Nanobody-based IgG simultaneously inhibit the allergenic and enzymatic activity of the dominant honeybee venom allergen

Domain: chemistry
Published: 2026-02-17
Editorial outcome: major_revision

Abstract
Insect venoms can cause severe allergic reactions, including anaphylaxis, in sensitized individuals. In this study, we aim at preventing anaphylaxis mediated by the most abundant and dominant honeybee venom allergen phospholipase A2 (Api m 1) by blocking its interaction with allergic patient IgE. Therefore, we characterize selected Api m 1-specific nanobodies and identify two high-affinity binders with non-overlapping epitopes. Crystal structures of Api m 1/nanobody complexes reveal diametrically opposed epitopes, one of which involves the active site of Api m 1. Based on this background, we develop mono- and bispecific nanobody-human IgG Fc, which exhibits pronounced blocking of IgE binding and effector cell activation in blood samples from honeybee venom allergic patients and reduces systemic reactions in a mouse model of allergen-induced anaphylaxis. This work provides a rationale for using nanobody-based inhibitors to prevent Api m 1-mediated anaphylaxis in honeybee venom allergy.

Introduction
Hymenoptera venom allergy (HVA) is a severe, potentially life-threatening condition and one of the most common causes of anaphylaxis1,2. The prevalence of systemic reactions to Hymenoptera stings is between 0.3 and 7.5% in the European population3. One of the main perpetrators of HVA is the honeybee (Apis mellifera). The response to honeybee venom (HBV) is a type I hypersensitivity reaction caused by allergen-induced cross-linking of IgE bound to the high-affinity receptor FcεRI on mast cells and basophils and subsequent release of mediators, which trigger immediate-type allergic reactions4,5.

The IgE response in HBV-allergic patients is directed against different venom allergens. The most abundant component is the cytolytic peptide melittin, which constitutes 50% of the venom dry weight6. The 16kDa allergen phospholipase A2 (Api m 1) constitutes 12% of the venom dry weight and has a reported sensitization rate of up to 97% of HBV-allergic patients6–8, thus being considered the dominant allergen in HBV. Most of the other allergens are of low abundance, comprising each less than 1–2% of venom dry weight, and have reported sensitization rates of >50%6,9. The individual clinical relevance of the lower-abundance allergens is not yet proven.

Api m 1 is in synergy with melittin, involved in the development of pain and inflammation induced by the sting. Api m 1 is a Ca2+-dependent phospholipase A2, which disrupts cellular membranes, resulting in release of pro-inflammatory mediators10–12. It comprises a conserved active site at the core of three α-helices, an N-terminal calcium-binding loop, and an asparagine-linked glycosylation site (N13)13. The carbohydrate moiety itself represents a prominent IgE epitope, which is considered to have rather limited clinical relevance14–17.

Currently, the only available treatment for HBV allergy that can limit further systemic reactions is venom immunotherapy (VIT)18. VIT can be performed with different treatment regimens, all of which aim to restore immune tolerance through subcutaneous injection of HBV extract over the course of 5 years, or even lifelong under certain circumstances including patients with a severe initial sting reaction or mastocytosis19. VIT has been reported to be effective in 77–94% of HBV-allergic patients19,20. However, VIT is also associated with a risk of systemic adverse effects19,21.

The dominant mechanism by which allergen immunotherapy (AIT) induces tolerance is through the production of allergen-specific IgG that block the interaction between the allergen and allergen-specific IgE22–24. Recent studies on passive immunotherapy in cat and birch pollen allergy revealed that a single dose of two or three monoclonal IgG antibodies against the major cat allergen Fel d 1 and the major birch pollen allergen Bet v 1, respectively, reduced clinical symptoms in allergic patients25–28. Passive immunization of HBV-allergic patients with purified IgG from tolerant beekeepers has been demonstrated to protect against systemic reactions when undergoing rush immunotherapy with HBV and increase the tolerated venom dose29,30. Thus, these studies underline the potential of using allergen-blocking IgG for passive immunotherapy as a treatment option for HBV allergy. In HBV allergy, stings occur unpredictably within a well-defined season, and passive immunotherapy with IgG having a half-life of 20–30 days31 could provide seasonal protection against severe symptoms, including anaphylaxis in case of a sting event.

In recent years, single-domain antibody therapeutics have gained ground. Single-domain antibodies, often called nanobodies, are isolated from heavy-chain-only antibodies found in camelid species32. Compared to conventional IgG antibodies, nanobodies are tenfold smaller and thus can easily be linked to other molecules, generating a variety of nanobody-based formats with numerous biotechnological and therapeutic applications33,34. Their slightly longer complementarity-determining region 3 (CDR3) and prolate shape render nanobodies particularly suited for targeting concave or cryptic structures such as active sites of enzymes35.

In this study, we generate Api m 1-specific nanobodies for interfering with the binding of HBV-allergic patient IgE to Api m 1 and assess a potential reduction of Api m 1-mediated anaphylaxis. We characterize the interaction between the nanobodies and the allergen structurally and biochemically and document the blocking potential of individual, combined, and bispecific nanobody-based hIgG Fc fusion proteins (nb-hIgG). The structural and functional data provide insights into the allergenicity of venom allergens as well as a proof-of-concept for using nanobody-based formats for blocking allergen-induced anaphylaxis in the context of HBV allergy.

Results
Establishment of high-affinity Api m 1-specific nanobodies
Api m 1-specific nanobodies were isolated by the establishment and selection of an immune library by phage display as recently described36. Selected nanobodies were initially characterized with respect to expression yield, affinity, and epitope overlap. On this background, two nanobodies, AM1-1 and AM1-4, were chosen for further studies. The two nanobodies are highly diverse in their CDR residues and length, in particular in the CDR3 (Supplementary Fig. 1). Both nanobodies were recombinantly expressed and purified at high yields from the culture supernatant. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the expected size of 15 kDa (Fig. 1a). Biolayer interferometry (BLI) demonstrated binding to immobilized Api m 1 with dissociation constant (KD) values in the nanomolar to sub-nanomolar range (Fig. 1b). Sequential BLI binding analysis further demonstrated independent, non-competitive binding to Api m 1, irrespectively of the order of nanobody binding, indicating two distinct epitopes of the nanobodies (Fig. 1c).

Structural analysis of Api m 1/nanobody interactions
In order to understand in detail the nanobody-allergen interactions, a ternary complex was constituted by mixing native Api m 1 (nApi m 1), AM1-1 and AM1-4 in equimolar amounts, and crystallized. X-ray diffraction data were obtained with a maximum resolution of 1.76 Å (Table 1). The structure revealed a 1:1:1 complex with the two nanobodies binding to diametrically opposing epitopes of the allergen (Fig. 2a, b). The architecture of the ternary complex corroborates the BLI data.

The structure of Api m 1 in complex with the two nanobodies is in close agreement with the known structure of unbound Api m 113, with a root mean square deviation <1 Å over 99 Cα atoms. Notably, the residues 1–5 and 11–23 in Api m 1, including the calcium-binding loop and the N-linked glycosylation site, could not be modeled in the Api m 1/ nanobody complex, suggesting that they are disordered.

Clear electron density is present for the residues at the Api m 1/nanobody interfaces (Supplementary Fig. 2). According to PISA analysis, the buried surface areas at the interface between Api m 1 and AM1-1 or AM1-4 are 1627.9 Å2 and 1473.3 Å2, respectively, which are common interface areas for antigen/nanobody complexes37. Both AM1-1 and AM1-4 recognize conformational epitopes on Api m 1 (Fig. 2c–e). As expected, the CDRs are responsible for the majority of the interactions (Supplementary Figs. 3 and 4). The relatively long CDR3 of AM1-4 reaches into a pocket between the α-helical segments and the C-terminal β-strands of Api m 1 (Fig. 2a, b, d, and Supplementary Fig. 4c). On the opposite hemisphere, the CDR3 of AM1-1 interact directly with the active site of Api m 1 (Figs. 2a–c and 3a, and Supplementary Fig. 4a). In addition to the CDR3, residues of the framework regions are also involved in direct and water-mediated contacts with Api m 1 (Supplementary Figs. 3 and 4b).

The nanobody AM1-1 binds to the active site of Api m 1 and blocks its enzymatic activity
The residues S102 and R104 of the AM1-1 CDR3 form hydrogen bonds with the residues H34, D35, and Y87 of Api m 1 (Fig. 3a, and Supplementary Fig. 4a). Together with D64, the residues H34 and Y87 form a catalytic network, while D35 is essential for Ca2+ binding13,38. Hence, our structure suggested that the binding of AM1-1 interferes with the enzymatic activity of Api m 1. Therefore, the enzymatic activity of Api m 1 was assessed by monitoring the cleavage of a fluorogenic substrate in the presence of the nanobodies. AM1-1 reduced the activity of Api m 1 to 50% whereas AM1-4 had no reducing effect (Fig. 3b). A reduction of the inhibitory effect upon combination might rely on minor conformational rearrangements affecting the binding of AM1-1. The observed inhibition supported the finding that AM1-1 interacts directly with the active site of Api m 1 and exhibits the potential to reduce the enzymatic activity of Api m 1, which contributes to the pain and inflammation of a honeybee sting.

The nanobody AM1-4 enables dimer formation and cellular activation
In parallel, an Api m 1/AM1-4 binary complex was crystallized (Table 1). For these crystals, diffraction data extending to a maximum resolution of 4.1 Å was obtained. The structure revealed a 1:1 complex in which an Api m 1 dimer is bound by two molecules of AM1-4 oriented in opposing directions in relation to the Api m 1 dimer axis (Fig. 3c). The position of AM1-4 in this complex is identical to the position in the complex of Api m 1 with both nanobodies. Also in this complex, the structure of Api m 1 is very similar to that of unbound Api m 113, with a root mean square deviation <1 Å over 116 Cα atoms.

Interestingly, in this complex Api m 1 forms a tight dimer in the crystal packing like the dimer observed for unbound Api m 113. The relationship between the two monomers differs by a rotation of 40° (Supplementary Fig. 5). Although the formation of dimers in solution has not been reported, Api m 1 might transiently form dimers at locally high concentrations, e.g., at cellular surfaces. Such propensity to dimer formation has been proposed as a mechanistic rationale for enhanced allergenicity of a large number of allergens39–41. Thus, a single IgE antibody clone recognizing one IgE epitope would be sufficient for cross-linking and cellular activation.

In order to investigate the potential formation of transient dimers, nanobody-human IgG Fc formats (nb-hIgE) of AM1-1 and AM1-4 were generated as previously described (Supplementary Fig. 6)36. The ability of the individual and combined nb-hIgE to activate effector cells upon Api m 1 stimulation was assessed using RBL-SX38 cells. Expectedly, the combination of AM1-1 hIgE and AM1-4 hIgE lead to allergen-dependent degranulation upon stimulation with Api m 1. Remarkably and in contrast to the AM1-1 hIgE, the AM1-4 hIgE alone was able to induce Api m 1-dependent cellular activation and degranulation to the same extent as the combination of two IgE (Fig. 3d). In contrast to cytokinergic IgE, which rely on spontaneous oligomerization of IgE42,43, the cellular activation was strictly allergen-dependent, and could be explained convincingly by the formation of Api m 1 dimers at the surface. This concept is supported by the structure showing that AM1-4 binding is compatible with dimer formation.

The binding of AM1-1 induces conformational rearrangements of Api m 1
Notably, the N-terminal calcium-binding loop and the N-linked glycosylation site of Api m 1 are disordered in the ternary Api m 1/nanobody complex. In contrast, in the Api m 1/AM1-4 binary complex, all Api m 1 residues including the first N-Acetylglucosamine (GlcNAc) of the glycan at N13 could be modeled (Fig. 3e). An overlay of the two Api m 1 complexes reveals a steric clash of the AM1-1 nanobody with the N-terminal loop and the glycan of Api m 1. Since the glycan is significantly larger than the first GlcNAc sugar that can be modeled in our structure, the clash between a bound AM1-1 nanobody and the glycan is likely to be pronounced. This finding suggests a displacement of the N-terminal loop including the calcium-binding loop and the glycan upon binding of AM1-1 to Api m 1 (Fig. 3f).

Generation of nanobody-human IgG
With the purpose of blocking the binding of allergen-specific IgE from HBV-allergic patients to Api m 1, monospecific nb-hIgG (AM1-1 hIgG and AM1-4 hIgG) comprising either of the nanobodies were generated by fusion of the nanobodies to the hinge region of human IgG Fc (Fig. 4a). In addition, a bispecific nb-hIgG format (AM1B-1-4 hIgG) comprising both of the nanobodies within a single molecule was generated by fusion of the nanobodies to human IgG Fc containing DEKK mutations for facilitated heterodimerization of the two different antibody chains44. The IgG format was chosen since IgG is an early blocking antibody arising in AIT24. The three nb-hIgG formats were expressed in mammalian cells, purified and analyzed by SDS-PAGE, which verified the expected molecular mass of a dimer glycosylated within the Fc domains (Fig. 4b). For the bispecific nb-hIgG, two bands under reducing conditions represented the two monomers of slightly different molecular weight due to the presence of specific epitope tags, suggesting efficient heterodimerization.

ELISA assessment verified the immunoreactivity of the different nb-hIgG (Supplementary Fig. 7a). ImmunoCAP analyzes further documented specificity for HBV without cross-reactivity to other Hymenoptera venom from species such as bumblebee, yellow jacket, and fire ant venom (Supplementary Fig. 7b). Notably, the bumblebee phospholipase A2, Bom t 1, shares a sequence identity of 54% and a conserved active site with Api m 1. Only one residue of the AM1-1 epitope is replaced, but no cross-reactivity with bumblebee venom was observed (Supplementary Fig. 7b, c).

BLI binding studies of the three immobilized nb-hIgG further revealed agreement of the kinetic data with those of the individual nanobodies, confirming that the Fc fusion has no impact on binding affinity (Fig. 4c). Notably, the dissociation rate of the bispecific nb-hIgG was significantly lower (<10−7 s−1), most likely reflecting increased avidity due to simultaneous binding of two Api m 1 epitopes. The change in the dissociation rate yielded an overall KD in the sub-picomolar range (<10−12 M).

Nanobody-hIgG formats block the binding of HBV-allergic patient IgE to Api m 1
The ability of the nb-hIgG formats to inhibit the binding of IgE from HBV-allergic patients to Api m 1 was assessed in an inhibition ELISA. Immobilized Api m 1 was incubated with the nb-hIgG followed by incubation with serum from HBV-allergic patients, and the remaining binding of allergen-specific IgE was analyzed. For this analysis, HBV-allergic patients with elevated specific IgE titers to Api m 1 and anaphylaxis grade ≥1 were chosen (Serological data Supplementary Table 1). Both individual monospecific nb-hIgG formats inhibited the binding of patient IgE partially (Fig. 5a, b). Varying ratios of inhibition with AM1-1 and AM1-4 were observed between different patients but in general AM1-4 hIgG showed a more pronounced inhibition (mean 69%) than AM1-1 hIgG (mean 49%). The inhibition could be significantly increased by using a combination of the two monospecific nb-hIgG formats (mean 91%) or the bispecific AM1B-1-4 nb-hIgG (mean 94%) (Fig. 5b). These data demonstrate that the individual and combined nb-hIgG formats can efficiently block the binding of IgE from HBV-allergic patients to immobilized Api m 1.

Nanobody-hIgG reduces Api m 1-induced basophil activation
The nb-hIgG formats were further evaluated for their ability to block Api m 1-induced activation of basophils from Api m 1-sensitized HBV-allergic patients (Supplementary Table 2).

It is well established that patient basophils respond to allergen stimulation with varying reactivity and sensitivity. Therefore, the response of basophils from nine HBV-allergic patients after stimulation with nApi m 1 was assessed as an initial step (Supplementary Fig. 8). Subsequently, the response in the presence of the nb-hIgG formats was analyzed. For seven out of nine patients, the nb-hIgG formats reduced basophil activation although to a varying degree (Fig. 6a, Supplementary Fig. 9, and Supplementary Table 3). For these seven patients, the AM1-4 hIgG format resulted in a more pronounced reduction than the AM1-1 hIgG format, which only exhibited a minor decrease in basophil reactivity. For most patients, an increased blocking was observed when using a combination of the two nb-hIgG formats. Notably, the bispecific format performed equally well as the combination of the two individual antibodies, and significantly reduced basophil activation compared to the isotype control (Fig. 6b). For two out of nine patients, the nb-hIgG formats had no inhibitory effect on basophil activation. One of these patients (patient N) showed a very low basophil response to Api m 1 at all, while the other patient (patient M) showed a very high response and highly elevated sIgE to Api m 1 (Fig. 6a, and Supplementary Fig. 8).

Overall, these data demonstrate the potential of the nanobody formats for blocking effector cell activation by Api m 1 stimulation. The data also confirms a large blocking variation, reflecting diverse IgE repertoires of the HBV-allergic patients.

Inhibition by the bispecific nb-hIgG format was further evaluated with HBV-stimulated basophils from four poly-sensitized patients, demonstrating the ability to significantly reduce basophil activation upon HBV stimulation (Fig. 6c, and Supplementary Fig. 10, Serological data in Supplementary Table 2). In three out of four patients, a clear reduction of above 34% was observed with a mean reduction of 35% for all patients. Thus, blocking the dominant allergen Api m 1 can reduce the activation of effector cells upon HBV-stimulation. Expectedly, a larger reduction of basophil activation was observed on Api m 1-stimulated basophils than on HBV-stimulated basophils, supporting a general contribution of other venom allergens to the overall basophil activation in HBV allergy.

Bispecific nanobody-hIgG reduces mast cell activation and anaphylaxis in vivo
The therapeutic potential of the bispecific nb-hIgG was evaluated in vivo using a mouse model of allergen-induced anaphylaxis. Sensitization with HBV extract resulted in the induction of HBV- and Api m 1-specific IgG and IgE (Fig. 7a and Supplementary Fig. 11). After sensitization, anaphylaxis was induced by injection of nApi m 1 (Fig. 7a). Anaphylaxis was evaluated as a drop in body temperature and mast cell activation was assessed by measuring serum concentrations of mast cell protease-1 (MCPT-1). Challenge with nApi m 1 resulted in an increased MCPT-1 serum concentration and a temperature drop in HBV-sensitized mice compared to non-sensitized mice, validating the model (Fig. 7b–d). Treatment with the bispecific nb-hIgG prior to Api m 1 challenge significantly reduced the Api m 1-induced MCPT-1 release and body temperature drop (Fig. 7b–d). The evaluation of treatment with the bispecific nb-hIgG prior to challenge with HBV showed, although non-significant, an average reduction of MCPT-1 release of 40%, which is comparable to the 35% reduction observed in HBV BAT (Supplementary Figs. 12 and 13). However, a pronounced body temperature drop (Supplementary Fig. 12) was also observed in non-sensitized mice, suggesting a non-allergic, mast cell-independent response to the venom.

Thus, the bispecific nb-hIgG format demonstrated the potential to reduce Api m 1-induced mast cell activation and Api m 1-mediated anaphylaxis in vivo in HBV-sensitized mice. The potential of the bispecific nb-hIgG format to reduce HBV-mediated anaphylaxis needs further investigation.

Methods
Ethical statement
The use of patient material was approved by the Ethics Committee of the Medical Faculty of Philipps University, Marburg, Germany (Az. 112/17). All patients provided written informed consent to participate in the study, including informed consent for the publication of potentially identifying individual-level data, as seen in Supplementary Tables 1 and 2.

All scientific procedures on living animals were approved by the regional council (RP Giessen Az: V54−19c2015h01 GI 20/4 Nr. G43/2024) in accordance with the German animal welfare law and the European legislation for the protection of animals used for scientific purposes (2010/63/EU).

Materials
Native Api m 1 was obtained from Latoxan.

Selection of Api m 1-specific nanobodies
Api m 1-specific nanobodies were selected previously using a nanobody library generated from peripheral blood mononuclear cells of a llama immunized with nApi m 136.

Recombinant expression of nanobodies, nb-hIgE, and nb-hIgG formats
Selected nanobodies were cloned into a pET-22b(+) vector (Novagen) containing a pelB signal sequence, a C-terminal polyhistidine tag, and a C-terminal V5-tag. Expression was performed in Rosetta (DE3) cells (Novagen).

