Serum polypeptide marker serpina5 for immune-related pneumonia and application and detection kit thereof

Abstract

The application discloses a serum polypeptide marker SERPINA5 of immune-related pneumonia and application and detection kit thereof.The accurate molecular weight of the serum polypeptide marker SERPINA5 is 3901.24 Dalton, the expression level of SERPINA5 in patients is quantitatively detected, and it is found that SERPINA5 presents specific high expression in serum detection of CIP patients, and can be used as a specific and efficient serum polypeptide marker for predicting CIP occurrence before immunotherapy of malignant tumor patients and screening high-risk groups of CIP.

Description

Technical Field

[0001] This invention belongs to the field of tumor marker technology, specifically relating to a serum polypeptide marker SERPINA5 for immune-related pneumonia, its application, and a detection kit. Background Technology

[0002] According to the World Health Organization, cancer is the second leading cause of death worldwide, and malignant tumors remain a significant disease burden for humanity. Since the approval of the first CTLA-4 antibody, ipilimumab, for the treatment of malignant melanoma in 2011, the field of tumor immunotherapy has experienced one great renaissance after another (Nat Rev Drug Discov, 2016, 15:235-247.). In recent years, numerous clinical trials of drugs have been widely conducted globally, demonstrating significant anti-tumor efficacy. Immune checkpoint inhibitors (ICIs), represented by PD-1 / PD-L1 inhibitors, have been widely used in various tumors, including non-small cell lung cancer (N Engl J Med, 2018, 379:2040-2051.) and malignant melanoma (N Engl J Med, 2017, 377:1345-1356.), achieving satisfactory results and ushering in a new era of anti-tumor therapy. However, in addition to the long-term benefits of ICIs treatment, a series of unavoidable adverse reactions have also emerged, and severe immune-related adverse reactions have greatly hindered the clinical application of immunotherapy.

[0003] Checkpoint immune pneumonitis (CIP) is one of the most common and deadliest adverse reactions (JAMA Oncol, 2016, 2:1346-1353). Previous reports and the applicant's earlier research show that the incidence of CIP can reach 10%-20% (J Thorac Oncol, 2018, 13:1930-1939), and the mortality rate of severe CIP is as high as 14%-35% (JAMA Oncol, 2018, 4:1721-1728). Severe CIP can cause irreversible damage to patients, shorten their survival time, incur huge costs, and consume a large amount of medical resources. In conclusion, CIP is a challenging clinical problem in immunotherapy for patients with malignant tumors.

[0004] Currently, the clinical diagnosis of CIP mainly relies on imaging examinations. However, the imaging changes in CIP are delayed, complex, and lack specificity, making early detection difficult and differentiation from other respiratory diseases challenging, leading to a high risk of missed diagnoses and increasing the risk of developing severe CIP. Therefore, early screening of high-risk CIP patients and actively exploring new, more specific, and predictive approaches are urgently needed. This is crucial for reducing the incidence and mortality of CIP and improving the safety of immunotherapy. As a research hotspot and challenge, multiple studies have attempted to explore the risk factors and biomarkers of CIP. Previous studies have shown that combination therapy, especially the combination of PD-1 / L1 inhibitors and CTLA-4 inhibitors, is more likely to cause CIP (JAMA Oncol, 2018, 4:1721-1728.). In addition, advanced age and smoking are also considered risk factors for increasing the risk of CIP (J Thorac Oncol, 2018, 13:1930-1939; J Thorac Oncol, 2018, 13:1138-1145.). Ryota Shibaki et al. found that men, smokers, patients with a history of interstitial lung disease, and patients with emphysema were also considered high-risk groups for CIP (Cancer Immunol Immunother, 2020, 69:15-22.). In hematological parameters, Professor Su Chunxia's team from Shanghai Pulmonary Hospital in my country found that an increase in baseline absolute eosinophil count during treatment was a strong indicator of CIP risk (Lung Cancer, 2020, 150:76-82.). Similarly, Ryosuke Matsukane et al. found a significant increase in the neutrophil-lymphocyte ratio, which reflects systemic inflammation, during CIP (Sci Rep, 2021, 11:1324.). While the aforementioned research results can predict the occurrence of CIP to some extent, the area under the ROC curve for identifying CIP is mostly below 0.8 (Lung Cancer, 2020, 150:76-82.; Clin Lung Cancer, 2019, 20:442-450.e4.), exhibiting drawbacks such as poor specificity and low stability in single-dimensional prediction. Furthermore, current research on predictive biomarkers for CIP is limited to clinical characteristics and non-specific hematological indicators, failing to achieve the goal of "precise prediction." Therefore, there is an urgent need to develop new biomarkers with high accuracy and specificity. In recent years, the rapidly developing proteomics analysis technology, with its significant advantages of high resolution, high accuracy, and high sensitivity, has brought proteomics-based clinical plasma biomarker screening to the forefront, becoming a new trend in precision medicine.This suggests that mass spectrometry analysis based on proteomics has the potential to become a comprehensive screening method for serum biomarkers closely related to the occurrence of CIP, thereby enabling a novel diagnostic and therapeutic approach for predicting the occurrence of CIP.

