Biomarker for monitoring kidney cancer immunotherapy responsiveness, kit and application

Abstract

The invention discloses a biomarker for monitoring kidney cancer immunotherapy responsiveness, a kit and application, and relates to the technical field of biomedicine. The invention provides a biomarker combination for monitoring the treatment responsiveness of a kidney cancer patient to an immune checkpoint inhibitor. The biomarker combination comprises CDH4 and HAVCR1; detecting the expression level or expression quantity of CDH4 and HAVCR1 in a sample to be detected, and monitoring the responsiveness of the kidney cancer patient to the treatment of the immune checkpoint inhibitor. Starting from the nature of tumor cell heterogeneity, the CDH4 + HAVCR1 + tumor cell subpopulation closely related to immunotherapy drug resistance is identified through a single cell sequencing technology, and a monitoring system developed based on the CDH4 + HAVCR1 + tumor cell subpopulation shows high accuracy in a verification queue and is superior to indexes such as traditional PD-L1 expression. A serological detection method based on peripheral blood is innovatively developed, serum detection has the advantages of being noninvasive, convenient and fast and capable of achieving dynamic monitoring, and the method is easier to popularize clinically and applied to rapid evaluation in treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a biomarker, reagent kit, and application for monitoring the response of renal cancer to immunotherapy. Background Technology

[0002] Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system, with clear cell RCC (ccRCC) accounting for the vast majority. Traditional radiotherapy and chemotherapy have limited effectiveness in treating advanced or metastatic renal cell carcinoma. In recent years, immune checkpoint inhibitors (ICIs), represented by inhibitors of programmed death receptor-1 (PD-1) and its ligand (PD-L1), have made groundbreaking progress in the treatment of advanced renal cell carcinoma, significantly prolonging the survival of some patients.

[0003] However, clinical practice shows that only about 20-40% of renal cell carcinoma patients can benefit from immunotherapy in the long term, with most patients exhibiting primary or secondary drug resistance. This leads to a waste of medical resources and may subject patients to unnecessary drug side effects, even delaying effective treatment. Therefore, finding biomarkers that can accurately and conveniently monitor the effectiveness of immunotherapy and achieve precise patient stratification is a key issue that urgently needs to be addressed in clinical practice.

[0004] Currently, biomarkers explored in clinical practice mainly include PD-L1 expression levels in tumor tissue, tumor mutational burden (TMB), and microsatellite instability (MSI). However, these biomarkers have limited value in renal cell carcinoma and are not suitable for monitoring. The high complexity of the tumor microenvironment (TME) is considered a core factor affecting immunotherapy response. From the perspective of tumor cell heterogeneity, identifying and quantifying tumor cell subsets with drug resistance characteristics holds promise for providing a more direct and reliable basis for drug resistance monitoring.

[0005] CDH4 (Cadherin-4, also known as R-cadherin) is a calcium-dependent cell adhesion molecule involved in intercellular connections and tissue structure maintenance. Its aberrant expression in various cancers is associated with invasion and metastasis. HAVCR1 (Hepatitis AVirus Cellular Receptor 1, also known as KIM-1 / TIM-1) is an immunomodulatory molecule highly expressed in damaged renal tubular epithelial cells and is also a biomarker of acute kidney injury. Currently, no studies have used the combination of CDH4 and HAVCR1 as a biomarker for immunotherapy in renal cell carcinoma.

[0006] The information disclosed above in this background section is only for enhancing the understanding of the background technology of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and provide a novel biomarker, detection method, and kit for more accurately monitoring the response of renal cell carcinoma patients, especially those with ccRCC, to immune checkpoint inhibitor therapy such as PD-1 / PD-L1 inhibitors. The purpose of this invention is to assist clinicians in identifying patients who may benefit from immunotherapy or who are at risk of drug resistance before treatment, thereby developing personalized treatment plans, identifying secondary drug resistance, avoiding ineffective treatment, and improving patient prognosis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention is the first to propose using the combination of CDH4 and HAVCR1 as a biomarker for monitoring the response to immunotherapy in renal cell carcinoma. By detecting the expression levels of CDH4 and HAVCR1 in the patient's tumor tissue, the patient's response to immune checkpoint inhibitor therapy can be monitored.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a combination of biomarkers for monitoring the response of renal cell carcinoma patients to immune checkpoint inhibitor therapy, including CDH4 and HAVCR1; the response of the renal cell carcinoma patients to the immune checkpoint inhibitor therapy is monitored by detecting the expression levels or amounts of CDH4 and HAVCR1 in tumor tissue.

