CN116819076A - A biomarker, application and method for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis accompanied by nasal polyps - Google Patents

Abstract

The present invention discloses indicators of eosinophil infiltration and activation in nasal polyp tissue as biomarkers for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibodies in the treatment of chronic sinusitis with nasal polyps, as well as applications and methods, specifically using ECP ⁺ Cell number/HPF was detected as a marker, and the final ROC curve for predicting and evaluating the patient's response to anti-IL-4Rα monoclonal antibody treatment showed AUC=0.824, a cutoff value of 33.013, a sensitivity of 100%, and a specificity of 76.5%. The present invention The markers can quickly and accurately predict or evaluate the efficacy of anti-IL‑4Rα monoclonal antibodies in the treatment of chronic sinusitis with nasal polyps, filling the gap in existing technology and having significant clinical and economic value.

Description

A prediction or evaluation of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps Biomarkers, applications and methods of efficacy

Technical field

The present invention relates to the technical field of immune analysis, and specifically to a biomarker and application for predicting the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis accompanied by nasal polyps.

Background technique

Currently, 4% of the world's population suffers from chronic rhinosinusitis with nasal polyps (CRSwNP), and its treatment remains a clinical challenge. Among them, eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) is the intrinsic type with the highest recurrence rate among refractory CRSwNP.

IL-4 is a key driver of signal transduction in type 2 inflammatory responses. Th2 cells lead to eosinophil differentiation through IL-4 cytokines. IL-4 cytokines and IL-4Rα receptors activate intracellular signal transduction. Transcription factors on the conduction pathway cause type 2 inflammatory response. As a biological agent targeting type 2 factors, anti-IL-4Rα monoclonal antibody (such as: Dupilumab or CM310) is a fully humanized monoclonal antibody targeting IL-4Rα. It blocks the interaction between IL-4Rα and IL-4Rα. -4 binding thereby inhibiting its biological activity. Anti-IL-4Rα monoclonal antibody has been approved by the US Food and Drug Administration and the US Drug Administration for the treatment of CRSwNP due to its excellent efficacy and safety. However, it has been found in clinical applications that there are still some patients who have poor efficacy after treatment with this type of monoclonal antibody, and these drugs are expensive and have few biomarkers that can be used to predict the response to anti-IL-4Rα monoclonal antibodies. In clinical studies using anti-IL-4Rα monoclonal antibodies (such as Dupilumab or CM310), no statistically significant changes in blood eosinophils were observed, and blood eosinophil infiltration cannot yet be used as an anti-IL-4Rα monoclonal antibody. An effective biomarker for -4Rα monoclonal antibody.

Therefore, it is crucial to explore indicators that can predict its efficacy to achieve precise treatment. This study aims to explore indicators that can be used to predict the efficacy of anti-IL-4Rα monoclonal antibodies.

Contents of the invention

The present invention first provides a biomarker for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis accompanied by nasal polyps. The marker is an indicator of eosinophil infiltration and activation in nasal polyp tissue, so The indicator is optionally ECP ⁺ cell number/HPF.

The present invention also provides an application of the marker according to claim 1, which application is a) preparing a detection reagent for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps; or b ) Prepare or evaluate a detection kit for predicting the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps; or c) Used to predict or evaluate the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps. Methods, optionally for non-diagnostic or non-therapeutic purposes.

The present invention also provides the application of a detection reagent for eosinophil infiltration and activation indicators in nasal polyp tissue in the preparation of a kit for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis accompanied by nasal polyps.

Preferably, the detection reagents include sample pretreatment reagents and reagents for detecting eosinophil infiltration and activation indicators in nasal polyp tissue;

Preferably, the reagents for detecting eosinophil infiltration and activation indicators in nasal polyp tissue include reagents suitable for at least one of the following methods: hematoxylin and eosin (H&E) staining and multiple immunofluorescence staining.

The samples were nasal secretions, nasal exfoliated cells and nasal polyp tissue.

