Natural and recombinant Fel d 1

Both natural Fel d 1 (nFel d 1; Indoor Biotech, Catalog #LTN-FD-1) and recombinant Fel d 1 (rFel d 1) were used in in vitro assays. Recombinant Fel d 1 was produced following the design of Kaiser et al.65 who showed that single-chain fusions were structurally and functionally equivalent to the natural Fel d 1 heterodimer. The Regeneron-produced recombinant proteins include amino acids 18–109 of Fel d 1 Chain 2 (NP_001041619.1) at the N-terminus fused directly in-line to amino acids 23–92 of Fel d 1 Chain 1 (NP_001041618.1) with a D27G mutation and a C-term myc–myc–hexahistidine (mmh) tag. The proteins were produced in Chinese Hamster Ovary (CHO) cells with either a monomeric (mmh) or a dimeric ((mouse IgG2a Fc (mFc)) C-terminal tag (rFel d 1.mmh and rFel d 1.mFc, respectively). In addition, FcεR1α (the high affinity receptor for IgE) extracellular domain protein was produced as a dimeric Fc fusion with a C-terminal mouse Fc tag (hFcεR1α.mFc) to support development of the ELISA-based competition assay.

Generation of REGN1908 and REGN1909

REGN1908 and REGN1909 are fully human monoclonal antibodies to Fel d 1 produced with IgG4P isotype Fc domains. The IgG4 constant domain contains a serine to proline amino acid substitution (S228P, EU numbering) in the hinge region that reconstructs the human IgG1 hinge sequence (CPPC) to promote stabilization of disulfide bonds between the two heavy chains19, therefore designated IgG4P. Briefly, VelocImmune mice17,18 were immunized with recombinant dimeric Fel d 1 (rFeld1.mFc). Hybridomas producing REGN1908 and REGN1909 were isolated and the variable regions were cloned onto plasmid vectors containing kappa light chain constant regions and hIgG4P heavy chain constant regions, respectively. The selected antibodies were produced in Regeneron’s proprietary cell line cloned from CHO cells. CHO-K1 cells were authenticated by isozyme testing according to ICH guidelines and have been tested for mycoplasma contamination. Lead antibodies were selected based on affinity and the ability to bind non-competitively to Fel d 1.

Isolation and concentration of IgG from patient sera

Serum from patients with cat allergy who underwent physician determined successful immunotherapy (Cat-SIT) and cat-allergic control patients (Non-SIT) was purchased from Dr. Jonathan Corren. Uses of human material were approved by Schulman Associates IRB. Cat allergy was physician diagnosed by allergy skin prick test and clinical symptoms. All patients with cat allergy were polyallergic. Immunotherapy was performed using cat extract diluted with human serum albumin in the absence of adjuvants with treatment success determined by the physician. Patient serum was separated from donor blood (purchased from Dr. Jonathan Corren) by centrifugation at 3000 rpm, passed through 0.22 μm filter, incubated overnight with 50 mL of Protein G sepharose beads at 4 °C, then poured into column and SIT IgG and eluted with Pierce IgG elution buffer. After neutralization of the eluate with 1 M Tris, pH 8.5, samples were dialyzed against PBS, pH 7.2. All samples were concentrated approximately 10-fold using an Amicon Ultracel with a 50 k MW cut off.

Quantitation of IgG from patient sera

Total and Fel d 1-specific IgG levels were quantitated in sera samples from human patients undergoing SIT therapy using a standard ELISA. Ninety-six-well microtiter plates (Thermo Scientific) were coated with 2 µg/mL of either natural Fel d 1 (LoTox Indoor Biotechnologies) or anti-human IgG (Jackson Immunoresearch) in phosphate-buffered saline (PBS, Irvine Scientific) overnight at 4 °C. The next day, plates were washed with PBS containing 0.05% Tween 20 (PBS-T, Sigma-Aldrich) four times using a plate washer (Molecular Devices). Plates were then blocked by incubation for 1 h at room temperature (RT) with 250 µL of 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. Sera or purified IgG from human patients undergoing SIT were serially diluted threefold in 0.5% BSA-PBS starting at 1:1000 (for Fel d 1 specific IgG) and 1:27,000 (for total IgG), added to the blocked plates in duplicate, and then incubated for 1 h at RT. The last two wells were left blank to be used as a secondary antibody alone control (background control). For total IgG quantitation, a standard curve was generated using human IgG (Thermo Scientific, # 31,154) starting at 1 µg/mL and diluted 3-fold across the plate. For Fel d 1-specific IgG quantitation, a standard curve was generated using an anti-Fel d 1 monoclonal antibody (H4H1238N) also starting at 1 µg/mL and diluted 3-fold across the plate. Goat anti-human IgG-Fc-Horse Radish Peroxidase (HRP) conjugated secondary antibody (Jackson Immunoresearch) was then added to the plates at 1:5000 dilution and incubated for 1 h at RT. Plates were washed with PBS-T in between each step of the protocol. To develop the colorimetric reaction, TMB/H 2 O 2 substrate was added to the plates and incubated for 20 min. The reaction was stopped using 2 N sulfuric acid (H 2 SO 4 , VWR). Absorbance was subsequently measured on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. Total and Fel d 1 specific IgG were computed from the respective standard curve plots using the Graphpad PRISM software.