[0005] However, there are currently no reports of studies that have obtained highly specific biomarkers for predicting CIP based on proteomics mass spectrometry analysis. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a serum polypeptide marker SERPINA5 for immune-related pneumonia, its application, and a detection kit.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention provides a specific predictive protein for the occurrence of CIP, obtained by serum proteomics mass spectrometry analysis, namely the serum polypeptide marker SERPINA5 for immune-associated pneumonia (CIP), whose amino acid sequence is: R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.- (human plasma serine protease inhibitor, SERPINA5), as shown in SEQ ID NO:1.

[0009] Preferably, serum proteins were extracted using a copper ion-type magnetic bead kit (IMAC-Cu), followed by primary mass spectrometry analysis using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain protein peptide peaks in the patient's serum. Statistical analysis was then performed to identify protein peptide peaks with significant differences between the experimental and control groups. The selected differentially expressed protein peptide peaks were then subjected to secondary proteomic identification to determine their corresponding proteins. Finally, the peptide with M / Z: 3901.24 Daltons was identified as SERPINA5 protein, which showed a highly significant difference between the experimental and control groups (p < 0.001), meaning the precise molecular weight of the serum peptide marker SERPINA5 is 3901.24 Daltons.

[0010] This invention also discloses the application of the serum polypeptide marker SERPINA5 of immune-associated pneumonia in the preparation of serum diagnostic reagents / kits for immune-associated pneumonia.

[0011] Preferably, the detection parameters of the serum polypeptide marker SERPINA5 in serum are 625.36–807.75 pg / mL.

[0012] Preferably, the serum diagnostic reagent / kit for immune-related pneumonia is a serum diagnostic reagent / kit for detecting immune-related pneumonia by ELISA.

[0013] Preferably, enzyme-linked immunosorbent assay (ELISA) was used for quantitative verification: the expression level of SERPINA5 protein in the experimental group and the control group was quantitatively detected by ELISA. It was found that SERPINA5 protein was highly expressed in CIP patients, and there was a highly significant difference between the experimental group and the control group (p < 0.001). The optimal cutoff value for the detection of the serum polypeptide marker SERPINA5 in serum was 642.27 pg / mL, and the corresponding AUC was 0.908, p < 0.0001.

[0014] The present invention also discloses the application of molecules that bind to the aforementioned serum polypeptide marker SERPINA5 for immune-associated pneumonia in the preparation of serum diagnostic reagents / kits for immune-associated pneumonia.

[0015] The present invention also discloses a serum diagnostic reagent for immune-associated pneumonia, which contains the aforementioned serum polypeptide marker SERPINA5 for immune-associated pneumonia, or includes a molecule that binds to the aforementioned serum polypeptide marker SERPINA5 for immune-associated pneumonia.