The present invention also provides a method for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps. The method is optionally used for non-diagnostic purposes or non-therapeutic purposes, which includes detecting nasal polyps. The steps of tissue eosinophil infiltration and activation index, the index is optionally ECP ⁺ cell number/HPF, and predicting or evaluating anti-IL-4Rα monoclonal antibody based on nasal polyp tissue eosinophil infiltration and activation index results Efficacy.

Preferably, the step of detecting eosinophil infiltration and activation indicators in nasal polyp tissue includes applying at least one of the following methods: hematoxylin and eosin (H&E) staining and multiple immunofluorescence staining.

Preferably, the hematoxylin and eosin (H&E) staining includes: washing the polyp tissue in PBS containing 1% penicillin, 1% streptomycin and 1% amphotericin, weighing and cutting into pieces weighing about 0.1 g of small sections, inject IL-4 or PBS with a working concentration of 10 μg/μL and a volume of 10 μL into each small section, and co-culture with leukocytes isolated from the same subject from whom the polyps were obtained for 96 hours (106 per well cells), and the degree of eosinophil infiltration in the tissue was assessed after hematoxylin and eosin (H&E) staining.

Preferably, the number of ECP ⁺ cells, ECP ⁺ CD63 ⁺ cells and ECP ⁺ CD69 ⁺ cells per high power field (HPF) in the baseline tissue of nasal polyps is assessed by multiplex immunofluorescence staining. Sections of 4 μm thickness were cut from formalin-fixed paraffin-embedded tissue, deparaffinized in xylene, and rehydrated through a graded alcohol series. Antigen recovery (microwave oven, 120°C, 20 minutes) was performed using ethylenediaminetetraacetic acid (EDTA, Dako, S2367) or AR6 buffer (AR600125ML, Akoya Biosciences). Multiplex immunofluorescence staining was performed using Opal 6-plex manual detection kit (NEL811001KT, Akoya Biosciences). After protein blocking (ARD1001EA, Akoya Biosciences), primary antibody was sequentially applied for 40 min incubation at 37°C, followed by secondary antibody incubation for 10 min, followed by tyramine signal amplification technology and Opal fluorescence detection for 10 min. Evaluation of multiple immunofluorescent markers of eosinophils includes: eosinophilic cationic protein (ECP, ab207429, 1:4000), CD63 (ZA-0694, undiluted), CD69 (ab233396, 1:1000). After each round of detection, heat-induced epitope retrieval was performed using antigen retrieval buffer. After the last round of immunofluorescence staining, 4',6-diamidino-2-phenylindole (DAPI, Akoya Biosciences, FP1490) was used for 5 min of incubation. Coverslips were mounted using ProLong Gold Antifade (ThermoFisher Scientific, P36935). Representative areas of multispectral images under 40x magnification were obtained using the VectraPolaris multispectral imaging system (Akoya Biosciences). Single-color and unstained sections were used as controls to generate a spectral library, and then multispectral unmixing was performed using the inForm software package (Akoya Biosciences, v2.6).

Preferably, the NPS score is used to divide the subjects into a response group and a non-response group based on whether the NPS change after anti-IL-4Rα monoclonal antibody treatment is greater than or equal to 2 points from the baseline NPS. 0 points: no polyps; 1 point: small polyps can be seen in the middle meatus, and the size does not exceed the lower edge of the middle turbinate; 2 points: polyps reach the lower edge of the middle turbinate; 3 points: polyps reach the lower edge of the inferior turbinate, or polyps with a score of 2 Also accompanied by a large polyp on the inner side of the middle turbinate; 4 points: The polyp completely or nearly completely blocks the nasal cavity, that is, to the bottom of the nose. The sum of the NPS scores on both sides is the final score.

Compared with the prior art, the present invention at least has the following beneficial effects:

The present invention has discovered the application of indicators of eosinophil infiltration and activation in nasal polyp tissue as biomarkers for predicting the efficacy of anti-IL-4Rα monoclonal antibodies. Specifically, ECP ⁺ cell number/HPF is used to predict the patient's response to anti-IL-4Rα monoclonal antibody treatment. The ROC curve shows AUC=0.824, the cut-off value is 33.013, the sensitivity is 100%, and the specificity is 76.5%. The marker of the present invention can quickly and accurately predict or evaluate the effectiveness of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis associated with The therapeutic effect on nasal polyps fills the gap in the existing technology and has great clinical and economic value.