Cat-SIT IgG binding studies

Cat-SIT-IgG binding studies were performed on Octet HTX biosensor and all the samples were prepared in a buffer containing 10 mM HEPES, 500 mM NaCl, 1 mg/mL BSA, 0.02% sodium azide, and 0.05% surfactant Tween-20 with pH 7.4 (HBS-BT). HIS1K Octet biosensors were first dipped in wells containing 2 µg/mL of rFel d 1.mmh for 60 s to capture 0.05 nm of rFel d 1.mmh followed by dipping Octet biosensors in wells containing 100 nM of concentrated Fel d 1-specific IgG present in different Protein G-purified Cat-SIT IgG samples. The specific binding sensorgrams were generated by a double referencing procedure by subtracting any interaction of Cat-SIT IgG over the reference surface (blank HIS1K Octet biosensor) from the Cat-SIT IgG binding to the Fel d 1.mmh captured surface; thereby removing any observed non-specific binding signal. In addition, rFel d 1.mmh captured biosensors were dipped in HBS-BT buffer to allow subtraction of signal changes resulting from the natural dissociation of captured rFel d 1.mmh from the HIS1K biosensor. Octet binding data were double reference subtracted and binding kinetic parameters were measured by fitting the data to a 1:1 binding model with mass transport limitation using Scrubber 2.0c.

SPR binding studies

Binding kinetics studies for REGN1908 and REGN1909 were performed on Biacore T200 using 10 mM HEPES, 150 mM NaCl, 3 mM MgCl 2 + 3 mM CaCl 2 , 0.05% (v/v) surfactant P20, pH 7.4 (HBSP+ +) as running buffer at a flow rate of 50 μL/min. Around 94–163 RU of REGN1908 or REGN1909 was first captured on different flow cells using the goat anti-human Fcγ coupled sensor surface. Different concentrations of nFel d 1 or rFel d 1.mmh, serially diluted by 2-fold in HBSP+ + buffer were later injected over the antibody captured surfaces for 2.5 min followed by 15 min dissociation phase. Specific Biacore kinetic sensorgrams were obtained by a double referencing procedure by first subtracting any interaction of nFel d 1 or rFel d 1.mmh over the reference surface (goat anti-human Fcγ coupled surface only) from the nFel d 1 or rFel d 1.mmh binding signal to REGN1908 or REGN1909 captured surfaces; thereby removing any refractive index changes. In addition, injections of HBSP+ + buffer were performed to allow subtraction of RU signal changes resulting from the natural dissociation of captured REGN1908 or REGN1909 from the goat anti-human Fcγ coupled surface. The binding kinetic parameters were obtained by globally fitting the double reference subtracted data to a 1:1 binding model with mass transport limitation using the Biacore T200 Evaluation Software, version 1.0.

Sequential binding competition studies were performed on Biacore T200 by immobilizing 4682–6683 RU of REGN1908 and REGN1909 over separate flow cells of a CM5 sensor chip using standard amine-coupling chemistry reported earlier. Freshly prepared and degassed HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20), pH7.4 was used as running buffer during the entire coupling steps. The coupled chip surface was then washed and treated with 10 mM glycine-HCl, pH 1.5 to remove uncoupled residual proteins. 200 nM nFel d 1 was first injected over the sensor surface coupled with REGN1908 or REGN1909 for 10 min followed by individual injections of REGN1908 and REGN1909 (25 μg/mL). The entire experiment was performed using HBS-P+ + running buffer at a flow rate of 10 μL/min.

