# Antibody-secreting cell repertoires hold high-affinity anti-rocuronium specificities that can induce anaphylaxis in vivo

Alice Dejoux, Qianqian Zhu, Adam Woolfe, Ophélie Godon, Sami Ellouze, Guillaume Mottet, Carlos Castrillon, Caitlin M. Gillis, Cyprien Pecalvel, Christelle Ganneau, et al.

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## To cite this version:

Alice Dejoux, Qianqian Zhu, Adam Woolfe, Ophélie Godon, Sami Ellouze, et al. Antibody-secreting cell repertoires hold high-affinity anti-rocuronium specificities that can induce anaphylaxis in vivo. Journal of Allergy and Clinical Immunology, 2025, 155 (5), pp.1557-1574. 10.1016/j.jaci.2025.01.025. pasteur-04961215

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# Antibody-secreting cell repertoires hold high-affinity anti-rocuronium specificities that can induce anaphylaxis in vivo

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Alice Dejoux, PhD, Qianqian Zhu, PhD, Adam Woolfe, PhD, Ophélie Godon, MS, Sami Ellouze, Guillaume Mottet, PhD, et al

## GRAPHICAL ABSTRACT

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**Capsule summary:** Immunizing mice with neuromuscular blocking agent rocuronium enabled identification from IgG-secreting cells of rocuronium-specific high-affinity antibodies. Expressed as human IgE, these antibodies trigger mast cell and basophil activation as well as systemic anaphylaxis in mouse models.
# Antibody-secreting cell repertoires hold high-affinity anti-rocuronium specificities that can induce anaphylaxis *in vivo*

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Alice Dejoux, PhD,<sup>a,b</sup> Qianqian Zhu, PhD,<sup>a,c</sup> Adam Woolfe, PhD,<sup>d‡</sup> Ophélie Godon, MS,<sup>a</sup> Sami Ellouze,<sup>d</sup>
Guillaume Mottet, PhD,<sup>a</sup> Carlos Castrillon, PhD,<sup>a</sup> Caitlin Gillis, PhD,<sup>a</sup> Cyprien Pecalvel,<sup>e</sup> Christelle Ganneau,<sup>f</sup>
Bruno Iannascoli,<sup>a</sup> Frédéric Lemoine, PhD,<sup>g</sup> Frederick Saul, PhD,<sup>h</sup> Patrick England, PhD,<sup>i</sup> Laurent L. Reber, PhD,<sup>e</sup>
Aurélie Gouel-Chéron, MD, PhD,<sup>a,j,k</sup> Luc de Chaisemartin, PharmD, PhD,<sup>c,l</sup> Ahmed Haouz, PhD,<sup>h</sup> Gaël A. Millot, PhD,<sup>a,g</sup>
Sylvie Bay, PhD,<sup>f</sup> Annabelle Gérard, PhD,<sup>d‡</sup> Friederike Jönsson, PhD,<sup>a,m</sup> Sylvie Chollet-Martin, MD, PhD,<sup>c,l</sup> and
Pierre Bruhns, PhD<sup>a,n</sup>
*Paris, Orsay, and Toulouse, France*

**Background:** Neuromuscular blocking agents (NMBAs) are muscle relaxants used to assist mechanical ventilation but lead in 1 per 10,000 anesthesia cases to severe acute hypersensitivity reactions—that is, anaphylaxis. Incidences vary between types of NMBAs. Rocuronium, a widely used nondepolarizing aminosteroid NMBA, induces among the highest anaphylaxis rates. Rocuronium-induced anaphylaxis is proposed to rely on preexisting rocuronium-binding antibodies, but no such antibodies have ever been identified.

**Objectives:** We sought to identify rocuronium-specific antibody repertoires from plasma cells or plasmablasts of rocuronium-immunized mice to determine the affinities, structures, and anaphylactogenic potential of these antibodies for rocuronium.

**Methods:** We engrafted rocuronium onto carrier proteins allowing immunization of mice against rocuronium, screening

for rocuronium-specific antibody responses, and sorting of rocuronium-specific plasma cells using droplet microfluids coupled to single-cell antibody gene (variable heavy chain [VH] and variable light chain [VL]) sequencing.

**Results:** The 2 different repertoires of >500 VH-VL pairs were oligoclonal, comprised 3 major clonal families, and displayed convergence. Expressed as human IgG1, these antibodies demonstrated subnanomolar affinities for rocuronium with families either monospecific for rocuronium or cross-reactive only for closely related NMBAs. Expressed as human IgE, they triggered human mast cell and basophil activation, and severe passive systemic anaphylaxis in mice humanized for the IgE receptor FceRI. Cocrystal structures between rocuronium and antibody representatives of 3 different VH-VL families revealed distinct interaction modes, with the ammonium group involved systematically in the binding interface.

**Conclusions:** This work identifies the epitopes of antibody reactivity to rocuronium, demonstrates anaphylactogenic potential of anti-rocuronium IgE, and establishes the first mouse model of NMBA anaphylaxis. (J Allergy Clin Immunol 2025;155:1557-74.)

**Key words:** Antibodies, allergy, rocuronium, neuromuscular blocking agents, B cells, anaphylaxis, plasma cells, therapy

Neuromuscular blocking agents (NMBAs) are used in the clinic to paralyze skeletal muscles during surgery conducted under general anesthesia.¹,² They help airway management in elective or urgent anesthesia by facilitating endotracheal intubation. NMBAs possess 2 tertiary and/or quaternary substituted ammonium groups at physiologic pH that enable them to compete at the neuromuscular junction with the quaternary ammonium group of acetylcholine for binding to muscle-type nicotinic receptors.³

The most dramatic adverse event after NMBA exposure is anaphylaxis—the severest form of allergic reaction, which can lead to death.⁴,⁵ In some countries, half of perioperative anaphylaxis cases are caused by NMBAs, with a global mortality rate of 4.1%.⁶ Currently it is thought that allergen-specific IgE antibodies, including anti-NMBA IgEs,⁷ lead to mast cell and basophil activation in allergen-sensitized patients.⁸ We reported earlier that indeed circulating anti-NMBA IgE levels, but also anti-NMBA IgG levels, correlate with the severity of anaphylaxis.⁹ In our view, 2 antibody-dependent pathways are involved in NMBA-induced anaphylaxis: the IgE pathway leading to

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<footnote>From <sup>a</sup>the Institut Pasteur, Université Paris Cité, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR1222, Antibodies in Therapy and Pathology, Paris;<sup>b</sup>the Collège Doctoral, Sorbonne Université, Paris;<sup>c</sup>the Université Paris-Saclay, INSERM, Inflammation Microbiome Immunosurveillance, Orsay;<sup>d</sup>HiFiBiO Therapeutics SAS, Paris;<sup>e</sup>the Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM UMR1291, Centre National de la Recherche Scientifique (CNRS) UMR5051, University Toulouse III, Toulouse;<sup>f</sup>the Institut Pasteur, Université Paris Cité, CNRS UMR3523, Chimie des Biomolécules, Paris;<sup>g</sup>the Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics Hub, Paris;<sup>h</sup>the Institut Pasteur, Université Paris Cité, CNRS UMR3528, Plate-forme Cristallographie-C2RT, Paris;<sup>i</sup>the Institut Pasteur, Université Paris Cité, CNRS UMR3528, Molecular Biophysics Core Facility, Paris;<sup>j</sup>the Anaesthesiology and Critical Care Medicine Department, DMU Parabol, Bichat-Claude Bernard Hospital, Assistance Publique-Hôpitaux de Paris, Paris;<sup>k</sup>the Université Paris Cité, Paris;<sup>l</sup>the Service d'immunologie, Hôpital Bichat, Assistance Publique Hôpitaux de Paris, Paris;<sup>m</sup>CNRS, F-75015, Paris; and <sup>n</sup>INSERM 1152, Département Hospitalo-Universitaire FIRE, Labex Inflamex, Université Paris Diderot, Paris.</footnote>

<footnote>‡Present affiliation: Saber Bio, part of Bio-Rad Laboratories, Paris, France</footnote>

<footnote>The first 2 authors contributed equally to this article, and both should be considered first author. The third and fourth authors contributed equally to this article and should be considered second author. The last 4 authors contributed equally to this article, and all should be considered senior author.</footnote>

<footnote>Received for publication July 30, 2024; revised January 15, 2025; accepted for publication January 27, 2025.</footnote>

<footnote>Available online January 30, 2025.</footnote>

<footnote>Corresponding author: Pierre Bruhns, PhD, Unit of Antibodies in Therapy and Pathology, Department of Immunology, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France. E-mail: bruhns@pasteur.fr.</footnote>

<footnote>The CrossMark symbol notifies online readers when updates have been made to the article such as errata or minor corrections</footnote>

<footnote>0091-6749</footnote>

<footnote>© 2025 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>

<footnote>https://doi.org/10.1016/j.jaci.2025.01.025</footnote>
Abbreviations used

BAT: Basophil activation test
BLI: Biolayer interferometry
CDR: Complementary determining region
HSA: Human serum albumin
IC: Immune complex
KLH: Keyhole limpet hemocyanin
MAT: Mast cell activation test
NMBA: Neuromuscular blocking agent
OD: Optical density
PBST: PBS-Tween 20
Roc: Rocuronium
scFv: Single-chain variable fragment
SPR: Surface plasmon resonance
VH: Variable heavy chain
VHH: Llama single domain antibody or nanobody
VL: Variable light chain

mast cell and basophil activation with histamine release as a major mediator, and the IgG pathway leading to neutrophil and monocyte activation with platelet-activating factor release as a main mediator.⁸ Both histamine and platelet-activating factor induce vasodilation, bronchoconstriction, and cardiac and pulmonary failure, with probable cumulative or potentiating effects when both mediators are released. The current treatment relies on adrenaline administration to induce vasoconstriction and bronchodilation, as well as antihistamines, which are administered mainly as a safety measure because they are inefficient at controlling anaphylactic shock.¹⁰ Intriguingly, the nondepolarizing aminosteroid NMBA rocuronium can be captured within minutes by the cyclodextrin sugammadex to reverse neuromuscular blockade in patients, leading in a few cases to amelioration of anaphylactic severity.¹¹,¹²