Description of the drawings

Figure 1. IL-4 promotes eosinophil infiltration and activation; 0.1 g of nasal polyp slice tissue is injected with a concentration of 10 μg/μL and a volume of 10 μL of IL-4 or PBS, and is incubated with leukocytes isolated from subjects who obtained the polyp tissue. After 96 hours, nasal polyp sections were collected and the number of eosinophils was assessed after hematoxylin and eosin (H&E) staining;

Figure 2 Potential biomarkers to distinguish the response group from the non-response group; (A) The eosinophil group of the response group: CD63, CD69, ECP; (B) The eosinophil group of the non-response group: CD63, CD69, ECP; (C) ROC curves of potential biomarkers predicting response and non-response groups.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

Example 1 Obtaining samples

A total of 22 patients were included in this study, and all patients must meet the following inclusion criteria: 1) age 18-70 years old; 2) have received systemic corticosteroids (SCS) treatment within 2 years before the lead-in period, or Have contraindications or intolerance to SCS treatment, or have undergone nasal polyp surgery 6 months before the lead-in period; 3) Nasal polyp score (NPS) is at least 5 points (at least 2 points for each nostril); 4) Have moderate or severe nasal congestion (0 = no symptoms, 1 = mild, 2 = moderate, 3 = severe), an average weekly nasal congestion score (NCS) of 2 or 3, and any other Symptoms, such as loss of smell or runny nose; 5) Patients need to use nasal spray corticosteroids stably and continuously for at least 4 weeks before screening, and throughout the study, patients receive mometasone furoate nasal spray (MFNS) as background treatment; 6 ) Peripheral blood eosinophil percentage in all patients ≥6.9% (without asthma) or ≥3.7% (with asthma) (4); 2) Absolute tissue eosinophil count ≥55 cells/high-power field by nasal polyp biopsy or eosinophil percentage ≥27% (2,3).

Patients were excluded if they: 1) had used IL-4Rα antagonists within 10 weeks or 5 half-lives (whichever was longer) before randomization, or had used IL-4Rα antagonists within 8 weeks or 5 half-lives (whichever was longer) whoever) has used biological therapy/systemic immunosuppressants within 130 days, or used anti-IgE monoclonal antibodies within 130 days; 2) experienced nasal polyp surgery within 6 months before the screening/lead-in period; 3) before screening Receive intermediate-acting and short-acting SCS treatment within 4 weeks, or receive long-acting SCS treatment within 6 weeks; 4) suffer from severe liver or kidney damage; 5) receive live vaccines or attenuated vaccines within 12 weeks before randomization, Or plan to receive live vaccines and attenuated vaccines during the study; 6) Patients with known or suspected immunosuppression, including but not limited to a history of invasive opportunistic infections (such as tuberculosis, histoplasmosis, listeriosis, Coccidiosis, pulmonary cyst, aspergillosis), even if the infection has resolved; 7) Women who are pregnant or breastfeeding, or women who plan to become pregnant during the study; 8) Any other medical or non-medical condition that the researcher considers inappropriate for participation in the study Medical conditions. This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (TREC2022-KY053.R1). Written informed consent was obtained prior to patient participation.

Example 2 Sample Detection

After the patients signed the informed consent form, they entered the 4-week screening/introduction period (V1). During this period, all patients received MFNS nasal spray, 100 μg per nasal cavity per day (total daily dose: 200 μg); after that, the patients received anti-IL-4Rα monoclonal antibody treatment. 16 weeks (first 600 mg subcutaneous injection, then 300 mg subcutaneous injection every 2 weeks, V2-V10). Patients underwent polyp collection at V1 for tissue eosinophil counter immunofluorescence staining, and NPS scoring at V2 and V10.

