This was an observational, retrospective, single-center study to collect data on adult patients with moderate/severe allergic rhinitis/rhinoconjunctivitis with or without controlled asthma due to sensitization to cat epithelium. Participants were recruited during routine visits to the Allergy Department of the Hospital Central de La Cruz Roja in Madrid, Spain. Patients in the effectiveness population were those who had received SCIT for at least 12 months of treatment. Patients in the safety population were those who had received at least one dose of the product. Inclusion criteria included: patients aged ≥18 years with a clinical history consistent with moderate/severe allergic rhinitis/rhinoconjunctivitis according to ARIA (Allergic Rhinitis and its Impact on Asthma) guideline criteria (27) with or without controlled asthma according to GINA (Global Initiative for Asthma) guideline criteria (28) for cat epithelium sensitization, as demonstrated by a positive skin prick test (≥3 mm) to cat epithelium and cat epithelium-specific IgE levels ≥0.35 KU/L; indication for SCIT in the context of routine clinical practice and patients on current or previous SCIT with depigmented, polymerized 100% cat extract for at least 12 months. Exclusion criteria included any contraindication to the use of allergen-specific SCIT in routine clinical practice or according to the product information.

The primary endpoint was to evaluate the effectiveness of SCIT with Dpg-pol-cat under real-life conditions, as measured by improvement in health-related quality of life (HRQoL) using the validated ESPRINT-15 questionnaire 24 months after treatment initiation. Secondary endpoints included i. to evaluate the effectiveness of SCIT with Dpg-pol-cat by changes in rhinitis HRQoL, symptoms, rescue medication use (as assessed by the daily medication score [dMS], calculated as the sum of oral antihistamines [1 point], topical corticosteroids [2 points] and topical antihistamine/corticosteroid use [3 points]), asthma control and forced expiratory volume (FEV₁) measurement (both in the asthmatic subgroup) after 6, 12, 18 and/or 24 months of treatment; ii. to evaluate the immunological response to the SCIT after 12 or 24 months of treatment (i.e., measurement of total IgE, cat-specific IgE, anti-Fel d 1 IgE, anti–Fel d 2 IgE, anti-Fel d 4 IgE, total IgG4, cat-specific IgG4, anti-Fel d 1 IgG4, anti-Fel d 2 IgG4 and anti-Fel d 4 IgG4 levels); and (iii) to evaluate the safety of Dpg-pol-cat. The safety assessment included: number of systemic reactions per injection administered [classified according to the 2010 World Allergy Organization (WAO) criteria] and time of occurrence (immediate/late); number of local reactions per injection administered, classified according to size and time of occurrence (immediate/late); number and description of other adverse reactions different from systemic or local.

Dpg-pol-cat (Depigoid® Cat) is a biologically standardized injectable suspension for subcutaneous administration, composed of depigmented, purified allergen extracts of 100% cat epithelium, polymerized with glutaraldehyde into allergoids, and adsorbed onto aluminum hydroxide. All patients received SCIT in a rush up-dosing schedule (i.e., a single day: two doses of 0.2 and 0.3 ml with a 30-min interval), followed by monthly maintenance injections of 0.5 ml. Treatment was administered for at least 24 months between May 2018 and December 2021. Throughout the study, the patient could have received any concomitant medication according to routine clinical practice or treatment deemed necessary to provide adequate supportive care.

Total IgE and IgG and specific IgE and IgG to cat allergens Fel d 1, Fel d 2 and Fel d 4 were measured using ImmunoCAP (Thermo Fisher Scientific®), following the manufacturer's instructions. The cut-off point for specific IgE was set at ≥0.35 kU/L.

The estimated sample size for the effectiveness analysis (primary endpoint) was estimated at 30 patients and was determined by the total number of patients who attended the Allergology Department of the Hospital between May 2018 and December 2021. Clinical data were collected at baseline and at visits at 6 months, 12 months and 18/24 months (i.e., final visit). The Last Observation Carried Forward (LOCF) imputation method was used to impute missing values. If information from the final visit was missing, the same information from the 12-month visit was imputed for the same patient. Data were recorded in a database specifically designed for the study. Descriptive analyses were carried out for all variables collected. Categorical variables were summarized by frequencies and percentages, whereas continuous variables were summarized using measures of central tendency and dispersion, which may include mean, standard deviation, median, first and third quartiles (Q1; Q3) and extreme values (minimum and maximum). Categorical variables were compared using the Chi-square or Fisher's exact test. For paired data, McNemar's test was used. Numerical variables were compared using the Student's T-test, ANOVA or the non-parametric Wilcoxon-Mann–Whitney U or Kruskal–Wallis tests. The significance threshold for all bivariate analyses was set at a two-sided α = 0.05. All analyses were performed using the statistical software suite SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