[0016] The present invention also discloses a kit for predicting immune-associated pneumonia, the kit comprising the serum polypeptide marker SERPINA5 of claim 1, or comprising a molecule that binds to the serum polypeptide marker SERPINA5 of claim 1.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention proposes a highly efficient serum polypeptide marker, SERPINA5, which can predict the occurrence of CIP. Its amino acid sequence is: R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-, and its precise molecular weight is 3901.24 Daltons. It is a member of the serine protease inhibitor protein family and plays an important role in tissue repair and immune regulation activities such as pro-inflammatory and anti-inflammatory reactions, and participates in the occurrence and development of CIP. This invention quantitatively detected the expression levels of SERPINA5 in CIP patients and non-CIP patients using ELISA. It was found that SERPINA5 was significantly highly expressed in CIP patients, with expression levels as follows: CIP patients vs. non-CIP patients: 738.25±58.2 pg / ml (625.36~807.75 pg / ml) vs. 609.24±27.19 pg / ml (558.67~675.35 pg / ml), p<0.001. The optimal cutoff value for identifying CIP was 642.27 pg / ml, corresponding to an AUC of 0.908 (p<0.0001). In other words, SERPINA5 is a highly efficient potential serum peptide biomarker for predicting CIP. It has significant advantages such as low detection cost, high predictive efficacy, and convenient clinical translation, providing a new diagnostic and treatment strategy for screening high-risk CIP populations and improving the safety of immunotherapy. Attached Figure Description

[0019] Figure 1 The image shows differentially expressed protein and peptide peaks in CIP patients compared to non-CIP patients, as identified by MALDI-TOF-MS mass spectrometry analysis.

[0020] Figure 2 The expression difference of protein peptide M / Z:3901.24 between CIP patients (red, curve with peak at the top) and non-CIP patients (green, curve with peak at the bottom);

[0021] Figure 3 The HPLC-MS / MS mass spectrometry identification chromatogram of SERPINA5;

[0022] Figure 4 The results of ELISA quantitative analysis of SERPINA5;

[0023] Figure 5 ROC curves for identifying CIP occurrences in SERPINA5. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0026] This invention provides a specific predictive protein for CIP occurrence obtained based on serum proteomics mass spectrometry analysis: human plasma serine protease inhibitor (Serpin family A member 5, SERPINA5).

[0027] Its amino acid sequence is: R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-.

[0028] Clinical serum sample collection and processing: This invention includes 139 patients with malignant tumors who underwent ICIs for the first time at the First Affiliated Hospital of Xi'an Jiaotong University and Tangdu Hospital of Air Force Medical University from January 1, 2019 to December 31, 2021. Baseline whole blood samples were collected from patients before ICIs treatment. Factors such as age, gender, collection time, consistency of storage conditions, and presence of underlying diseases were fully considered to ensure that baseline conditions were basically consistent. Blood was collected from subjects in the morning on an empty stomach using vacuum blood collection tubes (red cap, with insulating gel, and no added coagulants, anticoagulants, or other additives). The blood was incubated at 4°C for 4 hours, and centrifuged within 8 hours to obtain serum. Centrifugation conditions were: 4°C, 3.0 rpm, 20 minutes. The supernatant serum was aliquoted into 100 μL / tube and immediately stored at -80°C, avoiding repeated freeze-thaw cycles. All patients were followed up for a minimum of 6 months. Based on the "Expert Consensus on the Management of Immune Checkpoint Inhibitor-Related Adverse Reactions" published by Chinese experts and the international NCCN guidelines for the management of immunotherapy-related toxicities (2021 edition), three radiologists with extensive clinical experience (at the associate chief physician level or above) assessed the included patients for CIP (Critical Illness Inhibition). Patients were ultimately divided into an experimental group (i.e., patients who developed CIP) and a control group (i.e., patients who did not develop CIP). Exclusion criteria were: (1) prior immune-related or immune-mediated therapy for the target lesion; (2) history of more than one primary malignant tumor; (3) lack of baseline characteristics and imaging evidence preventing dynamic follow-up; and (4) poor blood sample quality, such as severe hemolysis or low blood concentration. Ultimately, this invention included 117 patients with malignant tumors (87 males and 30 females). Fifty-four patients were randomly selected as the training set for mass spectrometry analysis (control group: 36 patients; experimental group: 18 patients), and 63 patients were selected as the validation set for ELISA (control group: 41 patients; experimental group: 22 patients).