The degree of eosinophil infiltration in polyp tissue was assessed by hematoxylin and eosin (H&E) staining. The polyp tissue was rinsed in PBS containing 1% penicillin, 1% streptomycin and 1% amphotericin, weighed and cut into small sections weighing approximately 0.1 g. IL-4 or PBS with a working concentration of 10 μg/μL and a volume of 10 μL was injected into each small section, and co-cultured with leukocytes isolated from the same subject from whom the polyps were obtained for 96 hours (106 cells per well), in hematoxylin The degree of eosinophil infiltration in the tissue was evaluated after H&E staining.

The number of ECP ⁺ cells, ECP ⁺ CD63 ⁺ cells and ECP ⁺ CD69 ⁺ cells per high power field (HPF) in the baseline tissue of nasal polyps was assessed by multiplex immunofluorescence staining. Sections of 4 μm thickness were cut from formalin-fixed paraffin-embedded tissue, deparaffinized in xylene, and rehydrated through a graded alcohol series. Antigen recovery (microwave oven, 120°C, 20 minutes) was performed using ethylenediaminetetraacetic acid (EDTA, Dako, S2367) or AR6 buffer (AR600125ML, Akoya Biosciences). Multiplex immunofluorescence staining was performed using Opal 6-plex manual detection kit (NEL811001KT, Akoya Biosciences). After protein blocking (ARD1001EA, Akoya Biosciences), primary antibody was sequentially applied for 40 min incubation at 37°C, followed by secondary antibody incubation for 10 min, followed by tyramine signal amplification technology and Opal fluorescence detection for 10 min. Evaluation of multiple immunofluorescent markers of eosinophils includes: eosinophilic cationic protein (ECP, ab207429, 1:4000), CD63 (ZA-0694, undiluted), CD69 (ab233396, 1:1000). After each round of detection, heat-induced epitope retrieval was performed using antigen retrieval buffer. After the final round of immunofluorescence staining, 4',6-diamidino-2-phenylindole (DAPI, Akoya Biosciences, FP1490) was used for 5 min of incubation. Coverslips were mounted using ProLong GoldAntifade (ThermoFisher Scientific, P36935). Representative areas of multispectral images under 40x magnification were obtained using the VectraPolaris multispectral imaging system (Akoya Biosciences). Single-color and unstained sections were used as controls to generate a spectral library, and then multispectral unmixing was performed using the inForm software package (Akoya Biosciences, v2.6).

Specifically, the NPS score was used to divide the subjects into a response group and a non-response group based on whether the change in NPS after anti-IL-4Rα monoclonal antibody treatment was greater than or equal to 2 points from the baseline NPS. 0 points: no polyps; 1 point: small polyps can be seen in the middle meatus, and the size does not exceed the lower edge of the middle turbinate; 2 points: polyps reach the lower edge of the middle turbinate; 3 points: polyps reach the lower edge of the inferior turbinate, or polyps with a score of 2 Also accompanied by a large polyp on the inner side of the middle turbinate; 4 points: The polyp completely or nearly completely blocks the nasal cavity, that is, to the bottom of the nose. The sum of the NPS scores on both sides is the final score.

Specifically, SAS version 9.4 was used for statistical analysis. In general, continuous variables are summarized descriptively by number of subjects, mean, standard deviation, median, 25% quantile, 75% quantile, minimum and maximum values, and categorical variables are summarized by frequencies and percentages Giving a summary. In all statistical analyses, all statistical tests were performed with two-sided α = 0.05 unless otherwise specified. Comparisons between groups were performed by Mann-Whitney U test. A receiver operating characteristic (ROC) curve was generated, and the area under the ROC curve (AUC) was calculated to evaluate its predictive value. The predictive value of AUC >0.9 is high; the predictive value of 0.7<AUC≤0.9 is medium; the predictive value of 0.5<AUC≤0.7 is weak.

Example 3 Sample Test Results

Evaluating the number of eosinophils in nasal polyp tissue after hematoxylin and eosin (H&E) staining, the study found that IL-4 was injected into the nasal polyps and co-cultured with leukocytes isolated from the same subjects from whom the polyps were obtained for 96 hours. Afterwards, compared with the PBS injection group, there were a large number of eosinophils in the tissue sections of nasal polyps treated with IL-4, which could promote the infiltration and activation of eosinophils in local tissues (Figure 1).

