Sigma) and Eosin Y (Sigma) according to previously reported protocol [START_REF] Feldman | Tissue Processing and Hematoxylin and Eosin Staining[END_REF].

Immunofluorescent and H&E stained sections were scanned with a 20x objective using an AxiObserver Z1 microscope (Zeiss).

RESULTS

Characterization of the native and the mutant C4. First, we sought to verify that introducing the H310A and H435Q mutations in the Fc domain of the C4 anti-PD-L1 antibody (C4 Fc-MUT ) led to significant differences in affinities for the FcRn. Affinities for the FcRn of both IgG were measured by a competition assay. Incubation with the native C4 led to a concentration-dependent decrease in bioluminescent signal signing the interaction with human (IC 50 = 10.2 µg/mL) and murine FcRn (IC 50 = 5.2 µg/mL). No signal inhibition was observed with the C4 Fc-MUT , which confirmed the abolition of interaction with the FcRn induced by the mutation (Figure 1A-B).

Lifting Fc/FcRn interaction for brain PET imaging. We evaluated the potency of anti-PD-L1 IgGs with low affinity for the FcRn receptor for brain PET imaging by comparing the kinetics of 89 Zr-DFO-C4 and 89 Zr-DFO-C4 Fc-MUT in an orthotopic syngeneic mouse model of GBM. Post-contrast T1-weighted images depicted comparable tumor growth between the two mice groups (Figure S7).

As expected, the plasma clearance of 89 Zr-DFO-C4 Fc-MUT was faster compared with 89 Zr-DFO-C4 (Figure 1E). A bicompartmental model with a first-order elimination function was individually fitted to plasma kinetics. Parameters' estimations of the two C4 formats plasmatic kinetic are reported in Table 1. 89 Zr-DFO-C4 Fc-MUT is cleared from the central compartment promptly compared with 89 Zr-DFO-C4, therefore associated with a shorter mean terminal half-life of 89.7 h versus 176.4 h for 89 Zr-DFO-C4 (pvalue = 0.009). 89 Zr-DFO-C4 Fc-MUT peripheral distribution volume is twice higher than 89 Zr-DFO-C4, and its transfer rate from peripheral to the central compartment (k 21 ) is significantly lower than IgG C4's one (0.020±0.009 h -1 versus 0.055±0.032 h -1 ). Those differences in kinetics are consistent with higher retention of the low FcRn affinity antibody in the liver and the spleen (Figure S8). FUS enabled the entry into the brain of both radiolabeled antibodies. At 1h post-injection, 89 Zr-DFO-C4 and 89 Zr-DFO-C4 Fc-MUT mean concentrations in the brain reached 3.64±0.73 %ID/cc and 3.75±0.41 %ID/cc, respectively. Reflecting its blood kinetic, the 89 Zr-DFO-C4 Fc-MUT concentrations in the contralateral hemisphere decreased at a higher rate than the concentrations of 89 Zr-DFO-C4 (Figure 1F).

The tumor distribution of the two C4 formats differed heavily over time. After reaching the maximal concentration of 5.1±1.5 %ID/cc, 89 Zr-DFO-C4 Fc-MUT concentration in the GBM tumor decreased at a slower pace than in the contralateral hemisphere. It resulted in a significant difference of tissue to plasma ratio between the tumor and the contralateral hemisphere from 5h p.i. (Figure 1F). Tumoral uptake of 89 Zr-DFO-C4, on the contrary, increased from 48h post-injection to reach a maximal uptake 7 days postinjection. Over time, those differences in brain distribution led to the highest PET contrast in the tumor observed at 22h post-injection for 89 Zr-DFO-C4 Fc-MUT versus 7 days post-injection for the 89 Zr-DFO-C4.

FUS-aided immunoPET imaging of glioblastoma.