[0029] Proteomics mass spectrometry analysis and identification: Serum proteins were extracted using IMAC-Cu ion magnetic beads, followed by primary mass spectrometry analysis using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain protein peptide peaks in patient serum. Statistical analysis revealed protein peptide peaks with significant differences between the experimental and control groups. Secondary proteomic identification was then performed on the screened differentially expressed protein peptide peaks to determine their corresponding proteins. Finally, the peptide with M / Z: 3901.24 Daltons was identified as SERPINA5 protein, which showed a highly significant difference between the experimental and control groups (p < 0.001).

[0030] Enzyme-linked immunosorbent assay (ELISA) quantitative validation: The expression level of SERPINA5 protein in the experimental group and the control group was quantitatively detected by ELISA. It was found that SERPINA5 protein was highly expressed in CIP patients, and there was a highly significant difference between the experimental group and the control group (p<0.001). The predictive power was 0.908, which can be used as a specific biomarker for predicting the occurrence of CIP.

[0031] In summary, this invention utilizes MALDI-TOF-MS to capture the protein and peptide profiles of CIP patients and those without CIP, and employs ClinProTools 2.1 software to compare and analyze the differences in serum protein and peptide profiles between the two groups, identifying the differentially expressed protein and peptide scores between the groups. ELISA is then used to quantitatively verify the expression differences between CIP patients and those without CIP. Ultimately, SERPINA5 protein, which is significantly highly expressed in the serum of CIP patients, was identified as a serum predictive biomarker for CIP. This method offers significant advantages such as low detection cost, high predictive efficacy, and convenient clinical translation, providing a new diagnostic and treatment strategy for screening high-risk individuals for CIP and improving the safety of immunotherapy.

[0032] The present invention will now be described in further detail with reference to the accompanying drawings:

[0033] 1. Clinical serum sample collection and processing

[0034] This invention includes 139 patients with malignant tumors who underwent ICIs for the first time at the First Affiliated Hospital of Xi'an Jiaotong University and Tangdu Hospital of Air Force Medical University between January 1, 2019 and December 31, 2021. Baseline whole blood samples were collected from patients before ICIs treatment. Factors such as age, gender, collection time, consistency of storage conditions, and presence of underlying diseases were carefully considered to ensure consistent baseline conditions. Blood was collected from subjects in the morning on an empty stomach using vacuum blood collection tubes (red cap, with insulating gel, and no added coagulants, anticoagulants, or other additives). The blood was incubated at 4°C for 4 hours, and centrifuged within 8 hours to obtain serum. Centrifugation conditions were: 4°C, 3.0 rpm, 20 minutes. The supernatant serum was aliquoted into 100 μL tubes and immediately stored at -80°C, avoiding repeated freeze-thaw cycles. All patients were followed up for a minimum of 6 months. Based on the "Expert Consensus on the Management of Immune Checkpoint Inhibitor-Related Adverse Reactions" published by Chinese experts and the international NCCN guidelines for the management of immunotherapy-related toxicities (2021 edition), three radiologists with extensive clinical experience (at the associate chief physician level or above) assessed the included patients for CIP (Critical Illness Inhibition). Patients were ultimately divided into an experimental group (i.e., patients who developed CIP) and a control group (i.e., patients who did not develop CIP). Exclusion criteria were: (1) prior immune-related or immune-mediated therapy for the target lesion; (2) history of more than one primary malignant tumor; (3) lack of baseline characteristics and imaging evidence preventing dynamic follow-up; and (4) poor blood sample quality, such as severe hemolysis or low blood concentration. Ultimately, this invention included 117 patients with malignant tumors (87 males and 30 females). Fifty-four patients were randomly selected as the mass spectrometry training set (control group: 36 patients; experimental group: 18 patients), and 63 patients were randomly selected as the ELISA validation set (control group: 41 patients; experimental group: 22 patients).