Nanobody-hIgE formats were cloned as previously described36. Briefly, the nanobody sequences were cloned into a pcDNA3.1 vector (Invitrogen) between a human immunoglobulin-derived signal sequence and the human IgE Fc domains (CH2-4 domains) containing a C-terminal polyhistidine-tag. Nanobody-hIgG formats were generated similarly by cloning the nanobody sequences into the pcDNA3.1 vector between a human immunoglobulin-derived signal sequence and the hinge region of a human IgG Fc (Hinge, CH2, and CH3 domain) containing a C-terminal polyhistidine-tag as previously described64. Expression of the nb-hIgE and nb-hIgG formats was performed in transiently transfected mammalian FreeStyle 293-f cells (Gibco, Thermo Fisher Scientific, catalog number R79007).

Bispecific nb-hIgG formats were generated by introducing DEKK mutations into the human IgG Fc as previously described44. Briefly, two vectors were constructed, containing each one chain of the nb-hIgG with either a L351D and a L368E mutation or a L351K and a T366K mutation. The two chains contained different tags (polyhistidine-tag or BC2-tag) for purification and assessment of heterodimerization. The two cloning vectors contained genes for either Zeocin or Geneticin (G418) resistance for selection of double-transfected cells. The bispecific nb-hIgG formats were expressed in adherent FreeStyle 293-f cells.

Purification of nanobodies, nb-hIgE formats, and nb-hIgG formats from expression media was performed by IMAC using HisTrapTM Excel columns (Cytiva). After washing (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4), bound protein was eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole pH 7.4). Eluted proteins were further purified by SEC on either a HiLoad 16/600 superdex 200pg or 75pg column (Ge Healthcare) equilibrated in PBS pH 7.4. Purified proteins were assessed by SDS-PAGE and immunoreactivity of nb-hIgG was assessed by ELISA with immobilized nApi m 1. Bound nb-hIgG was detected with AP-conjugated anti-human IgG (Sigma).

Affinity determination and epitope binning
BLI measurements were performed at 30 °C on an Octet RED96 (ForteBio) using amine-reactive second-generation (AR2G) biosensors (Sartorius). Sensors were loaded in 20 µg/ml nApi m 1 in 10 mM sodium acetate pH 6.0 for 2 min. Sensors were quenched for 300 s in 1 M ethanolamine pH 8.5, and the baseline was obtained for 120 s in kinetic buffer (PBS pH 7.4, 0.02% Tween 20, 0.1% BSA). Association was performed over 300 s, and the dissociation phase was monitored for 1200 s, both in kinetic buffer. Samples without nanobody were subtracted as background. Curve fitting was performed with Octet software using a 1:1 binding model. For epitope binning 600 s association was followed by 120 s dissociation and then 600 s association of the second nanobody.

Affinity measurements of the nb-hIgG formats were performed using anti-hIgG Fc Capture (AHC) biosensors (Sartorius). Sensors were loaded in 2 nM nb-hIgG for 420 s, and a baseline was obtained for 300 s in kinetic buffer. Association was performed over 300 s with twofold dilution series of nApi m 1 from 160 to 5 nM (AM1-1 hIgG), 40 to 1.25 nM (AM1-4 hIgG) and 50 to 0.8 nM (AM1B-1-4 hIgG). Dissociation was measured for 1200 s in kinetic buffer. Samples with no Api m 1 were subtracted as background. Curve fitting was performed with Octet software using a 1:1 binding model.

Structure determination
For crystallization of the ternary complex, nApi m 1, and the two nanobodies were concentrated to 14.5 mg/ml in 20 mM HEPES, 150 mM NaCl, 10 mM CaCl2, pH 7.4 with a 1:1:1 molar ratio. The complex was crystallized by vapor diffusion in sitting drops formed by mixing protein solution and reservoir solution containing 0.2 M NaCl, 0.1 M Phosphate/citrate pH 4.2 and 20% w/v polyethyleneglycol (PEG) 8000 in a 1:1 ratio at 18 °C. Crystals were cryo-cooled in liquid nitrogen. Data were collected at the beamline P13 operated by EMBL at the PETRA III storage ring (DESY, Hamburg, Germany) and processed with the XDS package65. The structure was determined by molecular replacement with phenix.phaser66, using PDB ID: 1POC as search model for Api m 1 and 7NP9 for the nanobodies. The final structure was obtained by alternating manual model building in COOT67 and refinement with phenix.refine68. The complex was analyzed with the “protein interfaces, surfaces and assemblies” service PISA at the European Bioinformatic Institute69. Figures were prepared with the PyMOL Molecular Graphics System (Schrödinger LLC).

The binary complex of nApi m 1 and the nanobody AM1-4 was crystallized by the same approach. Briefly, crystals were obtained with a reservoir containing 0.1 M sodium citrate pH 5.5, and 15% w/v PEG 6000 at 4 °C. Data were collected at the beamline i24 at Diamond Light Source, UK. The structure was determined by molecular replacement using the subcomplex of Api m 1 and AM1-4 from the ternary complex as search model.

Activity assay
Inhibition of the enzymatic activity of nApi m 1 was evaluated using the EnzChek Phospholipase A2 Assay kit (Invitrogen) according to the manufacturer´s recommendation. Briefly, 2 µg/ml nApi m 1 was pre-incubated with twofold dilution series of the nanobodies and incubated for 1 h at room temperature shaking. The enzymatic reaction was started by adding 1:1 PLA2 substrate-lipid mix. Fluorescence (Excitation 467±7 and Emission 515±10 and 575±10) was measured after 10 min incubation on a ClarioStar Plus plate reader (BMG LABTECH). The fluorescence ratio (515 nm/575 nm) was determined, background (no Api m 1) subtracted, and the data normalized to a positive control (nApi m 1 without nanobody).

RBL assay
In vitro degranulation was analyzed using RBL-SX38 cells expressing the human FcεRI receptor70. RBL-SX38 cells were a generous gift from Jean-Pierre Kinet. Cells were cultured in Minimum Essential Medium (GIBCO) supplemented with Earle’s salts, 2 mM L-glutamine, penicillin/streptomycin, 1 mM sodium pyruvate, 15% fetal calf serum, and 1–2 mg/mL G418 at 37 °C and 5% CO2. For analysis, cells were seeded in 96-well culture plates, 1×105 cells per well overnight at 37 °C and 5% CO2. The cells were sensitized with 1 µg/ml nb-hIgE for 1 h at 37 °C. After washing with Tyrode’s buffer (10 mM HEPES pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA), the cells were incubated with dilution series of nApi m 1 and incubated for 1 h at 37 °C. As a reference, cross-linking was achieved by incubation with a 1:1000 dilution of polyclonal anti-human IgE antibody (Dako). Supernatants were saved before wash and lysis of cells with Tyrode’s buffer containing 0.1% triton-X. β-hexosaminidase release of viable versus lysed cells was assessed with 8 mM p-nitrophenyl N-acetyl-glucosaminide (Sigma). Results were calculated as percentage of total release.

Inhibition ELISA
Inhibition ELISA was performed using serum from 5 HBV-allergic patients sensitized to Api m 1. Clinical data of the patients with regard to HBV-specific allergic symptoms, sensitization profiles, age, and sex are reported in Supplementary Tables 1 and 2. High-binding 384 microwell plates (Greiner Bio-One) were coated o.n. at 4 °C with 5 µg/ml nApi m 1 diluted in PBS. After blocking for 30 min with TBS containing 4% BSA, the wells were incubated for 1 h at room temperature. shaking with 1 µM nb-hIgG diluted in TBS + 2% BSA. Serum was then added to the wells, and the plates were incubated for an additional 1 h. After washing, bound IgE was detected by 1 h incubation with AP-conjugated goat anti-hIgE antibody (Sigma) diluted 1:2500 in TBS + 2% BSA. The plates were developed with substrate solution (5 mg/mL 4-nitro-phenyl phosphate, Sigma) for 100 min and measured at 405 nm. Background was subtracted, and IgE binding was normalized to the binding observed in presence of an nb-hIgG isotype control.

Basophil activation test
BAT was performed using serum from HBV-allergic patients sensitized to Api m 1. Clinical data of the patients with regard to HBV-specific allergic symptoms, sensitization profiles, age, and sex are reported in Supplementary Tables 1 and 2. BAT was performed with the FlowCast kit (Bühlmann) according to the manufacturers´ recommendations. Peripheral EDTA blood of HBV-allergic patients was incubated with tenfold dilution series of nApi m 1. Staining with anti-CD63-FITC and anti-CCR3-PE was performed in a 37 °C water bath for 15 min. Erythrocytes were lysed for 7.5 min followed by centrifugation at 600×g for 5 min. Cell pellets were resuspended in 100 µl washing buffer, and basophil activation was evaluated by flow cytometry on a FACSCalibur (BD Biosciences) using FlowJo software (version 10.8.1) for analysis. Basophils were defined as CCR3+/SSClow cells and activated basophils were distinguished by being CD63 positive.

Based on the assay above, a fixed sub-maximal concentration of Api m 1 was chosen for each patient. Inhibition was assessed by pre-blocking Api m 1 with nb-hIgG (tenfold serial dilutions) in stimulation buffer for 1 h at room temperature. Peripheral EDTA blood from HBV-allergic patients was then incubated with the pre-blocked allergen, and activated basophils were stained and detected as mentioned before.

To assess inhibition of basophil activation upon HBV-stimulation versus Api m 1-stimulation, titrations of Api m 1 and HBV extract (Bühlmann) was performed as above. Based on the titration a fixed sub-maximal concentration of Api m 1 and a tenfold higher concentration of HBV was chosen for each patient. The inhibition assay was performed as described using a 20-fold molar excess of the bispecific nb-IgG compared to Api m 1.

ImmunoCAP measurements
Specific IgE and IgG antibody measurements were performed by ImmunoCAPs on a Phadia 200 and 250 instrument (Thermo Fisher Scientific).

Mouse experiments
Female BALB/c mice were purchased from Charles River Laboratories (Erkrath, Germany) and used at age 8–10 weeks. They were housed under specified pathogen-free conditions in filter-top cages and received a standard diet. Housing of single mice was avoided. All cages were provided with nesting material, mouse houses and chew toys. Mice were kept with a constant day/night rhythm of 14 h day and 10 h night (day beginning at 5 am winter time). Mice were allocated in groups and intraperitoneal (IP) injected three times (day 0, 7, 14) with either Dulbeccos phosphate buffered saline (DPBS) (n=12) as a control or 25 µg HBV (Latoxan) adsorbed to 2 mg aluminum hydroxide (Imject alum, Pierce, Thermo Fisher Scientific). For evaluation of the HBV- and Api m 1-specific IgE and IgG1 response, peripheral blood was taken on day 0 and 21, and the serum was stored at −20 °C until further processing.

Sensitized mice were challenged on day 28 IP with 75 µg of Api m 1 contained in 100 µl DPBS. The challenge with HBV was performed identically using 30 µg of HBV in DPBS.

Anaphylaxis was monitored by measuring the core body temperature with an infrared thermometer (Voltocraft/Conrad Electronic) on the abdomen every 15 min for a period of 90 min. Temperature-time curves were integrated, and the area under curve (AUC) indicated as a measure for anaphylaxis, the baseline for the AUC curve being the pre-challenge body temperature.

Sensitized mice were treated by IP injection of the bispecific nb-hIgG using doses of 1200 µg per animal in a total volume of 200 µl DPBS. Mice injected with DPBS (non-sensitized) and sensitized mice injected with DPBS were used as controls. To test the potential of the bispecific nb-hIgG in preventing anaphylaxis, sensitized and treated mice were challenged by IP injection of Api m 1 4 h later as described above.

90 min after provocation mice were sacrificed and bled by heart puncture. The serum was stored at −20 °C for further analysis.

Analysis of mouse IgG and IgE antibodies and MCPT-1
Specific IgG levels were quantified in mouse serum by ELISA. Briefly, high-binding 384-well plates were coated with 1 µg/ml antigen and blocked with 4% skimmed milk in DPBS. Dilutions of samples were performed in 4% skimmed milk and incubated for 2 h before the addition of biotinylated rat anti-mouse IgG (BD Pharmingen; 1:1000). The plates were incubated for 30 min with horseradish peroxidase-conjugated streptavidin (Merck-Millipore) before adding the tetramethylbenzidine (TMB) substrate. The reaction was stopped with sulphuric acid and the absorbance read at 450 nm.

For the detection of specific IgE, plates were coated with 10 µg/ml of antigen. To reduce IgG in the serum, samples were diluted 1:2 in NaCl and pre-incubated with Protein G agarose at a ratio of 1:5 (Santa Cruz Biotechnology, Dallas, TX) while shaking for 1 h at room temperature. Following a centrifugation for 1 min at 12,000×g the supernatant was used for the ELISA and the remaining Protein G agarose was discarded. Biotinylated rat anti-mouse IgE (BD Pharmingen) was used as secondary antibody. Blocking, detection, and incubation times were done according to the spec. IgG ELISA.

In addition, MCPT-1 levels in mice as marker for cellular degranulation and anaphylaxis were determined with a mouse MCPT-1 ELISA kit (Invitrogen). All steps were performed according to the manufacturer’s protocol.

Statistics & reproducibility
The quality of purified proteins was routinely assessed by SDS-PAGE. Kinetic data was fitted with a 1:1 binding model using the Octet System Data Analysis Software, Release 7.1. The activity assay and RBL assay were reproduced in two independent experiments. For experiments using patient serum, no statistical method was used to predetermine sample size. The number of patients was determined based on feasibility criteria and the availability of untreated patients with clinically confirmed HBV-allergy or sensitization in the Department of Dermatology and Allergology at Philipps University Marburg, Germany. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment, and no data were excluded from the analyses. Sex- and gender-based analysis of the samples was not performed.

For mice experiments, a temperature difference between two groups of at least 1.5 °C was considered as a clearly measurable difference, and significance was calculated with a one-tailed Wilcoxon–Mann–Whitney test, an alpha-error of 0.05 and an actual power of 0.8. This led to a sample size of 6 mice per group. Two independent experiments with 6 mice per group were performed. The investigators were not blinded to allocation during experiments and outcome assessment. Data from two mice had to be excluded: One mouse lost weight after the 3rd sensitization and had to be sacrificed 12 days before anaphylaxis provocation due to regional animal care regulations. The second mouse died during the anaphylaxis between 30 and 45 min after provocation. Both mice belonged to the group that was HBV sensitized, vehicle pre-treated and Api m 1 challenged.