The tertiary and quaternary substituted ammonium groups present in NMBAs have been proposed to be part of the epitope recognized by anti-NMBA antibodies in patients,⁷ as patient sera reacting with one or several NMBAs¹³ cross-react with unrelated molecules possessing a quaternary ammonium.¹⁴,¹⁵ Because anti-NMBA mAbs have never been reported in either people or animals, molecular or structural evidence is lacking to confirm these hypotheses. In addition, to add complications to our understanding of the allergenic epitope, different conformer species can coexist. Indeed, rocuronium ring A can adopt a chair or a twist-boat conformation,¹⁶ with the twist-boat conformation being favorable for the accommodation of rocuronium within the cyclodextrin sugammadex.¹⁷

Importantly, anaphylaxis occurs at the first exposure to aminosteroid NMBAs in half of patients,¹⁸ excluding NMBAs as primary sensitizing epitopes in these patients' history. In addition, NMBAs are small (molecular weight ≈ 300-1000 Da) and nonimmunogenic. Several families of compounds are, however, considered to sensitize toward NMBAs, including cough syrups containing pholcodine (a morphine-based compound), cosmetics, and cleaning and bleaching products.¹⁵,¹⁹⁻²¹ Routinely, anti-NMBA IgEs are quantified using a surrogate for NMBA in ImmunoCAP detection: a morphine group termed *quaternary ammonium molecule* (aka QAM) that resembles core structures of aminosteroid NMBAs.²²,²³ Large variations in serum reactivity profiles to NMBAs are found between individuals, from a single

NMBA to NMBAs belonging to the same chemical family, and even to all NMBAs in 4% of the cases.¹³ This suggests B-cell responses with one or multiple monospecificities each toward a particular compound, and/or cross-reactive B-cell responses toward different NMBAs.⁷ Understanding the reason for mono- or cross-reactivity in patients would require analyzing their antibody repertoire and studying representative antibodies to determine if cross-reactive mAbs do indeed exist in patients, or if a collection of monospecific mAbs targets different NMBAs instead. The antibody repertoire is classically studied from circulating memory B cells,²⁴,²⁵ but it can also be defined from tissue-resident plasma cells, which are proposed to harbor the highest-affinity antibodies.²⁶⁻²⁹

Rocuronium represents an advantageous study model of NMBA anaphylaxis because it has among the highest anaphylaxis incidences and is widely used.⁵,³⁰⁻³² In addition, rocuronium possesses a hydroxyl group that can be targeted chemically to allow engraftment on larger molecules for immunization or assay development. In this study, we analyzed the rocuronium-specific antibody repertoires from plasma cells of rocuronium-immunized mice using single-cell droplet-based microfluidic technologies.²⁶ Affinities for rocuronium were in the subnanomolar to nanomolar range with either monospecificity for rocuronium or cross-reactivity only to closely related aminosteroid NMBAs that could be explained by crystallographic structures. Anti-rocuronium antibodies were anaphylactogenic when expressed as human IgE *in vitro* and *in vivo* in mice. Identification of anti-rocuronium mAbs permitted the development of the first mouse model of rocuronium anaphylaxis.

## METHODS

### Mice

C57BL/6J mice were purchased from Charles River and used for experiments after maintaining the mice for at least 1 week in specific-pathogen-free conditions after arrival at Institut Pasteur's animal facility. Human FcεRI<sup>lg</sup> mouse FcεRI<sup>-/-</sup> mice bred on a C57BL/6 background have been described previously.³³ Mice were bred at Institut Pasteur and used for experiments when they were 7 to 12 weeks of age. All animal care and experiments were conducted in compliance with the guidelines and specific approval of the animal ethics committee at Institut Pasteur, Paris, France (CETEA no. 89, registered under 170043, 2013-0103, and 27465), and by the French Ministry of Research (agreement 00513.02).

### Production and characterization of rocuronium bioconjugates

Hapten-protein couplings were performed using active ester derivatives of rocuronium and keyhole limpet hemocyanin (KLH) or monomeric human serum albumin (HSA) as the carrier protein. A carboxylate function was first introduced by adding succinic anhydride to rocuronium bromide in the presence of 4-(dimethylamino)-pyridine in pyridine. After stirring overnight at room temperature under argon, the solvent was removed under vacuum. The crude compound was purified by reverse-phase flash chromatography, resulting in a rocuronium derivative with a carboxylic acid at the 3 position of the steroid scaffold. The compound was characterized by mass spectrometry and by nuclear magnetic resonance analysis. All quantities of products
were calculated relative to the carrier protein. The functionalized rocuronium (200 equivalent) was dissolved in 0.05 mol 2-(*N*-morpholino)ethanesulfonic acid (aka MES) with 0.5 mol NaCl buffer (pH 5.6) and combined with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (400 equivalent) in the presence of *N*-hydroxysulfo-succinimide (200 equivalent). After 5 minutes' stirring at room temperature, pH was adjusted to 7.2 with 2 mol NaOH. This solution was progressively added to the carrier protein dissolved in 100 mmol PBS (pH 7.2). After 3 hours of gentle agitation at room temperature, buffer exchange was performed in 20 mmol HEPES and 300 mmol NaCl with minitrap desalting columns. The exact measurement of the concentration of the bioconjugates was achieved by quantitative amino acid analysis. The average density of conjugated rocuronium derivatives was evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The HSA-rocuronium (HSA-Roc) conjugates used for all experiments were monomeric with an average engraftment of 32 or 10 rocuronium molecules per HSA molecule for sorting and characterization (ELISA and bioluminescence imaging [BLI]) experiments, respectively, determined with the mean of the gaussian distribution of the mass-to-charge ratio (m/z) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as we reported earlier.⁷,⁹

## Immunization of mice against rocuronium

Female C57Bl/6J were immunized subcutaneously with 10 µg KLH-rocuronium (KLH-Roc; or KLH only as control) combined with adjuvant alum 1:1 (vol/vol) and 20 ng pertussis toxin in physiologic saline 0.9% NaCl for 3 times at 3-week intervals. Mice were boosted with 10 µg KLH-Roc without adjuvant 3 weeks after the last immunization. Four days later, the superficial cervical lymph nodes were removed, and cells were resuspended as single cells for immediate sorting using droplet microfluidics.

## Plasma cell sorting using droplet microfluidics

Plasma cells and plasmablasts were sorted on the basis of their secretion of IgG antibodies binding to HSA-Roc using methods and instruments that we have reported earlier.²⁶ In brief, single cells from lymph nodes of immunized mice were encapsulated into microfluidic droplets with paramagnetic nanoparticles (Strep Plus, 300 nm, Ademtech) coated with biotinylated anti-mouse IgGκ V<sub>H</sub>H (llama single domain antibody or nanobody), 75 nmol rabbit F(ab')₂ anti-mouse IgG Fc specific (Alexa Fluor 647 labeled, Jackson ImmunoResearch Laboratories) and 50 nmol HSA-Roc (purified monomer, Alexa Fluor 488 labeled). Under the influence of a magnetic field, the nanoparticles align into a beadline within each droplet³⁴ and are flown through a droplet-sorting chip placed under a high-speed camera, lasers, and photomultipliers. Relocation of both Alexa Fluor 647 and fluorescein isothiocyanate fluorescence onto the beadline of one drop triggered sorting of these droplets by dielectrophoresis into a collection tube, followed by cell recovery, recompartmentalization in droplets with single barcoded hydrogel beads and lysis and reverse transcription reagents using a microfluidic device for subsequent cell lysis and cDNA synthesis. The sequencing library was generated by 2-step nested PCR with the barcoded cDNA. Final products were sequenced on an Illumina MiSeq (2 × 300 bp paired-end reads), which allowed sequencing of the entire variable heavy chain (VH) and variable

light chain (VL) domains as well as the barcode sequence, as described elsewhere.²⁶ A bioinformatics pipeline was developed to allow sequence read trimming, merging, barcode extraction and clustering, and antibody sequence characterization and filtering. Importantly, for each barcode cluster that contained productive VH and VL made up of at least 20 reads each, the most abundant VH and VL rearrangement were considered to be paired.

## Antibody repertoire analysis and representative mAb selection

For reproducibility matters,³⁵ all bioinformatic analyses of the batches of nucleotide sequence FASTA files, representing the raw single cell Sanger sequencing of the VH-VL IgG domains, were implemented in a Nextflow (10.1038/nbt.3820) workflow, with software environment managed by Apptainer (apptainer.org). The workflow is available at Gitlab (gitlab.pasteur.fr/gmillot/repertoire_profiler) and is detailed below. Most of the process is based on tools from the Immcantation Portal (immcantation.readthedocs.io/en/stable/), notably from the change-O tool set.³⁶ VH and VL sequences, while single cell paired, could only be analyzed independently (VH sequences, then VL sequences), because no method taking this parameter into account currently exists.

First, the 'AssignGenes.py igblast' tool was run to detect and annotate the VDJ complementary-determining region (CDR) and FWR domains in each sequence using the IMGT database (www.imgt.org/). Then 'MakeDb.py igblast' converted the result into a readable TSV file. The 'ParseDb.py select' tool kept productive sequences—that is, those for which: (1) the coding region has an open reading frame, (2) no defect are present in the start codon, splicing sites, or regulatory elements, (3) no internal stop codons were found, and (4) junction regions are in frame. The clustering of sequences into clonal groups was performed as follows: (1) grouping of the sequences according to the same V and J genes (allelic variation not considered) and the same length of the CDR3, (2) for each group, computation of the distance score $D = (\text{Hamming distance})/(\text{CDR3 length})$ between each 2 × 2 sequence, (3) determination of the *D* threshold, which determines whether or not 2 sequences derive from the same germline cell sequence, using the 'distToNearest'() function of the 'shazam' R package, and (4) clone assignment for each sequence (ie, same ID for sequences belonging to the same clonal group) with the 'DefineClones.py' tool. We note that a few sequences could be lost at that stage because they did not fit the criteria required by this tool. Then the putative germline sequence of each clonal group was inferred by the 'CreateGermlines.py' tool. Finally, a single 'all_passes_seq.tsv' file was obtained for each batch of VH or VL initial FASTA files, containing, notably, for each sequence the VDJ gene and allelic names returned by the 'AssignGenes.py igblast' tool, the clone ID, the putative germline sequence before somatic hypermutations, and the putative VDJ gene and allelic names of the germline sequence returned by the 'CreateGermlines.py' tool.