[0012] Furthermore, the detection of the expression levels of CDH4 and HAVCR1 in the sample to be tested includes detecting the expression of CDH4 and HAVCR1 at the protein level.

[0013] Furthermore, the detection of the expression levels of CDH4 and HAVCR1 in the sample to be tested includes detecting the concentration of HAVCR1 in serum or plasma.

[0014] Secondly, the present invention provides a detection method based on the above-mentioned combination of markers, comprising the following steps:

[0015] Step 1: Obtain biological samples from the patient with renal cell carcinoma to be tested, including but not limited to tumor tissue, single-cell suspensions prepared from tumor tissue, peripheral blood serum or plasma;

[0016] Step 2: Detect the expression levels or amounts of CDH4 and HAVCR1 in the biological sample;

[0017] Step 3: Compare the test results with clinically validated preset thresholds to determine the patient's secondary resistance to immune checkpoint inhibitor therapy: When the expression levels or amounts of CDH4 and HAVCR1 exceed the preset thresholds, it indicates that the patient has a high risk of resistance to immunotherapy; when they are below the preset thresholds, it indicates that the patient is more likely to benefit from immunotherapy and belongs to the potential response population.

[0018] Furthermore, flow cytometry is suitable for single-cell suspensions of tumor tissue biopsy specimens during patient immunotherapy. Cell surface antibody staining is performed (protected from light on ice), and data are acquired using a high-parameter flow cytometer (BD) and FlowJo software. Tumor cell subsets (live cells - single cells - CD45) are analyzed through continuous gating. - CD31 - CAIX + Identify and statistically analyze CDH4 + HAVCR1 + CAIX + CAIX + The percentage of tumor cells. Determined through receiver operating characteristic (ROC) curve analysis, CDH4 + HAVCR1 + CAIX + More than CAIX + The 27% probability of high resistance to immune checkpoint inhibitor therapy indicates a high odds ratio (OR) of 5.250, with a 95% confidence interval of 1.069–25.789 (p<0.001). The CAIX flow cytometry antibody was obtained from Invitrogen (MA5-16318), the CD45 flow cytometry antibody from Invitrogen (clone 58-0459-42), the CD31 flow cytometry antibody from Invitrogen (clone 14-0319-82), the CDH4 flow cytometry antibody from Invitrogen (clone 23GB5055), and the HAVCR1 flow cytometry antibody from Invitrogen (clone P365D).

[0019] Furthermore, immunohistochemistry (IHC) is applicable to paraffin-embedded or frozen tissue sections. Serial sections or the same section are stained with anti-CDH4 and anti-HAVCR1 antibodies to assess the proportion of CDH4 and HAVCR1 positive cells within the tumor region. A high proportion of CDH4 and HAVCR1 positive cells in the tumor tissue indicates a high risk of resistance to immune checkpoint inhibitor therapy. The CDH4 immunohistochemical antibody was derived from Invitrogen (OTI4H2), and the HAVCR1 immunohistochemical antibody was derived from CST (E1R9N).

[0020] Furthermore, serological testing is applicable to peripheral blood samples. The protein concentration of HAVCR1 in serum or plasma is quantitatively detected using a highly sensitive immunoassay method. An optimal concentration cutoff value for distinguishing between tumor regression and progression is determined by establishing a receiver operating characteristic (ROC) curve. Exemplary data show that serum HAVCR1 concentrations are significantly higher in the progression phase than in the response group.

[0021] Thirdly, the present invention also provides a dedicated kit for implementing the above detection method, the kit containing reagents capable of specifically detecting the expression level or amount of CDH4 and / or HAVCR1.

[0022] Furthermore, when used for tissue or cell sample testing, the kit comprises: a first antibody that specifically recognizes CDH4, a second antibody that specifically recognizes HAVCR1, and a corresponding detection system; the first antibody is an anti-CDH4 monoclonal antibody, and the second antibody is an anti-HAVCR1 monoclonal antibody; the detection system comprises an enzyme-labeled or fluorescently labeled secondary antibody and a corresponding substrate.