Statistical analysis was performed using GraphPad Prism 10. Data was tested for normality and statistical analyses were performed accordingly.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Type 1 hypersensitivity reactions to insect venoms are potentially fatal with honeybee venom a main cause. Standard of care involves avoidance, epinephrine use or allergen specific immunotherapy. Given the challenges and risks associated with the standard of care, new and effective treatment options are needed. Passive immunotherapy with allergen specific monoclonal antibodies is a promising new approach with cocktails of either fel d 1 specific or bet v 1 specific monoclonal antibodies demonstrating proof of concept in patients with cat and birch pollen allergy, specifically. Since 97% of Honey bee venom allergic patients have IgE to a 16kDa allergen phospholipase A2 (api m 1), the authors generated Api m 1-specific nanobodies to block HBV-allergic patient IgE to the allergen. Structural work was performed to elucidate the how the nanobodies interact with Api m 1 is well done and provides key information needed to help understand how the protein interacts with IgE to drive a response.\nMajor comments:\nThere are four major limitations to this study:\n1. While the authors demonstrate that the nanobody molecules can block Api m 1 binding to IgE in vitro using human IgE sera and basophils, the authors do not demonstrate that these molecules will block HBV. Suggestions around the ability this approach to prevent a reaction beyond Api m 1 and will broadly block HBV must be tempered. It is critical for the approach to show that the api m 1 nanobody molecules block a HBV driven response\n2. There are no in vivo data demonstrating that the api m 1 nanobody molecules can block a polyclonal IgE mediated response using either mouse sera and preferable human IgE (from HBV allergic patients). To this point, it is important to demonstrate this in vivo blockade using HBV and not just api m 1 as the challenge.\n3. The data using api m 1 challenge of human basophils was not robust with partial blockade and the non-normalized data in the supplemental figures. The non-normalized data should be moved to the main figures to capture the variability across HBV donors and also be demonstrated with HBV challenge.\n4. Statists should be shown for the data in the figures to validate the conclusions\nOther Comments:\nIn general, allergen should be changed to api m 1 throughout the manuscript, since this approach is specific to api m 1 rather than the HBV ‘allergen’ and it will avoid confusion.\nABSTRACT,\nline 40: please revise to honeybee venom allergic patient SAMPLES as blockade is not assessed in patients.\nINTRODUCTION\n-intro line 53: what is the other ~80% of the venom dry weight\n-intro line 54-55: the authors report that 97% of HBV-allergic patients are sensitized to Api m 1, but it is important to know the sensitization rates of the other Api proteins and not just IgE for Api m 1, to help put things into context. That is, what other Api m allergens may be drivers of the allergic response?\n-intro line 65: It is important to include a comment/reference about other immunotherapy regimens (RUSH and ultra-RUSH) that are not as lengthy\n-Intro line 70-83: these paragraphs can be combined. Also the authors comment about the‘one mechanism’ is generation of blocking antibodies, but it is really THE mechanism. In this paragraph they do refer to rush IT, but not in the line 65 paragraph.\n-intro line 77: Suggest citing Kamal et al., 2021 Clin Trans Sci reference here with a sentence referring to the half-life of monoclonal IgG being 20-30 days and therefore having the potential to provide protection within a well-defined season.\n-intro line 93: “allergen” should be changed to Api m 1 because we only see data for this one protein and not venom in general. NOTE: this should change throughout the paper.\nRESULTS\n-line 149: authors should temper language around “potential to reduce its toxicity” as evidence is not provided to show that this active site contributes to “toxicity” or that blocking it reduces ‘toxicity’\n-Figure 3B:AM1-1 binds to catalytic site and inhibits enzymatic activity, but when combined with 1-4 there is a shift to showing less of an effect with the combo. The authors should discuss why this might be happening\nline 174-179: this observation is in agreement with structural data, but not SEC which did not support the formation of Api m 1 dimers- this should be discussed. Also, is the dimer formation observed when bound to IgG1 or IgG4 format?\n-line 178: Suggest softening language around “can only be convincingly explained” because maybe it is an aggregation issue with the solution and not allergen dimers per se\nLine 209: remove qualifying language around “excellent” agreement\nLine 211:last sentence of the paragraph about increased avidity: this is purely speculative and the language could be tempered. One way to definitively show avidity driven results is to compare monomer vs dimer binding? This can also be done with varying concentrations of the allergen if in fact it is existing as a dimer in solution\nFigure 5: No statistics in the figure, please add statistics for the figure\nELISA:\n• Since the plate is coated with allergen (api m 1), it is possible to do the same experiment with HBV and prove that these nanobodies are preventing IgE binding to HBV\n• Line 217-219- description of ELISA assay is confusing here. Per the methods, immobilized Api m 1 was not pre-incubated with the nb-hIgG1. Also, it’s more an ability of patient IgE to bind to Api m 1 vs remaining immunoreactivity of allergen-specific IgE\nLine 224: the authors captured median’s with the range, but didn’t show any statistics to support the statement that 1-4 is better than 1-1 at inhibition.\nFigure 6: No statistics shown in the figure, please include statistics\n• Suppl S9 should be the main figure with the non-normalized data and discuss how the nanobody molecules work across all donors. Figure 6 should be supplement.\n• It is important to the unstim control and also the anti-FceR1 control for each person to capture the thresholds for the assays\n• The experiment should be repeated with HBV as the challenge. Even if it doesn’t work it proves that HBV allergy is more complicated and it would be important to the field to have this information.\n• Suggest calculating blocking percentage across donors, as it will be helpful to see the big picture\nLine 240: “1-4 had a more pronounce reduction than 1-1”: are there stats to support this statement?\nLine 277: “clear potential”-soften this language to reflect the results\nDISCUSSION:\n-line 254: half-life of 20-30 days and cite Kamal et al., 2021 as mentioned above\n-line 258: delete “only”\n-Line 279: delete ‘excellently”\n-line284: consider revising this to temper a bit, line 285 delete “excellent”\n-line 300: high affinity and epitope specificity\n-line 301: The role of Fcgr2b in IgG mAb blockade remains controversial: to date it has not been definitively demonstrated that engaging inhibitory receptors add value. Paolucci et al (ref 60) suggests that engagement of Fcgr2b is not a mechanism (along with Mckendry et al., 2021 Pediatr Allergy Immunol, and Liao et al., 2019 Clin and Exp Allergy) as opposed to Martin Bachmann research papers that suggest a role. In fact early clinical proof of concept has been demonstrated with cocktails of antibodies specific to fel d 1 and bet v 1 for cat and birch allergy using antibodies that do not engage inhibitory receptors (Orengo et al, 2018; Atanasio, et al 2022; de Blay, et al, 2022; Gevaert, et al. 2022)\n-Line 304: clarify that their results are specific for api m1 and not HBV\n-Line 315: clarify that their results are specific for api m1 and not HBV\n-Line 323: the concept that blocking one allergen might be sufficient has also been demonstrated for cat and birch allergy in people (Orengo et al, 2018; Atanasio, et al 2022; de Blay, et al, 2022; Gevaert, et al. 2022). The ara h 2 studies, while important are only in mice at this point and have not shown clinical efficacy.\nIn the discussion it would also be helpful to have a paragraph to help us understand the potential benefits or limitations of a nanobody vs a traditional bivalent antibody. This could be relevant for our interpretation of structural data in figure 3. i.e. what is the relevance of a nanobody inducing dimer formation?", "reviewer_2": "The potential use of nanobodies in immunotherapy as well in diagnostics or immunoassays has received increasing recognition in the latest years. Especially, the COVID-19 pandemic spurn the development of therapeutic nanobodies for use as rescue medication in severe cases. In allergy, the potential of nanobodies has followed recent developed of monoclonal antibodies targeting allergens. The submitted paper add to these developments by describing nanobodies specific to bee venom allergen Api m 1.\nThe study is well designed and the paper well written. The applied tests for physicochemical characterisation as well as the characterisation of their binding to its target protein are of the state of art and the data of good quality. However, the study would benefit from further biological testing. The pure fact that a nanobody (or monoclonal antibody) can bind target and prevent some of the protein functions in vitro does not necessarily guarantee good and relevant therapeutic function in vivo. Similarly, not even measurement of antibodies after immunotherapy enable good predictions of therapeutic efficacy. In the current study, this limitation is especially evident when testing the nanobodies on patient blood. Out of 9 patient samples, 2 samples were not sensitive to treatment, and another 4-5 samples shown only weak sensitivity. Additional biological testing, for instance application in an animal model of anaphylaxis, would have provided important information related to bio-distribution, if the nanobodies are able to block allergen in tissues, and on therapeutic potential.\nSpecific comments and suggestions.\nPage 2\nL30: two commas\nL35: Are these different from other know Ig-binding epitopes\nL36: one ore more residues?\nL40-41: Unclear meaning. Recognition in term of identifying epitopes or blocking by treatment?\nPage 3\nL55: 80% of patients diagnosed with bee venom allergy show a specific IgE binding reaction to Api m 10. It is considered to be the major bee venom allergen, although its content in venom is marginal (<1% dry weight). Comment on this.\nL65: delete “continuous” as it suggest non-stop and steady state.\nL67: Instead of “high risk of future stings”, rather in patients with mastocytosis, increased baseline serum tryptase levels, mast cell activation disorders, and a history of systemic anaphylaxis to stings, e.g. ref #17 (Sturm et al.).\nPage 4\nL73 and 80: no section breaks\nL89: explain \"extended\" CDR3. Is the geographical shape of CDR3 on nanobodies different from those on antibodies?\nPage 5\nL94: “HBV patients”\nPage 6\nL119: The Table 1 should be placed in the suppl. data\nL121-122: redundant sentence. Delete.\nPage 7\nL132-133: With five (AM1-4) versus two (AM1-1) H-bond sites, would you not expect AM1-4 to have a much stronger binding affinity to Api m1? Or is this the 10x stronger binding indicated in Fig. 1B?\nL147: 50%. Why is the activity not totally abrogated?\nL149: What is the clinical importance of this with relation to using AM1-1 in passiv IT?   Discussion\nPage 8\nL159: replace order of Fig. 3D and 3E (order of their appearance).\nL163: Are SEC data shown?\nL175: Since the two curves overlap, is the result of the combination a result of classical crosslinking (AMI1-1 and AMI1-4) or only the dimeric effect of the AMI1-4-binding epitope with AMI1-1 playing no role? Comment.\nPage 9\nL176: You are showing the dose-response of allergen. Rather, or in addition, you should demonstrate the dose-response of the nanobodies (titration). For the experimental condition of max degranulation (fix Api m1 concentration). How much can nanobodies reduce degranulation?\nL197: Why did the authors chose to use IgG1 and not IgG4? IgG4 is considered more relevant in allergen immunotherapy and also the mAbs used in clinical trials (which you reffered to earlier) are of IgG4 type. Especially in passive immunotherapy, IgG4 is considered much safer than complement-activating IgG1.\nL200: The gel in Figure 4B suggests rather 100-120 kDa.\nPage 10\nL202: Why are the two binds from bi-specific nb larger than the individual bands from the mono-specific nb?\nL211: impact on “what”?\"\nL212: What is the distance between the two diametrically distant epitopes, and is the bispecific nb able to cover that distance? Do you have crystal structures or representations of such binding, equivalent to Fig. 2.\nPage: 11\nL226: How do author explain the improved effect of bispecific over two monospecific?\nL242: Can you make a correlation plot between inhibition in ELISA and in basophil activation?\nL245: Include the two patients in Fig 6\nL248-249: Delete sentence. The patient variance has been known for decades.\nPage: 12\nL254: How long do you consider a season to be? How many injections of IgG would that require?\nL365-367: Does this mean that nbs are potentially better than natural abs? By consequence, VIT will have limited efficacy because of the lack of \"prolate shaped and extended CDR3\"?\nPage 13\nL294-295: What is the expected affinity of specific abs as compared to nbs? As abs are bigger, would their half-life be longer, hence, better candidates?\nL298: Regeneron and other developers of anti-allergen mAbs focus on IgG4. Comment on this in the discussion.\nPage 14\nL310-311: Discuss how will the authors deal with this clinical challenge or patient variance?\nL323: Discuss if Api m1 may represents such an immunodominant allergen\nPage 33\nFigure 6: If data are normalized, what does the black line of isotype control mean and why is it not a straight line at 100%?"}, "author_response": "Point-by-point reply to the reviewer comments, Aagaard et al.\nReviewer #1 (Remarks to the Author):\nMajor comments:\nThere are four major limitations to this study:\n1. While the authors demonstrate that the nanobody molecules can block Api m 1 binding to IgE in\nvitro using human IgE sera and basophils, the authors do not demonstrate that these molecules will\nblock HBV. Suggestions around the ability this approach to prevent a reaction beyond Api m 1 and\nwill broadly block HBV must be tempered. It is critical for the approach to show that the api m 1\nnanobody molecules block a HBV driven response\nReply: In this study we focus on the inhibition of Api m 1 responses and the molecular understanding\nof the mode of action. However, we agree with the reviewer that providing evidence for the efficacy\nof reducing a HBV-driven response by blocking Api m 1 is highly relevant. In the revised manuscript\nwe therefore followed different avenues to document these effects. We have:\n1) Tempered the wording as suggested.\n2) Established a mouse model of allergen-induced anaphylaxis to provide evidence for in vivo efficacy\n(see primarily point 2).\n3) Performed new basophil activation tests on HBV-allergic patients with a head-to-head comparison\nof Api m 1 and HBV stimulation.\nBriefly, the new data show a substantial reduction of HBV-induced activation of basophils in BAT upon\nblocking Api m 1. The data obtained in the mouse model reveal pronounced inhibition of anaphylaxis\nby the bispecific nanobody-IgG format.\nFor further details, see the individual points below.\n2. There are no in vivo data demonstrating that the api m 1 nanobody molecules can block a\npolyclonal IgE mediated response using either mouse sera and preferable human IgE (from HBV\nallergic patients). To this point, it is important to demonstrate this in vivo blockade using HBV and\nnot just api m 1 as the challenge.\nReply: As suggested by the reviewers we have established a mouse model of allergen-induced\nanaphylaxis to provide evidence for in vivo efficacy. This model is based on sensitization with HBV and\ntreatment with the bispecific nb-hIgG1. Challenge with Api m 1 reveals the impact of specific blocking\nthe targeted allergen.\nAs evident from these data, the inhibition of anaphylaxis is specific, profound and comparable in all\nanimals. The data also indicate that the in vivo model does not fully replicate the human response\nmirrored in the basophil activation testing. Hence, we addressed the impact of Api m 1 inhibition on\nthe allergic response upon stimulation with HBV in BAT for newly recruited HBV-allergic patients.\nNotably, the Api m 1 inhibition showed a reduction of HBV stimulation in all patients, some of which\nat a comparable level of inhibition for Api m 1. This finding suggests a major contribution of Api m 1\nto the cellular response in some of the patients. The relevance of Api m 1 was observed in patients co-\nsensitized to other HBV allergens, providing for the first time insights into the individual patients´\nreactivity profiles.\nThese new in vivo efficacy and BAT data are now included in the manuscript as novel parts of the\nresults (line 256-268 of the unmarked manuscript), and the supplementary data section including the\nnew figures (Fig. 7, S10, S11, and Table S2). The new data and the implications are discussed\naccordingly in the discussion section (line 362-369 of the unmarked manuscript).\n3. The data using api m 1 challenge of human basophils was not robust with partial blockade and\nthe non-normalized data in the supplemental figures. The non-normalized data should be moved to\nthe main figures to capture the variability across HBV donors and also be demonstrated with HBV\nchallenge.\nReply: As the reviewer suggested, we have moved the non-normalized data to the main text to better\ncapture the variability across the donors.\nFurthermore, as mentioned above, we have added substantial new BAT data addressing the\ncomparison of Api m 1 and HBV. These data have been described in the results (Line 247-254 of the\nunmarked manuscript) and added to the supplementary data of the manuscript as new Fig. S10 and\nTable S2.\n4. Statists should be shown for the data in the figures to validate the conclusions.\nReply: We agree with the reviewer and have included statistics for the data in the figures where\nmeaningful (see also below).\nOther Comments:\nIn general, allergen should be changed to api m 1 throughout the manuscript, since this approach is\nspecific to api m 1 rather than the HBV ‘allergen’ and it will avoid confusion.\nReply: We have addressed this relevant point accordingly throughout the manuscript in order to avoid\nconfusion.\nABSTRACT\n-line 40: please revise to honeybee venom allergic patient SAMPLES as blockade is not assessed in\npatients.\nReply: The reviewer is correct, hence we have adjusted the wording accordingly.\nINTRODUCTION\n-intro line 53: what is the other ~80% of the venom dry weight\nReply: We have added this information in brief (see also next point).\nThe sentence (now line 47) reads now as: “The most abundant component of HBV is the cytolytic\npeptide melittin with 50% of the venom dry weight.”\n-intro line 54-55: the authors report that 97% of HBV-allergic patients are sensitized to Api m 1, but\nit is important to know the sensitization rates of the other Api proteins and not just IgE for Api m 1,\nto help put things into context. That is, what other Api m allergens may be drivers of the allergic\nresponse?\nReply: The reviewer addresses an important aspect, hence we have provided a reference (Köhler et\nal.), in which we have determined the sensitization rates in a larger cohort of patients in detail.\nRoughly similar sensitization rates have been reported for different cohorts of patients. It has to be\nkept in mind, however, that the clinical relevance is not equivalent to the sensitization rate.\nThe text (now in line 50) reads now as: “Most of the other allergens are of low abundance comprising\neach less than 1-2% of venom dry weight, and have reported sensitization rates of >50% (Köhler et\nal)”.\n-intro line 65: It is important to include a comment/reference about other immunotherapy regimens\n(RUSH and ultra-RUSH) that are not as lengthy.\nReply: The reviewer is correct, modified regiments provide a quicker build-up phase of the treatment\nby which the maintenance dose can be reached more rapidly, although the duration of the entire\ntreatment does not change significantly. In order to provide this information, we have added a\nsentence highlighting these options.\nWe have changed the wording (now line 61) as follows:\n“VIT can be performed with different treatment regimens, all of which aiming aims to restore immune\ntolerance through continuous subcutaneous injection of HBV extract over the course of 5 years,\nindependently of the treatment regimen used, or even life-long under certain circumstances …”\n-Intro line 70-83: these paragraphs can be combined. Also the authors comment about the‘one\nmechanism’ is generation of blocking antibodies, but it is really THE mechanism. In this paragraph\nthey do refer to rush IT, but not in the line 65 paragraph.\nReply: We have re-organized and combined the paragraphs. We have also adjusted the wording.\nThe sentence (now line 67) reads now as: “The dominant mechanism by which AIT induces tolerance\nis through the production of allergen-specific IgG1, IgG4 and IgA that block the interaction between\nthe allergen and allergen-specific IgE.”\n-intro line 77: Suggest citing Kamal et al., 2021 Clin Trans Sci reference here with a sentence referring\nto the half-life of monoclonal IgG being 20-30 days and therefore having the potential to provide\nprotection within a well-defined season.\nReply: We agree with the reviewer and have added the information.\nThe sentence (now line 76) reads now as: “In HBV allergy, stings occur unpredictably within a well-\ndefined season, and passive immunotherapy with IgG having a half-life of 20-30 days (Kamal et al.)\ncould provide seasonal protection against severe symptoms including anaphylaxis in case of a sting\nevent.”\n-intro line 93: “allergen” should be changed to Api m 1 because we only see data for this one protein\nand not venom in general. NOTE: this should change throughout the paper.\nReply: We have adjusted the wording accordingly.\nRESULTS\n- line 149: authors should temper language around “potential to reduce its toxicity” as evidence is\nnot provided to show that this active site contributes to “toxicity” or that blocking it reduces\n‘toxicity’\nReply: The reviewer is correct, we do not provide evidence that Api m 1 contributes to toxicity. As\nalready stated in the introduction and discussion section, it is however common understanding that\nApi m 1 with its enzymatic activity together with melittin are the culprits for the toxic effects of the\nvenom.\nIn order to clarify, we have adjusted the wording in the results part. The sentence (now line 142) reads\nnow as: “This inhibition supported the finding that AM1-1 interacts directly with the active site of Api\nm 1 and exhibits the potential to reduce the enzymatic activity of Api m 1 which contributes to the\npain and inflammation of a honeybee sting.”\n-Figure 3B:AM1-1 binds to catalytic site and inhibits enzymatic activity, but when combined with 1-\n4 there is a shift to showing less of an effect with the combo. The authors should discuss why this\nmight be happening\nReply: When using the combination the concentration of AM1-1 is halved which can explain the shift.\nIt is also possible that the binding of AM1-4 introduces minor conformational rearrangements that\naffects the binding of AM1-1. In the end, both AM1-1 alone and in combination reaches an inhibition\nof 50%.\n-line 174-179: this observation is in agreement with structural data, but not SEC which did not\nsupport the formation of Api m 1 dimers- this should be discussed. Also, is the dimer formation\nobserved when bound to IgG1 or IgG4 format?\nReply: We have adjusted the wording.\nThe sentence (now line 156) reads now as: “Although formation of dimers in solution has not been\nreported, Api m 1 might transiently form dimers at locally high concentrations, e.g. at cellular\nsurfaces.”\nThe formation of dimers is further discussed in the discussion section on page 14 (line 309-316) in the\nrevised manuscript.\nWe do not have evidence of dimer formation of Api m 1 bound by IgG formats.\n-line 178: Suggest softening language around “can only be convincingly explained” because maybe\nit is an aggregation issue with the solution and not allergen dimers per se\nReply: We have softened the language accordingly.\nThe sentence (now line 168) reads now as: “… the cellular activation was strictly allergen-dependent,\nand could be explained convincingly by the formation of Api m 1 dimers at the surface.”