Epitope determination by hydrogen/deuterium exchange

In order to determine the epitopes of Fel d 1 recognized by REGN1908 and REGN1909, HDX studies were performed for each antibody co-complexed with rFel d 1.mmh. Amide protons on recombinant Fel d 1 (rFel d 1.mmh) were first exchanged in D 2 O, then the deuterated rFel d 1.mmh was complexed with either REGN1908 or REGN1909 prior to the back-exchange in H 2 O. The control experiment is that the complexed rFel d 1.mmh with either antibody was deuterated in D 2 O and back-exchanged in H 2 O. The solution was then quenched in cold (4 °C) acidic (pH 2.5) aqueous solution to minimize back-exchange and subjected to proteolysis and mass spectrometry analysis. Fel d 1-derived peptic peptides that exhibited increased mass (very likely due to retained deuterons from the antibody protection) greater than 0.2 mass units relative to control experiment were defined as the binding epitopes based on H/DX methodology.

REGN1909 binding to Fel d 1 by X-ray crystallography

A Fab fragment of REGN1909 was cloned and expressed in HEK293 cells, then purified by Protein A affinity chromatography. Recombinant Fel d 1 (chain 2(N50A)–chain 1(G31D), with a C-terminal myc–myc–His6 affinity tag) was purified as described above. rFel d 1.mmh and REGN1909 Fab were mixed in approximately 1:1 molar ratio, incubated at RT for 1 h, then the complex was separated from a slight excess of Fab by size exclusion chromatography. rFel d 1–Fab complex was concentrated to 20 mg/ml in HEPES-buffered saline, then crystallized against a reservoir solution of 0.2 M calcium acetate, 0.1 M sodium cacodylate pH 6.5, 40% (v/v) polyethylene glycol 300. Crystals were harvested directly from the mother liquor and frozen in liquid nitrogen.

Diffraction data to 2.9 Å were collected on a rFel d 1-Fab crystal at beamline 5.0.2 of the Advanced Light Source, Lawrence Berkeley National Laboratory. The structure was solved by molecular replacement using Phaser66, with PDB code 1PUO65 as the search model for the Fel d 1 component, and PDB code 2R8S67 for the Fab component. The structure was refined using refmac5, and rebuilt using coot68. Coordinates for the final rFel d 1-Fab structure have been deposited in the RCSB Protein Data Bank with accession code 5VYF. See Supplementary Table 9 for data and refinement statistics.

Allergen-specific blocking ELISA

The ability of anti-Fel d 1 monoclonal antibodies or purified IgG from SIT patient serum to block Fel d 1 binding to plate-captured IgE from allergic human donor plasma/sera was determined using a blocking ELISA. Microtiter plates were coated overnight at 4 °C with human FcεR1α (the high affinity receptor for IgE) extracellular domain protein with a C-terminal mouse Fc tag (hFcεR1a.mFc). Plates were blocked with 0.5% BSA (w/v) for 1 h at RT. Plasma from allergic donors was diluted 5-fold and total IgE was captured over the receptor-coated surface. A constant amount of recombinant Fel d 1.mmh (0.7 nM) was pre-mixed with serial dilutions of anti-Fel d 1 monoclonal antibodies and Fel d 1 specific SIT IgG starting from 10 µg/ml each in 3-fold serial dilution and incubated for 1 h at RT to allow Fel d 1–antibody interaction to reach equilibrium. The antibody–Fel d 1 mixture was then added to the IgE-coated plate for 1 h. Plates were subsequently washed and the amount of free Fel d 1.mmh bound to plate was detected using an anti-myc antibody (clone 9E10 produced in-house as a human IgG1 isotype) conjugated to HRP, and incubated at a 1:10,000 dilution, by incubating for 1 h at RT. Plates were washed with PBS-T between each step of the protocol. To develop the colorimetric reaction, TMB/H 2 O 2 substrate was added to the plates and incubated for 20 min at RT. The reaction was stopped using 2 N sulfuric acid (H 2 SO 4 ; VWR, #BDH3500-1). Absorbance was subsequently measured on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. The concentration of antibody required to inhibit the signal of a constant concentration of Fel d 1 by 50% (IC 50 ) was determined using the Prism software.