For phylogenetic circos plots (Fig E3), productive sequences returned by 'ParseDb.py,' as above, were converted into amino acid sequences. Groups of sequences having the same V and J were extracted from the dataset using Goalign (10.1093/nargab/lqab075) (command goalign subset). Sequences of each group were aligned using ABAlign (10.1093/nar/gkad400) (with options -i <basta>
<-al for light chain or -ah for heavy chain> -sp MU). Phylogenetic trees (Fig E3 and Fig E6) were then inferred using IQTree v2.2.5 (10.1093/molbev/msaa015) using an antibody-specific model of amino acid substitution (10.1093/molbev/msu340) (options -s <Alignment file> -m AB_model +I+R6—seed 123456789). Trees, along sequence annotations, were then uploaded to iTOL (10.1093/nar/gkae268) using Gotree (10.1093/nargab/lqab075) (command gotree upload itol).

The phylogenetic trees in Fig E3 and Fig E6 were obtained with the 'formatClones()' and 'getTrees()' functions of the 'dowser' R package,³⁷ and homemade wrapping functions of the 'ggtree' R package,³⁸ for clonal groups with at least 3 nonidentical sequences. Amino acid sequence alignments and percentage identity calculations were performed with Jalview software using Muscle (Multiple Sequence Comparison by Log-Expectation) and colored by percentage identity. Number of mutations was obtained using the IMGT database by comparing sequences with germlines and plotted as histograms in GraphPad Prism.

## Gene synthesis, cloning and mAbs production

VH and VL synthesis was performed by Synbio Technologies. VHs were cloned into pUC19 vectors containing the sequence encoding the constant regions of human IgG1 heavy chain (CH), effectorless human IgG1 heavy chain mutated N₂⁹⁷A, human IgE heavy chain. VLs were cloned into pUC19 vectors containing the sequence encoding the constant regions (CL) of human kappa light chain. Antibodies were produced by transient cotransfection of VH-CH and VL-CL expression plasmids into exponentially growing Freestyle HEK 293-F cells, which were cultured in serum-free Freestyle 293 Expression Medium (Life Technologies; Thermo Fisher Scientific) in suspension at 37°C in a humidified 8% CO₂ incubator on a shaker platform rotating at 110 rpm. Twenty-four hours before transfection, cells were collected by centrifugation at 300 × g for 5 minutes and resuspended in expression medium at a density of 1 × 10⁶ cells/mL, and cultured overnight in the same conditions as mentioned above. To produce mAbs, 40 µg of each VH- and VL-expressing plasmid was diluted in 80 µL of FectoPRO reagent (Polyplus) at a final DNA concentration of 0.8 µg/mL, then incubated for 10 minutes at room temperature before addition to the cells. Twenty-four hours after transfection, cells were diluted 1:1 with expression medium. Cells were cultured for 6 days after transfection; then supernatants were collected, centrifuged at 1800 × g for 40 minutes, and filtered (0.2 µm). Antibodies were purified by affinity chromatography using an AKTA pure FPLC instrument (GE Healthcare) on a HiTrap Protein A Column (GE Healthcare) for human IgG1 and human IgG1N₂⁹⁷A, and on a anti-human IgE omalizumab-bound affinity column for human IgE, and desalted on a HiTrap Desalting Column (GE Healthcare).

## Anti-rocuronium ELISA

Ninety-six-well plates (Costar) were coated with HSA-Roc (mean of 10 rocuronium molecules grafted on a HSA carrier molecule) or HSA at 1 µg/mL in 1× PBS (Gibco; Thermo Fisher Scientific) overnight at 4°C overnight, washed 3 times with PBS–Tween 20 0.5% (PBST), blocked with PBS–3% BSA, and washed 3 times with PBST. Mouse serum (diluted 1:250,000-1:5,000-1:100) or monoclonal antibodies were incubated for 2 hours at room temperature, and bound antibodies were detected with

horseradish peroxidase–conjugated goat anti-mouse or anti-human IgG (Bethyl Laboratories) at 1:10,000 dilution. The secondary antibody was revealed using OPD substrate (Sigma-Aldrich) and the reaction stopped with 2 mol H₂SO₄, and absorbance was subsequently recorded at 490 nm and corrected at 620 nm using a spectrophotometer (Biophotometer, Eppendorff). The final optical density (OD) reported in all graphs and tables corresponds to the subtraction of the OD value of the HSA-Roc ELISA minus the OD value of the HSA ELISA. OD values were considered positive if they were at least 3 times higher than the OD value of the negative control.

## Competition ELISA

To ascertain antibody reactivity to rocuronium, and not toward the linker used to attach rocuronium to HSA or a neoepitope formed between rocuronium, the linker, and HSA, a competitive inhibition ELISA was developed.⁷ This inhibition ELISA also enabled us to detect cross-binding of antibodies to a panel of different NMBAs. Ninety-six-well plates (Costar) were coated with 1 µg/mL HSA-Roc at 4°C overnight, washed 3 times with PBST, and blocked with PBST containing 3% BSA at room temperature for 2 hours. The mAbs (300 ng/mL) were preincubated with 5-fold serial dilution of free NMBA drug (rocuronium, vecuronium, pancuronium, cisatracurium, atracurium) or with NMBA surrogates devoid of quaternary ammoniums (5a-DHT or diisopentyl succinate) from 15,000 µmol to 0.96 µmol.

The antibody-NMBA mixture was added to the ELISA plates and incubated at room temperature for 2 hours, washed 3 times, and revealed with horseradish peroxidase–conjugated goat anti-mouse/human IgG (Bethyl Laboratories) at 1:10,000 dilution. Plates were revealed using an OPD substrate (Sigma-Aldrich). The reaction was stopped with 2 mol H₂SO₄, and absorbance was subsequently recorded at 490 nm and corrected at 620 nm using a spectrophotometer (Biophotometer, Eppendorff). For purified mAbs, the drug concentration causing 50% inhibition was measured using a nonlinear fit [Inhibitor] versus response – Variable slope (4 parameters) with GraphPad Prism.

## Affinity measurements using BLI

Affinity of IgG mAbs for rocuronium was tested by BLI with the OctetRED 384 system. Amine reactive second-generation (aka AR2G) biosensors were covalently immobilized with HSA-Roc mean of 10 rocuronium molecules per HSA carrier molecule, then inserted in wells containing different concentrations of antibodies for 1800 seconds (association phase). The antigen–antibody complex was dissociated by introducing the biosensor in the reference buffer (PBS + BSA). Curves were retrieved as raw data from the Octet Data Analysis software and processed in Scrubber2 software before analyses by BIAevaluation software. IgG-free buffer was used as reference and for background subtraction. Fitting curves and $K_D$ values were obtained using the 1:1 Langmuir model.

## Affinity measurements using surface plasmon resonance

CM5 chips were primed using PBS, then washed 3 times with NaOH (50 mmol) and SDS (0.1%) for 180 seconds each time (5 µL/min). At least 5000 RU of antibody was covalently bound to a
CM5 chip by activating using 1:1 NHS:EDC (6000 seconds), in-
jecting the antibody (900 seconds) in acetate buffer pH 5.5 and
quenching with EtNH2 (600 seconds). Free rocuronium was in-
jected (30 µL/min) in PBS-BSA (1 mg/mL) at different concen-
trations. Signal was subtracted from the signal of the reference
canal bound with a nonspecific antibody. K<sub>D</sub> was determined us-
ing a kinetic analysis (RI fixed to 0, model 1:1 binding) with
Biaeval software.

Fab preparation

Fab was prepared following the instructions of the Pierce Fab
Preparation Kit (Thermo Fisher Scientific) with 4-hour digestion
with papain in the presence of 4 mmol cysteine. After purification
on protein A columns, gel filtration was performed (Tris 50 mmol,
pH 7.4; 100 mmol NaCl).

Single-chain variable fragment production,
purification, and characterization

Rocuronium-specific VH and VL sequences were built into
single-chain variable fragments (scFvs) with a polyglycine-
serine linker (GGGGS) between the VH and VL sequences.⁴
The VH-linker-VL sequences were synthesized at GenScript
with codon optimization for expression in *Drosophila* S2 cells,
cloned into pT-350 vector using *BglII* and *BstBI* restriction sites
(the vector was the kind gift of Dr Felix Rey, Institut Pasteur,
Paris). For transfection, 5 × 10⁶ *Drosophila* S2 cells were seeded
in a T25 conical flask containing 5 mL Schneider *Drosophila* me-
dium (Gibco) complemented with 10% FBS and incubated at
28°C overnight. Twenty-four hours later, 2 µg of pMT-Roc-
scFv-Strep plasmid were cotransfected with 0.1 µg (ratio 20:1)
of selection plasmid pCoBlast carrying a gene that confers resis-
tance to puromycin. Effectene transfection kit was used (Qiagen)
according to the vendor's protocol. Forty-eight hours after trans-
fection, the selection process was started by addition of puromy-
cin (InvivoGen) to the cells at a final concentration of 7 µg/mL.
Transfected cells were collected every 4 or 5 days by centrifuga-
tion at 90 × g for 5 minutes, and cell pellets were resuspended in
fresh medium containing 7 µg/mL puromycin. Cell propagation
was initiated about 2 weeks after transfection. Puromycin-
selected cells were adapted to serum-free HyClone Insect cell cul-
ture media (GE Healthcare Life Sciences) and were amplified in
large volume flasks as needed. Protein expression was induced
with 0.5 mmol CuSO₄ when the cell density reached
~7.5 × 10⁶ cells/mL. Seven days after induction, S2 cell suspen-
sion was centrifuged for 30 minutes at 15,000 × g to remove the
cells and the supernatant was collected. Avidin was added at 15
mg/L to sequester any biotin present in the medium. The superna-
tant, cleared by centrifugation at 20,000 × g for 30 minutes and
filtration, was loaded onto a Strep-Tactin Superflow high-
capacity 5 mL column (IBA) using a peristaltic pump or
AKTA. The column was washed with 15 mL of 0.1 mol Tris
(pH 8), 0.15 mol NaCl, and 1 mmol EDTA, and the protein was
eluted with 8 mL of the same buffer containing 2.5 mmol
desthiobiotin.

Crystallization and X-ray data collection

Crystallization screening trials were carried out by the vapor
diffusion method using a Mosquito nanodispensing system

(STPLabtech) following established protocols.³⁹ Briefly, we set
up crystallization sitting drops of 400 nL containing a 1:1 mixture
of protein sample (Fab or scFv) in complex with 10 mmol of ro-
curonium and crystallization solutions (672 different commer-
cially available conditions) equilibrated against 150 µL of
reservoir solution in multiwell plates (Greiner Bio-One). The
crystallization plates were stored at 18°C in a RockImager (For-
mulatrix) automated imaging system to monitor crystal growth.
Manual optimization was performed in Linbro plates by the
hanging-drop method by mixing 2 µL of protein samples with
2 µL of reservoir solution. The best crystals were obtained with
the conditions shown in Table E1 in this article's Online Reposi-
tory available at www.jacionline.org. The crystals were flash
cooled in liquid nitrogen for data collection using the crystalliza-
tion solution as cryoprotectant. X-ray diffraction data were
collected on beamlines PROXIMA-1 and PROXIMA-2A at the
Synchrotron SOLEIL (Saint-Aubin, France).