To validate the benefits of applying FUS to target PD-L1 with 89 Zr-DFO-C4 Fc-MUT , immunoPET imaging was performed in a GBM model with (FUS group) and without (sham group) FUS-induced BBB permeabilization before the injection. A similar imaging protocol to the one described earlier was applied and we verified that tumor growth was similar between the sham and the FUS group on post-contrast T1-weighted MRI (Figure S7). In addition to the brain, FUS protocol had a significant impact on the spleen uptake of the 89 Zr-DFO-C4 Fc-MUT (Figure S8). As expected, BBB permeabilization by FUS significantly enhanced the brain uptake of 89 Zr-DFO-C4 Fc-MUT (Figure 2A). Onehour post-injection, brain uptake of 89 Zr-DFO-C4 Fc-MUT reached 3.75±0.41%ID/cc with FUS versus 1.92±0.45%ID/cc without FUS. The difference between the two groups remained significant up to 168h post-injection, even in the contralateral hemisphere (Figure 2B). Immunostaining with a secondary antibody targeting the 89 Zr-DFO-C4 Fc-MUT confirmed the specificity of the signal observed (Figure 2C). The injected antibody was only detectable in the tumor when FUS were applied, even though PD-L1 was expressed. No evidence of brain damage was observed into the brain after FUS protocol on hematoxylin/eosin staining (Figure S9).

Kinetic modeling of 89 Zr-labeled C4 brain PET data.

To further decipher the role of FcRn at the BBB and characterize the impact of FUS on the entry of antibodies in the brain, compartmental modelling was performed (Figure 3A). As no specific binding is expected in the contralateral hemisphere, a 1-Tissue compartment model was chosen. For the tumoral volume, a 2-Tissue compartment model was fitted (Figure 3B). Mean predicted and observed data of 89 Zr-DFO-C4 and 89 Zr-DFO-C4 Fc-MUT versus time in each brain volume are displayed in figures 3C and 3D, respectively.

Our structural model introduced two supplemental parameters, t FUS which describes the time for the BBB to recover integrity after FUS, and K FUS , which describes the perfusion-dependent transfer rate of IgG across the permeabilized BBB (Figure 3B). K FUS and t FUS estimates did not differ between the two 89 Zr-DFO-C4 formats or across brain regions (Table 2). FUS enabled the entry of the two antibodies at a mean rate of 0.163±0.071 mL/h/g of tissue for 12.1±4.6 minutes after the end of the FUS protocol.

The impaired interaction of 89 Zr-DFO-C4 Fc-MUT with FcRn significantly decreased the efflux rate constant (k 2 ) from the healthy brain tissue to plasma (0.015±0.027 h -1 ) compared with 89 Zr-DFO-C4 (0.300±0.218 h -1 , p-value = 0.002). It also impacted the influx rate constant (K 1 ) post-FUS, with a 10-fold decrease in the rate constant for transfer from plasma to tissue (Table 2). However, in the tumoral volumes, there was no significant difference between both 89 Zr-DFO-C4 formats' K 1 and k 2 estimates.

DISCUSSION

Focused ultrasound is an emerging technology that could change the paradigms of glioblastoma treatment. If associated with immunotherapy, there is a need for a companion methodology to select patients that will respond to such therapy. Improving the kinetic of antibodies in brain tumors is essential for therapy and imaging purposes. The difficulty in addressing personalized therapy for GBM prompted us to propose a theranostic approach based on quantifying PD-L1 expression in brain lesions.