[0035] This study was conducted in accordance with the Declaration of Helsinki (2013 revision). All patients or their legal representatives voluntarily signed written informed consent before participating in the study. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (Approval No.: XJTU1AF2021LSK-001).

[0036] 2. Proteomics mass spectrometry analysis and identification

[0037] 2.1 Reagents and Instruments

[0038] Serum proteins were extracted using the magnetic bead kit "copper ion type" (IMAC-Cu) from Xiamen Primagene Biotechnology Co., Ltd., along with spectroscopically pure (HPLC grade) acetonitrile, trifluoroacetic acid (Merck, Germany), and α-cyano-4-hydroxycinnamic acid (HCCA) (Sigma, USA).

[0039] Magnetic bead separator, 600 / 384 AnchorChip target plate, and AutoFlex III matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker Daltonics, Germany).

[0040] 2.2 Preparation of serum protein samples

[0041] The specific steps for capturing serum protein peptides using copper ion-modified (IMAC-Cu) magnetic beads are as follows:

[0042] ①Use a mixer to thoroughly mix the magnetic bead suspension for 1 minute;

[0043] ② Add 10 μL of IMAC-Cu binding solution and 10 μL of IMAC-Cu magnetic beads to the PCR tube, mix well, then add 5 μL of serum, mix at least 5 times, and let stand for 5 min.

[0044] ③ Place the PCR tube into the magnetic column separator, allow the magnetic beads to adhere to the wall for 1 minute, and discard the supernatant after the liquid becomes clear;

[0045] ④ Add 100 μL of IMAC-Cu washing buffer, move the PCR tube back and forth 10 times on the magnetic column separator, discard the supernatant after the magnetic beads adhere to the wall, and repeat steps ③ and ④ twice.

[0046] ⑤ Add 5 μL of IMAC-Cu elution buffer to wash the adhered magnetic beads and repeatedly blow them 10 times. The magnetic beads adhered to the wall for 2 minutes. Transfer the supernatant into a clean centrifuge tube.

[0047] ⑥ Add 5 μL of IMAC-Cu stabilizing solution to the centrifuge tube and mix well. The extracted protein peptides can be used for direct MALDI-TOF-MS detection or frozen in a -20℃ freezer for up to 24 hours for mass spectrometry analysis.

[0048] 2.3 Mass Spectrometry Analysis

[0049] 1 μL of the isolated protein sample was mixed with 10 μL of the matrix α-cyano-4-hydroxycinnamic acid. 1 μL of the mixture was then spotted onto an Anchorchip target plate (Bruker, Germany), with three spots spotted for each sample for three replicates. After drying at room temperature, the target plate was placed in a mass spectrometer for analysis. Standard calibration was performed using FlexControl 2.0 software (Bruker, Germany) before sample detection began. Each sample underwent a total of 300 laser targeting cycles (5 spotting cycles, 2 × 30 cycles per cycle) to generate a mass spectrum, obtaining protein-peptide spectra composed of different mass-to-nucleus ratios (M / Z). ClinProTools 2.1 software (Bruker, Germany) combined with biostatistical and bioinformatics methods, including genetic algorithms, was used to analyze the protein-peptide spectra of the two serum samples. The total ion chromatogram was normalized and smoothed to eliminate chemical and electrophysical noise; differentially expressed proteins between groups were analyzed and the magnitude of the differences was calculated. The proteins were sorted from largest to smallest difference to identify the peaks of proteins with significant differences in expression between groups (p<0.05).

[0050] Serum samples from the CIP experimental group and the control group without CIP were processed using a magnetic bead separation system and then analyzed by MALDI-TOF-MS. Protein and peptide profiles were plotted for each sample from both groups. A total of 88 protein and peptide peaks were detected within the molecular weight range of 1000 Da to 10000 Da. The stability of the three replicates for each sample was high. Figure 1 As shown.