Basophil activation assessed by phosphorylated Erk

Blood drawn from patients with cat allergy (n = 9 patients, 10 total samples based on one repeat visit) was shipped at RT for same-day delivery (Bioreclamation IVT). PBMCs were purified by centrifugation on a Ficoll layer, washed three times in pre-warmed RPMI media (Gibco), resuspended in pre-warmed serum-free X-Vivo 15 media (Lonza) and plated on a 96-well plate (as single points). The cells were then incubated at 37 °C for 30 min. In parallel, a 2× stimulation plate was prepared that included a dose response of purified nFel d 1 as well as dose responses of anti-Fel d 1 antibodies (2.56 fM-200 nM) mixed with a constant dose (final concentration 200 pM) of purified natural Fel d 1 (Indoor Biotechnologies). The stimulation plate was also incubated for 30 min at 37 °C. The cells were then stimulated at 37 °C using a 96-well multichannel pipette, and the stimulation was stopped after precisely 5 min by adding 1 volume of pre-warmed Cytofix (BD). After 15 min of fixation, the cells were washed twice in MACS buffer and made permeable by resuspending and storing them in ice-cold methanol overnight at −20 degrees. The cells were then washed three times with MACS buffer, resuspended for 10 min in human Fc blocker (ebioscience) and then stained with an antibody cocktail containing pErk-Alexa 488 (Cell Signaling, Catalog #13214s, clone 197G2), CD123-BUV395 (BD, Catalog #564195,clone 7G3), and HLA-DR-APC (BD Catalog #559866, clone G46-6) antibodies for 30 min. Cells were washed twice with MACS buffer, incubated for 15 min with Cytofix (BD) diluted 1:4 in PBS to fix the stain, resuspended in MACS buffer and acquired in an LSR-Fortessa instrument. Data were analyzed by calculating the median fluorescence intensity (MFI) of phosphorylated Erk staining within the basophil gate. Percent Max Inhibition was calculated as

$$100-(({\mathrm{100}} \times {\mathrm{Maximum}}\,{\mathrm{Antibody}}\,{\mathrm{Response}})/{\mathrm{Isotype}}\,{\mathrm{Response}})$$

Maximum antibody response was the average MFI of phosphorylated Erk in the top three doses of antibody in the dose–response curve (plateau of the curve) minus the baseline MFI (average of replicate unstimulated samples), and isotype response is the average of all the MFI values in the dose response of a Regeneron-produced IgG4P isotype control antibody minus the baseline MFI.

Basophil activation assessed by CD203/CD63 in cat allergy patients

To evaluate antibody-mediated inhibition of basophil activation, a 20 pM final constant concentration of Fel d 1 (Indoor Biotechnologies) was pre-incubated for 30 min at 37 °C with REGN1908–1909 combination or IgG4P isotype control antibody at final concentrations ranging from 0.8 fM to 1.0 μM. Concurrently with the Fel d 1 and antibody pre-incubation, PBMCs (BioreclamationIVT) were purified from fresh whole blood from allergic donors by Ficoll density gradient centrifugation. The purified PBMCs were washed, resuspended in X-VIVO 15 media (Lonza), and plated in duplicate columns in a v-bottom, polypropylene, 96-well plate (approximately 5 × 105 cells/well). To prime the basophils contained in the overall PBMC population for activation, hIL-3 (R&D Systems, 0.3 nM) was added to the cell suspension and the plate was incubated at 37 °C for 10 min. The pre-incubated antibodies and Fel d 1 were then added to the primed PBMC. Cells without the addition of antibody were included as negative control samples and a dose response of Fel d 1 ranging from final concentrations of 7.8 aM to 10.0 nM was included as a positive control. The cells were then incubated at 37 °C for 20 min to facilitate basophil activation. Activation was subsequently stopped by incubation at 4 °C for 5 min. Basophil activation was then evaluated by flow cytometry. The cells were stained at 4 °C for 20 min with either an antibody cocktail containing anti-HLA-DR-FITC (Beckman Coulter, Catalog #IM0463U, clone B8.12.2), anti-CD123-APC (BD, clone 7G3, Catalog #560087), and anti-CD203c-PE (Beckman Coulter, Catalog #IM3575, clone 97A6) or a cocktail containing anti-HLA-DR-PE-Cy7 (Biolegend, Catalog #307616, clone L243), anti-CD123-APC (BD, Catalog #560087,clone 7G3), and anti-CD63-FITC (Beckman Coulter Catalog #IM1165U clone CLBGran/12). After staining, the cells were washed in a 1:20 dilution of MACS BSA Stock Solution (Miltenyi Biotec) in autoMACS Rinsing Solution (Miltenyi Biotec), fixed in Cytofix (BD) diluted 1:4 in Dulbecco’s phosphate-buffered saline (Gibco), and analyzed on a flow cytometer (BD LSRFortessa X-20). The gating strategy (Supplementary Fig. 6c) was used to identify basophils within the larger population of PBMC and to determine levels of basophil activation. Samples were run to collect approximately 1000 events determined to be basophils. Basophils were defined as singlet, lymphoid, HLA-DR-, and CD123+. Activated basophils were further defined as CD203cHi or CD63Hi. To specify a baseline level of activation, gates were set so that 10% of basophil events from hIL-3-primed, unstimulated samples (no Fel d 1) were positive for activation (CD203cHi or CD63hi). These gates were then applied to all other experimental conditions to determine the relative level of basophil activation.