Antibody design with low FcRn affinity has been described as a promising strategy to overcome some pharmacokinetic limitations of immunoPET imaging. Bouleau et al. demonstrated that using an H310A/H435Q mutant-IgG offers more advantages for immunoPET imaging than using smaller objects, which mainly aims to decrease the plasma half-life. They obtained significantly higher tumoral uptake of the 89 Zr-C4 Fc-MUT compared with the corresponding 89 Zr-Fab while benefiting from a fairly fast blood clearance resulting in high contrast PET images at only 24h post-injection [START_REF] Bouleau | Optimizing immunoPET imaging of tumor PD-L1 expression: pharmacokinetics, biodistribution and dosimetric comparisons of 89Zr-labeled anti-PD-L1 antibody formats[END_REF]. Although human IgG1 has a stronger affinity for murine FcRn than for human FcRn, which could impact the prediction of a human antibody pharmacokinetic from mice models, the H310A/H435Q mutation has a translational value as it abolishes IgG1 interaction with both the human and the murine FcRn [START_REF] Pyzik | The Neonatal Fc Receptor (FcRn): A Misnomer? Front Immunol[END_REF][START_REF] Kim | Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn[END_REF]. Moreover, the importance of FcRn functions on the transcytosis and recycling of IgGs is preserved across species [START_REF] Ober | Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies[END_REF].

The overall brain exposure of 89 Zr-DFO-C4 Fc-MUT was lower than that of 89 Zr-DFO-C4. This decrease in brain disposition was similarly observed by Chang et al., who compared the brain kinetics of the i.v administered human IgG1 trastuzumab and its (I253A/H310A/H435A) mutant, which is unable to bind FcRn [START_REF] Chang | A translational platform PBPK model for antibody disposition in the brain[END_REF][START_REF] Chang | Effect of the Size of Protein Therapeutics on Brain Pharmacokinetics Following Systematic Administration[END_REF]. The retention of the mutated C4 due to the binding to PD-L1, enabled by FUS, did not compensate for the reduced brain disposition compared with the 89 Zr-DFO-C4. The dose-normalized simulation of both C4 formats kinetic in the tumor considering a single intravenous injection and FUSinduced BBB disruption shows that tumor IgG exposure overtime was higher for the 89 Zr-DFO-C4 (AUC 0- 168h, C4 =433 μg*h/mL) compared to the mutated IgG (AUC 0-168h, C4(Fc-MUT) =247 μg*h/mL) (Figure 4). Therefore, using IgG with a modified Fc to reduce their affinity for FcRn does not appear advantageous for therapeutic purposes. These results are the consequence of the significantly reduced plasma half-life of 89 Zr-DFO-C4 Fc-MUT due to the abolition of FcRn-mediated transcytosis and recycling. In fact, the high brain exposure of 89 Zr-DFO-C4 might result from the BBB disruption associated with GBM at an advanced stage at 21 days post-implementation, in addition to the long plasma half-life of IgGs [START_REF] Maes | Experimental immunotherapy for malignant glioma: lessons from two decades of research in the GL261 model[END_REF][START_REF] Oh | Immunocompetent murine models for the study of glioblastoma immunotherapy[END_REF]. 89 Zr-DFO-C4 Fc-MUT foremost benefits from improved kinetic properties for PET imaging of brain tumors expressing PD-L1 as it generates an optimal contrast between the brain tumor and the contralateral hemisphere at 22h p.i. for the 89 Zr-DFO-C4 Fc-MUT against 168h for 89 Zr-DFO-C4. Kinetic modeling was used to decipher the non-specific and the specific binding contribution to the observed PET signal. It enlightens the potency of 89 Zr-DFO-C4 Fc-MUT to measure target engagement with the specific binding being predominant as soon as 12h postinjection, versus 48h post-injection for the full IgG, 89 Zr-DFO-C4 (Figure 4). Moreover, the low disposition of 89 Zr-DFO-C4 Fc-MUT in the brain without FUS-induced BBB disruption, associated with its fast plasma clearance, implies that the PET image predominantly reflects PD-L1 expression at the time of injection.

The same assumption cannot be made for the 89 Zr-DFO-C4 which uptake may depend on the growth and evolution of the tumor between the time of injection and optimal time for PET acquisition.