All methods and analyses were performed in compliance with local legal and regulatory requirements, as well as generally accepted research practices described in the latest version of the Declaration of Helsinki and the International Conference of Harmonization-Good Clinical Practice Guidelines. All patients provided written informed consent to participate in the study. All data included in this study were collected as part of routine clinical practice and analyzed retrospectively. In compliance with Organic Law 3/2018, of December 5, on the Protection of Personal Data and the Guarantee of Digital Rights, and Regulation (EU) 2016/679 of the European Parliament and of the Council, of April 27, 2016, all data included in this study were anonymized, a fundamental regulation of patient autonomy and rights and obligations regarding clinical information and documentation, as well as current and applicable regulations.

Ultimately, data from 28 patients were collected, instead of the 30 initially estimated as the sample size. The median (Q1; Q3) age was 35 (28.3; 39.5) years. There were more women than men (67.9% vs. 32.1%), resulting in a male to female ratio of 0.47. On average, patients had a body mass index (BMI) within the normal range, with a median (Q1; Q3) score of 23.2 (21.1; 25.9). Eight patients (28.6%) were current or former smokers. Most patients with respiratory disease had mild or moderate symptoms, including those with rhinoconjunctivitis (92.9%; 26/28), rhinitis (85.7%; 24/28) and allergic asthma (100.0%; 27/27). All patients with allergic asthma (n = 27) had controlled or partially controlled disease, according to clinical criteria. The percentage of patients with atopic dermatitis and food allergy were low, 3.6% and 10.7%, respectively. At baseline, the median (Q1; Q3) levels of cat epithelium-specific IgE were 42.5 (10.5; 78.0) kU/L and total IgE levels were 294.0 (127.0; 726.0) kU/L. All patients had been exposed to cats, and the median (Q1; Q3) exposure time was 36.0 (24.0; 84.0) months. Four patients (14.3%) had been exposed to other animals, in all 4 cases to dogs (Table 1). When analyzing the allergen sensitization profile as determined by skin prick test (SPT), more than half of the population was sensitized to dogs (60.7%) and pollen (75.0%), whereas only a small percentage was allergic to house dust mites (14.3%), molds (7.1%), or pan-allergens (7.1%), including profilin (Pho d 12, 3.6%) and LTP (Pru p 3, 3.6%). The most common pollen allergies identified were to Lolium perenne (60.7%), Phleum pratense (60.7%), Olea europea (53.6%), ash tree (50.0%), and Cupressus arizonica (42.9%) (Supplementary Table S1).

All patients had a rapid, 1-day rush build up schedule. The median (Q1; Q3) time of SCIT with Dpg-pol-cat was 21.8 (18.1; 25.2) months. SCIT treatment resulted in a sustained, statistically significant improvement in allergic rhinitis HRQoL (i.e., patients' perception of rhinitis symptoms) as measured by the ESPRINT-15 questionnaire, from 6 months to the final visit. The median (Q1; Q3) questionnaire score decreased significantly from 36.0 (18.0; 52.0) points at baseline, to 35.0 (10.0; 45.0) at 6 months (p = 0.0052) and to 13.0 (8.0; 35.0) at 12 months (p = 0.0002), with a slight recovery to 16.0 (7.0; 31.0) points at the final visit (baseline vs. final visit, p = 0.0009; 12 months vs. final visit, p = 0.0470). Overall, the median questionnaire score decreased by 20 points from baseline to the final visit (p = 0.0004). When the patients were asked to rate their overall HRQoL, considering only their rhinitis, the proportion of patients who reported feeling better increased progressively during SCIT treatment. The percentage of patients feeling good, very good or excellent increased from 76.8% at baseline to 81.8% at 6 months (p = 0.0009), to 89.5% at 12 months (p = 0.0006), and to 90.9% at the final visit (p = 0.2211). Overall, the proportion of patients feeling better after SCIT treatment increased by approximately 13% from baseline to the LOCF (78.6% to 88.9%, p = 0.0082) (Table 2).