Mouse experiments

Female, 7, 8 week old Balb/c mice from Jackson Laboratories were used for all mouse studies.

For the entire duration of the experiment, animals remained housed in the Regeneron animal facility under standard conditions, and were allowed to acclimate for at least 7 days prior to being placed on study. All animal experiments were performed in accordance with the guidelines for the Institutional Animal Care and Use Committee at Regeneron. For preclinical mouse studies, no statistical methods were used to predetermine sample size. Mice were randomly assigned to treatment groups without predefined criteria and blinding was not able to be performed due to obvious color change of the ear.

Passive cutaneous anaphylaxis mouse model

Antisera used for passive intradermal (ID) administration were previously generated by immunizing Balb/c mice with either Fel d 1, standardized cat hair extract, or crude peanut allergen extract (irrelevant control antisera) using alum adjuvant. Sera from 10 to 20 mice were pooled and used for passive administration of allergen-specific IgE. On day 1, groups of Balb/c mice (exact “n” noted in the figure legend of the corresponding figure) received a SC injection of REGN1908, REGN1909, REGN1908–1909, concentrated Cat-SIT IgG or Non-SIT IgG, or a Regeneron generated IgG4P isotype control antibody. Three days later, antisera generated to Fel d 1 or cat hair extract, or peanut (negative control) was injected ID into the right and left ears, respectively, allowing allergen-specific IgE to bind FcεR on mast cells. Each anti-serum was standardized to contain 1–25 ng IgE per injection (exact concentration noted in respective experiment). Twenty-four hours after local administration of allergen-specific antisera, mice were challenged by intravenous (IV) injection of 0.25–1μg Fel d 1 (exact concentration noted in respective experiment) or 250 Bioequivalent allergy units (BAU) cat hair extract diluted in PBS containing 0.5% Evans blue dye (Sigma, Catalog #E2129). One hour after allergen challenge, mice were sacrificed, Evans blue dye was extracted from ear tissue and spectrophotometrically quantitated using a standard curve. Ears were then dried and weighed.

Data are presented as ng/mg with value obtained from each peanut ear subtracted from the corresponding value for the Fel d 1 challenge ear. Circulating human antibody levels were measured at the day of sacrifice by ELISA. Microtiter plates (VWR, Catalog # 62409-024) were coated overnight at 4 °C with 1 µg/mL goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog #109-005-098), washed four times with a plate washer (Molecular Devices), and blocked with 0.5%BSA (w/v). Two-fold dilutions of mouse serum were added in duplicate wells, and incubated for 1 h at RT. Plates were washed 4 times and bound antibodies detected using goat anti-human IgG Fc HRP (Jackson ImmunoResearch, Catalog #109-035-098) and BD Opt EIA TMB Substrate (BD Pharmingen, Catalog #555214). The reaction was stopped using 2 N sulfuric acid (Sigma) and absorbance at 450 nm read on a spectrophotometer (Molecular Devices).

Preclinical statistical analysis

Statistical analysis was assessed using GraphPad Prism 6. Analysis of the differences between two groups was assessed using the Student’s paired or Student’s unpaired two-tailed t test.