Structure determination and model refinement

The diffraction images for Fab-IP8 and scFv-IP11 were
processed with XDS,⁴⁰ and for Fab-IP2 with autoPROC,⁴¹
XDS, and STARANISO.⁴² Crystallographic calculations were
carried out with programs from the CCP4 program suite.⁴³

The structures of anti-rocuronium Fab and scFv in complex
with rocuronium were solved by molecular replacement with
Phaser⁴⁴ using the structures of the catalytic antibody Fab7A1
(pdb 2AJU) as search model. The final models of the complexes
were obtained through interactive cycles of manual model build-
ing with Coot⁴⁵ and reciprocal-space refinement with Refmac5⁴⁶
for the Fab-IP8 and scFv-IP11 complexes, and with Buster⁴⁷ for
the Fab-IP2 complex. X-ray data collection and model refinement
statistics are summarized in Table E1. Figures showing the crys-
tallographic structures were generated by PyMOL (Schrödinger).

Human mast cell activation test

CD34⁺ precursor cells were isolated from peripheral blood
mononuclear cells of healthy donors (provided by the French
Blood Bank EFS). CD34⁺ cells were maintained for 1 week under
serum-free conditions using StemSpan medium (9655 STEM-
CELL Technologies) supplemented with recombinant human
IL-6 (50 ng/mL; 200-06 PeproTech), human IL-3 (10 ng/mL;
200-03 PeproTech), 3% supernatant of CHO transfectants
secreting mouse SCF (a gift from Dr P. Dubreuil, Marseille,
France, 3% [corresponding to ~50 ng/mL SCF]), and ciproflox-
acin (10 ng/mL; 17850-5G-F Sigma-Aldrich). Thereafter, the
cells were maintained at least 7 weeks in Iscove modified Dul-
becco medium–GlutaMAX I (31980048), 2-mercaptoethanol
(31-350-010), insulin-transferrin selenium (2506865) (all from
Gibco), sodium pyruvate (S8636 Sigma-Aldrich), 0.5% BSA
(A2153 Sigma-Aldrich), penicillin/streptomycin (100 U/mL/
100 µg/mL; Life Technologies), IL-6 (50 ng/mL 200-06 Pepro-
Tech) and 3% supernatant of CHO transfectants secreting mouse
SCF. Before their use in experiments, mast cells were tested for
phenotype by flow cytometry (CD117⁺, FcεRI⁺). Cells were
ready for experiments after ~10 weeks in culture (at which
time >95% of all cells were CD117⁺FcεRI⁺).

Human mast cells were sensitized overnight with anti-
rocuronium human IgE antibodies at 1 µg/mL. Cells were then
washed and stimulated with increasing doses of HSA-Roc in
Tyrode buffer. Mast cell degranulation was measured by flow cytometry using fluorescent avidin (5 µg/mL; A2170, Invitrogen), which binds to heparin contained in mast cell granules. Data were acquired with a MACSQuant MQ10 flow cytometer (Miltenyi Biotec) and analyzed by FlowJo v10.8.1 software (Becton Dickinson).

## Hulman BAT

Human whole blood was preincubated for 3 hours at room temperature with anti-rocuronium IgE at 10 µg/mL. According to manufacturers' instructions (Flow CAST, FK-CCR, Buhlmann), 50 µL of blood was incubated with HSA or HSA-Roc for 15 minutes at 37°C with staining reagents (anti-CCR3 phycoerythrin and anti-CD63 fluorescein isothiocyanate) in an IL-3-containing buffer. Red blood cells were lysed and the cells analyzed by flow cytometry. Basophils were gated as SSClo/CCR3hi cells, and the percentage of CD63⁺ cells was used as the activation readout.

## Neutrophil activation tests

Neutrophils were isolated from fresh human EDTA-drawn blood using the MACSxpress Neutrophil Isolation kit (Miltenyi Biotec). Immune complexes (ICs) were formed for 30 minutes at 37°C in HBSS/2% FCS. For flow cytometry, 1 × 10⁵ neutrophils were stimulated with ICs at a final concentration of 30 µg/mL HSA-Roc and 100 µg/mL anti-Roc IgG in 200 µL in HBSS/2% FCS for 30 minutes at 37°C, washed, and stained with anti-CD62L (clone DREG-56) and anti-CD11b (clone M1/70) (both BD Biosciences) for 15 minutes at 4°C, washed, and analyzed on MACSquant16 (Miltenyi Biotec). For reactive oxygen species production measurements, 5 × 10⁴ neutrophils in 100 µL HBSS containing 2% FCS were incubated with or without stimuli (10 µg/mL HSA-Roc, or indicated preformed ICs at a final concentration of 10 µg/mL HSA-Roc and 100 µg/mL anti-Roc IgG) in the presence of 50 µmol luminol (Sigma-Aldrich) in a 96-well View-Plate (PerkinElmer). Chemiluminescence was measured every 240 seconds using a Spark microplate reader (Tecan) for 2 hours. The area under the curve was calculated for each sample tested in triplicate.

## Passive systemic anaphylaxis in mice

The hFcεRI¹ g mFcεRI⁻/⁺ mice³³ were injected intravenously with 5 µg of anti-rocuronium human IgE mAbs at time 0 hours and challenged 24 hours later with 2 µg of HSA-Roc. Specifically for anti-rocuronium low-affinity mAb IP12, 20 µg of human IgE mAb IP12 was injected intravenously twice at 0 hours and 24 hours to favor human IgE biodistribution,⁴⁸ and mice were challenged 24 hours later with 100 µg of HSA-Roc. Control mice were injected with irrelevant human IgE and physiologic serum. Central temperature was monitored with a digital thermometer with a rectal probe (YSI), and time of death was recorded.

## Statistical analyses

The R environment v4.3.1 was used for all analyses (R Project; www.r-project.org). Data were neither averaged nor normalized before analyses. Response variables were log₂ converted when required for better adjustment to linear models. Mixed models using the 'lmer'() function of the 'lme4' package was used in order to consider the time series (Fig 5, B-D), or the random effect of the

cell samples (Fig 4, B and C) or donors (Fig 4, D). In time series (Fig 5, B-D), the fixed effect of each treatment was compared to the reference (physiologic serum) by *t* test. Otherwise (Fig 4, B-D), effects of treatment were 2 × 2 compared (contrast comparisons) using the 'emmeans()' function of the 'emmeans' package. Statistical significance was set to *P* ≤ .05. In each panel, type I error was controlled for by correcting the *P* values according to the Benjamini-Hochberg method ('BH' option in the 'p-adjust()' function of R).

## RESULTS

### Anti-rocuronium antibody repertoires in mice are oligoclonal

To generate the first anti-rocuronium mAbs, we decided to immunize mice against rocuronium and to use rocuronium-specific plasma cells as a source of the highest-affinity mAbs.⁴⁹,⁵⁰ Because rocuronium injections in adjuvant failed to be immunogenic (data not shown), we immunized mice with rocuronium coupled to KLH, as a large carrier protein that favors bystander activation. Cervical lymph nodes were collected from 2 mice displaying the highest anti-rocuronium titer (Fig 1, A), and their cells were encapsulated into microfluidic droplets with fluorescent bioassay reagents and magnetic beads coated with an anti-mouse kappa light chain (Igκ) nanobody (VH_H) (Fig 1, B). Immediately after droplet generation, these were subjected to a magnetic field that induced magnetic beads within each droplet to form a line (beadline) (Fig 1, C) and serve as a physical surface for a double-fluorescent sandwich ELISA, revealing IgG secretion from the cell using Alexa Fluor 647-labeled Fc-specific anti-mouse IgG, and specificity of that IgG for rocuronium using Alexa Fluor 488-labeled rocuronium-coupled HSA (see Fig E1 and Fig E2, A, in the Online Repository available at www.jacionline.org). The spatial distribution of fluorescence in the droplets was analyzed by reinjecting them into a second microfluidic chip where each droplet was scanned with superimposed laser lines and epifluorescence was detected using photomultiplier tubes. Secreted IgG and antigen binding were determined from red and green fluorescence localization to the beadline, respectively, and droplets that contained a cell of interest were sorted by fluorescence-activated dielectrophoretic sorting; 3162 and 3213 droplets that contained secreted IgG with detectable binding to HSA-Roc were sorted from lymph node cells of Mouse1 and Mouse2, respectively (Fig E2, B). Single-cell barcoded RNA sequencing was immediately performed (Fig 1, D) to retrieve the sequences encoding the variable heavy (VH) and light (VL) chains of the IgG secreted by the sorted plasma cell, as we reported earlier.²⁶ Sequencing data were processed computationally to identify cell-specific barcodes and to identify cognate VH-VL pairs (one pair per barcode, selecting the most probable VH and VL pair) and were corrected for errors introduced by PCR or sequencing by creation of consensus sequences.²⁶

Using the 'repertoire_profiler' bioinformatic pipeline, we established the anti-rocuronium antibody repertoire profile of the 2 immunized mice. We obtained a total of 628 VH and VL pairs from Mouse1 and 421 VH and VL pairs from Mouse2 (Fig 1, E).