[0051] Serum protein and peptide profiles of CIP experimental group and non-CIP control group captured by mass spectrometry were analyzed using ClinProTools 2.1 software. The serum peptide profiles of CIP patients and non-CIP patients were compared. A protein peptide peak with a molecular weight of 3901.24 Daltons was detected as significantly highly expressed in the serum of CIP patients (CIP patients vs. non-CIP patients: 1.66±0.63 vs. 1.37±0.40, p=0.044). The results are as follows: Figure 2 As shown, the expression of M / Z:3901.24 in CIP patients (red, curve with peak at the top) and non-CIP patients (green, curve with peak at the bottom) was compared. It was found that its protein peptide peak was significantly highly expressed in the serum of CIP patients. Therefore, further sequence identification was carried out to identify it as the preferred biomarker.

[0052] 2.4 Sequence identification of potential CIP serum biomarkers

[0053] Specifically, liquid chromatography coupled with mass spectrometry (LC-MS) was used to identify the serum peptide marker M / Z:3901.24 in patients with CIP. Two-dimensional gel chromatography was used to separate the remaining serum protein peptides collected after magnetic bead separation and mass spectrometry loading. 15–30 peptide fractions were collected, and the target protein was detected in the collected solution. The sequence of the upregulated protein peptide M / Z:3901.24 in the serum of CIP patients was then identified using a Thermo Fisher LTQ Orbitrap XL mass spectrometry system.

[0054] The specific operating steps are as follows:

[0055] 2.4.1 Sample Pretreatment

[0056] Combine the extracted protein samples, reflux at 1300 rpm for 10 minutes, collect the supernatant, and freeze-dry to a final volume of 50 μL to obtain liquid A. Concentrate liquid A using an Agilent Ziptip extraction column. Treatment method: ① Activate the Ziptip column by blowing and aspirating it 5 times with 100% acetonitrile; ② Repeat the blowing and aspirating process 10 times with the activated Ziptip in liquid 1, minimizing bubble formation; ③ Wash the Ziptip column 3 times with a 50% ACN and 0.1% TFA aqueous solution; ④ Elute the sample by repeatedly blowing and aspirating the Ziptip column in 0.1% TFA to obtain eluent 2; ⑤ Repeat steps 1-4 above 30 times; ⑥ Combine the 30 eluents 2, freeze-dry to 10 μL, and use for mass spectrometry identification.

[0057] 2.4.2 Chromatographic separation:

[0058] Add 10 μL of mobile phase A to the original sample and transfer it to a vial, for a total of 20 μL.

[0059] One-dimensional ultra-high performance liquid chromatography system: Waters Nano Aquity UPLC (Waters Corporation, Milford, USA). Column:

[0060] Trapping column: C18,5μm,180μm×20mm,nanoAcquity TM Column

[0061] Analysis column: C18,3.5μm,75μm×150mm,nanoAcquity TM Column

[0062] Mobile phase A: an aqueous solution of 5% acetonitrile and 0.1% formic acid.

[0063] Mobile phase B: 95% acetonitrile, 0.1% formic acid aqueous solution; all solutions were HPLC grade.

[0064] The capture flow rate was 15 μL / min, the capture time was 3 min, the analysis flow rate was 400 nL / min, the analysis time was 60 min, the column temperature was 35 ℃, and the injection was performed in Partial Loop mode with an injection volume of 18 μL.

[0065] The gradient elution procedure is shown in Table 1 below:

[0066] Table 1 Gradient elution program

[0067]

[0068] MS / MS mass spectrometry identification chromatograms based on gel chromatography separation results are as follows: Figure 3 As shown in the figure. The horizontal axis of the chromatogram represents the sample elution time, and the vertical axis represents the relative abundance of peptides. The chromatographic setting time is 60 min, and the fractions are collected starting from 10 min. The peptide components are mainly separated after 15 min and gradient elution is used to improve the elution efficiency. The set capture time for collecting fractions is 15 to 30 peptide fractions.