FUS were necessary to image GBM with the 89 Zr-DFO-C4 Fc-MUT . Although BBB disruption was induced on both brain hemispheres, the binding of 89 Zr-DFO-C4 Fc-MUT was mostly observed in the GBM tumor volume, thus confirming the specificity of the PET signal for PD-L1 expression. The maximum concentration in the GBM tumor was attained at 5h post-injection versus 1h post-injection in the contralateral hemisphere. This delay reflects the time of association of 89 Zr-DFO-C4 Fc-MUT liberated in the interstitial fluid of the brain parenchyma to its target, PD-L1. Safety of the FUS protocol used was previously validated by contrast-enhanced T1w and T2w MRI in addition to histological analysis [START_REF] Tran | 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[END_REF][START_REF] Gerstenmayer | Ultrasound Induced Blood-brain Barrier Opening on Rodents : from Nanoparticles Delivery to a Therapy for Alzheimer's Disease[END_REF][START_REF] Porret | Enhanced therapeutic effect of anti-EGFR cetuximab antibody on glioblastoma by focused ultrasound in preclinical model (+ Running poster)[END_REF]. Concordantly, no structural impact of FUS was observed on the brain of mice on H&E staining. Kinetic modeling of the impact of FUS on the brain kinetics of a radiolabeled compound was previously described using a classic 1-tissue compartment model [START_REF] Hugon | 18F]2-Fluoro-2-deoxy-sorbitol PET Imaging for Quantitative Monitoring of Enhanced Blood-Brain Barrier Permeability Induced by Focused Ultrasound[END_REF]. This model implies that the entry rate of the radiotracer is constant throughout the observation period. In this study, we considered a discontinuous entry function to match the transient character of FUS-induced BBB disruption. To this end, we introduced two additional parameters related to the molecule uptake, K FUS and t FUS , that corresponds respectively to the clearance and to the time of BBB/BTB sealing after the transient disruption with FUS.

The similarity of these two parameters in all brain regions where FUS were applied reinforces the hypothesis that they only are related to the dynamics of BBB permeation/recovery rather than the intrinsic kinetic properties of tested antibodies. Considering all brain regions and for both C4 formats, the mean K FUS is 0.163±0.071 mL/h/g of tissue. The same order of magnitude with the uptake clearance calculated for 89 Zr-Cetuximab passage has been observed by Tran et al. (CL up Cetuximab = 0.78±0.36 mL/h/g of tissue) [START_REF] Tran | 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[END_REF]. The mean t FUS was determined to be 10.4±4.6 min, which is consistent with the theoretical closure half-life of the BBB from Marty et al. [START_REF] Marty | Dynamic study of blood-brain barrier closure after its disruption using ultrasound: a quantitative analysis[END_REF]. Their estimation, based on semi-empirical observation with MRI revealed a half-life of 17.8 min after FUS-enhanced delivery of particles with a hydrodynamic diameter similar to a diameter of an antibody (≈10 nm). Additionally, in our case, implementing a discontinuous permeability function improved the model's fit with the observed concentrations in the tumor volume delineated by contrast-enhanced T1w-MRI. In this particular volume, the BBB is already disrupted by the GBM presence, allowing diffusion of gadoteric acid in the tumor. This suggests that FUS transiently induced a stronger disruption of the BTB than the one induced by this particular tumor model. Enhanced BTB permeability induced by FUS remains to be further investigated in other preclinical models. Of note, this was not observed by Brighi et al., who performed FUS-aided immunoPET imaging of a patient-derived glioma model in mice [START_REF] Brighi | MR-guided focused ultrasound increases antibody delivery to nonenhancing high-grade glioma[END_REF].