Analyses of V(D)J gene usage demonstrated an oligoclonal antibody repertoire profile for both mice. Indeed, for Mouse1, 2 V_HJ_H and 2 V_LJ_L rearrangements encoded >91% and >77% of all the VH and VL rearrangements, respectively. Among these, VH1-
<figure><img src="image_7.png" /><figcaption>**FIG 1.** Generation of rocuronium-specific antibody repertoires from immunized mice. (A) Scheme of mice immunization strategy and blood sampling (top) and IgG anti-rocuronium ELISA results from sera of mice immunized with KLH-Roc or KLH (bottom). (B-D) Scheme of droplet microfluidic steps: (B) droplet generation, (C) droplet sorting, and (D) in-droplet single-cell reverse transcription and barcoding. PMTs, Photomultipliers. (E and F) Donut plots of VJ gene usage among (E) all VH (left) and VL (right) or among (F) clonal groups (identical VJ genes and CDR3 length) identified from Mouse1 (top) and Mouse2 (bottom). Central value indicates number of sequences. VH-VL pairs corresponding to mAbs further characterized in this work are indicated (IP1-IP12 for Mouse1 and IP21-IP33 for Mouse2). Five most representative VJ families are colored and labeled; others are shades of gray. (G) Circos plots of pairing of VH clonal groups from (F) with their VL rearrangement for Mouse1 (left) and Mouse2 (right).</figcaption></figure>
7_J2/VK2-112_J2 represented 48% and VH1-55_J2/VK4-68_J5 represented 44% of all VH-VL pairs. Strikingly, the latter rearrangement also represented 26% of all VH-VL pairs for Mouse2 (Fig 1, E). Clonal groups were defined as at least 3 distinct VH-VL pairs with identical VJ gene rearrangement and identical CDR3 length, leading to 2 VH clonal groups and 2 VL clonal groups for Mouse1, and 4 VH clonal groups and 15 VL clonal groups for Mouse2. The V1-55_J2 rearrangement represented 33% and 74% of the VH clonal groups, and the V4-68_J5 rearrangement 65% and 51% of the VL clonal groups of Mouse1 and Mouse2, respectively (Fig 1, F). Circos plot representation of the VH-VL pairing highlights these clonal groups among the total repertoire, with a given VH_JH rearrangement pairing systematically with a given VL_JL rearrangement, with a few exceptions (Fig 1, G). For each VH clonal group, a phylogenic analysis of the VH amino acid sequence was performed, and 15 and 13 VH-VL pairs from the major clonotypes of Mouse1 and Mouse2, respectively, were selected (see Fig E3 in the Online Repository available at www.jacionline.org) and expressed as chimeric human IgG1,κ for further characterization and validation as anti-rocuronium mAbs.

## Anti-rocuronium mAbs interact with rocuronium at high affinity

Using ELISA, we confirmed that 11 (73%) of 15 and 7 (54%) of 13 of mAbs selected from Mouse1 and Mouse2, respectively (Fig 2, A, Table I) bound HSA-Roc. No mAbs among IP6 (V1-75_J3), IP13 (V1-62-2/71_J2), IP14 (V1-85_J2), or IP15 (V3-8/3_J3) from Mouse1, and IP28 and IP29 (V3-6_J3), IP30 and IP31 (HV1-18_J2), IP32 (HV1-26_J3), and IP33 (HV1-39_J1) from Mouse2 bound HSA-Roc, suggesting that these VH-VL pairs instead bound fluorophores used for cell sorting. To ensure that the other 18 mAbs bound rocuronium and not the linker used to engraft rocuronium onto HSA, we performed competition ELISA in the presence of free NMBA molecules or analogs (Fig 2, B). Nine of these mAbs demonstrated exquisite specificity for rocuronium: IP7, IP8, IP9, IP10, IP12, IP21, IP22, and IP23 (Fig 2, C, and see Fig E4 in the Online Repository available at www.jacionline.org). Indeed, only free rocuronium, but not free molecules with the same aminosteroid scaffold (ie, closely related homologs vecuronium and pancuronium, NMBAs atracurium, and cisatracurium) or 5α-dihydrotestosterone and diisopentyl succinate, which lack quaternary ammonium groups, could inhibit their interaction with HSA-Roc. The mAbs IP5, IP11, IP24, and IP25 cross-reacted to rocuronium and vecuronium, whereas the mAbs IP1, IP2, IP3, and IP4 cross-reacted to rocuronium, vecuronium, and pancuronium (Fig 2, C, and Fig E4). Estimated drug concentrations causing 50% inhibition of all these cross-reactions indicated 100- to 1000-fold better affinities for rocuronium than for vecuronium and pancuronium, respectively (Table I). However, IP26 and IP27 (V3-6_J3) from Mouse2 bound neither free rocuronium nor HSA itself but rather the linker used to couple rocuronium to HSA, or a neoepitope created by this chemical modification. Thus, competitive inhibition ELISA confirmed that only 11 (73%) of 15 and 5 (38%) of 13 of mAbs selected initially from Mouse1 and Mouse2, respectively (Table I), bound free rocuronium.

Identification of these confirmed anti-rocuronium mAbs allowed us to reanalyze the antibody repertoires by only considering the clonal groups whose VH or VL rearrangement corresponded to a VH_JH or VL_JL used in a mAb confirmed to

bind free rocuronium. These “functional” repertoires were reduced to 3 VH and 2 VL rearrangements for Mouse1 and a single VH and VL rearrangement for Mouse2 (see Fig E5, A and B, in the Online Repository available at www.jacionline.org), with a mutational load compared to germline showing log-normal-like distributions for Mouse1 and Mouse2 with an average of ~5 to 7 nucleotide mutations in the VH and ~3 to 5 mutations in the VL (Fig E5, C). These functional repertoires unequivocally highlight the absolute pairing of HV1-55_J2 with KV4-68_J5, and of HV1-7_J2 with KV2-112_J2. Even if both mice generated large numbers of HV1-55_J2 encoded sequences that almost exclusively paired to KV4-68_J5, suggestive of a public VH repertoire (identical rearrangements with similar/identical CDR3 size and sequence), CDR sequences diverged largely between the plasma cells from the 2 mice and clustered separately (see Fig E6 in the Online Repository).

As a first exploration of the interaction of these 16 mAbs with rocuronium, we used BLI to measure their avidity toward rocuronium haptenized to HSA with an average molar ratio of 15:1. The avidities of these mAbs were very high toward rocuronium-HSA and ranged from 10 pmol to 1.5 nmol, except for mAb IP12 at 220 nmol (see Fig E7 in the Online Repository available at www.jacionline.org and Table I). The very small size of rocuronium (530 Da) is incompatible with the relatively poor sensitivity of BLI but remains inside the lower limit of detection for surface plasmon resonance (SPR). SPR thus allowed to measure the affinity ($K_D$) between immobilized mAbs and free rocuronium in solution ranging from 1.2 to 56 nmol for all mAbs except mAb IP12, which exhibited a poor $K_D$ of 1.9 mmol, in a transitory mode with a very fast association and dissociation (Fig 2, D, Table I, and see Table E2 in the Online Repository).

## Anti-rocuronium mAbs interact differently with rocuronium

Rocuronium ring A exists in 2 different conformations, twist boat (Fig 3, A) or chair (Fig 3, B),¹⁶ with the sterically less demanding twist-boat conformation being favorable for its accommodation within sugammadex.¹⁷ We thus wondered (1) if anti-rocuronium mAbs would also bind preferentially to one of these conformations, (2) if the ammonium groups of rocuronium would be essential for the binding as suggested earlier,¹⁴,¹⁵ and (3) if differences in binding modalities existed among the collection of mAbs we identified. We thus performed crystallographic studies with either Fab fragments or scFvs of representative mAbs from different clonal groups: IP2 (HV1-55_J2 KV4-68_J5), IP8 (HV1-7_J2 KV2-112_J2), and IP11 (HV1-81_J2 KV2-112_J2) representing the 3 clonal groups of Mouse1 and the clonal group of Mouse2, and mAbs cross-binding rocuronium and vecuronium (IP2, IP11) and a mAb with exclusive binding to rocuronium (IP8). These antibody fragments and rocuronium cocrystalized efficiently, and we solved the structure of Fab-IP2, Fab-IP8, and scFv-IP11 complexed with rocuronium, respectively, at 2.4, 1.7, and 1.2 Å resolution (Fig 3, C-E, and Table E1). Coordinates and structure factors of Fab-IP2, Fab-IP8, and scFv-IP11 have been deposited in the Protein Data Bank (www.wwpdb.org) under accession nos. 9G6S, 9G6R, and 9G6Q, respectively.

In the Fab-IP2 complex, rocuronium interacts with VH and VL like an arrow in a target (Fig 3, C). The allylpyrrolidinium group encompassing the quaternary ammonium is completely buried in
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

**FIG 2.** Characterization of rocuronium-specific mAbs from immunized mice. (A) Rocuronium-specific ELISA of representative IgG mAbs from indicated mice. (B) Structural formula of indicated molecules. (C) Competition ELISA of anti-rocuronium IgG ELISA at fixed concentration of mAbs but increasing concentrations of indicated molecules free in solution. 5a-DHT, 5-Alpha-dihydrotestosterone; DS, diisopentyl succinate; QA, quaternary ammonium. (D) Left, Scheme of SPR affinity measurements. Right, SPR results for interaction between immobilized mAbs and varying concentrations of free rocuronium in solution. KD was obtained by fitting curves using heterogenous ligand model. RU, Resonance unit.

the binding pocket, and the morpholino with the hydroxyl group is exposed to the solvent. This allows vecuronium and pancuronium to bind, as their methylpiperidinium substituent is outside the binding pocket and exposed to the solvent. The conformation of the androstane ring A of rocuronium (chair or twist boat) cannot be precisely determined as a result of mobility of this region in the structure.

In the cocrystal structure of Fab-IP8 (Fig 3, D), rocuronium is in the twist-boat conformation and interacts differently compared to Fab-IP2—that is, the Fab-IP8 VH and VL form a pocket inside of which the androstane component of rocuronium binds flat, the hydroxyl group is exposed to the solvent, and the acetoxyl group is buried inside the binding pocket. The specificity for rocuronium and not vecuronium or pancuronium could be explained by the fact that no space remained in the hydrophobic pocket formed by the VH and the VL to accommodate the methylpiperidinium

substituent of vecuronium and pancuronium, and by numerous hydrophobic contacts made by the allyl group of rocuronium.

scFv-IP11 bound similarly to rocuronium as Fab-IP8, but the binding of rocuronium in its twist-boat conformation induced a rearrangement of the binding site (Fig 3, E). The androstane moiety is stacked between VH residues Trp98 and Tyr56, but unlike IP2 and IP8, the hydroxyl group makes a hydrogen bond with VH Ser54. Interestingly, the stacking interaction with Trp98-H and hydrogen bond with Ser54-H appear to sequester rocuronium in the binding site.