[0069] 2.4.3 LTQ-Orbitrap XL mass spectrometry analysis:

[0070] A Thermo Fisher LTQ Obitrap XL mass spectrometry system was used. A nano ion source (Michrom Bioresources, Auburn, USA) was used with a spray voltage of 1.8 kV. The mass spectrometry scan time was 60 min. The experimental modes were data-dependent and dynamic exclusion. The precursor ion was cascaded twice within 10 seconds and then added to the exclusion list for 90 seconds. The scan range was 400-2000 m / z. The primary scan (MS) used the Obitrap with a resolution of 100,000. The CID and secondary scans used the LTQ. The 10 strongest ions from the MS spectrum were selected as single isotopes as precursor ions for MS / MS (single charge exclusion, not considered as precursor ions). The detection results are as follows: Figure 3 As shown.

[0071] Data Analysis: Using Bioworks Browser 3.3.1SP1 data analysis software for Sequest TMSearch. The parent ion error was set to 100 ppm, the fragment ion error was set to 1 Da, the enzyme digestion method was non-enzymatic digestion, and the variable modification was M (Methionine) methionine oxidation. The search result parameter was set to deltacn>=0.10. The search results were: (1) m / z: 3901.24; protein ID: P05154; Gene Symbol=SERPINA5; the sequence was R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-, which is a member of the serpin gene cluster located on the q arm of chromosome 14, and the protein encoded by this gene is a member of the serine protease inhibitor protein family. The isolated protein, M / Z: 3901.24, is designated as human plasma serine protease inhibitor (SERPINA5). Its precise molecular weight is 3901.24 Daltons, and its sequence is R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-. This protein belongs to the serine protease inhibitor family, whose members are glycoproteins widely expressed in multiple organs of the human body. They can inhibit various serine proteases, including protein C and various plasminogen activators and kallikrein, playing a role in physiological activities such as tissue repair and hemostasis. It also participates in the expression of pro-inflammatory and anti-inflammatory factors, thereby regulating the body's immune processes. SERPINA5 is an effective inhibitor of activated protein C (APC), an anti-inflammatory protein, indicating that SERPINA5 has a pro-inflammatory effect. Furthermore, SERPINA5 also participates in the innate immune response, regulating the expression of inflammatory factors and thus modulating the body's immune processes. Furthermore, during tissue repair and regeneration, the thrombin cascade involving SERPINA5 is crucial for activating growth factors in tissue damage responses. Increased thrombin production, along with the generation of pro-inflammatory cytokines and growth factors, can stimulate pulmonary vascular inflammation and thickening of the vessel walls, leading to vascular leakage and pulmonary tissue remodeling. This process may also play a role in CIP.

[0072] The results above suggest that SERPINA5 is a protein specifically associated with CIP and can serve as a potential predictive biomarker for CIP. Further quantitative detection using ELISA can be used to verify its clinical value as a predictive biomarker.

[0073] 3. Quantitative Validation Analysis Using Enzyme-Linked Immunosorbent Assay (ELISA)

[0074] 1) Serum samples: Serum samples were collected from 22 patients with CIP (14 males and 8 females) and 41 patients without CIP (27 males and 14 females) for ELISA quantitative validation analysis. All serum samples were obtained from the First Affiliated Hospital of Xi'an Jiaotong University and Tangdu Hospital of Air Force Medical University, and were collected from January 2019 to December 2021.

[0075] 2) Detection Method: Serum SERPINA5 expression levels in patients with CIP and those without CIP were detected using an enzyme-linked immunosorbent assay (ELISA). The kit was purchased from R&D Corporation, USA. The kit uses a one-step sandwich ELISA with double antibodies: The sample, standard, and HRP-labeled detection antibody were added sequentially to microwells pre-coated with anti-human SERPINA5 protein antibody, followed by incubation and thorough washing. The substrate TMB was used for color development; TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the amount of SERPINA5 protein in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the sample concentration was calculated. Specific experimental procedures were performed according to the kit instructions, and the positive judgment criteria were defined as per the kit instructions.