At baseline, most patients had moderate or severe symptoms of rhinitis (85.7%), rhinorrhea (82.2%), nasal itchiness (71.4%), nasal obstruction (71.4%), dyspnea (70.3%), asthma (66.7%), and ocular itchiness (53.6%). After SCIT treatment with Dpg-pol-cat, although the changes from baseline were not statistically significant except for wheezing (p = 0.041), most patients were asymptomatic or had mild symptoms at the LOCF, including asthma (92.3%), rhinitis (81.5%), conjunctivitis (66.3%), dyspnea (96.3%), and wheezing (100.0%) (Figure 1 and Supplementary Table S2). Comparing the presence of clustered symptoms at baseline (including mild, moderate, and severe symptoms) with that at the LOCF, the percentage of asymptomatic patients increased from none to 11.1% (n = 3) for nasal symptoms (including rhinorrhea, nasal itching and nasal obstruction), from 3.6% to 40.7% (n = 1 vs. 11) for ocular symptoms (including eye itching, tearing and eye redness), and from 3.6% to 29.6% (n = 1 vs. 8) for bronchial symptoms (including cough, dyspnea and wheezing) (Figure 2). For symptom improvement, defined as a decrease in symptom severity rating, regardless of step, data were available for 27 patients at the end of the study (1 patient lost to follow-up). Comparing baseline and LOCF data, most patients experienced improvement in all symptoms, with statistically significant changes in tearing (63%; p = 0.031), eye redness (48.1%; p = 0.001) and wheezes (70.4%; p = 0.006) (Supplementary Table S3).

All patients used medication for rhinitis prior to starting SCIT with Dpg-pol-cat to manage symptoms. The mean dMS, which was estimated at 1.79 (SD 0.50) before SCIT, significantly decreased after treatment to 0.96 (SD 0.79) at the LOCF (p < 0.0001). Regarding the use of specific medications, antihistamines were reduced by 31.0% (p = 0.1610), antileukotrienes by 14.3% (p = 0.0015), short-acting B2 agonists (SABAs) by 3.6% (p < 0.0001), nasal steroids by 36% (p = 0.0202), and inhaled steroids plus long-acting B2 agonists (LABAs) by 28.6% (p = 0.0052) (Table 3 and Supplementary Table S4).

Asthma control was assessed in asthmatic patients using the validated Asthma Control Test (ACT). The ACT mean score, which was estimated to be 19.07 (SD: 4.68) at baseline, progressively and significantly improved during the SCIT treatment with Dpg-pol-cat, reaching mean scores of 21.63 (SD: 2.87) at 6 months (p = 0.0051), 21.95 (SD 3.40) at 12 months (p = 0.0201) and 22.00 (SD: 3.20) at the LOCF (p = 0.0121). Overall, the percentage of patients with good asthma control (i.e., ACT score ≥20) increased from 40.7% at baseline to 80.0% at the LOCF, whereas the percentage of patients with partial (i.e., 15 < ACT score < 20) and bad (i.e., ACT score ≤15) control decreased from 25.9% to 8.0% and from 33.3% to 12.0%, respectively (Table 3).

Compared to baseline, a slight increase in FEV₁ was observed at 12 months, final visit and LOCF, although values were close to initial values [92.6% (SD: 8.46), 96.9% (SD: 4.91) and 95.1% (SD: 8.08), respectively]. Nevertheless, a decrease was observed at 6 months, probably due to the high number of missing data, as patients were treated during the COVID-19 pandemic and spirometry data were not available (Table 3).

IgE levels were measured at baseline and at each follow-up visit. There were no significant changes during the study period in total IgE, cat epithelium-specific IgE, anti-Fel d 1, anti-Fel d 2, or anti-Fel d 4 IgE antibodies. No significant associations were found between specific IgE levels (Fel d 1, Fel d 2, Fel d 4; mono- or oligosensitized) and the severity of asthma, rhinitis, or atopic dermatitis, nor with the classification of asthma or rhinitis (data for Fel d 2 and Fel d 4 not shown). Cat epithelium-specific IgG4 antibodies increased significantly from baseline to the final visit, from 3.84 to 4.43 kU/L (p = 0.0092). No significant changes were observed in total IgG4, anti-Fel d 1, anti-Fel d 2 or anti-Fel d 4 IgG4 antibodies (Supplementary Table S5).

Thirteen out of the 28 patients (46.4%) had a total of 20 adverse reactions after initiation of SCIT with Dpg-pol-cat, of which 16 were local reactions (80.0%) and 2 were systemic reactions (15.5%). One patient (5.0%) had both local and systemic reactions. All local reactions had a late onset, with 10 (62.5%) occurring during the up-dosing and 6 (37.5%) during the maintenance phase. Most local reactions (n = 15; 93.8%) were mild (n = 7; 43.8%) or moderate (n = 8; 50.0%), with only one reaction (6.2%) classified as severe. Three additional systemic reactions occurred during the maintenance phase, for a total of five systemic reactions. In most cases, no treatment adjustment was required during follow-up. Only 1 patient required a dose reduction and 7 patients received premedication with antihistamines. According to the 2010 WAO criteria, there were 2 grade I cases (40.0%) and 3 grade II cases (60.0%), and no grade III-IV reactions. All patients recovered from systemic reactions and only one patient discontinued the treatment (Table 4).