According to whether the change in NPS from baseline NPS after anti-IL-4Rα monoclonal antibody treatment was greater than or equal to 2 points, 22 patients were divided into a response group (n=17) and a non-response group (n=5). The basic characteristics of the patients are shown in Table 1.

Table 1 Characteristics of patients in the response group and non-response group

Multiple immunofluorescence staining (ECP Opal 520, CD69 Opal 690 and CD63 Opal 620) was performed on eosinophils in paraffin-embedded nasal polyp tissue, and ECP ⁺ cells, Number of ECP ⁺ CD63 ⁺ cells and ECP ⁺ CD69 ⁺ cells. The results showed that the number of ECP-positive cells in the non-responsive group was smaller than that in the responsive group (Table 2).

Table 2. Potential biomarkers distinguishing responder from non-responder groups

The ROC curve using ECP ⁺ cell number/HPF to predict patient response to anti-IL-4Rα monoclonal antibody treatment showed AUC=0.824, a cutoff value of 33.013, a sensitivity of 100%, and a specificity of 76.5% (Figure 2).

The above is the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (8)

1. A biomarker for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis accompanied by nasal polyps, characterized in that the marker is an indicator of eosinophil infiltration and activation in nasal polyp tissue, so The indicator is optionally ECP ⁺ cell number/HPF. 2. An application of the marker according to claim 1, characterized in that the application is a) preparing a detection reagent for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps; or b) Prepare or evaluate a test kit for predicting the efficacy of anti-IL-4Rα monoclonal antibodies in the treatment of chronic sinusitis with nasal polyps; or c) Use to predict or evaluate the efficacy of anti-IL-4Rα monoclonal antibodies in the treatment of chronic sinusitis with nasal polyps. Therapeutic methods, optionally for non-diagnostic or non-therapeutic purposes. 3. Application of a marker detection reagent according to claim 1 in the preparation of a kit for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis accompanied by nasal polyps. 4. Application of the detection reagent according to claim 3 in the preparation of a kit for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps, characterized in that the detection reagent includes Sample pretreatment reagents and reagents for detecting eosinophil infiltration and activation indicators in nasal polyp tissue; the samples are nasal secretions, nasal exfoliated cells and nasal polyp tissue. 5. A method for predicting or evaluating the efficacy of anti-IL-4Rα monoclonal antibody in the treatment of chronic sinusitis with nasal polyps, characterized in that the method is optionally used for non-diagnostic purposes or non-therapeutic purposes, and it includes detecting nasal polyps. The steps of using an indicator of eosinophil infiltration and activation in polyp tissue, the indicator optionally being ECP ⁺ cell number/HPF, and using the indicator result of eosinophil infiltration and activation in nasal polyp tissue to predict or evaluate anti-IL- Efficacy of 4Rα monoclonal antibody. 6. The application according to claims 2-4, characterized in that the detection reagents for eosinophil infiltration and activation indicators in nasal polyp tissue include reagents suitable for at least one of the following methods: hematoxylin and eosin. (H&E) staining, multiplex immunofluorescence staining. 7. The method according to claim 5, wherein the step of detecting indicators of eosinophil infiltration and activation in nasal polyp tissue includes being suitable for at least one of the following methods: hematoxylin and eosin (H&E) staining, multiple immunofluorescence staining. 8. The method according to claim 7, wherein the hematoxylin and eosin (H&E) staining comprises: staining the polyp tissue in a solution containing 1% penicillin, 1% streptomycin and 1% amphotericin. Rinse in PBS, weigh and cut into small slices weighing about 0.1g. Inject IL-4 or PBS with a working concentration of 10 μg/μL and a volume of 10 μL into each of the small slices, respectively, from the same subject who obtained the polyps. The isolated leukocytes were cultured for 96 hours (106 cells per well), and the degree of eosinophil infiltration in the tissue was evaluated after hematoxylin and eosin (H&E) staining.