[0023] Furthermore, when used for serum or plasma sample testing, the kit is a chemiluminescent immunoassay kit based on a double-antibody sandwich method, and its main components include:

[0024] Solid-phase carriers coated with anti-HAVCR1 monoclonal antibodies (capture antibodies), such as magnetic microparticles and microplates;

[0025] A chemiluminescent anti-HAVCR1 monoclonal antibody (detection antibody) is labeled with a chemiluminescent material to recognize epitopes different from those of the capture antibody; the chemiluminescent label is selected from acridinium ester, alkaline phosphatase, horseradish peroxidase, and ruthenium tripyridine.

[0026] Chemiluminescent substrate solution (corresponding to the chemiluminescent label, when the label is AE, the substrate solution is preferably hydrogen peroxide and sodium hydroxide; when the label is ALP, the substrate solution is preferably AMPPD and its derivatives; when the label is HRP, the substrate solution is preferably a mixture of luminol and its derivatives with hydrogen peroxide; when the label is ruthenium terpyridine, the substrate solution is preferably a mixture containing tripropylamine and other co-reactants), washing solution, sample diluent, and a series of HAVCR1 calibrators of known concentrations are used to establish a standard curve and quantify unknown samples.

[0027] Fourthly, the present invention also provides the use of the biomarker combination or kit in the preparation of in vitro diagnostic products for monitoring the response to immunotherapy for renal cell carcinoma.

[0028] Fifthly, the present invention also provides a non-volatile computer-readable storage medium storing a computer program, which, when executed by a processor, implements step 3 of the above prediction method, namely, outputting a prediction result of the response to immune checkpoint inhibitor treatment based on the input expression data of CDH4 and HAVCR1.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. This invention, starting from the essence of tumor cell heterogeneity, identifies CDH4, which is closely related to immunotherapy resistance, through single-cell sequencing technology. + HAVCR1 + The prediction system developed based on tumor cell subpopulations showed high prediction accuracy in the validation cohort.

[0031] 2. This invention not only provides a detection scheme based on tumor tissue, but also innovatively develops a serological detection method based on peripheral blood. Serum detection has the advantages of being non-invasive, convenient, and dynamically monitorable, making it easier to promote in clinical practice and apply to the evaluation of treatment.

[0032] 3. This invention provides specific detection methods, detailed antibody information, and validated discrimination thresholds. Through this invention, the regression phase and the progression phase can be effectively distinguished, enabling clinicians to prioritize immunotherapy for patients who may benefit, while exploring other combination treatment options or alternative therapies for high-risk drug-resistant patients. This truly achieves precision medicine for renal cell carcinoma, saves medical costs, and improves patients' quality of life. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0034] Figure 1 CDH4 identified by single-cell transcriptome sequencing analysis + HAVCR1 + A schematic diagram of tumor cell subsets (UMAP diagram).

[0035] Figure 2 This is a schematic diagram of flow cytometry analysis results, showing CDH4 levels in tumor tissues of patients in the regression and progression phases before treatment. + HAVCR1 + CAIX + Differences in cell proportions.

[0036] Figure 3This is a schematic diagram of the immunohistochemical staining results, comparing representative tissue section images from patients with positive CDH4 and HAVCR1 expression with those of patients with double negative expression.

[0037] Figure 4 The high predictive power of HAVCR1 concentration changes at different stages of treatment and its receiver operating characteristic curves were demonstrated. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] Example 1: CDH4 + HAVCR1 + Single-cell transcriptome identification of drug-resistant tumor subsets

[0040] We collected paired samples from 10 patients with advanced clear cell renal cell carcinoma: Group A consisted of fresh tumor tissue obtained by biopsy when the immune checkpoint inhibitor (anti-PD-1 monoclonal antibody) treatment was effective; Group B consisted of fresh tumor tissue obtained by re-biopsy when the same patient's disease progressed (drug resistance) after treatment.

[0041] Immediately after ex vivo, the tissues were placed in pre-cooled tissue preservation solution. Enzymatic digestion and gentle mechanical disruption were performed using a GentleMACS tissue dissociator and a tumor tissue dissociation kit. Single-cell suspensions were obtained after filtration through a 70 μm filter and then subjected to single-cell transcriptome sequencing.