The robustness of the estimation of K FUS and t FUS allowed the characterization of our two C4 formats brain kinetic to assess the influence of FcRn affinity quantitatively. In the contralateral hemisphere devoid of tumoral tissue, the 89 Zr-DFO-C4 Fc-MUT is associated with a lower efflux rate between the brain and plasma. The influx rate constant was also lower when affinity with FcRn is lost. These results suggest that antibody-FcRn interaction controls the mAbs transcytosis across the BBB in both directions. This conclusion is consistent with the slower clearance from the brain of IgG with reduced affinity observed when the IgG is intracranially injected [START_REF] Cooper | Efflux of monoclonal antibodies from rat brain by neonatal Fc receptor, FcRn[END_REF][START_REF] Zhang | Mediated efflux of IgG molecules from brain to blood across the bloodbrain barrier[END_REF]. In regions associated with GBM tumoral tissues, transfer rate constants of 89 Zr-DFO-C4 Fc-MUT were higher than those in the contralateral hemisphere and were similar to that observed with the unmodified 89 Zr-DFO-C4. The absence of exchange constants difference between the PET contrast-enhanced and the T1w MRI contrast enhanced volume indicates that mAbs tumoral distribution does not relate to antibody-FcRn interaction or BBB integrity before FUS application but mainly on the antigen presence.

Abolishing Fc-FcRn interaction confers improved kinetic properties to 89 Zr-DFO-C4 Fc-MUT for immunoPET imaging with a better contrast obtained sooner in brain tumoral tissues. FUS-aided BBB/BTB disruption enables quantitative imaging of PD-L1 expression by glioblastoma tumors within the brain and allowed us to study the effect of Fc-FcRn interaction on the brain distribution of antibodies.

This study demonstrates the potency of FUS-aided BBB disruption with the smart design of radiolabeled antibodies to enable quantitative immunoPET imaging of PD-L1 within the brain.. Immunofluorescence signal is overlaid on DAPI images (blue). Magnification of a cryo-section stained with rat anti-mouse-PD-L1 IgG and an AF546-goat-anti-rat IgG (orange) or AF546-goat-anti-human IgG (red) to detect the injected antibody. Immunofluorescence signal is overlaid on DAPI images (blue), GL261 cells express GFP (green). Supplementary immunofluorescence staining are shown in SI Appendix (Figure S8). Parameters used for the simulation were the mean of individual estimated parameters.

Tables

  Blood pharmacokinetic. Plasmatic concentrations were calculated from image-derived blood activity concentration drawn in the left cardiac ventricle. Considering that antibodies are restricted to the serum, a blood-to-plasma concentration ratio of 0.55 was used. A bicompartmental model with a first-order elimination function was individually fitted using Phoenix WinNonlin (v.8.3.1, Certara®). Parameters and equations of the model are detailed in supplemental materials. Parameters of the plasma kinetic were intraindividually fixed to apply the same input function to all brain VOI. Brain kinetics. Brain kinetic parameters were obtained by fitting regional TACs to either a modified 1tissue compartment model (m1TCM) or a modified 2-tissue compartment model (m2TCM) in Phoenix WinNonlin (v.8.3.1, Certara®). These two models were modified to match the transient character of FUSinduced BBB disruption. Parameters and equations of the model are detailed in SI App. Goodness-to-fit of models was determined by generating the Akaike Information Criterion, plots of residuals over time, and plots of individual prediction versus observed concentration (Figure S3-6). The influence of C4 format on kinetic parameters estimations was assessed with a two-factor ANOVA followed by pairwise comparison of mean with Bonferroni's p-value adjustment in R v.4.0.2. Significance was set to 95%.

  Abbreviations: BBB: blood-brain-barrier; BTB: blood-tumotal barrier; CNS: central nervous system; FcRn: neonatal Fc receptor; FUS: focused ultrasound; GBM: glioblastoma; HPLC: high-performance liquid chromatography; ICI: immune checkpoint inhibitor; iTLC: immunoglobuline G; MB: microbubble; MRI: magnetic resonance imaging; PD1: programmed cell death protein 1; PD-L1: programmed death ligand 1; PET: positron emission tomography.Fundings:We thank ITMO Cancer-Aviesan for their financial support (funds administered by the lnserm, Project IM2FUS). This work was performed on an imaging platform member of the France Life Imaging network (grant ANR-11-INBS-0006).

FiguresScheme 1 .

 1 Figures

Figure 1 .