For multiple group animal studies, normality of the data was evaluated using the Shapiro–Wilk if n > 7 or the Kolmogorov–Smirnov test if n <7. If data passed the normality test, and standard deviations of the different groups were not statistically different from each other as assessed by the Brown–Forsythe test, results were interpreted by one-way analysis of variance followed by the Tukey post hoc test for multiple comparisons. If data failed to pass the normality test, or standard deviations were significantly different, results were interpreted using the Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons. Differences were considered to be statistically significant when p < 0.05.

Clinical study design

R1908–1909-ALG-1325.03 (NCT02127801) was a multicenter phase 1b, randomized, double-blind, placebo-controlled, single SC dose, proof-of-mechanism study conducted in six study centers in Europe and Asia-Pacific. The first subject visit was September 10, 2014 and the last subject completed day 85 on December 15, 2015.

Study participants eligible for randomization were stratified in two blocks: the London site (Quintiles phase I Unit), and all other study sites combined, implemented through a central interactive voice response system. Study participants were randomized 1:1 on day 1 to receive a single SC dose of REGN1908–1909 (600 mg total, 1:1 antibody ratio; n = 37) or placebo (n = 36). Study participants, the principal investigators, and study site personnel remained blinded to all randomization assignments throughout the study. The Regeneron study director, medical monitor, study monitor, and any other Regeneron and contract research organization personnel who were in regular contact with the study site remained blinded to all subject randomization assignments. A Study Manual was developed to standardize techniques across the sites. Personnel from all sites underwent centralized training on the Manual and all study procedures at Quintiles Unit in London. The number of staff at each site performing specific assessments was limited to minimize the inter-operator variability.

The protocol was approved by the appropriate ethics committees/institutional review boards, and each patient gave written consent at screening visit 1. The study was conducted in compliance with institutional review board regulations, the International Conference on Harmonization Guidelines for Good Clinical Practice, and the Declaration of Helsinki. This study and all uses of human material were approved by the following national competent authorities: Centrale Commissie Mensgebonden Onderzoek (CCMO), Netherlands; Medsafe, New Zealand; Läkemedelsverket Medical Products Agency, Sweden; and Medicines and Healthcare products Regulatory Agency (MHRA), United Kingdom.

Patient population

Eligible participants were 18–55 years of age with cat-induced allergic rhinitis and cat sensitization confirmed at screening. To confirm cat-sensitization, participants underwent screening at two visits, day −28 and day −14 (±2 days). At screening visit 1, participants were screened for allergen-specific IgE, underwent a skin prick test with cat hair extract (cat-SPT, Aquagen, ALK-Abello) and other allergen extracts, and were tested for lung function (FEV1). Participants were eligible for screening visit 2 based on IgE titers specific for Fel d 1 and cat hair extract >0.35kAU/l each, cat-SPT mean wheal diameter of >3 mm compared to a negative control SPT, and normal lung function. Patients were excluded if they had prior history of SIT or vaccination with cat allergen, or anti-IgE therapy; SIT to other allergens within 3 months prior to screening; or were living with or chronically exposed to a cat.

At screening visit 2, a single NAC was performed using increasing doses of cat hair extracts (100-33,000 SQ-U/ml). Briefly, cat hair extract was applied intranasally every 10 min for 1 h, or until a TNSS >7 was reached. TNSS (measured on a 0–12 scale) is a composite patient symptom assessment of congestion, itching, and rhinorrhea (each graded on 0–3 scale, 3 being severe), and sneezing (3 being >5 sneezes). Cat-sensitized patients were eligible for enrollment based on having a TNSS <2 prior to the screening NAC (time 0), and peak TNSS >7 within one hour of NAC initiation. PNIF (measured in nasal patency, l/min) was also measured at screening visit 2.

Eligible study participants were randomized to receive study drug or placebo on study day 1 (14 days after screening visit 2). On study days 8, 29, 57, and 85, study participants underwent NAC using the same allergen titration required for each individual subject to reach TNSS >7 at their 2nd screening visit, not to exceed the maximum dose established in the screening visit, regardless of whether TNSS>7 was reached. At each study visit, TNSS, and PNIF and were measured pre-NAC, then at 10, 30, and 60 min during the first hour, and once per hour for 8 h to measure the LPR. Serum samples were collected at each study visit, and a repeat cat-SPT was performed on study days 29 and 85.