[0042] Cells were grouped based on gene expression profiles using bioinformatics analysis. By comparing the gene expression profiles of tumor cell populations in groups A and B, and employing machine learning algorithms, gene features significantly enriched in post-drug-resistant samples were identified. Ultimately, a tumor cell subpopulation simultaneously highly expressing CDH4 and HAVCR1 (KIM-1) was identified. This subpopulation showed a significant increase in proportion in post-treatment drug-resistant samples (e.g., ...). Figure 1 (As shown).

[0043] Example 2: Flow cytometry validation and determination of critical values

[0044] A separate validation cohort of 31 patients with advanced ccRCC who were ineligible for surgery and had received first-line anti-PD-1 / PD-L1 therapy was also included. Pre-treatment tumor biopsy specimens were collected from 13 patients in remission, and post-treatment tumor biopsy specimens were collected from 20 patients whose disease progressed.

[0045] Take single-cell suspensions and perform surface staining with the following antibody combinations (protected from light on ice):

[0046] Lineage antibodies: FITC-labeled mouse anti-human CD45 antibody (clone 58-0459-42, Invitrogen) and PerCP-Cy5.5-labeled mouse anti-human CD31 antibody (clone 14-0319-82, Invitrogen).

[0047] Tumor marker antibody: APC-labeled mouse anti-human CAIX antibody (clone MA5-16318, Invitrogen).

[0048] Target antibodies: PE-labeled mouse anti-human CDH4 antibody (clone 23GB5055, Invitrogen) and BV421-labeled mouse anti-human HAVCR1 antibody (clone P365D, Invitrogen).

[0049] At the same time, isotype control and monochromatic compensation control were set up.

[0050] Data were acquired using a high-parameter flow cytometer. Analysis was performed using FlowJo software. The gating strategy was as follows: first, viable cells were delineated (dead cells were excluded based on live / dead staining), and single cells were delineated (FSC-A vs FSC-H); second, CD45 was excluded from single cells. + immune cells and CD31 + Endothelial cells, to obtain CD45 - CD31 - The stromal cell population; finally, CAIX cells were delineated within this population. + The tumor cells were analyzed, and CDH4 was found within them. + HAVCR1 + The proportion of double-positive cells (e.g.) Figure 2 (As shown).

[0051] According to RECIST 1.1 criteria, efficacy was assessed 3-6 months after treatment, and patients were divided into an objective response group (OR, including CR, PR, and SD) and a disease progression group (PD). The levels of CDH4 in tumors were compared between the two groups. + HAVCR1 + CAIX + Differences in cell proportions. Using the Mann-Whitney U test, the results showed that the proportion in the PD group was significantly higher than that in the OR group (p<0.01). ROC curves were plotted, and the optimal cutoff value was determined using the maximum Yoden index. The results indicate that when CDH4... + HAVCR1 + CAIX + Cells account for all CAIX +When the proportion of tumor cells exceeded 27%, the odds ratio (OR) for immunotherapy resistance was 5.250, with a 95% confidence interval (1.069–25.789), p<0.001.

[0052] Example 3: Immunohistochemical detection kit

[0053] An immunohistochemical assay kit contains:

[0054] Rabbit anti-human CDH4 monoclonal antibody (clone E1R9N, CST) and mouse anti-human HAVCR1 monoclonal antibody (clone OTI4H2, Invitrogen).

[0055] HRP polymer secondary antibodies corresponding to different species, and tyramine signal amplification reagents for signal amplification and labeling with different fluorescent dyes;

[0056] Antigen retrieval solution, antibody dilution solution, washing buffer, mounting medium, and known positive and double-negative control tissue sections.

[0057] Serial sections of paraffin-embedded renal cell carcinoma tissue were obtained from patients during treatment and subjected to standard dewaxing, hydration, and antigen retrieval. CDH4 primary antibody and the corresponding HRP secondary antibody were incubated sequentially, followed by microwave inactivation of HRP; then HAVCR1 primary antibody and the corresponding HRP secondary antibody were incubated sequentially.