 1 Figure 1. In vitro and in vivo characterization of the C4 formats for brain PET PD-L1 imaging. (A, B) Dose-dependent inhibition curves of both C4 formats with human and murine FcRn. Data represent the mean ± SD of triplicate readings. (C) Timeline of the MRI and PET imaging protocol. (D) Representative brain PET-T1w MRI images of GL261-GFP bearing C57Bl/6 mice injected with 89 Zr-DFO-C4 (n=6) or 89 Zr-DFO-C4 Fc-MUT (n=8) at 22h and 168h p.i. Time activity curves in plasma (E) and in the brain (F) of the two C4 formats. TACs in the tumor and contralateral hemisphere are differentiated. (F) Tissue to plasma ratio of the two C4 radioligands in tumor and contralateral hemisphere.

Figure 2 .

 2 Figure 2. Brain kinetic of 89 Zr-DFO-C4 Fc-MUT after FUS-induced BBB permeabilization. (A) Representative PET images overlaid on post-contrast T1-weighted MRI from the same mouse at different time points (1h, 5h, 22h, 46h, 70h and 168h after injection) for the sham (top) and the FUS (bottom) groups. (B) Time activity curves of the 89 Zr-DFO-C4 Fc-MUT in the tumor enhanced on T1-weighted MRI and in the contralateral hemisphere in the sham (red, n= 5) and the FUS (blue, n=8) groups. All data are represented as mean ± SD. (C) Immunofluorescence staining of brain sections of C4 Fc-MUT injected mice. Adjacent 10µm cryo-sections were stained with either a rat anti-mouse-PD-L1 IgG and an AF546-goatanti-rat IgG (orange) or AF546-goat-anti-human IgG (red) to detect the injected antibody.

Figure 3 .

 3 Figure 3. Brain kinetic modeling to elucidate FcRn's role at the BBB and the impact of FUS-induced BBB disruption on antibodies delivery. (A) Representative example of VOI identifying the contralateral hemisphere (yellow), the tumor enhanced on post-contrast T1-weighted MRI (pink) and the tumor enhanced on PET images (red). (B) Structure of the 2-tissue compartment model applied on tumoral VOI. Observed (markers) and predicted (solid lines) brain concentration of 89 Zr-DFO-C4 (n=4) (C) and 89 Zr-DFO-C4 Fc-MUT (n=7) (D) according to the different VOI. All data are represented as mean ± SD. K FUS and K1: perfusion dependent entry constant from plasma to free/non-specifically bound compartment. t FUS : time of significant closure of the BBB. k2: transfer constant from free/non-specifically bound compartment to plasma. k3 and k4: transfer constant between free/non-specifically bound compartment and specifically bound compartment. FUS +MB: Focused ultrasound on microbubbles.

Figure 4 .

 4 Figure 4. Simulated concentrations of the two C4 formats in GBM tumor with a preserved BBB integrity on T1w post-contrast MRI. Concentrations were normalized to an injected dose of 70µg of antibody. The PET signal simulated is the resultant of the sum of the non-specific and specific fixation.

Table 1 . Plasma pharmacokinetic parameters of the 89 Zr-DFO-C4 and the 89 Zr-DFO-C4 Fc-MUT .

 1

		89 Zr-DFO-C4	89 Zr-DFO-C4 Fc-MUT
	Parameter	Mean	Sd	Mean	Sd	p-value
	V c (mL)	1.5	0.1	1.7	0.3	0.45
	k 10 (h-1)	0.014	0.004	0.076	0.011	7.4E-06
	k 12 (h-1)	0.119	0.058	0.094	0.047	0.48
	k 21 (h-1)	0.055	0.032	0.02	0.009	0.03
	V p (mL)	3.4	0.5	7.8	2.7	0.01
	Initial t 1/2 (h)	4.3	1.5	4.2	1.3	0.84
	Terminal t 1/2 (h) AUC 0  ∞ (µg.h.mL -1 )	176.4 5954	50 1411	89.7 569	30.9 108	0.009 1.1E-05
	Mean parameters estimates and associated standard errors obtained by individually fitting a
	bicompartmental kinetic to plasmatic concentration of 89 Zr-DFO-C4 (n=6) and 89 Zr-DFO-C4 Fc-MUT (n=8). p-
	value shown are results from Student comparison of mean. V