\nThe cellular activation is only observed with the AM1-4 IgE and not the AM1-1 IgE. This specific and\nquick effect would argue against any aggregation phenomenon.\nLine 209: remove qualifying language around “excellent” agreement\nReply: We reviewer is right, we have removed the qualifying language.\nLine 211: last sentence of the paragraph about increased avidity: this is purely speculative and the\nlanguage could be tempered. One way to definitively show avidity driven results is to compare\nmonomer vs dimer binding? This can also be done with varying concentrations of the allergen if in\nfact it is existing as a dimer in solution\nReply: We appreciate the comment but think that it is based on a misunderstanding. The avidity that\nwe refer to is not caused by Api m 1 dimers. We are referring to the bi-epitopic binding of the bispecific\nantibody to Api m 1. Each Api m 1 molecule is thus bound at two epitopes at once resulting in the\nhigher dissociation constant compared to the monospecific IgG.\nWe have adjusted the wording in order to make this clearer. The sentence (now line 206) reads now\nas: “Notably, the dissociation rate of the bispecific nb-hIgG1 was significantly lower (<10-7 s-1),\nreflecting increased avidity due to simultaneous binding of two Api m 1 epitopes.”\nFigure 5: No statistics in the figure, please add statistics for the figure\nReply: The reviewer is correct. We have added statistics for the figure.\nELISA:\n•Since the plate is coated with allergen (api m 1), it is possible to do the same experiment with HBV\nand prove that these nanobodies are preventing IgE binding to HBV\nReply: The reviewer is correct, that would be possible. However, we see neither the need nor the\nbenefit as we want to demonstrate the nanobodies capacity to inhibit Api m 1. If the plates were\ncoated with HBV, any additional reactivity of serum IgE to other allergens would overlay the inhibition.\n• Line 217-219- description of ELISA assay is confusing here. Per the methods, immobilized Api m 1\nwas not pre-incubated with the nb-hIgG1. Also, it’s more an ability of patient IgE to bind to Api m 1\nvs remaining immunoreactivity of allergen-specific IgE.\nReply: Immobilized Api m 1 is first incubated with nb-hIgG1 and thereafter with patient serum. We\nhave adjusted the wording in order to facilitate understanding.\nThe sentence (now line 213) reads now as: “Immobilized Api m 1 was incubated with the nb-hIgG1\nfollowed by incubation with serum from HBV-allergic patients and the remaining binding of allergen-\nspecific IgE was analyzed.”\nLine 224: the authors captured median’s with the range, but didn’t show any statistics to support\nthe statement that 1-4 is better than 1-1 at inhibition.\nReply: The reviewer is correct. We have added statistics to Fig. 5 and changed the wording in the result\nsection (now lines 219-222) accordingly.\nFigure 6: No statistics shown in the figure, please include statistics\nReply: The figure shows data from individual patients and therefore, we do not think that adding\nstatistics is meaningful.\n• Suppl S9 should be the main figure with the non-normalized data and discuss how the nanobody\nmolecules work across all donors. Figure 6 should be supplement.\nReply: As the reviewer suggested, we have moved the non-normalized data to the main text and the\nnormalized data to the supplement.\n• It is important to the unstim control and also the anti-FceR1 control for each person to capture\nthe thresholds for the assays\nReply: We do not see a clear benefit in providing this information, but we followed the suggestion and\nhave included the data in the new Table S3 and added the reference to the sentence in line 236.\n• The experiment should be repeated with HBV as the challenge. Even if it doesn’t work it proves\nthat HBV allergy is more complicated and it would be important to the field to have this information.\nReply: In order to address the point we have performed new BAT analyses and added the requested\ndata. Please see the more detailed comments and reply above.\n• Suggest calculating blocking percentage across donors, as it will be helpful to see the big picture\nReply: We do not think that this would be meaningful as there is a large variation between the\nindividual patients. We think that it is better to visualize the individual patients’ responses.\nLine 240: “1-4 had a more pronounce reduction than 1-1”: are there stats to support this statement?\nReply: The statement is based on the observation that 1-4 has a stronger inhibitory effect in all\npatients. We do not provide statistics on this observation.\nIn order to clarify we have changed the wording. The sentence (now line 236) reads now as: “For all\npatients, the AM1-4 hIgG1 format resulted in a more pronounced reduction than the AM1-1 hIgG1\nformat.”\nLine 277: “clear potential”-soften this language to reflect the results\nReply: We have adjusted the wording.\nThe sentence (now line 244) reads now as: “These data demonstrate the potential of the nanobody\nformats for blocking effector cell activation by Api m 1 stimulation.”\nDISCUSSION:\n-line 254: half-life of 20-30 days and cite Kamal et al., 2021 as mentioned above\nReply: We have added the information and the suggested reference (line 273-276).\n-line 258: delete “only”\nReply: We have adjusted the wording. The sentence (line 276) reads now as: “Moreover, imperative\nis the reduction of symptom severity and thereby the prevention of potentially life-threatening\nanaphylaxis.”\n-Line 279: delete ‘excellently”\nReply: We have adjusted the phrase (now line 301) accordingly.\n-line284: consider revising this to temper a bit, line 285 delete “excellent”\nReply: We have deleted the sentence.\n-line 300: high affinity and epitope specificity\nReply: The reviewer is correct.\nThe sentence (now line 323) reads now as: “Instead, high affinity and epitope specificity are crucial for\nthe neutralizing effects”\n-line 301: The role of Fcgr2b in IgG mAb blockade remains controversial: to date it has not been\ndefinitively demonstrated that engaging inhibitory receptors add value. Paolucci et al (ref 60)\nsuggests that engagement of Fcgr2b is not a mechanism (along with Mckendry et al., 2021 Pediatr\nAllergy Immunol, and Liao et al., 2019 Clin and Exp Allergy) as opposed to Martin Bachmann research\npapers that suggest a role. In fact early clinical proof of concept has been demonstrated with\ncocktails of antibodies specific to fel d 1 and bet v 1 for cat and birch allergy using antibodies that\ndo not engage inhibitory receptors (Orengo et al, 2018; Atanasio, et al 2022; de Blay, et al, 2022;\nGevaert, et al. 2022)\nReply: We agree with the opinion of the reviewer on the controversy of the engagement of FcgR2B,\nhence we have removed the sentence.\n-Line 304: clarify that their results are specific for api m1 and not HBV\nReply: We have adjusted the wording.\nThe sentence (now line 326) reads now as: “The nb-hIgG1 formats were able to inhibit both the binding\nof patient IgE to Api m 1 and the subsequent basophil activation.”\n-Line 315: clarify that their results are specific for api m1 and not HBV\nReply: We have adjusted the wording.\nThe sentence (now line 326) reads now as: “but the fact that we could observe a clear reduction in Api\nm 1-induced basophil activation supports the concept of an efficient mode of action of the nb-hIgG1\nassessed here.”\n-Line 323: the concept that blocking one allergen might be sufficient has also been demonstrated\nfor cat and birch allergy in people (Orengo et al, 2018; Atanasio, et al 2022; de Blay, et al, 2022;\nGevaert, et al. 2022). The ara h 2 studies, while important are only in mice at this point and have\nnot shown clinical efficacy.\nReply: We agree with the reviewer and have rewritten the text (now line 344-349) to include the cat\nand birch study (Orengo et al, 2018; Atanasio, et al 2022). We also do think that the Ara h 2 study in\nmice, in particular in the context of our new mouse data, is relevant as it demonstrates the potential\nto reduce anaphylaxis.\nIn the discussion it would also be helpful to have a paragraph to help us understand the potential\nbenefits or limitations of a nanobody vs a traditional bivalent antibody. This could be relevant for\nour interpretation of structural data in figure 3. i.e. what is the relevance of a nanobody inducing\ndimer formation?\nReply: The point is truly valid, hence we have expanded the information on benefits of nanobodies on\npage 13 (now lines 278-290) and included i.a. a very recent nanobody-based candidate for treatment\nof asthma.\nAs the nanobody is not inducing dimerization, we do not see the last point mentioned by the reviewer.\nReviewer #2 (Remarks to the Author):\nThe potential use of nanobodies in immunotherapy as well in diagnostics or immunoassays has\nreceived increasing recognition in the latest years. Especially, the COVID-19 pandemic spurn the\ndevelopment of therapeutic nanobodies for use as rescue medication in severe cases. In allergy, the\npotential of nanobodies has followed recent developed of monoclonal antibodies targeting\nallergens. The submitted paper add to these developments by describing nanobodies specific to bee\nvenom allergen Api m 1.\nThe study is well designed and the paper well written. The applied tests for physicochemical\ncharacterisation as well as the characterisation of their binding to its target protein are of the state\nof art and the data of good quality. However, the study would benefit from further biological testing.\nThe pure fact that a nanobody (or monoclonal antibody) can bind target and prevent some of the\nprotein functions in vitro does not necessarily guarantee good and relevant therapeutic function in\nvivo. Similarly, not even measurement of antibodies after immunotherapy enable good predictions\nof therapeutic efficacy. In the current study, this limitation is especially evident when testing the\nnanobodies on patient blood. Out of 9 patient samples, 2 samples were not sensitive to treatment,\nand another 4-5 samples shown only weak sensitivity.\nAdditional biological testing, for instance application in an animal model of anaphylaxis, would have\nprovided important information related to bio-distribution, if the nanobodies are able to block\nallergen in tissues, and on therapeutic potential.\nReply: See also comment of reviewer 1 and the reply above. We agree with the reviewer that providing\nevidence for the efficacy of the molecules in an animal model of anaphylaxis would be highly\nbeneficial. In the revised manuscript we therefore followed different avenues to document these\neffects.\nAs suggested by the reviewers, we have established a mouse model of allergen-induced anaphylaxis\nto provide evidence for in vivo efficacy. This model is based on the sensitization with HBV and\nchallenge with Api m 1 to reveal the impact of specific blocking the targeted allergen. As evident from\nthese data, the inhibition of anaphylaxis is specific, profound and comparable in all animals. Moreover,\nthe data obtained in the mouse model revealed pronounced inhibition of Api m 1-induced anaphylaxis\nby the nanobody-IgG formats.\nThese new in vivo efficacy data are now included in the manuscript as novel parts of the results (line\n256-268 of the unmarked manuscript), and the supplementary data section including the new figures\n(Fig. 7 and S11). The new data and the implications are discussed accordingly in the discussion section\n(line 362-369 of the unmarked manuscript).\nSpecific comments and suggestions.\nPage 2\nL30: two commas\nReply: We have adjusted accordingly.\nL35: Are these different from other know Ig-binding epitopes\nReply: We do not know if the epitopes are different for IgG or IgE as no molecular information on IgG\nand IgE epitopes is available. These aspects are discussed on page 13 of the revised manuscript (lines\n291-300).\nL36: one or more residues?\nReply: In order to facilitate the reading we have adjusted the sentence.\nThe sentence (now line 30) reads now as: “Crystal structures of Api m 1/nanobody complexes revealed\ndiametrically opposed epitopes, one of which involved the active site of Api m 1.”\nL40-41: Unclear meaning. Recognition in term of identifying epitopes or blocking by treatment?\nReply: Valid point. As we have included more data, the abstract has been rewritten and the sentence\nbecame obsolete.\nPage 3\nL55: 80% of patients diagnosed with bee venom allergy show a specific IgE binding reaction to Api\nm 10. It is considered to be the major bee venom allergen, although its content in venom is marginal\n(<1% dry weight). Comment on this.\nReply: A very interesting point raised by the reviewer. According to the majority of studies, Api m 10\nsensitization has a prevalence of 24-75% of allergic patients. In our own study identifying Api m 10 as\nmajor allergen, the sensitization rate was 62%. The sensitization rate is consistently lower than the\none of Api m 1. This distribution is also evident from the patients we have included in this study. We\nhave shown recently that predominant sensitization to Api m 10 might increase the rate of treatment\nfailures and that Api m 10 is lacking in purified therapeutic HBV preparations. However, the clinical\nrelevance of Api m 10 and its potential absence in VIT products is controversially debated and no\nconclusive information is yet available. As the reviewer points out, the abundance of Api m 10 and\nmost other allergens is rather low.\nBasic information on sensitization rates has been added to the introduction (line 50-53; see also points\nabove).\nL65: delete “continuous” as it suggest non-stop and steady state.\nReply: We have adjusted the text accordingly.\nL67: Instead of “high risk of future stings”, rather in patients with mastocytosis, increased baseline\nserum tryptase levels, mast cell activation disorders, and a history of systemic anaphylaxis to stings,\ne.g. ref #17 (Sturm et al.).\nReply: We agree with the reviewer and have adjusted the sentence accordingly.\nThe sentence reads now as: “… or even life-long under certain circumstances including patients with\na severe initial sting reaction or mastocytosis (Sturm et al.).”\nPage 4\nL73 and 80: no section breaks\nReply: We have rearranged the text and removed the section brakes as suggested.\nL89: explain \"extended\" CDR3. Is the geographical shape of CDR3 on nanobodies different from\nthose on antibodies?\nReply: The number of residues within the CDR is often elevated, making the CDR slightly longer as\ncompared to regular antibodies. This also changes the shape of the CDR3. The position of the CDR\nwithin the framework regions is not changed as such.\nTo improve the understanding, we have replaced the term extended by the term longer (now line 84).\nPage 5\nL94: “HBV patients”\nReply: We have adjusted the text accordingly.\nThe sentence (now line 87) reads now as: “In this study, we generated Api m 1-specific nanobodies for\ninterfering with the binding of HBV-allergic patient IgE to Api m 1 and thereby reducing the risk of\nanaphylaxis in HBV-allergic patients.”\nPage 6\nL119: The Table 1 should be placed in the suppl. Data\nReply: Table 1 comprises important information for structural biology readers, hence we prefer to\nkeep the table in the main document.\nL121-122: redundant sentence. Delete.\nReply: The reviewer is fully correct, hence we have shorted the sentences accordingly.\nThe shortened sentence (now line 116) reads now as: “The architecture of the ternary complex\ncorroborates the BLI data”.\nPage 7\nL132-133: With five (AM1-4) versus two (AM1-1) H-bond sites, would you not expect AM1-4 to have\nmuch stronger binding affinity to Api m1? Or is this the 10x stronger binding indicated in Fig. 1B?\nReply: As can be seen in supplement Fig. S2, there are much more H-bonds than the reviewer\nmentions. But the reviewer is also fully correct, the 10-fold higher affinity is shown in Fig. 1B.\nL147: 50%. Why is the activity not totally abrogated?\nReply: This is definitely an interesting aspect, and it is open why we do not see a full inhibition. We\ncan only speculate about the affinity of the interaction and the potential effect of the conformational\nconsequences of AM1-1 binding on the monomeric form of Api m 1, but this is not substantiated by\nany data, so we excluded these speculations.\nL149: What is the clinical importance of this with relation to using AM1-1 in passiv IT?  Discussion\nReply: The enzymatic activity of Api m 1 together with the presence of Api m 4 is known to contribute\nto the pain perception and inflammation of a honeybee sting. In addition, the toxicity of HBV is mostly\nattributed to Api m 1 (see also reply to the comment of reviewer 1). Thus, inhibition of the enzymatic\nactivity will most likely reduce the adverse reactions, even beyond blocking the IgE binding. In case of\nmultiple stings, this effect will be even more important.\nWe have added a sentence in the discussion to emphasize the potential benefit in the clinical setting\n(now lines 304-308).\nPage 8\nL159: replace order of Fig. 3D and 3E (order of their appearance).\nReply: We are grateful for the comment, the order is indeed different. Hence, we have reorganized\nand adjusted the text and the appearance of the figures in the text.\nL163: Are SEC data shown?\nReply: The text relating to the SEC data has been modified.\nThe sentence (lines 156-157) reads now as follows: “Although the formation of dimers in solution has\nnot been reported, Api m 1 might transiently form dimers at locally high concentrations, e.g. at cellular\nsurfaces.”\nL175: Since the two curves overlap, is the result of the combination a result of classical crosslinking\n(AMI1-1 and AMI1-4) or only the dimeric effect of the AMI1-4-binding epitope with AMI1-1 playing\nno role? Comment.\nReply: The reviewer is correct, in this type of assay we cannot discriminate between the two\nexplanations, which is similar to any assay format using a cellular read-out and any effect in basophil\nactivation testing. Hence, we avoid making a clear-cut statement on the molecular basis of cross-\nlinking. Both explanations, however, would be interesting with respect to the molecular features of\nthe allergen.\nPage 9\nL176: You are showing the dose-response of allergen. Rather, or in addition, you should\ndemonstrate the dose-response of the nanobodies (titration). For the experimental condition of\nmax degranulation (fix Api m1 concentration). How much can nanobodies reduce degranulation?\nReply: The point might arise from a misunderstanding. In this assay, which is established in the field,\nthe dose-response curve of the allergen is based on the fact that the nb-hIgE format is bound to the\ncellular surface and the allergen is added in increasing concentrations. The nanobodies used here as\nIgE format do not reduce degranulation, they mediate degranulation.\nL197: Why did the authors chose to use IgG1 and not IgG4? IgG4 is considered more relevant in\nallergen immunotherapy and also the mAbs used in clinical trials (which you referred to earlier) are\nof IgG4 type. Especially in passive immunotherapy, IgG4 is considered much safer than complement-\nactivating IgG1.\nReply: We discuss and address this point already in the discussion section (lines 294-303 in the revised\nmanuscript). In brief, we and others agree that IgG1 corresponds better to the early VIT-induced\neffects. Thus, for mirroring the biological effect of blocking antibodies, IgG1 might be a preferable\nformat. In general, however, our approach is not limited to IgG1. Re-engineering into IgG4-based\nformats is state-of-the-art and even multi-specific, non-Fc-fused formats might become an attractive\napproach.\nL200: The gel in Figure 4B suggests rather 100-120 kDa.\nReply: Yes, the molecule is observed at a slightly higher molecular weight, which is very typical and\nessentially due to the glycosylation of the Fc. Hence, we have adjusted the sentence.\nThe sentence (now lines 192-194) reads now as: “The three nb-hIgG1 formats were expressed in\nmammalian cells, purified and analyzed by SDS-PAGE verifying the expected molecular mass of a\nglycosylated format, suggesting proper folding and dimerization”.\nPage 10\nL202: Why are the two binds from bi-specific nb larger than the individual bands from the mono-\nspecific nb?\nReply: The observation is correct. The minor difference is due to addition of an epitope tag at the C-\nterminus of the chains allowing assessment of heterodimerization. This information has been added\nto the method section in the revised manuscript on page 18.\nL211: impact on “what”?\"\nReply: We have adjusted the sentence.\nThe sentence (now lines 204-206) reads now as: “BLI binding studies of immobilized nb-IgG1 further\nrevealed agreement of the kinetic data with those of the individual nanobodies, confirming that the\nFc fusion has no impact on binding affinity.”\nL212: What is the distance between the two diametrically distant epitopes, and is the bispecific nb\nable to cover that distance? Do you have crystal structures or representations of such binding,\nequivalent to Fig. 2.\nReply: We do interpret the point as a question about the ability of a bispecific antibody to bind\nsimultaneously to the epitopes of one and the same allergen. This is a difficult point, but several lines\nof evidence suggest a simultaneous binding.\nMost importantly, the BLI analysis of the bispecific versus monospecific analysis shows a significant\nincrease of affinity in the set-up where the bispecific antibody is immobilized on the sensor. The\nobtained results suggest that simultaneous binding to the two epitopes is occurring efficiently. Since\nthe nb-IgG1 formats do contain hinge regions of IgG1, molecular flexibility and ability to bridge\ndistances is given. Non-simultaneous binding would be reflected in an average affinity and re-binding\nis negligible, as evident from the monospecific antibodies. This information is available in the results\nsection (now lines 206-209).\nWe cannot fully rule out that stable immune complexes might also be formed in solution by\nintermolecular interaction, resulting in larger linear or circular complexes. However, similar complexes\ncan also be expected for a combination of monospecific antibodies and are also formed during AIT.\nWe do not have structural data supporting either type of interactions.\nPage: 11\nL226: How do author explain the improved effect of bispecific over two monospecific?\nReply: Based on the comments of reviewer 1 we have added statistics to Fig. 5B and do not see a\nstatistical significance for the bispecific compared to the mix of the two monospecific nb-hIgG1.\nNevertheless, a slightly improved effect in our opinion would be a result of increased steric hindrance\nby a bispecific format.\nL242: Can you make a correlation plot between inhibition in ELISA and in basophil activation?\nReply: We are not able to do so, since the samples used for the ELISA and the BAT were not from the\nsame patients. The BATs were performed using EDTA blood samples from patients prior to VIT coming\nin occasionally. The sera for the inhibition ELISA were selected based on a sufficiently high sIgE level\nto Api m 1.\nL245: Include the two patients in Fig 6\nReply: As suggested by reviewer 1 we have moved the non-normalized data to the main text. These\ndata include now the two patients in whom no reduction of basophil activation was observed.\nL248-249: Delete sentence. The patient variance has been known for decades.\nReply: We agree with the reviewer that the patient variance is known, hence we have adjusted the\nsentence.\nThe sentence (now line 245) reads now as: “The data also confirms a large blocking variation, reflecting\ndiverse IgE repertoires of the HBV-allergic patients.”\nPage: 12\nL254: How long do you consider a season to be? How many injections of IgG would that require?\nReply: The honeybee season is very dependent on the geographical distribution, but typically a period\nof 3 month. Given an IgG half-life of 20-30 days, a window of 90 days could be covered with a single\ninjection. In addition, half-life extension strategies like the introduction of mutations increasing the\naffinity to FcRn and other concepts are state of the art, hence we believe that a season could be fully\ncovered with one injection.\nWe have added these considerations to the revised manuscript (see also comments of reviewer 1).\nL365-367: Does this mean that nbs are potentially better than natural abs? By consequence, VIT will\nhave limited efficacy because of the lack of \"prolate shaped and extended CDR3\"?