Of note, the hydroxyl group, which served as the attachment point to the rocuronium-carrier proteins we used for immunization and screening, is not involved in the binding with IP2 and IP8, whereas the acetoxyl group is buried inside the binding pocket of all 3 antibodies. Importantly, these cocrystal structures revealed that the allylpyrrolidine substituent containing the quaternary
<table><caption>TABLE I. Characteristics of mAbs expressed from repertoires of Mouse1 and Mouse2</caption><thead><tr><th rowspan="2">Mouse</th><th rowspan="2">mAb</th><th rowspan="2">VH gene</th><th colspan="4">Sanger sequencing</th><th rowspan="2">BLI<br>Avidity for<br>HSA-Roc<br>(nmol)</th><th rowspan="2">SPR<br>Affinity<br>for free<br>R<br>(nmol)</th><th colspan="4">Competition ELISA</th><th colspan="2">Mutations</th></tr><tr><th>JH gene</th><th>VL gene</th><th>JL gene</th><th>Specificity</th><th>IC<sub>50</sub> R<br>(µmol)</th><th>IC<sub>50</sub> V<br>(µmol)</th><th>IC<sub>50</sub> P<br>(µmol)</th><th>VH aa ≠</th><th>VL aa ≠</th></tr></thead><tbody><tr><td rowspan="17">Mouse1</td><td>IP1</td><td>V1-55†</td><td>J2†</td><td>KV4-68†</td><td>KJ5†</td><td>0.9</td><td>NT</td><td>R-V-P</td><td>0.3</td><td>177</td><td>6,995</td><td>3</td><td>5</td></tr><tr><td>IP2</td><td>V1-55†</td><td>J2†</td><td>KV4-68†</td><td>KJ5†</td><td>0.6</td><td>NT</td><td>R-V-P</td><td>3.8</td><td>20</td><td>787</td><td>3</td><td>4</td></tr><tr><td>IP3</td><td>V1-55†</td><td>J2†</td><td>KV4-68†</td><td>KJ5†</td><td>1.2</td><td>NT</td><td>R-V-P</td><td>1.3</td><td>110</td><td>737</td><td>3</td><td>4</td></tr><tr><td>IP4</td><td>V1-55†</td><td>J2†</td><td>KV4-68†</td><td>KJ5†</td><td>0.9</td><td>NT</td><td>R-V-P</td><td>3.1</td><td>240</td><td>3,635</td><td>7</td><td>3</td></tr><tr><td>IP5</td><td>V1-55†</td><td>J2†</td><td>KV4-68†</td><td>KJ5†</td><td>0.4</td><td>NT</td><td>R-V</td><td>289</td><td>615</td><td>—</td><td>6</td><td>3</td></tr><tr><td>IP6*</td><td>V1-75*</td><td>J3*</td><td>KV6-25*</td><td>KJ1*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>6*</td><td>7*</td></tr><tr><td>IP7</td><td>V1-7†</td><td>J2†</td><td>KV2-112†</td><td>KJ2†</td><td>0.82</td><td>NT</td><td>R</td><td>4.4</td><td>—</td><td>—</td><td>9</td><td>3</td></tr><tr><td>IP8</td><td>V1-7†</td><td>J2†</td><td>KV2-112†</td><td>KJ2†</td><td>0.46</td><td>NT</td><td>R</td><td>3.3</td><td>—</td><td>—</td><td>3</td><td>3</td></tr><tr><td>IP9</td><td>V1-7†</td><td>J2†</td><td>KV2-112†</td><td>KJ2†</td><td>0.8</td><td>NT</td><td>R</td><td>3.5</td><td>—</td><td>—</td><td>4</td><td>2</td></tr><tr><td>IP10</td><td>V1-7†</td><td>J2†</td><td>KV2-112†</td><td>KJ2†</td><td>0.86</td><td>NT</td><td>R</td><td>5.0</td><td>—</td><td>—</td><td>8</td><td>5</td></tr><tr><td>IP11</td><td>V1-81</td><td>J2</td><td>KV2-112†</td><td>KJ2†</td><td>0.53</td><td>56</td><td>R-V</td><td>5.0</td><td>548</td><td>—</td><td>5</td><td>1</td></tr><tr><td>IP12</td><td>V1-81</td><td>J2</td><td>KV4-68§</td><td>KJ5§</td><td>220</td><td>1,990,000</td><td>R</td><td>2,572</td><td>—</td><td>—</td><td>4</td><td>4</td></tr><tr><td>IP13*</td><td>V1-62-2/71*</td><td>J2*</td><td>KV19-93*</td><td>KJ2*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>5*</td><td>7*</td></tr><tr><td>IP14*</td><td>V1-85*</td><td>J2*</td><td>KV6-13*</td><td>KJ2*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>9*</td><td>7*</td></tr><tr><td>IP15*</td><td>V3-8/3*</td><td>J1*</td><td>KV14-111*</td><td>KJ1*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>5*</td><td>8*</td></tr><tr><td rowspan="17">Mouse2</td><td>IP21</td><td>V1-55¶</td><td>J2¶</td><td>KV4-6¶8</td><td>KJ5¶</td><td>0.13</td><td>NT</td><td>R</td><td>26</td><td>—</td><td>—</td><td>10</td><td>6</td></tr><tr><td>IP22</td><td>V1-55¶</td><td>J2¶</td><td>KV4-68¶</td><td>KJ5¶</td><td>0.2</td><td>NT</td><td>R</td><td>14</td><td>—</td><td>—</td><td>6</td><td>1</td></tr><tr><td>IP23</td><td>V1-55¶</td><td>J2¶</td><td>KV4-68¶</td><td>KJ5¶</td><td>NT</td><td>NT</td><td>R</td><td>109</td><td>—</td><td>—</td><td>9</td><td>8</td></tr><tr><td>IP24</td><td>V1-55¶</td><td>J2¶</td><td>KV4-68¶</td><td>KJ5¶</td><td>0.01</td><td>12</td><td>R-V</td><td>18</td><td>NT</td><td>—</td><td>6</td><td>1</td></tr><tr><td>IP25</td><td>V1-55¶</td><td>J2¶</td><td>KV4-68¶</td><td>KJ5¶</td><td>NT</td><td>NT</td><td>R-V</td><td>20</td><td>682</td><td>—</td><td>2</td><td>1</td></tr><tr><td>IP26*</td><td>V3-6*</td><td>J3*</td><td>KV12-89*</td><td>GKJ4*</td><td colspan="6">Binding linker or neoepitope*</td><td>3*</td><td>0*</td></tr><tr><td>IP27*</td><td>V3-6*</td><td>J3*</td><td>KV12-89*</td><td>GKJ4*</td><td colspan="6">Binding linker or neoepitope*</td><td>7*</td><td>1*</td></tr><tr><td>IP28*</td><td>V3-6*</td><td>J3*</td><td>KV10-94*</td><td>KJ2*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>3*</td><td>4*</td></tr><tr><td>IP29*</td><td>V3-6*</td><td>J3*</td><td>KV10-94*</td><td>KJ2*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>6*</td><td>4*</td></tr><tr><td>IP30*</td><td>HV1-18*</td><td>J2*</td><td>KV6-17*</td><td>KJ5*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>1*</td><td>7*</td></tr><tr><td>IP31*</td><td>HV1-18*</td><td>J2*</td><td>KV6-17*</td><td>KJ5*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>4*</td><td>10*</td></tr><tr><td>IP32*</td><td>HV1-26*</td><td>J3*</td><td>KV12-46*</td><td>J2*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>12*</td><td>4*</td></tr><tr><td>IP33*</td><td>HV1-39*</td><td>J1*</td><td>KV12-44*</td><td>KJ2*</td><td>NB*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>NT*</td><td>5*</td><td>3*</td></tr></tbody></table>

The mAbs were measured for binding to HSA-Roc by BLI (avidity) and to free rocuronium by SPR (affinity), and evaluated for cross-reactivity to rocuronium (R), vecuronium (V), and pancuronium (P) by competition in HSA-Roc ELISA (specificity and IC<sub>50</sub> values). Number of amino acid (aa) mutation differences (≠) compared to germline sequence are indicated for VH and VL.

IC<sub>50</sub>, Half-maximal inhibitory concentration; NB, not binding; NT, not tested.

*V(D)J rearrangements that do not translate into anti-rocuronium mAb.

†‡§¶Identical gene families used in V-J rearrangements.

ammonium is completely buried and makes numerous van der Waals contacts with the antibody (Fig 3, C-E), emphasizing the important role of this group in the rocuronium binding mode, in agreement with hypotheses extrapolated from serum cross-reactivity in patients.<sup>13,51</sup> These data demonstrate that IP2, IP8, and IP11 complexes with rocuronium display an essential role of the quaternary ammonium group for the interaction with antibodies, in a twist-boat or chair conformation, with the hydroxyl group O atom making a hydrogen bond with Ser54H in the VH of IP11. The ring A twist-boat rocuronium conformation observed in IP8 and IP11 may be due to induced fit, as described for rocuronium-cyclodextrin complexes.<sup>17</sup>

## Anti-rocuronium mAbs allow to model rocuronium anaphylaxis *in vivo*

Next, we wondered if anti-rocuronium mAbs expressed as human IgEs could sensitize and promote human mast cell and basophil activation in the presence of rocuronium. Human mast cells and basophils express the high-affinity IgE receptor FcεRI that allows them to be sensitized (prearmed) with IgE *in vivo* for immediate cell activation after IgE engagement with allergens, leading to rapid anaphylactogenic mediator release. Human mast cells derived from peripheral blood mononuclear cells of healthy donors were used to develop a mast cell activation test (MAT), and whole blood from healthy donors was used to develop

a basophil activation test (BAT) (Fig 4, A). Mast cells sensitized with IP2, IP8, or IP11 human IgEs degranulated in the presence of HSA-Roc but not in the presence of free rocuronium or HSA alone. IP12 human IgE weakly degranulated mast cells and only at elevated doses of HSA-Roc, as expected from IP12's poor affinity for rocuronium (Fig 4, B). Similarly, basophils sensitized with IP2, IP8, or IP11, but not IP12, human IgEs became dose-dependently activated in the presence of HSA-Roc but not in the presence of free rocuronium (Fig 4, C). Thus, MAT and BAT were positive only in the presence of haptenized, but not free, rocuronium, as previously reported.<sup>52</sup> Because we previously reported that NMBA anaphylaxis in humans can engage an IgE pathway but also an IgG pathway leading to neutrophil activation by IgG receptors,<sup>9</sup> we investigated neutrophil activation by immune complexes (ICs) made of anti-rocuronium human IgG mAbs and HSA-Roc. Neutrophils from healthy donors expressed elevated levels of CD11b, low levels of CD62L, and liberated reactive oxygen species when stimulated with ICs made of anti-rocuronium human IgG1, but not with ICs made of their effectorless variants (N<sub>297</sub>A mutation of the IgG1 heavy chain), and HSA-Roc compared to unstimulated controls (Fig 4, D, and see Fig E8 in the Online Repository available at www.jacionline.org).