c : volume of the central compartment; k 10 : elimination constant from the central comportment; k 12 : transfer constant from the central to the peripheral compartment; k 21 : transfer constant from peripheral to central compartment; Vp: volume of the peripheral compartment; Initial t 1/2 : initial half-life; Terminal t 1/2 : terminal half-life.

Table 2 . Mean estimates and standard deviation of parameters of the different models applied to 542 brain VOI data to characterize 89 Zr-DFO-C4 and 89 Zr-DFO-C4 Fc-MUT kinetic.

 2

	543
				89 Zr-DFO-C4	89 Zr -DFO-C4 Fc-MUT
				Mean		Mean
	Brain region	Parameter	Estimate	Sd	Estimate	Sd	p-value
	Contralateral
		K FUS (mL/h/g of tissue)	0.167	0.156	0.094	0.037	ns
		t FUS (h)	0.165	0.099	0.215	0.065	ns
		K 1 (mL/h/g of tissue)	0.030	0.019	0.003	0.005	0.005
		k 2 (h -1 )	0.300	0.218	0.015	0.027	0.002
		vB		0.064	0.006	0.077	0.014	ns
	T1w MRI CE
		K FUS (mL/h/g of tissue)	0.179	0.046	0.200	0.087	ns
		t FUS (h)	0.142	0.077	0.179	0.102	ns
		K 1 (mL/h/g of tissue)	0.053	0.021	0.062	0.028	ns
		k 2 (h -1 )	0.566	0.188	0.601	0.308	ns
		vB		0.052	0.013	0.053	0.009	ns
		k 3 (h -1 )	0.027	0.017	0.111	0.063	0.031
		k 4 (h -1 )	0.005	0.006	0.020	0.011	0.039
	PET CE (wo T1w MRI CE)
		K FUS (mL/h/g of tissue)	0.155	0.049	0.188	0.077	ns
		t FUS (h)	0.150	0.037	0.161	0.070	ns
		K 1 (mL/h/g of tissue)	0.069	0.033	0.051	0.026	ns
		k 2 (h -1 )	0.742	0.393	0.491	0.298	ns
		vB		0.058	0.010	0.063	0.013	ns
		k 3 (h -1 )	0.017	0.013	0.077	0.047	0.036
		k 4 (h -1 )	0.000	0.000	0.015	0.007	0.003
	544	K FUS and K1: perfusion dependent entry constant from plasma to free/non-specifically bound compartment.
	545	t FUS : time of significant closure of the BBB. k 2 : transfer constant from free/non-specifically bound
	546	compartment to plasma. k 3 and k 4 : transfer constant between free/non-specifically bound compartment and
		specifically bound compartment. vB: fraction of blood in the tissue.

Acknowledgments: Figures were created with BioRender.com and GraphPad Software, Inc. Authors' Contributions: C.C., A.N., N.T., B.M., H.N & C.T. designed research; C.C., A.N., A.D., S.D., D.K., E.S., B.J., H.N., C.T. performed research; C.C., A.N., A.D., S.D., D.K., E.S., B.J., B.L., H.N., C.T. contributed new reagents/analytic tools, C.C., N.T., S.D., D.K., E.S., B.J., B.M., H.N., C.T., analyzed data and, C.C., A.N., N.T., B.M., H.N., C.T. wrote the paper;

Competing interest statement:

The authors declare no competing interest.


Keywords: AChBP, antagonism, macrocylic imine toxin, nicotinic ACh receptor, structure-function relationships

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Diversity in the binding interactions of nicotinic ligands to the nAChRs and associated conformational fluctuations -Insights into the core motif dictating antagonism

Yves Bourne, Gerlind Sulzenbacher, Zoran Radić, Laurent Chabaud, Rómulo Aráoz, Evelyne Benoit, Armen Zakarian, Denis Servent, Catherine Guillou, Palmer Taylor, et al.