\nReply: In this paragraph we only claim that nanobodies are powerful alternatives. Based on their\ncharacteristics, nanobodies of tailored specificity might even perform better in some settings than\nmost of naturally induced antibodies. There is, however, broad evidence for the existence of canonical\nantibodies with extended CDR3, implying that natural antibodies of appropriate characteristics are\ngenerally capable of having a similar binding mode and thus efficacy. The frequency of occurrence of\nsuch antibodies, however, might be lower, hence a propensity of nanobodies targeting efficiently such\nepitopes is likely to be higher.\nPage 13\nL294-295: What is the expected affinity of specific abs as compared to nbs? As abs are bigger, would\ntheir half-life be longer, hence, better candidates?\nReply: This is an important point with implications on the general applicability. In general, it is\nextensively documented that nanobodies do have similar affinities as regular antibodies. We mention\nthe comparability of the affinities in the discussion section (now lines 324-326).\nLikewise, the half-life of antibodies and engineered nanobodies providing Fc domains can be expected\nto be in the same range. Moreover, a plethora of half-life extension approaches can be pursued,\nmostly similar to the ones applied to IgG formats. As also included in the revised version of the\nmanuscript (line 286), the pentavalent nanobody fusion molecule Lunsekimig exhibits an extended\nhalf-life by including an HSA-targeting nanobody. Thus, nanobody-based formats are not per ser\ninferior to regular formats and can be expected to have similar performance.\nL298: Regeneron and other developers of anti-allergen mAbs focus on IgG4. Comment on this in the\ndiscussion.\nReply: Please see the reply to the comment of reviewer 1.\nThe reviewer is correct, typically IgG4 is chosen for blocking approaches, essentially due to the\nsomewhat erroneous understanding of IgG4 as an AIT-induced subclass. However, we discuss and\naddress this point in the discussion section (lines 294-303 in the revised manuscript). In brief, we and\nothers agree that IgG1 corresponds better to the early VIT-induced effects. Thus, for mirroring the\nbiological effect of blocking antibodies, IgG1 might be a preferable format. In general, however, our\napproach is not limited to IgG1. Re-engineering into IgG4-based\nformats is state-of-the-art and even multi-specific, non-Fc-fused formats might become an attractive\napproach.\nPage 14\nL310-311: Discuss how will the authors deal with this clinical challenge or patient variance?\nReply: The reviewer touches an important aspect. Currently, VIT uses a “one fits all” approach with\nentire venom even for treatment of patients sensitized to potentially only a single allergen.\nIntroducing precision by targeting selected allergens could provide a benefit but comes with the\nlimitation that treatment needs to be adjusted to the individual patient.\nIn the case of HBV allergy, it remains open so far, which allergens and at what concentrations drive or\nadd to the anaphylactic reaction.\nA robust inhibition of the dominant allergen(s) might provide exactly the lever to limit anaphylaxis and\nprovide protection. As stated in the discussion, additional epitopes and additional allergens might be\nneeded to achieve this level of inhibition.\nStratification of patients for the protective effect upon passive immunization might be another option\n(see also point below).\nL323: Discuss if Api m1 may represents such an immunodominant allergen\nReply: This is of course a very important point. As also touched upon in the reply to the previous\ncomment, it is not fully clear which of the venom allergens are the drivers of severe reactions. Api m\n1 is the most abundant major allergen and has a molecular weight which allows for efficient use of\nepitopes in proximity. In comparison, the venom hyaluronidase Api m 2 is typically considered less\nallergenic. Serological data of the patients in this study show a frequent dominant sensitization to Api\nm 1 with higher sIgE levels as compared to the other allergens. However, this does not necessarily\nimply clinical relevance.\nNotably, our novel BAT data included in the results and discussion section provide first insights into\nthe level of basophil activation of individual allergens, here Api m 1, and thus provide initial answers.\nA relatively large inhibition of HBV-stimulated basophils suggest that the response is dominantly\ndriven by the inhibited allergen (Api m 1). This finding implies that the other 12 allergens combined,\nall at lower abundance, have a more limited effect. However, the other allergens do also contribute\nto basophil activation. To what extent these contributions are clinically relevant, remains open. These\ninitial findings need more robust analyses in future studies revealing and differentiating the impact of\nsingle allergens to the clinical picture.\nAs mentioned before, this interpretation is presented in the context of the new BAT data in the revised\nmanuscript.\nPage33: Figure 6: If data are normalized, what does the black line of isotype control mean and why\nis it not a straight line at 100%?\nReply: The data are normalized to the average of the isotype control across all measurements. Thus,\nthe black line shows the deviation of the single measurements from the average. This information has\nbeen added to the figure legend. The normalized data have been moved to the supplement. See also\nthe comments above.\nPoint by point reply:\nWe would like to thank the reviewers for their appreciation of the additional experiments we\nhave included in the revised version of the manuscript and the comments on important aspects\nof the manuscript, which we took as stimulus to further improve the manuscript.\nAll the comments raised by the reviewer were addressed in detail below.\nOne general aspect mentioned is the varying capacity of Api m 1-specific nanobodies to inhibit\nan Api m 1- or HBV-induced allergic reaction. In relation to this aspect and the comments of\nreviewer 1, we would like to emphasize that our manuscript is a proof-of-concept and feasibility\nstudy for the use of nanobody-based antibody formats and the concept described should\ntherefore not be considered a final treatment approach for patients with honeybee venom\nallergy.\nPlease find the detailed response below.\nReviewer #1 (Remarks to the Author):\nPoint 1. The ex-vivo Api-m 1-driven BAT data remains highly variable and\nsubjective as currently presented.\n• The addition of anti-Fcer1 control values is appreciated but highlights that the stim\nconcentrations of Api m 1 used may not be achieving max activation potential.\no Specifically, in 6 out of the 9 donors, the anti-Fcer1a is at least >25% higher than\nthe isotype control group.\no In 2 out of the 3 donors that achieve “max activation” (H, K amd M), partial\n(~40%) or no blockade is observed.\n• The addition of untsim control values is also appreciated but demonstrates that the\nApi m 1-based nanobody IgG1 molecules block basophil activation to levels of unstim\ncontrol in only 1 out of the 9 samples evaluated.\nReply: We agree with the reviewer that the data on ex vivo Api m 1-specific BAT shows high\nvariability, which in our opinion reflects the situation in patients with honeybee venom allergy.\nThis means that activation of basophils is dependent on several factors like e.g. the epitope\ndiversity and cell surface density of IgE, the varying affinity of IgE for the different allergen\nepitopes, the complexity and valency of the allergen as well as intrinsic levels of Syk\nexpression. Consequently, we do not expect a single allergen to cause as high activation as\na stimulation control for basophils such as anti-FcεR1. Moreover, the differences in the ability\nof the nb-hIgG1 formats to inhibit IgE binding of the three donors showing almost identical\nactivation by anti-FcεR1 and Api m 1 (patients H, K and M) indicate that blockade depends\nlargely on the individual composition of the targeted epitopes, which emphasizes the\ndependency of both basophil activation and blockade on the above-mentioned factors.\nIn addition, and more in general we would like to emphasize that successful treatment of HBV\nallergy by VIT over a course of several years offers protection against anaphylaxis. It is,\nhowever, well established in the field that in tolerant patients after VIT, blocking antibodies only\nreduce basophil activation to a minor extent and with variability. Inversely, the observed partial\nreduction in inhibition BAT achieved by VIT is still indicative for functional protection (as\nrelevant references the following might serve: Arzt et al., Allergy 2018 PMID: 29171032; Erzen\net al., Allergy 2012 PMID: 22469017).\nThis information was already briefly mentioned in the discussion section on page 15, now\nlines 335-340, but we have reworded and extended the sentences to facilitate the\ninterpretation of the data. We hope that this explanation makes our results more clear.\nIn this context, we are convinced that the BAT inhibition we do observe corresponds fully to\nthe picture observed for VIT treated allergic patients.\n• As previously suggested, blocking percentages should be calculated for each donor\nand summary data graphed with statistical analyses run\no Although the authors state a preference to show individual patient responses,\nquantification of blocking percentages to be graphed alongside individual plot\ndata would more accurately represent the dataset\no As blocking percentages are provided for the newly added HBV-BAT (fig. S10),\nfor consistency blocking percentages should also be provided for the Api m 1-\nBAT\nReply: As suggested by the reviewer, we have added graphs displaying the individual and\nsummarized blocking effect and we do agree that the information is actually explanatory and\nbeneficial for the reader.\nThe paired graph is now included as figure 6B highlighting the inhibition by the bispecific nb-\nhIgG1. The summary of data for the bispecific nb-hIgG1 and the blocking percentages are\nshown in figure S9, B and C. For both graphs, statistical analysis is added. The figures are\nreferenced accordingly in the text of the results section on page 11, line 240.\nPoint 2. For this approach to be clinically relevant, the ability of the nanobody\nmolecules to block HBV-induced allergic effector cell activation needs to be\nbetter supported.\no Blockade of HBV-induced basophil activation data is not robust; Line 251: >34% is\nnot a “large” reduction- it is partial at best\nReply: We have reworded the phrase accordingly. In addition, we have modified the figure 6\nand figure S10 by showing a graph that displays the individual blocking effect upon stimulation\nwith Api m 1 and HBV. The graph is implemented as figure 6C and referenced accordingly in\nthe text of the results section on page 12, line 244-246.\no it would have been nice to follow the same assay format utilized for Api m 1-BAT\n(dose-dependent treatment vs single-point/fixed molar ratio).\nReply: We do understand the point of the reviewer. However, the data in Fig. 6 and in Fig. 10\nclearly suggest that differences in inhibition between Api m 1 and HBV are rather limited and\nnot expected. Hence, we think that the approach taken in Fig. S10 is sufficient and we do not\nexpect a significant gain of additional insights from another assay format.\n• Despite sensitization with HBV in the in-vivo model, challenge with Api m 1 allergen\nis supportive only of the ability to block exposure to Api m 1 and data with an HBV\nchallenge is required to support this approach and demonstrate efficacy against HBV\nchallenge.\nReply: The reviewer is correct; the challenge with Api m 1 demonstrates the efficacy of the\napproach for blocking allergen-induced cellular activation and anaphylaxis for Api m 1 only.\nThis has been the clear focus of this proof-of-concept study.\nObviously, HBV contains additional allergens, but also lower molecular weight compounds\nwith a variety of functional activities. Thus, we do agree that using HBV for the challenge might\nprovide more detailed support of the approach.\nHence, we have performed a new set of experiments using HBV for both sensitization and\nchallenge of sensitized mice. These experiments show a reduction of serum MCPT-1 levels in\ntreated animals as compared to non-treated controls. This reduction, although not significant,\nshows a mean value of MCPT-1 levels, which is 38% lower as compared to the non-treated\ncontrol. This is a lower effect as compared to the BAT data, which shows significantly reduced\nactivation by HBV, but the sensitization with HBV might also be broader than observed in\npatients.\nHowever, the challenge with HBV at the concentrations used in this experiment was\naccompanied by a clear-cut body temperature drop even in non-sensitized mice. The absence\nof mast cell activation as documented by lack of MCPT-1 induction in non-sensitized mice\nindicates a response beyond an IgE-mediated allergic reaction which is most likely caused by\nthe toxicity of the venom.\nAlthough the observed toxic effect might limit the benefit of the entire data obtained, we think\nthat the information is important and supports our concept and thus is important to show.\nTherefore, these data are now included in the supplementary data as novel figure S12 and\nS13 and as text in the results and discussion section on page 12, line 263-267 and 17, line\n269-273.\nMinor comments:\n1. Line 61- edit to “all of which aim to restore immune tolerance…”\nReply: We have corrected the phrase on now page 3 accordingly.\n2. Line 67- blocking ability of IgA remains controversial/poorly understood: suggest\nediting to “through the production of allergen-specific IgG that block the interaction…”\nReply: The reviewer is correct with respect to the blocking ability of IgA. We have reworded\nthe sentence on now page 4 accordingly.\n3. Figure 3B: AM1-1 binds to catalytic site and inhibits enzymatic activity, but when\ncombined with 1-4 there is a shift to showing less of an effect with the combo. The\nauthors should include text related to the response provided below in the text of the\nmanuscript and not just in the reviewer response document\n• Reply: When using the combination the concentration of AM1-1 is halved which can\nexplain the shift. It is also possible that the binding of AM1-4 introduces minor\nconformational rearrangements that affects the binding of AM1-1. In the end, both AM1-\n1 alone and in combination reaches an inhibition of 50%.\nReply: We do agree with the suggestion of the reviewer, hence we have introduced a short\nstatement on now page 7.\n4. Methods and Figure S11: The HBV- and Api m 1-specific IgE measurements are quite\nlow. This is likely due to IgG:allergen binding as allergen-specific IgG is more abundant\nthan IgE. To avoid interference of IgG binding to allergen, plates should be coated with\nan anti-mouse IgE, followed by sample and then a biotinylated HBV extract or Api m 1\nallergen. Detection with streptavidin-HRP and substrate can then be utilized for a more\naccurate allergen-specific IgE measurement.\nReply: The reviewer is correct in the lower intensity of the signals in the IgE measurements. It\nis a quite common phenomenon, which is mostly based on the generally low IgE levels.\nIn order to avoid interference with IgG binding and in accordance with established protocols,\nwe have used protein G for removal of IgG before performing the assays. This approach is\nalready described in the methods section. In order to underline the point and emphasize the\nremoval of IgG we have reworded the relevant sentences in the methods section on now page\n24.\nPoint by point reply revision 3:\nWe would like to thank reviewer 1 for the comments on important aspects of the manuscript, which we took as\nstimulus to further improve the manuscript.\nAll the comments raised by the reviewer are addressed in detail below.\nReviewer #1 (Remarks to the Author):\nGeneral:\nThe additional data and revisions have improved the manuscript, but there is still the major gap to this work. As the\nauthors note, there is varying capacity to block Api m1 or HBV induced responses. While the concept is interesting\nand the authors have demonstrated that nanobody based antibody formats can bind to an allergenic protein, it is\nprecisely just that: an approach to bind to and block Api m 1, a protein in honey bee venom. If the authors want to\nconsider this as a proof of concept and not a final treatment approach, the language throughout the manuscript\nshould be modified further to reflect this. for example, the abstract says.\n“This work provides a rationale for using nanobody-based inhibitors of Api m 1 for seasonal protection against ana-\nphylaxis in honeybee venom allergy by passive immunotherapy.”\nIt should be modified to reflect the finding that the nanobody based inhibitors of Api m 1 can protect against Api m\n1 and there should be no suggestion that this approach would work for honey bee venom allergy until data show that\nthis approach will sufficiently block and HBV driven response.\nReply: We do in principle agree with the reviewer and the title of our manuscript essentially states that we inhibit the\nallergenic activity of Api m 1, not of HBV. Moreover, we have already earlier stated that this manuscript is a proof-of-\nconcept study and not meant as a final treatment approach. To better reflect this, we carefully went through the man-\nuscript, identified the wording suggesting efficacy for treatment of HBV allergy and modified the following sentences.\nThe sentences from the abstract, the introduction and the discussion are listed below.\nPage 2, line 26-28 reads now: “We aimed at preventing anaphylaxis mediated by the most abundant and dominant\nhoneybee venom allergen phospholipase A2 (Api m 1) by blocking its interaction with allergic patient IgE.”\nPage 2, line 34-36 reads now: “This work provides a rationale for using nanobody-based inhibitors to prevent Api m 1-\nmediated anaphylaxis in honeybee venom allergy.\nPage 5, line 86-88 reads now: In this study, we generated Api m 1-specific nanobodies for interfering with the binding\nof HBV-allergic patient IgE to Api m 1 and assessing a potential reduction of Api m 1-mediated anaphylaxis.\nPage 5, line 90-93 reads now: “The structural and functional data obtained provide insights into the allergenicity of\nvenom allergens as well as a proof-of-concept for using nanobody-based formats for blocking allergen-induced anaphy-\nlaxis in the context of HBV allergy.”\nPage 13, line 277-279 reads now: “In this study, we provide proof-of-concept for using nanobody-based formats to\ninhibit IgE binding to the major HBV allergen Api m 1 and the subsequent activation of effector cells in order to reduce\nthe risk of Api m 1-mediated anaphylaxis.”\nPage 18, line 405-407 reads now: “The nanobody-based inhibitors of both enzymatic and allergenic activity of Api m 1\nprovide a rationale for future development of broadly blocking antibody combinations for seasonal protection against\nanaphylaxis in HVA by passive immunotherapy.”\nFurthermore, we have added a sentence on page 13, line 272-274 stating that “The potential of the bispecific nb-hIgG\n1\nformat to reduce HBV-mediated anaphylaxis needs further investigation.”\nApart from adjusting the wording in the above-mentioned sentences, we have emphasized the potential problems,\nchallenges and perspectives in a new paragraph in the discussion section on page 17-18, lines 384-396. Here we sum-\nmarize points briefly mentioned throughout the manuscript and put them into an overall perspective. The paragraph\nis kept as concise as possible to avoid redundancy and reads now as follows:\n“The results of this study show proof-of-concept for the use of blocking antibodies, but also suggest potential challenges\nand limitations, also mentioned above. Firstly, blocking additional epitopes on Api m 1 might be required for increased\nblocking of effector cell activation for those donors showing limited reduction of activation. Secondly, the antibodies\nwith inhibitory characteristics, such as AM1-1 might have limited blocking capacity. Thirdly, blocking only one allergen\nmight not be sufficient for preventing HBV-mediated anaphylaxis donors with pronounced sensitization to other aller-\ngens than Api m 1. Although Api m 1 apparently is a dominant driver of the effector cell response and anaphylaxis,\nfurther studies are needed to understand and differentiate the impact of other allergens on the development of ana-\nphylaxis. Fourthly, the correlation of reduction of effector cell activation with protection against anaphylaxis remains\nunclear. VIT-treated patients are protected despite having a limited reduction of effector cell activation and other bi-\nomarkers for treatment success are currently not available.\nTo overcome these factors, a dual strategy could be useful. The development of further blocking antibodies against Api\nm 1 and other venom allergens could enable a deeper dissection of the impact of the individual allergens and their\nblocking on the allergic activation. Simultaneously, purified or recombinant allergens might be used to generate a tailor-\nmade “synthetic” venom which would allow for detailed comparison of allergenic activity in absence of selected aller-\ngens and a more reliable use in in vivo experiments.“\nWe do hope that these adjustments are in line with the considerations of the reviewer.\nAdditional major considerations:\nPoint 1: As noted by the authors, the variability in the BAT reflects real life variability. At the same time, this is pre-\ncisely the point, it is important to show two results with this assay:\n1- AM1B-1-4 can significantly block Ap1 m1 activation of human basophils on the majority of donors tested\n2- AM1B1-4 can significantly block HBV activation of human basophils on the majority of donors tested.\nReply: We agree with the reviewer that these results are important.\n1- Therefore, we show in figure 6B that the bispecific nanobody-IgG significantly blocks Api m 1 activation of\n1\nhuman basophils. We have added a sentence in the result section page 11 line 236-238 to emphasize this. The\nsentence now reads: “Notably, the bispecific format performed equally well as the combination of the two\nindividual antibodies, and significantly reduced basophil activation compared to the isotype control (Fig. 6B)”.\n2- Moreover, figure 6C as a matter of fact demonstrates that the bispecific nanobody-IgG significantly reduces\non page 12 line 247-250 to make this even more clear. The sentence now reads: “In three out of four patients\na clear reduction of above 34% was observed with a mean reduction of 35% for all patients. Thus, blocking the\ndominant allergen Api m 1 can reduce the activation of effector cells upon HBV-stimulation”\nIt is however important to recapitulate that the reviewer in the first revision stated that “the experiment should be\nrepeated with HBV as the challenge. Even if it doesn’t work it proves that HBV allergy is more complicated and it would\nbe important to the field to have this information.”.\nWe now reach substantial reduction similar or even superior to the reduction observed in successfully VIT-treated pa-\ntients. Hence, we show in Figure 6C, that the approach does work in the majority of patients tested here, which we\nconsider important information for the field. We do not state that this approach works for all patients.\nPoint 2: For figure 6B-can the authors clarify if this is the quantification of Figure 6A. Figure 6A has 9 curves and Figure\n6B has 7, so it is not clear if they are from the same data set. If they are from the same data set, has one sample been\nomitted?\nReply: We are not fully sure about the comments of the reviewer, this might be a misunderstanding. Correct, figure 6B\nis the quantification of figure 6A. However, both figures do contain data from the same nine patients. We are not aware\nof a figure with 7 curves only, and no data has been omitted, although this would have been two, not one. However, in\norder to facilitate the visual and contextual interpretation of the figure, we have enlarged the figure and reworded the\nfigure legend on page 39 line 863-864. We hope this is what the reviewer had in mind.\nOverall, by eye, it seems there is less than 50% blockade across less than 50% of the donors-please confirm?\nReply: We assume that the reviewer means less than 50% blockade in more than 50% of the donors. As evident from\nthe data presented in the supplement, we have provided earlier, and as we state in the result section page 11 line 231-\n233 we see inhibition in 7 out of 9 patients although to a varying degree. As referenced in the text, the individual inhi-\nbition percentage for the bispecific AM1B-1-4 hIgG can be found in figure S9. Inhibition of >50% is seen in 3 patients.\nyears offers protection against anaphylaxis. There is currently no meaningful cut-off of 50% established in the field.