To test the anaphylactogenic potential of IP2, IP8, IP11, and IP12 human IgEs *in vivo*, we used mice expressing human FcεRI, sensitized them with anti-rocuronium IgEs, and challenged them with HSA-Roc in a classical model of passive systemic
<figure><img src="image_17.png" /><figcaption>**FIG 3.** Crystal structures of mAbs in complex with rocuronium. (A) Rocuronium twist-boat conformation. (B) Rocuronium chair conformation. (C-E) Top, Surface representation of cocrystal structures of rocuronium-specific (C) Fab-IP2, (D) Fab-IP8, and (E) scFv-IP11. Bottom, Superposition of rocuronium and key residues of binding site. Rocuronium molecule is shown in yellow; antibody heavy chain and light chain are green and cyan, respectively. O atoms are in red and N atoms in blue.</figcaption></figure>

anaphylaxis (Fig 5, A). Rocuronium bound to HSA was used instead of free rocuronium because by itself, the latter induces hypothermia and paralysis. Mice sensitized with increasing doses of IP8 human IgE and subsequently challenged with 2 µg HSA-Roc dose-dependently displayed a pronounced drop in central body temperature (Fig 5, B), a hallmark of anaphylaxis in mice.⁵³ Mice sensitized with 5 µg of IP2, IP8, or IP11 human IgEs, but not unsensitized mice, and challenged with 2 µg HSA-Roc displayed a similar drop in central body temperature (Fig 5, C). Thus, although identified from IgG-secreting plasma cells or plasmablasts, the IP2, IP8, and IP11 VH-VL rearrangements generate rocuronium-specific antibodies of high affinity that demonstrate potential as human IgE for human mast cell and basophil activation *in vitro* and induction of anaphylaxis *in vivo* in mice humanized for FcεRI. To induce an IgE-mediated passive systemic anaphylaxis of similar intensity with the lowest affinity antirocuronium mAb we identified herein, mAb IP12, it was necessary to increase sensitization 8-fold (40 µg IP12 human IgE) and antigen dose 70-fold (140 µg) (Fig 5, D).

DISCUSSION

In this work, we selected rocuronium, one of the most widely used NMBAs, to investigate the rocuronium-specific antibody response in immunized mice and devise the first mouse model of rocuronium anaphylaxis. A cutting-edge high-throughput microfluidic droplet sorting system²⁶ allowed us to identify hundreds of rocuronium-specific antibodies from immunized mice. These antibodies were derived from limited $V_H-V_L$ rearrangements and showed convergence between the 2 immunized mice. The representative antibodies from different rearrangements show high avidity and affinity for rocuronium, and for some examples also cross-reactivity to rocuronium chemical family members vecuronium and pancuronium, but not any other NMBA. Cocrystal structures revealed different binding modes of rocuronium by these mAbs, with the ammonium group of rocuronium always involved in the binding. Expressed as human IgE or IgG1 antibodies and aggregated by rocuronium, these antibodies demonstrated *in vitro* efficacy to activate human mast cells and basophils, and human neutrophils, respectively. Passively
transferred to mice expressing human IgE receptor FcεRI, high- and low-affinity anti-rocuronium IgE antibodies sensitized mice that developed systemic anaphylaxis on injection of rocuronium coupled to HSA.

NMBA are small molecules of less than 1 kDa (rocuronium, 530 Da). The antibody repertoire against low-molecular-weight drugs (<1 kDa) has never been investigated, which may be due to the technical difficulty in identifying the scarce drug-specific B cells elicited by poorly or nonimmunogenic small compounds. Here we used for B-cell screening and sorting plasma cells and plasmablasts from draining lymph nodes that secrete high amounts of IgG, and a bioassay setup and duration that allows accumulation of these IgG onto a physical surface before detection and sorting. Even if we obtained several hundred VH-VL pairs of potential anti-rocuronium mAbs using this approach, we cannot compare it to standard methods for this type of small drug because no other repertoire is currently available. However, by expressing representative VH-VL pairs from the clonal families of these repertoires, and considering only those families with confirmed members binding to free rocuronium, we predict that this method allowed us to obtain about 700 potential anti-rocuronium mAbs from 2 immunized mice. The same method applied to large, highly immunogenic protein antigens, demonstrated earlier, that from 150 to 530 mAbs could be identified from a single immunized mouse.²⁶

Bioinformatic analyses of the initial $V_H$-$V_L$ repertoires identified only 2 major $V_H$-$V_L$ rearrangements from both mice, with one (HV1-55_J2/KV4-68_J5) shared between the 2 mice. Further analyses on representative $V_H$-$V_L$ pairs expressed as IgG mAbs and extrapolations thereof demonstrated that only these 2 clonal families (HV1-7_J2 and HV1-55_J2) and the HV1-81_J2 family represented the functional anti-rocuronium repertoire from these immunized mice. These 3 families represented >90% of the $V_H$-$V_L$ sequences of Mouse1 but only 25% of the VH-VL sequences of Mouse2. This might be a consequence of largely higher “noise” in the Illumina sequencing of Mouse2 cells compared to Mouse1 cells. In both cases, however, the functional repertoires were oligoclonal and monoclonal, respectively, which might be a consequence of (1) the small size of rocuronium, (2) the restricted number of mouse germline $V_H$-$V_L$ sequence able to bind this drug, and/or (3) the effect of the linker attached to the hydroxyl group of rocuronium that may even further restrict the number of these germline sequences. The mono/oligoclonal nature of the repertoire is not due to the cell population (plasma cells and plasmablasts) chosen or to the droplet microfluidic method used; we know this because we previously reported several highly diverse repertoires of IgG-secreting plasma cells against tetanus toxoid, glucose-6-phosphate isomerase, or tetraspanin-8 covering multiple V-gene families.²⁶ The mono/oligoclonal nature of the anti-rocuronium repertoires is thus due to the size and nature of the antigen rather than to the method used to identify these repertoires.

A strong rearrangement convergence was observed between the 2 mouse repertoires in this study. Convergent (or public) clonotypes (identical rearrangements with similar/identical CDR3 size and sequence) in unrelated individuals with viral infection or vaccination has been reported in particular for immunoglobulin kappa and lambda chains as a result of the lack of combination diversity and junction diversity during light chain rearrangement,⁵⁴,⁵⁵ but less so for IGH.⁵⁶,⁵⁷ These public VLs were able to pair with diverse VHs in multiple donors.⁵⁸

In our work, 2 $V_L$-$V_L$ rearrangements (KV2-112_J2 and KV4-68_J5) paired with over 90% of VH sequences in the 2 mice analyzed. Within the functional repertoire we identified, HV1-55_J2 paired exclusively with KV4-68_J5 and HV1-7_J2 paired exclusively with KV2-112_J2, suggesting that these pairings are required for rocuronium binding. Indeed, exchanging light chains between HV1-55_J2 and HV1-7_J2 mAbs (ie, between IP2 and IP8, or between IP5 and IP7) led to a loss of binding to rocuronium (data not shown). For the HV1-81_J2 clonal group, we expressed 2 mAbs that shared the same VH rearrangement with identical CDRH1 and CDRH3, and only 2 amino acid mutations in the CDRH2, 1 mAb with light chain KV2-112_J2 (IP11), and 1 mAb with light chain KV4-68_J5 (IP12); these demonstrated a ~35,000-fold difference in affinity for rocuronium in favor of IP11. Because of the barcoding method used (split-and-pool synthesis) in the single-cell sequencing method and the probabilistic pairing of the most abundant $V_H$ and $V_L$ associated to a given barcode, we wondered if IP12 was not the consequence of a $V_H$-$V_L$ mispairing that led to the recombinant expression of this antibody, whereas it should have been paired with a more “favorable” light chain for this heavy chain rearrangement (ie, KV2-112_J2). Indeed, when we expressed the $V_H$ of IP11 with the $V_L$ of IP12, the avidity for HSA-Roc was equivalent to that of IP12, and when we expressed the $V_H$ of IP12 with the $V_L$ of IP11, the avidity was equivalent to that of IP11 (data not shown). The less common $V_L$ pairings to $V_H$ found in these repertoires therefore may mostly or all result in lower-affinity antibodies or loss of binding, and one could propose to choose in the probability ranking the first “favorable” $V_L$ (ie, a KV4-68_J5 VL systematically for a V1-55_J2 and a KV2-112_J2 $V_L$ systematically for a V1-7_J2 $V_H$ or a V1-81_J2 $V_H$). This method may result in ameliorating affinity for rocuronium but would reduce the diversity in these repertoires by excluding rare VH-VL pairings.

The ability of the anti-rocuronium mAbs we identified either to bind specifically rocuronium or to cross-bind vecuronium and pancuronium may be explained by the 3 cocrystal structures of antibody fragments of IP2, IP8, and IP11 in complex with rocuronium. The 3 different structures we identified represent the binding mode of one representative of each VH family V1-55_J2, V1-7_J2, and V1-81_J2, and all reveal that both VH and VL of each mAb is involved in the binding, and that the allylpyrrolidinium substituent containing the quaternary ammonium of rocuronium is completely buried in the binding cleft of the antibodies. These cocrystal structures of mAbs with rocuronium, even if these mAbs were raised in mice immunized with rocuronium haptenized onto a carrier protein, are thus in agreement with hypotheses extrapolated from serum cross-reactivity in patients suggesting that the ammonium group is part of the binding interaction with antibodies.¹³,⁵¹ In the same manner as in rocuronium-sugammadex complexes, rocuronium bound IP2, IP8, and IP11 in a twist-boat or chair conformation, which might be due to induced fit, as reported for rocuronium-sugammadex complexes¹⁷ or by the selection among the 2 rocuronium conformers. Cross-reactivity with vecuronium and pancuronium required that their different substituents (compared to rocuronium) would remain outside the binding pocket and be exposed to the solvent. Cross-binding was reported decades ago for IgE antibodies in the serum of patients with NMBA hypersensitivity to alcuronium; their binding to alcuronium was strongly inhibited by tubocurarine, modestly by
A

Mast cell Activation test

Primary mast cells derived from CD34+
blood progenitors from healthy donors

<figure><img src="image_18.png" /></figure>

Flow cytometry
(fluorescent avidin)

Basophil Activation test

Whole blood incubation with anti-roc IgE

<figure><img src="image_19.png" /></figure>

Sensitization with anti-roc IgE
unspecific IgE

<figure><img src="image_20.png" /></figure>

Flow cytometry
(CD63 activation)

Neutrophil Activation test

<figure><img src="image_21.png" /></figure>

Flow cytometry
(CD62L shedding; CD11b activation)