To cite this version:

Yves 18

Diversity in the binding interactions of nicotinic ligands and toxins to the nAChRs and associated conformational fluctuations -Insights into the core motif dictating antagonism

Yves BOURNE 1 , Gerlind SULZENBACHER 1 , Zoran RADIĆ 2 , Laurent CHABAUD 3 , Rómulo ARÁOZ 4 , Evelyne BENOIT 4 , Armen ZAKARIAN 5 , Denis SERVENT 4 , Catherine GUILLOU 3 , Palmer TAYLOR 2 , Jordi MOLGÓ 4 , Pascale MARCHOT *Corresponding author, e-mail: pascale.marchot@univ-amu.fr

The pentameric ACh-binding proteins (AChBP) from water snails are soluble structural and pharmacological surrogates of the extracellular, ligand-binding domain of nicotinic ACh receptors (nAChRs) 1 . As such they offer pertinent models for studying the modes of binding of nicotinic effectors and the associated conformational changes, and correlate them with functional alteration of the nAChR channel. Crystal structures of AChBP complexes revealed that nicotinic agonists and competitive antagonists bind primarily within a nest of aromatic side chains, contributed by loops C and F located on opposing faces of each subunit interface, and induce a range of loop C conformations that modulate the size and shape of the binding pocket 2-4 .

The macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Binding and voltage-clamp recordings on muscle-type and neuronal nAChRs revealed subnanomolar affinities dictated by slow dissociation, potent antagonism, and varying levels of nAChR subtype selectivity. Crystal structures of the complexes showed that common AChBP determinants imbedded within the aromatic nest confer high affinity binding to the toxins, while distinctive determinants brought about by loop F and located within the nest, or extending outside the nest towards apical, radial or 'membrane' subsites of the interface, dictate either broad or narrow nAChR subtype selectivity by the toxins [5][6][7] .

Based on these data, new organic compounds aimed at pinpointing the minimal chemical motif that dictates antagonism were designed, synthetized and analyzed relative to nAChRs and AChBP 8 . Structural analysis of the complexes showed that the spiroimine core common to these compounds is the major component of their mode of binding, while the surrounding substituents are involved into nAChR subtype specificity.

These data identify distinctive ligands, functional determinants and binding sites for the design of new drugs targeting disease-associated nAChR subtypes.

Works supported by grants from the CNRS-DREI (YB, PM), FRISBI (AFMB lab), NIH/NIGMS (AZ), USPHS (PT), ANR (JM).

  Bourne, Gerlind Sulzenbacher, Zoran Radić, Laurent Chabaud, Rómulo Aráoz, et al.. Diversity in the binding interactions of nicotinic ligands to the nAChRs and associated conformational fluctuations -Insights into the core motif dictating antagonism. RT26 -26th Meeting of the French Society of Toxinology -Bioengineering of Toxins, French Society of Toxinology (SFET), Dec 2019, Paris, France. hal-04469851 Bioingénierie des Toxines -Bioengineering of Toxins 26 èmes Rencontres en Toxinologie -26 th Meeting on Toxinology Paris, 4-5 décembre 2019 -Paris, December 4-5, 2019

  1,* 1 'Architecture et Fonction des Macromolécules Biologiques', CNRS/AMU, Faculté des Sciences -Campus Luminy, Marseille. 2 Skaggs School of Pharmacy and Pharmaceutical Sciences, Dept. Pharmacology, UC San Diego, La Jolla, CA, USA. 3 Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France. 4 Institut des sciences du vivant Frédéric Joliot, CEA Paris-Saclay, Gif-sur-Yvette, France. 5 Dept. Chemistry and Biochemistry, UCSB, Santa Barbara, CA, USA.