\nIt is, however, established in the field that in tolerant patients after VIT, blocking antibodies only reduce basophil acti-\nvation to a minor extent and with variability. Inversely, the observed partial reduction in inhibition BAT achieved by VIT\nis still indicative for functional protection (as relevant references the following might serve: Arzt et al., Allergy 2018\nPMID: 29171032; Erzen et al., Allergy 2012 PMID: 22469017). This information is discussed in the discussion section\npage 15 line 340-344. Furthermore, we state on page 16 line 349-350 as well as in the newly introduced paragraph\nmentioned above that blocking of additional epitopes might be needed for sufficient blocking in all patients.\nPoint 3: For Figure 6C, it is not clear what curves these data are from. Is the Ap1m1 stimulation from a different set\nof curves from Figure 6A. If the HBV stimulation in Figure 6C is shown, the curves from those experiments must be\nshown so the reader can understand the relationship between HBV and blockade and determine the % blockade\nacross donors.\nReply: The data in figure 6C are from 4 different patients than the patient in figure 6A. We have reworded the figure\ntext on page 39 line 865-866 to make this more clear. These data have been added to the manuscript earlier and the\nreviewer had pointed out earlier that a better representation of the data in the manuscript might be beneficial. Titra-\ntions of Api m 1 and HBV for these four patients can be found in figure S10 together with the inhibition percentage\nacross the four donors.\nPoint 4: In the new S12, the authors do not demonstrate that that AM1B-1-4 blocks an HBV driven response. This\nhighlights he biggest gap of the paper, the data are not convincing that AM1B-1-4 can block HBV driven response. \nPerhaps the following suggestions will help demonstrate whether or not the AMB1-1-4 can block an HBV response.\nThis is one of the most important experiments in the manuscript. and should not be in supplemental\nReply: As the experiments demonstrate a non-allergic, mast cell independent response to the venom, and therefore\nprovide limited additional information on the blocking of IgE-mediated anaphylaxis, we do not see a clear benefit of\nincluding it in the main manuscript. Although we are confident that the observed MCPT-1 reduction is in line with a\nreduced mast cell activation, this information is available from the text and the supplementary data."}, {"round": 2, "reviewer_comments": {"reviewer_1": "It is appreciated that the authors want to emphasize blockade of Api-m 1-induced allergy using nanobody molecules.\nIndeed, the in-vitro data package including structural data, binding kinetics and blocking ELISA nicely demonstrate the\nability of the Api m 1 nanobody molecules to bind non-competitively with high affinity to Api m 1 thus preventing Api m 1-\nspecific IgE:allergen engagement. Additionally, the HBV-induced BAT assays and the mouse model of HBV allergy are nice\nadditions to this manuscript. That said, the authors did not demonstrate that blocking Api m 1 alone is sufficient to block an\nHBV allergic response. Specifically, the authors demonstrate partial blockade of HPV induced activation of human\nbasophils but blockade of Ap1 m 1 induced activation is greater than what was observed with HPV induced activation.\nThese data clearly demonstrate that this approach is not sufficient to block HPV driven IgE response. Similarly, in the\nabsence of a HBV challenge, the mouse model only demonstrates that the Api m 1 nanobody can block a Ap1 m 1 induced\nresponse (no data are shown to show an in vivo effect on an HBV challenge). In the absence data demonstrating that the Api\nm 1 nanobody can block an HPV driven response, the relevance of this approach is not clear.\nThus, major issues remain.\n1. The ex-vivo Api-m 1-driven BAT data remains highly variable and subjective as currently presented.\n• The addition of anti-Fcer1 control values is appreciated but highlights that the stim concentrations of Api m 1 used may not be achieving max activation potential.\no Specifically, in 6 out of the 9 donors, the anti-Fcer1a is at least >25% higher than the isotype control group.\no In 2 out of the 3 donors that achieve “max activation” (H, K amd M), partial (~40%) or no blockade is observed.\n• The addition of untsim control values is also appreciated but demonstrates that the Api m 1-based nanobody IgG1\nmolecules block basophil activation to levels of unstim control in only 1 out of the 9 samples evaluated.\n• As previously suggested, blocking percentages should be calculated for each donor and summary data graphed with\nstatistical analyses run\no Although the authors state a preference to show individual patient responses, quantification of blocking percentages to be\ngraphed alongside individual plot data would more accurately represent the dataset\no As blocking percentages are provided for the newly added HBV-BAT (fig. S10), for consistency blocking percentages\nshould also be provided for the Api m 1-BAT\n2. For this approach to be clinically relevant, the ability of the nanobody molecules to block HBV-induced allergic effector\ncell activation needs to be better supported.\no Blockade of HBV-induced basophil activation data is not robust; Line 251: >34% is not a “large” reduction- it is partial at\nbest\no it would have been nice to follow the same assay format utilized for Api m 1-BAT (dose-dependent treatment vs single-\npoint/fixed molar ratio).\n• Despite sensitization with HBV in the in-vivo model, challenge with Api m 1 allergen is supportive only of the ability to\nblock exposure to Api m 1 and data with an HBV challenge is required to support this approach and demonstrate efficacy\nagainst HBV challenge..\nMinor comments:\n1. Line 61- edit to “all of which aim to restore immune tolerance…”\n2. Line 67- blocking ability of IgA remains controversial/poorly understood: suggest editing to “through the production of\nallergen-specific IgG that block the interaction…”\n3. Figure 3B: AM1-1 binds to catalytic site and inhibits enzymatic activity, but when combined with 1-4 there is a shift to\nshowing less of an effect with the combo. The authors should include text related to the response provided below in the text\nof the manuscript and not just in the reviewer response document\n• Reply: When using the combination the concentration of AM1-1 is halved which can explain the shift. It is also possible that\nthe binding of AM1-4 introduces minor conformational rearrangements that affects the binding of AM1-1. In the end, both\nAM1-1 alone and in combination reaches an inhibition of 50%.\n4. Methods and Figure S11: The HBV- and Api m 1-specific IgE measurements are quite low. This is likely due to\nIgG:allergen binding as allergen-specific IgG is more abundant than IgE. To avoid interference of IgG binding to allergen,\nplates should be coated with an anti-mouse IgE, followed by sample and then a biotinylated HBV extract or Api m 1 allergen.\nDetection with streptavidin-HRP and substrate can then be utilized for a more accurate allergen-specific IgE measurement.", "reviewer_2": "The authors have addressed all my previous questions and comments to my satisfaction. They have provided detailed and\nthoughtful responses to my concerns, demonstrating a high level of scientific rigor and attention to detail. Furthermore, they\nhave included new data that significantly strengthen their findings, including an important animal experiment that\nconvincingly demonstrates the potential therapeutic effect of their drug development. This addition is a major enhancement\nto the manuscript and provides strong support for their conclusions.\nThe work now represents an important contribution to the field, particularly at a time when anti-allergen monoclonal\nantibodies are seeing significant developments. The authors' innovative use of nanobody-based inhibitors of Api m 1 to\nblock IgE binding and effector cell activation, as well as their demonstration of reduced systemic reactions in a mouse\nmodel, highlight the potential of this approach for passive immunotherapy in honeybee venom allergy. The combination of\nstructural insights, high-affinity nanobody binders, and preclinical efficacy data makes this study a noteworthy advancement\nin the field of allergen immunotherapy."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_1": "The additional data and revisions have improved the manuscript, but there is still the major gap to this work. As the authors note, there is varying capacity to block Api m1 or HBV induced responses. While the concept is interesting and the authors have demonstrated that nanobody based antibody formats can bind to an allergenic protein, it is precisely just that: an approach to bind to and block Api m 1, a protein in honey bee venom. If the authors want to consider this as a proof of concept and not a final treatment approach, the language throughout the manuscript should be modified further to reflect this.\nfor example, the abstract says:\n“This work provides a rationale for using nanobody-based inhibitors of Api m 1 for seasonal protection against anaphylaxis in honeybee venom allergy by passive immunotherapy.”\nIt should be modified to reflect the finding that the nanobody based inhibitors of Api m 1 can protect against Api m 1 and there should be no suggestion that this approach would work for honey bee venom allergy until data show that this approach will sufficiently block and HBV driven response.\nAdditional major considerations:\nAs noted by the authors, the variability in the BAT reflects real life variability. At the same time, this is precisely the point, it is important to show two results with this assay:\n1- AM1B-1-4 can significantly block Ap1 m1 activation of human basophils on the majority of donors tested\n2- AM1B1-4 can significantly block HBV activation of human basophils on the majority of donors tested.\nFor figure 6B-can the authors clarify if this is the quantification of Figure 6A. Figure 6A has 9 curves and Figure 6B has 7, so it is not clear if they are from the same data set. If they are from the same data set, has one sample been omitted? Overall, by eye, it seems there is less than 50% blockade across less than 50% of the donors-please confirm?\nFor Figure 6C, it is not clear what curves these data are from. Is the Ap1m1 stimulation from a different set of curves from Figure 6A. If the HBV stimulation in Figure 6C is shown, the curves from those experiments must be shown so the reader can understand the relationship between HBV and blockade and determine the % blockade across donors.\nIn the new S12, the authors do not demonstrate that that AM1B-1-4 blocks an HBV driven response. This highlights he biggest gap of the paper, the data are not convincing that AM1B-1-4 can block HBV driven response. Perhaps the following suggestions will help demonstrate whether or not the AMB1-1-4 can block an HBV response. This is one of the most important experiments in the manuscript. and should not be in supplemental\nThere are two potential follow ups:\n1- Pretreat the mice with anti-Fcg (clone 24G2) to block Fc receptors and prevent any IgG mediated anaphylaxis that is confounding the result. The authors noted in their response that they are concerned about a non-IgE mediated response driving the result. Using this antibody to block Fc Receptors will alleviate this concern.\n2- Consider giving the nanobody a day before (not just 4 hours before HBV challenge)"}, "author_response": "Point-by-point reply to the reviewer comments, Aagaard et al.\nReviewer #1 (Remarks to the Author):\nMajor comments:\nThere are four major limitations to this study:\n1. While the authors demonstrate that the nanobody molecules can block Api m 1 binding to IgE in\nvitro using human IgE sera and basophils, the authors do not demonstrate that these molecules will\nblock HBV. Suggestions around the ability this approach to prevent a reaction beyond Api m 1 and\nwill broadly block HBV must be tempered. It is critical for the approach to show that the api m 1\nnanobody molecules block a HBV driven response\nReply: In this study we focus on the inhibition of Api m 1 responses and the molecular understanding\nof the mode of action. However, we agree with the reviewer that providing evidence for the efficacy\nof reducing a HBV-driven response by blocking Api m 1 is highly relevant. In the revised manuscript\nwe therefore followed different avenues to document these effects. We have:\n1) Tempered the wording as suggested.\n2) Established a mouse model of allergen-induced anaphylaxis to provide evidence for in vivo efficacy\n(see primarily point 2).\n3) Performed new basophil activation tests on HBV-allergic patients with a head-to-head comparison\nof Api m 1 and HBV stimulation.\nBriefly, the new data show a substantial reduction of HBV-induced activation of basophils in BAT upon\nblocking Api m 1. The data obtained in the mouse model reveal pronounced inhibition of anaphylaxis\nby the bispecific nanobody-IgG format.\nFor further details, see the individual points below.\n2. There are no in vivo data demonstrating that the api m 1 nanobody molecules can block a\npolyclonal IgE mediated response using either mouse sera and preferable human IgE (from HBV\nallergic patients). To this point, it is important to demonstrate this in vivo blockade using HBV and\nnot just api m 1 as the challenge.\nReply: As suggested by the reviewers we have established a mouse model of allergen-induced\nanaphylaxis to provide evidence for in vivo efficacy. This model is based on sensitization with HBV and\ntreatment with the bispecific nb-hIgG1. Challenge with Api m 1 reveals the impact of specific blocking\nthe targeted allergen.\nAs evident from these data, the inhibition of anaphylaxis is specific, profound and comparable in all\nanimals. The data also indicate that the in vivo model does not fully replicate the human response\nmirrored in the basophil activation testing. Hence, we addressed the impact of Api m 1 inhibition on\nthe allergic response upon stimulation with HBV in BAT for newly recruited HBV-allergic patients.\nNotably, the Api m 1 inhibition showed a reduction of HBV stimulation in all patients, some of which\nat a comparable level of inhibition for Api m 1. This finding suggests a major contribution of Api m 1\nto the cellular response in some of the patients. The relevance of Api m 1 was observed in patients co-\nsensitized to other HBV allergens, providing for the first time insights into the individual patients´\nreactivity profiles.\nThese new in vivo efficacy and BAT data are now included in the manuscript as novel parts of the\nresults (line 256-268 of the unmarked manuscript), and the supplementary data section including the\nnew figures (Fig. 7, S10, S11, and Table S2). The new data and the implications are discussed\naccordingly in the discussion section (line 362-369 of the unmarked manuscript).\n3. The data using api m 1 challenge of human basophils was not robust with partial blockade and\nthe non-normalized data in the supplemental figures. The non-normalized data should be moved to\nthe main figures to capture the variability across HBV donors and also be demonstrated with HBV\nchallenge.\nReply: As the reviewer suggested, we have moved the non-normalized data to the main text to better\ncapture the variability across the donors.\nFurthermore, as mentioned above, we have added substantial new BAT data addressing the\ncomparison of Api m 1 and HBV. These data have been described in the results (Line 247-254 of the\nunmarked manuscript) and added to the supplementary data of the manuscript as new Fig. S10 and\nTable S2.\n4. Statists should be shown for the data in the figures to validate the conclusions.\nReply: We agree with the reviewer and have included statistics for the data in the figures where\nmeaningful (see also below).\nOther Comments:\nIn general, allergen should be changed to api m 1 throughout the manuscript, since this approach is\nspecific to api m 1 rather than the HBV ‘allergen’ and it will avoid confusion.\nReply: We have addressed this relevant point accordingly throughout the manuscript in order to avoid\nconfusion.\nABSTRACT\n-line 40: please revise to honeybee venom allergic patient SAMPLES as blockade is not assessed in\npatients.\nReply: The reviewer is correct, hence we have adjusted the wording accordingly.\nINTRODUCTION\n-intro line 53: what is the other ~80% of the venom dry weight\nReply: We have added this information in brief (see also next point).\nThe sentence (now line 47) reads now as: “The most abundant component of HBV is the cytolytic\npeptide melittin with 50% of the venom dry weight.”\n-intro line 54-55: the authors report that 97% of HBV-allergic patients are sensitized to Api m 1, but\nit is important to know the sensitization rates of the other Api proteins and not just IgE for Api m 1,\nto help put things into context. That is, what other Api m allergens may be drivers of the allergic\nresponse?\nReply: The reviewer addresses an important aspect, hence we have provided a reference (Köhler et\nal.), in which we have determined the sensitization rates in a larger cohort of patients in detail.\nRoughly similar sensitization rates have been reported for different cohorts of patients. It has to be\nkept in mind, however, that the clinical relevance is not equivalent to the sensitization rate.\nThe text (now in line 50) reads now as: “Most of the other allergens are of low abundance comprising\neach less than 1-2% of venom dry weight, and have reported sensitization rates of >50% (Köhler et\nal)”.\n-intro line 65: It is important to include a comment/reference about other immunotherapy regimens\n(RUSH and ultra-RUSH) that are not as lengthy.\nReply: The reviewer is correct, modified regiments provide a quicker build-up phase of the treatment\nby which the maintenance dose can be reached more rapidly, although the duration of the entire\ntreatment does not change significantly. In order to provide this information, we have added a\nsentence highlighting these options.\nWe have changed the wording (now line 61) as follows:\n“VIT can be performed with different treatment regimens, all of which aiming aims to restore immune\ntolerance through continuous subcutaneous injection of HBV extract over the course of 5 years,\nindependently of the treatment regimen used, or even life-long under certain circumstances …”\n-Intro line 70-83: these paragraphs can be combined. Also the authors comment about the‘one\nmechanism’ is generation of blocking antibodies, but it is really THE mechanism. In this paragraph\nthey do refer to rush IT, but not in the line 65 paragraph.\nReply: We have re-organized and combined the paragraphs. We have also adjusted the wording.\nThe sentence (now line 67) reads now as: “The dominant mechanism by which AIT induces tolerance\nis through the production of allergen-specific IgG1, IgG4 and IgA that block the interaction between\nthe allergen and allergen-specific IgE.”\n-intro line 77: Suggest citing Kamal et al., 2021 Clin Trans Sci reference here with a sentence referring\nto the half-life of monoclonal IgG being 20-30 days and therefore having the potential to provide\nprotection within a well-defined season.\nReply: We agree with the reviewer and have added the information.\nThe sentence (now line 76) reads now as: “In HBV allergy, stings occur unpredictably within a well-\ndefined season, and passive immunotherapy with IgG having a half-life of 20-30 days (Kamal et al.)\ncould provide seasonal protection against severe symptoms including anaphylaxis in case of a sting\nevent.”\n-intro line 93: “allergen” should be changed to Api m 1 because we only see data for this one protein\nand not venom in general. NOTE: this should change throughout the paper.\nReply: We have adjusted the wording accordingly.\nRESULTS\n- line 149: authors should temper language around “potential to reduce its toxicity” as evidence is\nnot provided to show that this active site contributes to “toxicity” or that blocking it reduces\n‘toxicity’\nReply: The reviewer is correct, we do not provide evidence that Api m 1 contributes to toxicity. As\nalready stated in the introduction and discussion section, it is however common understanding that\nApi m 1 with its enzymatic activity together with melittin are the culprits for the toxic effects of the\nvenom.\nIn order to clarify, we have adjusted the wording in the results part. The sentence (now line 142) reads\nnow as: “This inhibition supported the finding that AM1-1 interacts directly with the active site of Api\nm 1 and exhibits the potential to reduce the enzymatic activity of Api m 1 which contributes to the\npain and inflammation of a honeybee sting.”\n-Figure 3B:AM1-1 binds to catalytic site and inhibits enzymatic activity, but when combined with 1-\n4 there is a shift to showing less of an effect with the combo. The authors should discuss why this\nmight be happening\nReply: When using the combination the concentration of AM1-1 is halved which can explain the shift.\nIt is also possible that the binding of AM1-4 introduces minor conformational rearrangements that\naffects the binding of AM1-1. In the end, both AM1-1 alone and in combination reaches an inhibition\nof 50%.\n-line 174-179: this observation is in agreement with structural data, but not SEC which did not\nsupport the formation of Api m 1 dimers- this should be discussed. Also, is the dimer formation\nobserved when bound to IgG1 or IgG4 format?\nReply: We have adjusted the wording.\nThe sentence (now line 156) reads now as: “Although formation of dimers in solution has not been\nreported, Api m 1 might transiently form dimers at locally high concentrations, e.g. at cellular\nsurfaces.”\nThe formation of dimers is further discussed in the discussion section on page 14 (line 309-316) in the\nrevised manuscript.\nWe do not have evidence of dimer formation of Api m 1 bound by IgG formats.\n-line 178: Suggest softening language around “can only be convincingly explained” because maybe\nit is an aggregation issue with the solution and not allergen dimers per se\nReply: We have softened the language accordingly.\nThe sentence (now line 168) reads now as: “… the cellular activation was strictly allergen-dependent,\nand could be explained convincingly by the formation of Api m 1 dimers at the surface.”\nThe cellular activation is only observed with the AM1-4 IgE and not the AM1-1 IgE. This specific and\nquick effect would argue against any aggregation phenomenon.\nLine 209: remove qualifying language around “excellent” agreement\nReply: We reviewer is right, we have removed the qualifying language.\nLine 211: last sentence of the paragraph about increased avidity: this is purely speculative and the\nlanguage could be tempered. One way to definitively show avidity driven results is to compare\nmonomer vs dimer binding? This can also be done with varying concentrations of the allergen if in\nfact it is existing as a dimer in solution\nReply: We appreciate the comment but think that it is based on a misunderstanding. The avidity that\nwe refer to is not caused by Api m 1 dimers. We are referring to the bi-epitopic binding of the bispecific\nantibody to Api m 1. Each Api m 1 molecule is thus bound at two epitopes at once resulting in the\nhigher dissociation constant compared to the monospecific IgG.\nWe have adjusted the wording in order to make this clearer. The sentence (now line 206) reads now\nas: “Notably, the dissociation rate of the bispecific nb-hIgG1 was significantly lower (<10-7 s-1),\nreflecting increased avidity due to simultaneous binding of two Api m 1 epitopes.”\nFigure 5: No statistics in the figure, please add statistics for the figure\nReply: The reviewer is correct. We have added statistics for the figure.\nELISA:\n•Since the plate is coated with allergen (api m 1), it is possible to do the same experiment with HBV\nand prove that these nanobodies are preventing IgE binding to HBV\nReply: The reviewer is correct, that would be possible. However, we see neither the need nor the\nbenefit as we want to demonstrate the nanobodies capacity to inhibit Api m 1. If the plates were\ncoated with HBV, any additional reactivity of serum IgE to other allergens would overlay the inhibition.\n• Line 217-219- description of ELISA assay is confusing here. Per the methods, immobilized Api m 1\nwas not pre-incubated with the nb-hIgG1. Also, it’s more an ability of patient IgE to bind to Api m 1\nvs remaining immunoreactivity of allergen-specific IgE.\nReply: Immobilized Api m 1 is first incubated with nb-hIgG1 and thereafter with patient serum. We\nhave adjusted the wording in order to facilitate understanding.