[0076] 3) Statistical methods: One-way ANOVA and independent samples t-tests were performed using GraphPad.Prism.v8 (GraphPad Software, La Jolla, CA, USA); ROC analysis and optimal cutoff value analysis were performed using IBM SPSS Statistics 25 (SPSS, Inc., Chicago, IL, USA).

[0077] 4) Quantitative Results Analysis: ELISA quantitative analysis showed that the expression levels of SERPINA5 in CIP patients and non-CIP patients were as follows: CIP patients vs. non-CIP patients: 738.25±58.2 pg / ml (625.36~807.75 pg / ml) vs. 609.24±27.19 pg / ml (558.67~675.35 pg / ml), p<0.001. Specific results are shown in Table 2. Figure 4 As shown above, SERPINA5 is a protein closely related to CIP.

[0078] Table 2. Serum SERPINA15 protein expression levels in different groups

[0079]

[0080] 5) Analysis of the optimal cutoff value: SPSS software was used to plot the ROC curve of SERPINA5, and the optimal cutoff value of SERPINA5 was determined based on the maximum Youden index to obtain the differential diagnostic efficacy of SERPINA5 between CIP patients and non-CIP patients (Youden index = sensitivity + specificity - 1). The final optimal cutoff value of SERPINA5 was 642.27 pg / ml, with a corresponding AUC of 0.908 (p < 0.0001). See below for detailed results. Figure 5 Patients were divided into high-level and low-level groups based on this boundary, and the risk of CIP was compared between the two groups. The results showed a statistically significant difference in the risk of CIP between the two groups (p < 0.001, 95% CI = 19.535–821.297), with an OR of 126.667. Specific results are shown in Table 3. These results indicate that SERPINA5 is a reliable biomarker for predicting the occurrence of CIP, with good disease differentiation ability. Individuals with high expression levels had a 126.667-fold increased risk of developing CIP compared to those with low expression levels.

[0081] Table 3. Regression analysis of SERPINA5 expression levels predicting CIP occurrence

[0082]

[0083]

[0084] In summary, this invention discloses a predictive serum peptide biomarker for the risk of CIP in patients with malignant tumors before immunotherapy and its clinical application. Its amino acid sequence is R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-, and the protein molecule is SERPINA5 with a precise molecular weight of 3901.24 Daltons. Quantitative detection of SERPINA5 expression levels in patients revealed its specific high expression in the serum of CIP patients. Therefore, it can serve as a specific and efficient predictive serum peptide biomarker for predicting the occurrence of CIP and screening high-risk individuals for CIP in patients with malignant tumors before immunotherapy.

[0085] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The application of SERPINA5, a serum polypeptide marker for immune-associated pneumonia, in the preparation of serum diagnostic reagents / kits for immune-associated pneumonia, characterized in that, The amino acid sequence of the serum polypeptide marker SERPINA5 is shown in SEQ ID NO:1, and its detection parameters in serum are 625.36~807.75 pg / mL.

2. The application as described in claim 1, characterized in that, The optimal cutoff value for the detection of the serum polypeptide marker SERPINA5 in serum was 642.27 pg / mL, corresponding to an AUC of 0.

908. p <0.0001.

3. The application as described in claim 1, characterized in that, The aforementioned serum diagnostic reagent / kit for immune-related pneumonia is a serum diagnostic reagent / kit for detecting immune-related pneumonia via ELISA.

4. The application as described in claim 3, characterized in that, The expression level of SERPINA5 protein was quantitatively detected using the ELISA method. SERPINA5 protein was highly expressed in CIP patients, with extremely significant differences.

5. The application of a molecule that binds to SERPINA5, a serum polypeptide marker of immune-associated pneumonia, in the preparation of serum diagnostic reagents / kits for immune-associated pneumonia, characterized in that, The amino acid sequence of the serum polypeptide marker SERPINA5 is shown in SEQ ID NO:1, and its detection parameters in serum are 625.36~807.75 pg / mL.

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