B

Mast cell activation test with IgE

<figure><img src="image_22.png" /></figure>

C

Basophil activation test with IgE

<figure><img src="image_23.png" /></figure>

D

CD11b

<figure><img src="image_24.png" /></figure>

CD62L

<figure><img src="image_25.png" /></figure>

%CD62Lhi/CD11bint
Unstimulated neutrophils

<figure><img src="image_26.png" /></figure>

FIG 4. Anti-rocuronium mAbs activate human mast cells, basophils, and neutrophils after aggregation by rocuronium. (A) Scheme of activation tests. (B) Human mast cell and (C) human BAT using hlgE for sensitization, followed by challenge with free rocuronium (Free Roc), HSA, or HSA-Roc, with degranulation measured using fluorescent avidin or anti-CD63 staining, respectively. Mean ± standard deviation of results from different donors (1 dot represents 1 donor) are indicated (n ≥ 3 for 1-100 ng/mL and n = 1 for 0.01-0.1 ng/mL). Each antibody was analyzed separately, comparing contrast between (B) HSA and HSA-Roc or (C) contrast between each HSA-Roc concentration and control medium without HSA-Roc after mixed linear modeling and multiple-test P value adjustment. ***P < .001. (D) Human neutrophil activation test using
pancuronium, weakly by decamethonium and gallamine, and even less so by succinylcholine.¹⁸ If these types of cross-bindings represent the property of a single mAb in each patient's serum or that of different populations of antibodies within each serum that each recognize a different NMBA with little or no cross-reactivity with other NMBA remains unknown. Surprisingly, however, the 3 cocrystal structures we report here highlighted 3 different binding modalities for such a small antigen and such restricted repertoire, with only 3 VHs and 2 VLs, with mAbs IP8 and IP11 using the same VL rearrangement KV2-112_J2 but binding to rocuronium with different modalities. Whether our results are predictive of antibodies involved in NMBA hypersensitivity in humans remains to be clarified, as the first human anti-rocuronium antibody repertoires we recently reported for 3 pateints allergic to rocuronium were highly polyclonal with no evidence of clonal families.⁷

This first description of anti-rocuronium mAbs isolated from plasma cells and plasmablasts allowed us to demonstrate their ability to induce cell activation when aggregated by rocuronium. As we reported earlier for NMBA hypersensitivity in humans, at least 2 activation pathways coexist that lead to severe anaphylactic reaction to NMBAs: the IgE pathway leading to mast cell and basophil activation, and the IgG pathway leading to neutrophil and platelet activation.⁵⁹ All the high-affinity anti-rocuronium mAbs expressed as human IgEs activated *in vitro* human mast cells and basophils, and expressed as human IgG1s activated human neutrophils, in the presence of haptenized rocuronium but not free rocuronium. A previous study using IgE-stripped human basophils sensitized with patient serum containing polyclonal anti-rocuronium IgE also reported that only haptenized rocuronium but not free rocuronium induced basophil activation *in vitro*.⁵² From the cocrystals of these mAbs with rocuronium, it appears unlikely that a single rocuronium molecule could be bound by 2 different mAbs; rocuronium is engulfed in the antibody binding site of a single antibody, providing no epitope for a second antibody to bind. A molecular partner may thus exist that allows rocuronium haptenization to induce cell activation and anaphylaxis in humans. Alternatively, patients may produce particular antibodies that could bind at least in pairs to a single rocuronium molecule to induce FcεRI aggregation and cell activation, but our recent study on anti-rocuronium mAbs from human memory B cells do not favor this latter hypothesis.⁷ Importantly, once passively transferred into mice expressing human IgE receptors, both high- and low-affinity anti-rocuronium IgE mAbs led to severe anaphylactic reactions *in vivo* on exposure to haptenized rocuronium. Rocuronium is indeed devoid of neuromuscular blocking properties once haptenized onto a larger protein that cannot freely diffuse into the neuromuscular junction. This strategy allowed us to generate for the first time a model of rocuronium-induced anaphylaxis in mice and to follow anaphylaxis by central body temperature. More complex setups including anesthesia, intubation, and artificial ventilation could be envisioned to model anaphylaxis with free rocuronium, but these would require using other parameters than body

temperature or cardiac rhythm—which are uncontrolled during anesthesia—to monitor anaphylaxis, and potentially larger animals like nonhuman primates to increase the relevance to human physiopathology.

This work could not have been performed without chemically modifying rocuronium, which represents a limitation of this study. We used rocuronium derivatives by extending the OH group with a functionalized linker for grafting on carrier proteins KLH and HSA that were used for B-cell sorting, ELISA, avidity measurements, and functional assays. This strategy allowed for identification of anti-rocuronium antibodies with no cross-reactivity for NMBAs from other families outside aminosteroids—not even to dihydrotestosterone, a rocuronium mimetic devoid of ammonium groups. Validated by other groups, these anti-rocuronium IgE or IgG mAbs could be used as internal standards and references in routine assays like skin prick tests, BATs, or the more recently developed MATs.⁶⁰ Another limitations is the use of rocuronium to immunize mice, whereas patients become sensitized in most cases without any exposure to rocuronium but to other ammonium-containing compounds present in cough syrup, cosmetics, hair dyes, household cleaning products, or drugs at the probable initiation of the immune reaction toward rocuronium.⁷,⁶¹

In conclusion, this work reports on the underlying mechanisms of anaphylactic reactions to NMBAs by identifying the first anti-rocuronium antibody repertoires from immunized mice, suggests novel diagnostic tools for rocuronium hypersensitivity, and provides proof of concept for modeling NMBA hypersensitivity *in vivo* in mice.

## DISCLOSURE STATEMENT

Work in Bruhns's laboratory was supported by the Institut Pasteur, Institut National de la Santé et de la Recherche Médicale (INSERM), Fondation pour la Recherche Médicale, Paris (Programme Equipe FRM grant EQU202203014631 to P.B.), Accélérateur de l'innovation de l'Institut Pasteur (grant rocuCEPT to P.B.), Agence Nationale de la Recherche (grants ANR-14-CE16-0011 "DROPmAbs," ANR-16-CARN-0023-01 and ANR-21-CE15-0027 "CURAREP" to P.B.), European Research Council (ERC)-Seventh Framework Program (ERC-2013-CoG 616050 to P.B.) and INSERM International Research Program (grant ALLERGYMINE to P.B.). A.D. was supported by a doctoral fellowship from Sorbonne Université, followed by a fellowship from the Fondation pour la Recherche Médicale (grant FDT202304016860). Q.Z. received a PhD scholarship from the Chinese Science Council. A.G.C. was a recipient of a *poste d'accueil* in 2017 from Institut Pasteur-Assistance Publique des Hôpitaux de Paris, and was supported by a grant provided by INSERM, Société Française d'Anesthésie et de Réanimation (SFAR), and Société de Réanimation de Langue Française (SRLF) through the "Bourse de Recherche du Comité d'interface INSERM-SFAR-SRLF 2012" to A.G.C. C.M.G. was supported in part by a stipend from the Pasteur-Paris University international
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**FIG 5.** IgE-mediated passive systemic anaphylaxis in hFcεRI-transgenic mice. (A) Scheme of induction of passive systemic anaphylaxis in hFcεRI-transgenic mice, with (top) one or (bottom) two sensitizations. (B and C) Passive systemic anaphylaxis measured by changes in body temperature (ΔTemperature) in hFcεRI-transgenic mice sensitized (B) once with hlgE IP8 at indicated doses (n = 3 independent experiments) or (C) once with 5 µg of hlgE IP2, IP8, or IP11 (n = 6 independent experiments), and challenged with 2 µg HSA-Roc. (D) Passive systemic anaphylaxis in hFcεRI-transgenic mice sensitized with 40 µg of hlgE IP12 once at time 0 hours and once at 24 hours, and challenged intravenously at 48 hours with 140 µg HSA-Roc (n = 3). (B-D) Means ± standard deviations of 3 mice per group are indicated. Effect of each antibody compared to control was tested after mixed linear modeling and multiple-test *P* value adjustment.
***P < .001, **P < .01, *P* < .05. For (D), each of 3 injection effects was analyzed separately.

PhD program and by the Institut Carnot Pasteur Maladies Infec-
tieuses. C.C. acknowledges financial support from CONCYTEC,
Peru. P.B. benefited from an additional support from Assistance
Publique-Hôpitaux de Paris through “Contrat Local d’Interface
2014” and Département Hospitalo-Universitaire FIRE.

Disclosure of potential conflict of interest: P. Bruhns reports consulting fees from Regeneron Pharmaceuticals outside the present work. L. de Chaisemartin reports lecture fees from MSD France outside the present work. L. L. Reber reports grants and/or consulting fees from Neovacs, CEVA, Novartis, and Argenx outside the present work. A. Gérard is a cofounder and employee of Saber Bio SAS and former HiFiBiO Therapeutics SAS employee. Patents have been filed on some aspects of this work and future development of the platform, and the inventors may

receive payments related to exploitation of these under their em-
ployer's rewards to inventors scheme. The rest of the authors
declare that they have no relevant conflicts of interest.

We thank our colleagues from Institut Pasteur, Paris, for their help and advice: Delphine Brun and Annalisa Meola (Structural Virology Unit) for scFv generation and production; Bertrand Raynal, Sébastien Brulé, and Sylviane Hoos for assistance with affinity measurements (Molecular Biophysics Core Facility), Fabrice Agou for access to the BLI instrument (Plateforme de Criblage Chémogénomique et Biologique; PF-CCB), Michel Perez for advice and support (Direction des Applications de la Recherche et des Relations Industrielles; DARRI), Odile Richard-Le Goff for training, and Henna Maulaboksh for administrative help. We thank the staff of the crystallography platform at Institut Pasteur for crystallization screening, and we acknowledge
Synchrotron SOLEIL (Saint-Aubin, France) for granting access to their facility and the staff of Proxima1 and Proxima2A for helpful assistance. The MATS performed via flow cytometry were performed at the INFINITY INSERM UMR1291 Core Facility connected to the Toulouse Réseau Imagerie network, a member of the France BioImaging national infrastructure supported by the French National Research Agency (ANR 10 INBS 04).

Key messages

* Generation of plasma cell and plasmablast antibody repertoires was performed to represent circulating antibody pools.

* The first high-affinity anti-rocuronium antibodies were used to demonstrate their anaphylactogenic potential.

* Generation of the first animal model of rocuronium anaphylaxis will permit testing novel drugs.

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