\nThe sentence (now line 213) reads now as: “Immobilized Api m 1 was incubated with the nb-hIgG1\nfollowed by incubation with serum from HBV-allergic patients and the remaining binding of allergen-\nspecific IgE was analyzed.”\nLine 224: the authors captured median’s with the range, but didn’t show any statistics to support\nthe statement that 1-4 is better than 1-1 at inhibition.\nReply: The reviewer is correct. We have added statistics to Fig. 5 and changed the wording in the result\nsection (now lines 219-222) accordingly.\nFigure 6: No statistics shown in the figure, please include statistics\nReply: The figure shows data from individual patients and therefore, we do not think that adding\nstatistics is meaningful.\n• Suppl S9 should be the main figure with the non-normalized data and discuss how the nanobody\nmolecules work across all donors. Figure 6 should be supplement.\nReply: As the reviewer suggested, we have moved the non-normalized data to the main text and the\nnormalized data to the supplement.\n• It is important to the unstim control and also the anti-FceR1 control for each person to capture\nthe thresholds for the assays\nReply: We do not see a clear benefit in providing this information, but we followed the suggestion and\nhave included the data in the new Table S3 and added the reference to the sentence in line 236.\n• The experiment should be repeated with HBV as the challenge. Even if it doesn’t work it proves\nthat HBV allergy is more complicated and it would be important to the field to have this information.\nReply: In order to address the point we have performed new BAT analyses and added the requested\ndata. Please see the more detailed comments and reply above.\n• Suggest calculating blocking percentage across donors, as it will be helpful to see the big picture\nReply: We do not think that this would be meaningful as there is a large variation between the\nindividual patients. We think that it is better to visualize the individual patients’ responses.\nLine 240: “1-4 had a more pronounce reduction than 1-1”: are there stats to support this statement?\nReply: The statement is based on the observation that 1-4 has a stronger inhibitory effect in all\npatients. We do not provide statistics on this observation.\nIn order to clarify we have changed the wording. The sentence (now line 236) reads now as: “For all\npatients, the AM1-4 hIgG1 format resulted in a more pronounced reduction than the AM1-1 hIgG1\nformat.”\nLine 277: “clear potential”-soften this language to reflect the results\nReply: We have adjusted the wording.\nThe sentence (now line 244) reads now as: “These data demonstrate the potential of the nanobody\nformats for blocking effector cell activation by Api m 1 stimulation.”\nDISCUSSION:\n-line 254: half-life of 20-30 days and cite Kamal et al., 2021 as mentioned above\nReply: We have added the information and the suggested reference (line 273-276).\n-line 258: delete “only”\nReply: We have adjusted the wording. The sentence (line 276) reads now as: “Moreover, imperative\nis the reduction of symptom severity and thereby the prevention of potentially life-threatening\nanaphylaxis.”\n-Line 279: delete ‘excellently”\nReply: We have adjusted the phrase (now line 301) accordingly.\n-line284: consider revising this to temper a bit, line 285 delete “excellent”\nReply: We have deleted the sentence.\n-line 300: high affinity and epitope specificity\nReply: The reviewer is correct.\nThe sentence (now line 323) reads now as: “Instead, high affinity and epitope specificity are crucial for\nthe neutralizing effects”\n-line 301: The role of Fcgr2b in IgG mAb blockade remains controversial: to date it has not been\ndefinitively demonstrated that engaging inhibitory receptors add value. Paolucci et al (ref 60)\nsuggests that engagement of Fcgr2b is not a mechanism (along with Mckendry et al., 2021 Pediatr\nAllergy Immunol, and Liao et al., 2019 Clin and Exp Allergy) as opposed to Martin Bachmann research\npapers that suggest a role. In fact early clinical proof of concept has been demonstrated with\ncocktails of antibodies specific to fel d 1 and bet v 1 for cat and birch allergy using antibodies that\ndo not engage inhibitory receptors (Orengo et al, 2018; Atanasio, et al 2022; de Blay, et al, 2022;\nGevaert, et al. 2022)\nReply: We agree with the opinion of the reviewer on the controversy of the engagement of FcgR2B,\nhence we have removed the sentence.\n-Line 304: clarify that their results are specific for api m1 and not HBV\nReply: We have adjusted the wording.\nThe sentence (now line 326) reads now as: “The nb-hIgG1 formats were able to inhibit both the binding\nof patient IgE to Api m 1 and the subsequent basophil activation.”\n-Line 315: clarify that their results are specific for api m1 and not HBV\nReply: We have adjusted the wording.\nThe sentence (now line 326) reads now as: “but the fact that we could observe a clear reduction in Api\nm 1-induced basophil activation supports the concept of an efficient mode of action of the nb-hIgG1\nassessed here.”\n-Line 323: the concept that blocking one allergen might be sufficient has also been demonstrated\nfor cat and birch allergy in people (Orengo et al, 2018; Atanasio, et al 2022; de Blay, et al, 2022;\nGevaert, et al. 2022). The ara h 2 studies, while important are only in mice at this point and have\nnot shown clinical efficacy.\nReply: We agree with the reviewer and have rewritten the text (now line 344-349) to include the cat\nand birch study (Orengo et al, 2018; Atanasio, et al 2022). We also do think that the Ara h 2 study in\nmice, in particular in the context of our new mouse data, is relevant as it demonstrates the potential\nto reduce anaphylaxis.\nIn the discussion it would also be helpful to have a paragraph to help us understand the potential\nbenefits or limitations of a nanobody vs a traditional bivalent antibody. This could be relevant for\nour interpretation of structural data in figure 3. i.e. what is the relevance of a nanobody inducing\ndimer formation?\nReply: The point is truly valid, hence we have expanded the information on benefits of nanobodies on\npage 13 (now lines 278-290) and included i.a. a very recent nanobody-based candidate for treatment\nof asthma.\nAs the nanobody is not inducing dimerization, we do not see the last point mentioned by the reviewer.\nReviewer #2 (Remarks to the Author):\nThe potential use of nanobodies in immunotherapy as well in diagnostics or immunoassays has\nreceived increasing recognition in the latest years. Especially, the COVID-19 pandemic spurn the\ndevelopment of therapeutic nanobodies for use as rescue medication in severe cases. In allergy, the\npotential of nanobodies has followed recent developed of monoclonal antibodies targeting\nallergens. The submitted paper add to these developments by describing nanobodies specific to bee\nvenom allergen Api m 1.\nThe study is well designed and the paper well written. The applied tests for physicochemical\ncharacterisation as well as the characterisation of their binding to its target protein are of the state\nof art and the data of good quality. However, the study would benefit from further biological testing.\nThe pure fact that a nanobody (or monoclonal antibody) can bind target and prevent some of the\nprotein functions in vitro does not necessarily guarantee good and relevant therapeutic function in\nvivo. Similarly, not even measurement of antibodies after immunotherapy enable good predictions\nof therapeutic efficacy. In the current study, this limitation is especially evident when testing the\nnanobodies on patient blood. Out of 9 patient samples, 2 samples were not sensitive to treatment,\nand another 4-5 samples shown only weak sensitivity.\nAdditional biological testing, for instance application in an animal model of anaphylaxis, would have\nprovided important information related to bio-distribution, if the nanobodies are able to block\nallergen in tissues, and on therapeutic potential.\nReply: See also comment of reviewer 1 and the reply above. We agree with the reviewer that providing\nevidence for the efficacy of the molecules in an animal model of anaphylaxis would be highly\nbeneficial. In the revised manuscript we therefore followed different avenues to document these\neffects.\nAs suggested by the reviewers, we have established a mouse model of allergen-induced anaphylaxis\nto provide evidence for in vivo efficacy. This model is based on the sensitization with HBV and\nchallenge with Api m 1 to reveal the impact of specific blocking the targeted allergen. As evident from\nthese data, the inhibition of anaphylaxis is specific, profound and comparable in all animals. Moreover,\nthe data obtained in the mouse model revealed pronounced inhibition of Api m 1-induced anaphylaxis\nby the nanobody-IgG formats.\nThese new in vivo efficacy data are now included in the manuscript as novel parts of the results (line\n256-268 of the unmarked manuscript), and the supplementary data section including the new figures\n(Fig. 7 and S11). The new data and the implications are discussed accordingly in the discussion section\n(line 362-369 of the unmarked manuscript).\nSpecific comments and suggestions.\nPage 2\nL30: two commas\nReply: We have adjusted accordingly.\nL35: Are these different from other know Ig-binding epitopes\nReply: We do not know if the epitopes are different for IgG or IgE as no molecular information on IgG\nand IgE epitopes is available. These aspects are discussed on page 13 of the revised manuscript (lines\n291-300).\nL36: one or more residues?\nReply: In order to facilitate the reading we have adjusted the sentence.\nThe sentence (now line 30) reads now as: “Crystal structures of Api m 1/nanobody complexes revealed\ndiametrically opposed epitopes, one of which involved the active site of Api m 1.”\nL40-41: Unclear meaning. Recognition in term of identifying epitopes or blocking by treatment?\nReply: Valid point. As we have included more data, the abstract has been rewritten and the sentence\nbecame obsolete.\nPage 3\nL55: 80% of patients diagnosed with bee venom allergy show a specific IgE binding reaction to Api\nm 10. It is considered to be the major bee venom allergen, although its content in venom is marginal\n(<1% dry weight). Comment on this.\nReply: A very interesting point raised by the reviewer. According to the majority of studies, Api m 10\nsensitization has a prevalence of 24-75% of allergic patients. In our own study identifying Api m 10 as\nmajor allergen, the sensitization rate was 62%. The sensitization rate is consistently lower than the\none of Api m 1. This distribution is also evident from the patients we have included in this study. We\nhave shown recently that predominant sensitization to Api m 10 might increase the rate of treatment\nfailures and that Api m 10 is lacking in purified therapeutic HBV preparations. However, the clinical\nrelevance of Api m 10 and its potential absence in VIT products is controversially debated and no\nconclusive information is yet available. As the reviewer points out, the abundance of Api m 10 and\nmost other allergens is rather low.\nBasic information on sensitization rates has been added to the introduction (line 50-53; see also points\nabove).\nL65: delete “continuous” as it suggest non-stop and steady state.\nReply: We have adjusted the text accordingly.\nL67: Instead of “high risk of future stings”, rather in patients with mastocytosis, increased baseline\nserum tryptase levels, mast cell activation disorders, and a history of systemic anaphylaxis to stings,\ne.g. ref #17 (Sturm et al.).\nReply: We agree with the reviewer and have adjusted the sentence accordingly.\nThe sentence reads now as: “… or even life-long under certain circumstances including patients with\na severe initial sting reaction or mastocytosis (Sturm et al.).”\nPage 4\nL73 and 80: no section breaks\nReply: We have rearranged the text and removed the section brakes as suggested.\nL89: explain \"extended\" CDR3. Is the geographical shape of CDR3 on nanobodies different from\nthose on antibodies?\nReply: The number of residues within the CDR is often elevated, making the CDR slightly longer as\ncompared to regular antibodies. This also changes the shape of the CDR3. The position of the CDR\nwithin the framework regions is not changed as such.\nTo improve the understanding, we have replaced the term extended by the term longer (now line 84).\nPage 5\nL94: “HBV patients”\nReply: We have adjusted the text accordingly.\nThe sentence (now line 87) reads now as: “In this study, we generated Api m 1-specific nanobodies for\ninterfering with the binding of HBV-allergic patient IgE to Api m 1 and thereby reducing the risk of\nanaphylaxis in HBV-allergic patients.”\nPage 6\nL119: The Table 1 should be placed in the suppl. Data\nReply: Table 1 comprises important information for structural biology readers, hence we prefer to\nkeep the table in the main document.\nL121-122: redundant sentence. Delete.\nReply: The reviewer is fully correct, hence we have shorted the sentences accordingly.\nThe shortened sentence (now line 116) reads now as: “The architecture of the ternary complex\ncorroborates the BLI data”.\nPage 7\nL132-133: With five (AM1-4) versus two (AM1-1) H-bond sites, would you not expect AM1-4 to have\nmuch stronger binding affinity to Api m1? Or is this the 10x stronger binding indicated in Fig. 1B?\nReply: As can be seen in supplement Fig. S2, there are much more H-bonds than the reviewer\nmentions. But the reviewer is also fully correct, the 10-fold higher affinity is shown in Fig. 1B.\nL147: 50%. Why is the activity not totally abrogated?\nReply: This is definitely an interesting aspect, and it is open why we do not see a full inhibition. We\ncan only speculate about the affinity of the interaction and the potential effect of the conformational\nconsequences of AM1-1 binding on the monomeric form of Api m 1, but this is not substantiated by\nany data, so we excluded these speculations.\nL149: What is the clinical importance of this with relation to using AM1-1 in passiv IT?  Discussion\nReply: The enzymatic activity of Api m 1 together with the presence of Api m 4 is known to contribute\nto the pain perception and inflammation of a honeybee sting. In addition, the toxicity of HBV is mostly\nattributed to Api m 1 (see also reply to the comment of reviewer 1). Thus, inhibition of the enzymatic\nactivity will most likely reduce the adverse reactions, even beyond blocking the IgE binding. In case of\nmultiple stings, this effect will be even more important.\nWe have added a sentence in the discussion to emphasize the potential benefit in the clinical setting\n(now lines 304-308).\nPage 8\nL159: replace order of Fig. 3D and 3E (order of their appearance).\nReply: We are grateful for the comment, the order is indeed different. Hence, we have reorganized\nand adjusted the text and the appearance of the figures in the text.\nL163: Are SEC data shown?\nReply: The text relating to the SEC data has been modified.\nThe sentence (lines 156-157) reads now as follows: “Although the formation of dimers in solution has\nnot been reported, Api m 1 might transiently form dimers at locally high concentrations, e.g. at cellular\nsurfaces.”\nL175: Since the two curves overlap, is the result of the combination a result of classical crosslinking\n(AMI1-1 and AMI1-4) or only the dimeric effect of the AMI1-4-binding epitope with AMI1-1 playing\nno role? Comment.\nReply: The reviewer is correct, in this type of assay we cannot discriminate between the two\nexplanations, which is similar to any assay format using a cellular read-out and any effect in basophil\nactivation testing. Hence, we avoid making a clear-cut statement on the molecular basis of cross-\nlinking. Both explanations, however, would be interesting with respect to the molecular features of\nthe allergen.\nPage 9\nL176: You are showing the dose-response of allergen. Rather, or in addition, you should\ndemonstrate the dose-response of the nanobodies (titration). For the experimental condition of\nmax degranulation (fix Api m1 concentration). How much can nanobodies reduce degranulation?\nReply: The point might arise from a misunderstanding. In this assay, which is established in the field,\nthe dose-response curve of the allergen is based on the fact that the nb-hIgE format is bound to the\ncellular surface and the allergen is added in increasing concentrations. The nanobodies used here as\nIgE format do not reduce degranulation, they mediate degranulation.\nL197: Why did the authors chose to use IgG1 and not IgG4? IgG4 is considered more relevant in\nallergen immunotherapy and also the mAbs used in clinical trials (which you referred to earlier) are\nof IgG4 type. Especially in passive immunotherapy, IgG4 is considered much safer than complement-\nactivating IgG1.\nReply: We discuss and address this point already in the discussion section (lines 294-303 in the revised\nmanuscript). In brief, we and others agree that IgG1 corresponds better to the early VIT-induced\neffects. Thus, for mirroring the biological effect of blocking antibodies, IgG1 might be a preferable\nformat. In general, however, our approach is not limited to IgG1. Re-engineering into IgG4-based\nformats is state-of-the-art and even multi-specific, non-Fc-fused formats might become an attractive\napproach.\nL200: The gel in Figure 4B suggests rather 100-120 kDa.\nReply: Yes, the molecule is observed at a slightly higher molecular weight, which is very typical and\nessentially due to the glycosylation of the Fc. Hence, we have adjusted the sentence.\nThe sentence (now lines 192-194) reads now as: “The three nb-hIgG1 formats were expressed in\nmammalian cells, purified and analyzed by SDS-PAGE verifying the expected molecular mass of a\nglycosylated format, suggesting proper folding and dimerization”.\nPage 10\nL202: Why are the two binds from bi-specific nb larger than the individual bands from the mono-\nspecific nb?\nReply: The observation is correct. The minor difference is due to addition of an epitope tag at the C-\nterminus of the chains allowing assessment of heterodimerization. This information has been added\nto the method section in the revised manuscript on page 18.\nL211: impact on “what”?\"\nReply: We have adjusted the sentence.\nThe sentence (now lines 204-206) reads now as: “BLI binding studies of immobilized nb-IgG1 further\nrevealed agreement of the kinetic data with those of the individual nanobodies, confirming that the\nFc fusion has no impact on binding affinity.”\nL212: What is the distance between the two diametrically distant epitopes, and is the bispecific nb\nable to cover that distance? Do you have crystal structures or representations of such binding,\nequivalent to Fig. 2.\nReply: We do interpret the point as a question about the ability of a bispecific antibody to bind\nsimultaneously to the epitopes of one and the same allergen. This is a difficult point, but several lines\nof evidence suggest a simultaneous binding.\nMost importantly, the BLI analysis of the bispecific versus monospecific analysis shows a significant\nincrease of affinity in the set-up where the bispecific antibody is immobilized on the sensor. The\nobtained results suggest that simultaneous binding to the two epitopes is occurring efficiently. Since\nthe nb-IgG1 formats do contain hinge regions of IgG1, molecular flexibility and ability to bridge\ndistances is given. Non-simultaneous binding would be reflected in an average affinity and re-binding\nis negligible, as evident from the monospecific antibodies. This information is available in the results\nsection (now lines 206-209).\nWe cannot fully rule out that stable immune complexes might also be formed in solution by\nintermolecular interaction, resulting in larger linear or circular complexes. However, similar complexes\ncan also be expected for a combination of monospecific antibodies and are also formed during AIT.\nWe do not have structural data supporting either type of interactions.\nPage: 11\nL226: How do author explain the improved effect of bispecific over two monospecific?\nReply: Based on the comments of reviewer 1 we have added statistics to Fig. 5B and do not see a\nstatistical significance for the bispecific compared to the mix of the two monospecific nb-hIgG1.\nNevertheless, a slightly improved effect in our opinion would be a result of increased steric hindrance\nby a bispecific format.\nL242: Can you make a correlation plot between inhibition in ELISA and in basophil activation?\nReply: We are not able to do so, since the samples used for the ELISA and the BAT were not from the\nsame patients. The BATs were performed using EDTA blood samples from patients prior to VIT coming\nin occasionally. The sera for the inhibition ELISA were selected based on a sufficiently high sIgE level\nto Api m 1.\nL245: Include the two patients in Fig 6\nReply: As suggested by reviewer 1 we have moved the non-normalized data to the main text. These\ndata include now the two patients in whom no reduction of basophil activation was observed.\nL248-249: Delete sentence. The patient variance has been known for decades.\nReply: We agree with the reviewer that the patient variance is known, hence we have adjusted the\nsentence.\nThe sentence (now line 245) reads now as: “The data also confirms a large blocking variation, reflecting\ndiverse IgE repertoires of the HBV-allergic patients.”\nPage: 12\nL254: How long do you consider a season to be? How many injections of IgG would that require?\nReply: The honeybee season is very dependent on the geographical distribution, but typically a period\nof 3 month. Given an IgG half-life of 20-30 days, a window of 90 days could be covered with a single\ninjection. In addition, half-life extension strategies like the introduction of mutations increasing the\naffinity to FcRn and other concepts are state of the art, hence we believe that a season could be fully\ncovered with one injection.\nWe have added these considerations to the revised manuscript (see also comments of reviewer 1).\nL365-367: Does this mean that nbs are potentially better than natural abs? By consequence, VIT will\nhave limited efficacy because of the lack of \"prolate shaped and extended CDR3\"?\nReply: In this paragraph we only claim that nanobodies are powerful alternatives. Based on their\ncharacteristics, nanobodies of tailored specificity might even perform better in some settings than\nmost of naturally induced antibodies. There is, however, broad evidence for the existence of canonical\nantibodies with extended CDR3, implying that natural antibodies of appropriate characteristics are\ngenerally capable of having a similar binding mode and thus efficacy. The frequency of occurrence of\nsuch antibodies, however, might be lower, hence a propensity of nanobodies targeting efficiently such\nepitopes is likely to be higher.\nPage 13\nL294-295: What is the expected affinity of specific abs as compared to nbs? As abs are bigger, would\ntheir half-life be longer, hence, better candidates?\nReply: This is an important point with implications on the general applicability. In general, it is\nextensively documented that nanobodies do have similar affinities as regular antibodies. We mention\nthe comparability of the affinities in the discussion section (now lines 324-326).\nLikewise, the half-life of antibodies and engineered nanobodies providing Fc domains can be expected\nto be in the same range. Moreover, a plethora of half-life extension approaches can be pursued,\nmostly similar to the ones applied to IgG formats. As also included in the revised version of the\nmanuscript (line 286), the pentavalent nanobody fusion molecule Lunsekimig exhibits an extended\nhalf-life by including an HSA-targeting nanobody. Thus, nanobody-based formats are not per ser\ninferior to regular formats and can be expected to have similar performance.\nL298: Regeneron and other developers of anti-allergen mAbs focus on IgG4. Comment on this in the\ndiscussion.\nReply: Please see the reply to the comment of reviewer 1.\nThe reviewer is correct, typically IgG4 is chosen for blocking approaches, essentially due to the\nsomewhat erroneous understanding of IgG4 as an AIT-induced subclass. However, we discuss and\naddress this point in the discussion section (lines 294-303 in the revised manuscript). In brief, we and\nothers agree that IgG1 corresponds better to the early VIT-induced effects. Thus, for mirroring the\nbiological effect of blocking antibodies, IgG1 might be a preferable format. In general, however, our\napproach is not limited to IgG1. Re-engineering into IgG4-based\nformats is state-of-the-art and even multi-specific, non-Fc-fused formats might become an attractive\napproach.\nPage 14\nL310-311: Discuss how will the authors deal with this clinical challenge or patient variance?\nReply: The reviewer touches an important aspect. Currently, VIT uses a “one fits all” approach with\nentire venom even for treatment of patients sensitized to potentially only a single allergen.