Efficacy endpoints

The primary efficacy endpoint was change in TNSS AUC between pretreatment and day 8 NAC over the first hour of challenge (0–1 h, early phase allergic response). Secondary endpoints were percent change in TNSS AUC from pretreatment to day 8 NAC over the first hour; change and percent change in TNSS AUC from pretreatment NAC to days 29, 57, and 85 over the first hour; and change and percent change in TNSS AUC from hours 1 to 8 post-challenge (LPR) from pretreatment NAC to days 8, 29, 57, and 85. Exploratory endpoints included change and percent change from pretreatment NAC to days 8, 29, 57, and 85 in peak TNSS, and peak PNIF; as well as PNIF AUC from 0 to 1 h, and 1 to 8 h. Responder analysis was performed ad hoc. Pharmacokinetic parameters were also measured.

Percentage change from baseline to day 29 or day 85 in mean wheal diameter AUC for the titrated cat hair extract skin prick test (100-33,000 SQ-U/ml) measured 15 min post-application was an additional exploratory endpoint. The test was performed with duplicate serial dilutions of Aquagen (ALK-Abello) cat hair extract using six dose titrations ranging from 100 to 33,000 SQ-U/ml. Single probes with a negative control solution (saline) and a positive histamine control were administered in duplicate simultaneously with cat hair extract probes on the opposite arm. 15 min after application, the wheal diameters were recorded. Mean wheal diameters were calculated by adding the longest diameter to the longest orthogonal diameter and dividing by 2. For each of the duplicate skin prick tests, the longest and longest orthogonal diameters should be recorded, and the mean diameter of each wheal calculated to two decimal places. The mean wheal diameters from the duplicate skin pricks were then averaged.

For the titrated cat skin prick test, the formula used to calculate the normalized average wheal diameter AUC was

$$\begin{array}{l}\left[{{(t1-t0)}}\left( {{{D1}} + {{D0}}} \right)/{{2}} + {{(t2 - t1)}}({{D2}} + {{D1}})/{{2}} + {{(t3 - t2)}}({{D3}} + {{D2}})/{{2}}\right.\\ \left.+ {{(t4 - t3)}}({{D4}} + {{D3}})/{{2}} + {{(t5 - t4)}}({{D5}} + {{D4}})/2\right]/{{(t5}} - {{t0)}}\end{array}$$

in which t i is the concentration (in SQU/ml) for which D i is measured;

$${{t0}} = 100,\,{{t}}1 = 330,\,{{t}}2 = 1000,\,{{t}}3 = 3300,\,{{t}}4 = 10,000,\,{{\rm and}}\,{{t}}5 = 33,000;$$

D i is the average wheal diameter obtained at concentration t i .

The negative control was not subtracted from this value.

Safety

Safety assessments included rates of TEAEs or serious AEs (SAEs) through day 85 as reported by investigators, along with vital signs and laboratory tests. Adverse events were described at the Medical Dictionary for Regulatory Activities (MedDRA; version 17.0) to lowest level terms.

Clinical statistical analysis

A sample size of approximately 70 patients with cat allergy was needed to provide at least 90% power to detect the expected mean differences in primary endpoint between the two treatment groups, with an assumption of a TNSS AUC mean value of 5 for the placebo group and 3 for the treatment group during the course of 0–1-h post-challenge. The assumed standard deviation of 2.43 is consistent with the effect of topical nasal corticosteroids69,70. Primary efficacy analyses were conducted in the FAS which includes all randomized patients who received any study drug on day 1 and had TNSS evaluation results on day 8 (n = 36 and 34, for placebo and REGN1908–1909 groups, respectively). Efficacy endpoints were analyzed using analysis-of-covariance (ANCOVA) model with treatment group as a factor and baseline value as a covariate. The results of the ANCOVA model included the summary of least-squares (LS) mean for each treatment group with corresponding standard error (SE), the LS mean difference between treatment group with corresponding SE and 95% confidence interval (CI), and the p-value corresponding to the between-treatment-group difference. Sensitivity analyses were performed for all efficacy analyses by excluding four patients from a study site terminated early due to non-compliance, as determined by the investigator/sponsor, and for the primary efficacy endpoint by imputing three missing TNSS AUC(0–1 h) values at day 8 with baseline values. Secondary and exploratory efficacy endpoints were analyzed using the same ANCOVA model as the primary analysis; no control for multiplicity was performed for secondary and exploratory endpoints, therefore p values are considered nominal. The safety analysis set included all randomized patients who received any study drug on day 1, based on the treatment received.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its supplementary information files, or are available upon reasonable requests to the authors.