\nIntroducing precision by targeting selected allergens could provide a benefit but comes with the\nlimitation that treatment needs to be adjusted to the individual patient.\nIn the case of HBV allergy, it remains open so far, which allergens and at what concentrations drive or\nadd to the anaphylactic reaction.\nA robust inhibition of the dominant allergen(s) might provide exactly the lever to limit anaphylaxis and\nprovide protection. As stated in the discussion, additional epitopes and additional allergens might be\nneeded to achieve this level of inhibition.\nStratification of patients for the protective effect upon passive immunization might be another option\n(see also point below).\nL323: Discuss if Api m1 may represents such an immunodominant allergen\nReply: This is of course a very important point. As also touched upon in the reply to the previous\ncomment, it is not fully clear which of the venom allergens are the drivers of severe reactions. Api m\n1 is the most abundant major allergen and has a molecular weight which allows for efficient use of\nepitopes in proximity. In comparison, the venom hyaluronidase Api m 2 is typically considered less\nallergenic. Serological data of the patients in this study show a frequent dominant sensitization to Api\nm 1 with higher sIgE levels as compared to the other allergens. However, this does not necessarily\nimply clinical relevance.\nNotably, our novel BAT data included in the results and discussion section provide first insights into\nthe level of basophil activation of individual allergens, here Api m 1, and thus provide initial answers.\nA relatively large inhibition of HBV-stimulated basophils suggest that the response is dominantly\ndriven by the inhibited allergen (Api m 1). This finding implies that the other 12 allergens combined,\nall at lower abundance, have a more limited effect. However, the other allergens do also contribute\nto basophil activation. To what extent these contributions are clinically relevant, remains open. These\ninitial findings need more robust analyses in future studies revealing and differentiating the impact of\nsingle allergens to the clinical picture.\nAs mentioned before, this interpretation is presented in the context of the new BAT data in the revised\nmanuscript.\nPage33: Figure 6: If data are normalized, what does the black line of isotype control mean and why\nis it not a straight line at 100%?\nReply: The data are normalized to the average of the isotype control across all measurements. Thus,\nthe black line shows the deviation of the single measurements from the average. This information has\nbeen added to the figure legend. The normalized data have been moved to the supplement. See also\nthe comments above.\nPoint by point reply:\nWe would like to thank the reviewers for their appreciation of the additional experiments we\nhave included in the revised version of the manuscript and the comments on important aspects\nof the manuscript, which we took as stimulus to further improve the manuscript.\nAll the comments raised by the reviewer were addressed in detail below.\nOne general aspect mentioned is the varying capacity of Api m 1-specific nanobodies to inhibit\nan Api m 1- or HBV-induced allergic reaction. In relation to this aspect and the comments of\nreviewer 1, we would like to emphasize that our manuscript is a proof-of-concept and feasibility\nstudy for the use of nanobody-based antibody formats and the concept described should\ntherefore not be considered a final treatment approach for patients with honeybee venom\nallergy.\nPlease find the detailed response below.\nReviewer #1 (Remarks to the Author):\nPoint 1. The ex-vivo Api-m 1-driven BAT data remains highly variable and\nsubjective as currently presented.\n• The addition of anti-Fcer1 control values is appreciated but highlights that the stim\nconcentrations of Api m 1 used may not be achieving max activation potential.\no Specifically, in 6 out of the 9 donors, the anti-Fcer1a is at least >25% higher than\nthe isotype control group.\no In 2 out of the 3 donors that achieve “max activation” (H, K amd M), partial\n(~40%) or no blockade is observed.\n• The addition of untsim control values is also appreciated but demonstrates that the\nApi m 1-based nanobody IgG1 molecules block basophil activation to levels of unstim\ncontrol in only 1 out of the 9 samples evaluated.\nReply: We agree with the reviewer that the data on ex vivo Api m 1-specific BAT shows high\nvariability, which in our opinion reflects the situation in patients with honeybee venom allergy.\nThis means that activation of basophils is dependent on several factors like e.g. the epitope\ndiversity and cell surface density of IgE, the varying affinity of IgE for the different allergen\nepitopes, the complexity and valency of the allergen as well as intrinsic levels of Syk\nexpression. Consequently, we do not expect a single allergen to cause as high activation as\na stimulation control for basophils such as anti-FcεR1. Moreover, the differences in the ability\nof the nb-hIgG1 formats to inhibit IgE binding of the three donors showing almost identical\nactivation by anti-FcεR1 and Api m 1 (patients H, K and M) indicate that blockade depends\nlargely on the individual composition of the targeted epitopes, which emphasizes the\ndependency of both basophil activation and blockade on the above-mentioned factors.\nIn addition, and more in general we would like to emphasize that successful treatment of HBV\nallergy by VIT over a course of several years offers protection against anaphylaxis. It is,\nhowever, well established in the field that in tolerant patients after VIT, blocking antibodies only\nreduce basophil activation to a minor extent and with variability. Inversely, the observed partial\nreduction in inhibition BAT achieved by VIT is still indicative for functional protection (as\nrelevant references the following might serve: Arzt et al., Allergy 2018 PMID: 29171032; Erzen\net al., Allergy 2012 PMID: 22469017).\nThis information was already briefly mentioned in the discussion section on page 15, now\nlines 335-340, but we have reworded and extended the sentences to facilitate the\ninterpretation of the data. We hope that this explanation makes our results more clear.\nIn this context, we are convinced that the BAT inhibition we do observe corresponds fully to\nthe picture observed for VIT treated allergic patients.\n• As previously suggested, blocking percentages should be calculated for each donor\nand summary data graphed with statistical analyses run\no Although the authors state a preference to show individual patient responses,\nquantification of blocking percentages to be graphed alongside individual plot\ndata would more accurately represent the dataset\no As blocking percentages are provided for the newly added HBV-BAT (fig. S10),\nfor consistency blocking percentages should also be provided for the Api m 1-\nBAT\nReply: As suggested by the reviewer, we have added graphs displaying the individual and\nsummarized blocking effect and we do agree that the information is actually explanatory and\nbeneficial for the reader.\nThe paired graph is now included as figure 6B highlighting the inhibition by the bispecific nb-\nhIgG1. The summary of data for the bispecific nb-hIgG1 and the blocking percentages are\nshown in figure S9, B and C. For both graphs, statistical analysis is added. The figures are\nreferenced accordingly in the text of the results section on page 11, line 240.\nPoint 2. For this approach to be clinically relevant, the ability of the nanobody\nmolecules to block HBV-induced allergic effector cell activation needs to be\nbetter supported.\no Blockade of HBV-induced basophil activation data is not robust; Line 251: >34% is\nnot a “large” reduction- it is partial at best\nReply: We have reworded the phrase accordingly. In addition, we have modified the figure 6\nand figure S10 by showing a graph that displays the individual blocking effect upon stimulation\nwith Api m 1 and HBV. The graph is implemented as figure 6C and referenced accordingly in\nthe text of the results section on page 12, line 244-246.\no it would have been nice to follow the same assay format utilized for Api m 1-BAT\n(dose-dependent treatment vs single-point/fixed molar ratio).\nReply: We do understand the point of the reviewer. However, the data in Fig. 6 and in Fig. 10\nclearly suggest that differences in inhibition between Api m 1 and HBV are rather limited and\nnot expected. Hence, we think that the approach taken in Fig. S10 is sufficient and we do not\nexpect a significant gain of additional insights from another assay format.\n• Despite sensitization with HBV in the in-vivo model, challenge with Api m 1 allergen\nis supportive only of the ability to block exposure to Api m 1 and data with an HBV\nchallenge is required to support this approach and demonstrate efficacy against HBV\nchallenge.\nReply: The reviewer is correct; the challenge with Api m 1 demonstrates the efficacy of the\napproach for blocking allergen-induced cellular activation and anaphylaxis for Api m 1 only.\nThis has been the clear focus of this proof-of-concept study.\nObviously, HBV contains additional allergens, but also lower molecular weight compounds\nwith a variety of functional activities. Thus, we do agree that using HBV for the challenge might\nprovide more detailed support of the approach.\nHence, we have performed a new set of experiments using HBV for both sensitization and\nchallenge of sensitized mice. These experiments show a reduction of serum MCPT-1 levels in\ntreated animals as compared to non-treated controls. This reduction, although not significant,\nshows a mean value of MCPT-1 levels, which is 38% lower as compared to the non-treated\ncontrol. This is a lower effect as compared to the BAT data, which shows significantly reduced\nactivation by HBV, but the sensitization with HBV might also be broader than observed in\npatients.\nHowever, the challenge with HBV at the concentrations used in this experiment was\naccompanied by a clear-cut body temperature drop even in non-sensitized mice. The absence\nof mast cell activation as documented by lack of MCPT-1 induction in non-sensitized mice\nindicates a response beyond an IgE-mediated allergic reaction which is most likely caused by\nthe toxicity of the venom.\nAlthough the observed toxic effect might limit the benefit of the entire data obtained, we think\nthat the information is important and supports our concept and thus is important to show.\nTherefore, these data are now included in the supplementary data as novel figure S12 and\nS13 and as text in the results and discussion section on page 12, line 263-267 and 17, line\n269-273.\nMinor comments:\n1. Line 61- edit to “all of which aim to restore immune tolerance…”\nReply: We have corrected the phrase on now page 3 accordingly.\n2. Line 67- blocking ability of IgA remains controversial/poorly understood: suggest\nediting to “through the production of allergen-specific IgG that block the interaction…”\nReply: The reviewer is correct with respect to the blocking ability of IgA. We have reworded\nthe sentence on now page 4 accordingly.\n3. Figure 3B: AM1-1 binds to catalytic site and inhibits enzymatic activity, but when\ncombined with 1-4 there is a shift to showing less of an effect with the combo. The\nauthors should include text related to the response provided below in the text of the\nmanuscript and not just in the reviewer response document\n• Reply: When using the combination the concentration of AM1-1 is halved which can\nexplain the shift. It is also possible that the binding of AM1-4 introduces minor\nconformational rearrangements that affects the binding of AM1-1. In the end, both AM1-\n1 alone and in combination reaches an inhibition of 50%.\nReply: We do agree with the suggestion of the reviewer, hence we have introduced a short\nstatement on now page 7.\n4. Methods and Figure S11: The HBV- and Api m 1-specific IgE measurements are quite\nlow. This is likely due to IgG:allergen binding as allergen-specific IgG is more abundant\nthan IgE. To avoid interference of IgG binding to allergen, plates should be coated with\nan anti-mouse IgE, followed by sample and then a biotinylated HBV extract or Api m 1\nallergen. Detection with streptavidin-HRP and substrate can then be utilized for a more\naccurate allergen-specific IgE measurement.\nReply: The reviewer is correct in the lower intensity of the signals in the IgE measurements. It\nis a quite common phenomenon, which is mostly based on the generally low IgE levels.\nIn order to avoid interference with IgG binding and in accordance with established protocols,\nwe have used protein G for removal of IgG before performing the assays. This approach is\nalready described in the methods section. In order to underline the point and emphasize the\nremoval of IgG we have reworded the relevant sentences in the methods section on now page\n24.\nPoint by point reply revision 3:\nWe would like to thank reviewer 1 for the comments on important aspects of the manuscript, which we took as\nstimulus to further improve the manuscript.\nAll the comments raised by the reviewer are addressed in detail below.\nReviewer #1 (Remarks to the Author):\nGeneral:\nThe additional data and revisions have improved the manuscript, but there is still the major gap to this work. As the\nauthors note, there is varying capacity to block Api m1 or HBV induced responses. While the concept is interesting\nand the authors have demonstrated that nanobody based antibody formats can bind to an allergenic protein, it is\nprecisely just that: an approach to bind to and block Api m 1, a protein in honey bee venom. If the authors want to\nconsider this as a proof of concept and not a final treatment approach, the language throughout the manuscript\nshould be modified further to reflect this. for example, the abstract says.\n“This work provides a rationale for using nanobody-based inhibitors of Api m 1 for seasonal protection against ana-\nphylaxis in honeybee venom allergy by passive immunotherapy.”\nIt should be modified to reflect the finding that the nanobody based inhibitors of Api m 1 can protect against Api m\n1 and there should be no suggestion that this approach would work for honey bee venom allergy until data show that\nthis approach will sufficiently block and HBV driven response.\nReply: We do in principle agree with the reviewer and the title of our manuscript essentially states that we inhibit the\nallergenic activity of Api m 1, not of HBV. Moreover, we have already earlier stated that this manuscript is a proof-of-\nconcept study and not meant as a final treatment approach. To better reflect this, we carefully went through the man-\nuscript, identified the wording suggesting efficacy for treatment of HBV allergy and modified the following sentences.\nThe sentences from the abstract, the introduction and the discussion are listed below.\nPage 2, line 26-28 reads now: “We aimed at preventing anaphylaxis mediated by the most abundant and dominant\nhoneybee venom allergen phospholipase A2 (Api m 1) by blocking its interaction with allergic patient IgE.”\nPage 2, line 34-36 reads now: “This work provides a rationale for using nanobody-based inhibitors to prevent Api m 1-\nmediated anaphylaxis in honeybee venom allergy.\nPage 5, line 86-88 reads now: In this study, we generated Api m 1-specific nanobodies for interfering with the binding\nof HBV-allergic patient IgE to Api m 1 and assessing a potential reduction of Api m 1-mediated anaphylaxis.\nPage 5, line 90-93 reads now: “The structural and functional data obtained provide insights into the allergenicity of\nvenom allergens as well as a proof-of-concept for using nanobody-based formats for blocking allergen-induced anaphy-\nlaxis in the context of HBV allergy.”\nPage 13, line 277-279 reads now: “In this study, we provide proof-of-concept for using nanobody-based formats to\ninhibit IgE binding to the major HBV allergen Api m 1 and the subsequent activation of effector cells in order to reduce\nthe risk of Api m 1-mediated anaphylaxis.”\nPage 18, line 405-407 reads now: “The nanobody-based inhibitors of both enzymatic and allergenic activity of Api m 1\nprovide a rationale for future development of broadly blocking antibody combinations for seasonal protection against\nanaphylaxis in HVA by passive immunotherapy.”\nFurthermore, we have added a sentence on page 13, line 272-274 stating that “The potential of the bispecific nb-hIgG\n1\nformat to reduce HBV-mediated anaphylaxis needs further investigation.”\nApart from adjusting the wording in the above-mentioned sentences, we have emphasized the potential problems,\nchallenges and perspectives in a new paragraph in the discussion section on page 17-18, lines 384-396. Here we sum-\nmarize points briefly mentioned throughout the manuscript and put them into an overall perspective. The paragraph\nis kept as concise as possible to avoid redundancy and reads now as follows:\n“The results of this study show proof-of-concept for the use of blocking antibodies, but also suggest potential challenges\nand limitations, also mentioned above. Firstly, blocking additional epitopes on Api m 1 might be required for increased\nblocking of effector cell activation for those donors showing limited reduction of activation. Secondly, the antibodies\nwith inhibitory characteristics, such as AM1-1 might have limited blocking capacity. Thirdly, blocking only one allergen\nmight not be sufficient for preventing HBV-mediated anaphylaxis donors with pronounced sensitization to other aller-\ngens than Api m 1. Although Api m 1 apparently is a dominant driver of the effector cell response and anaphylaxis,\nfurther studies are needed to understand and differentiate the impact of other allergens on the development of ana-\nphylaxis. Fourthly, the correlation of reduction of effector cell activation with protection against anaphylaxis remains\nunclear. VIT-treated patients are protected despite having a limited reduction of effector cell activation and other bi-\nomarkers for treatment success are currently not available.\nTo overcome these factors, a dual strategy could be useful. The development of further blocking antibodies against Api\nm 1 and other venom allergens could enable a deeper dissection of the impact of the individual allergens and their\nblocking on the allergic activation. Simultaneously, purified or recombinant allergens might be used to generate a tailor-\nmade “synthetic” venom which would allow for detailed comparison of allergenic activity in absence of selected aller-\ngens and a more reliable use in in vivo experiments.“\nWe do hope that these adjustments are in line with the considerations of the reviewer.\nAdditional major considerations:\nPoint 1: As noted by the authors, the variability in the BAT reflects real life variability. At the same time, this is pre-\ncisely the point, it is important to show two results with this assay:\n1- AM1B-1-4 can significantly block Ap1 m1 activation of human basophils on the majority of donors tested\n2- AM1B1-4 can significantly block HBV activation of human basophils on the majority of donors tested.\nReply: We agree with the reviewer that these results are important.\n1- Therefore, we show in figure 6B that the bispecific nanobody-IgG significantly blocks Api m 1 activation of\n1\nhuman basophils. We have added a sentence in the result section page 11 line 236-238 to emphasize this. The\nsentence now reads: “Notably, the bispecific format performed equally well as the combination of the two\nindividual antibodies, and significantly reduced basophil activation compared to the isotype control (Fig. 6B)”.\n2- Moreover, figure 6C as a matter of fact demonstrates that the bispecific nanobody-IgG significantly reduces\non page 12 line 247-250 to make this even more clear. The sentence now reads: “In three out of four patients\na clear reduction of above 34% was observed with a mean reduction of 35% for all patients. Thus, blocking the\ndominant allergen Api m 1 can reduce the activation of effector cells upon HBV-stimulation”\nIt is however important to recapitulate that the reviewer in the first revision stated that “the experiment should be\nrepeated with HBV as the challenge. Even if it doesn’t work it proves that HBV allergy is more complicated and it would\nbe important to the field to have this information.”.\nWe now reach substantial reduction similar or even superior to the reduction observed in successfully VIT-treated pa-\ntients. Hence, we show in Figure 6C, that the approach does work in the majority of patients tested here, which we\nconsider important information for the field. We do not state that this approach works for all patients.\nPoint 2: For figure 6B-can the authors clarify if this is the quantification of Figure 6A. Figure 6A has 9 curves and Figure\n6B has 7, so it is not clear if they are from the same data set. If they are from the same data set, has one sample been\nomitted?\nReply: We are not fully sure about the comments of the reviewer, this might be a misunderstanding. Correct, figure 6B\nis the quantification of figure 6A. However, both figures do contain data from the same nine patients. We are not aware\nof a figure with 7 curves only, and no data has been omitted, although this would have been two, not one. However, in\norder to facilitate the visual and contextual interpretation of the figure, we have enlarged the figure and reworded the\nfigure legend on page 39 line 863-864. We hope this is what the reviewer had in mind.\nOverall, by eye, it seems there is less than 50% blockade across less than 50% of the donors-please confirm?\nReply: We assume that the reviewer means less than 50% blockade in more than 50% of the donors. As evident from\nthe data presented in the supplement, we have provided earlier, and as we state in the result section page 11 line 231-\n233 we see inhibition in 7 out of 9 patients although to a varying degree. As referenced in the text, the individual inhi-\nbition percentage for the bispecific AM1B-1-4 hIgG can be found in figure S9. Inhibition of >50% is seen in 3 patients.\nyears offers protection against anaphylaxis. There is currently no meaningful cut-off of 50% established in the field.\nIt is, however, established in the field that in tolerant patients after VIT, blocking antibodies only reduce basophil acti-\nvation to a minor extent and with variability. Inversely, the observed partial reduction in inhibition BAT achieved by VIT\nis still indicative for functional protection (as relevant references the following might serve: Arzt et al., Allergy 2018\nPMID: 29171032; Erzen et al., Allergy 2012 PMID: 22469017). This information is discussed in the discussion section\npage 15 line 340-344. Furthermore, we state on page 16 line 349-350 as well as in the newly introduced paragraph\nmentioned above that blocking of additional epitopes might be needed for sufficient blocking in all patients.\nPoint 3: For Figure 6C, it is not clear what curves these data are from. Is the Ap1m1 stimulation from a different set\nof curves from Figure 6A. If the HBV stimulation in Figure 6C is shown, the curves from those experiments must be\nshown so the reader can understand the relationship between HBV and blockade and determine the % blockade\nacross donors.\nReply: The data in figure 6C are from 4 different patients than the patient in figure 6A. We have reworded the figure\ntext on page 39 line 865-866 to make this more clear. These data have been added to the manuscript earlier and the\nreviewer had pointed out earlier that a better representation of the data in the manuscript might be beneficial. Titra-\ntions of Api m 1 and HBV for these four patients can be found in figure S10 together with the inhibition percentage\nacross the four donors.\nPoint 4: In the new S12, the authors do not demonstrate that that AM1B-1-4 blocks an HBV driven response. This\nhighlights he biggest gap of the paper, the data are not convincing that AM1B-1-4 can block HBV driven response. \nPerhaps the following suggestions will help demonstrate whether or not the AMB1-1-4 can block an HBV response.\nThis is one of the most important experiments in the manuscript. and should not be in supplemental\nReply: As the experiments demonstrate a non-allergic, mast cell independent response to the venom, and therefore\nprovide limited additional information on the blocking of IgE-mediated anaphylaxis, we do not see a clear benefit of\nincluding it in the main manuscript. Although we are confident that the observed MCPT-1 reduction is in line with a\nreduced mast cell activation, this information is available from the text and the supplementary data."}]

Decision letter