[0058] High-resolution imaging was performed using a full-slide fluorescence scanner. Tumor regions were automatically identified and CDH4 counts were performed using image analysis software. + and HAVCR1 + The tumor cells. When the percentage of positive tumor cells exceeds 28.7% under multiple high-power fields, the patient is considered to be at high risk of immunotherapy resistance (e.g., ...). Figure 3 (As shown).

[0059] Example 4: Serum HAVCR1 Chemiluminescence Detection Kit

[0060] A double-antibody sandwich assay kit based on magnetic microparticles and acridil ester chemiluminescence, comprising:

[0061] Capture antibody: Mouse anti-human HAVCR1 monoclonal antibody was covalently coupled to magnetic microparticles;

[0062] Antibody detection: Another mouse anti-human HAVCR1 monoclonal antibody that recognizes a different epitope was labeled with acridinium ester;

[0063] Calibrators: Recombinant human HAVCR1 protein was formulated into 6 calibrators at known concentrations;

[0064] Washing solution, sample diluent, chemiluminescent substrate solution A (containing H2O2), and chemiluminescent substrate solution B (containing NaOH).

[0065] Testing steps:

[0066] a. Mix 50 μL of calibrator or 1:10 diluted serum with magnetic microparticles coated with capture antibody and incubate at 37°C for 30 minutes.

[0067] b. Wash the particles three times with washing solution on the magnetic separator;

[0068] c. Add acridinium ester-labeled detection antibody and incubate at 37°C for 30 minutes;

[0069] d. Wash again 3 times;

[0070] e. Resuspend the microparticles in a mixture of chemiluminescent substrate solutions A and B, and immediately measure the relative luminescence unit value on a chemiluminescent immunoassay analyzer;

[0071] f. Plot the calibrator concentration on the x-axis and the RLU value on the y-axis to fit a four-parameter logistic curve. Calculate the HAVCR1 concentration from the standard curve based on the RLU value of the sample to be tested.

[0072] Serum samples were collected from 33 patients with advanced renal cell carcinoma at different stages of immunotherapy. The results showed that the median serum HAVCR1 concentration in the progressive stage (PD) was 1645 pg / mL (interquartile range: 1032.5-5530 pg / mL), which was significantly higher than that in the tumor regression stage (CR / PR / SD) at 220 pg / mL (interquartile range: 135-300 pg / mL), p<0.001 (Mann-Whitney U test).

[0073] Plotting ROC curves (e.g.) Figure 4 (As shown). The area under the curve was 0.989 (95% confidence interval: 0.9740–1.000), p < 0.0001. The optimal cutoff value was determined to be 695 pg / mL, at which the sensitivity for monitoring drug resistance was 88.24% and the specificity was 91.18%. This indicates that an increase in serum HAVCR1 concentration during treatment suggests treatment resistance and is a highly promising non-invasive predictive indicator.

[0074] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A combination of biomarkers for monitoring the response to immunotherapy in renal cell carcinoma, characterized in that: The biomarker combination includes CDH4 and HAVCR1; the response of the renal cell carcinoma patients to the immune checkpoint inhibitor therapy is monitored by detecting the expression levels or amounts of CDH4 and HAVCR1 in the test samples.

2. A biomarker for monitoring the response to immunotherapy in renal cell carcinoma according to claim 1, characterized in that: The detection of the expression levels of CDH4 and HAVCR1 in the sample to be tested includes detecting the co-expression of CDH4 and HAVCR1 at the protein level.

3. A biomarker for monitoring the response to immunotherapy in renal cell carcinoma according to claim 1, characterized in that: The detection of the expression levels of CDH4 and HAVCR1 in the sample to be tested includes detecting the concentration of HAVCR1 in serum or plasma.

4. A method for monitoring the response of renal cell carcinoma patients to immune checkpoint inhibitor therapy, characterized in that, Includes the following steps: Step 1: Obtain biological samples from the patient with renal cell carcinoma to be tested, including but not limited to tumor tissue, single-cell suspensions prepared from tumor tissue, peripheral blood serum or plasma; Step 2: Detect the expression levels or amounts of CDH4 and HAVCR1 in the biological sample; Step 3: Compare the test results with clinically validated preset thresholds to determine the patient's resistance to immune checkpoint inhibitor therapy: when the expression levels or amounts of CDH4 and HAVCR1 are higher than the preset thresholds, it indicates that the patient has a high risk of resistance to immunotherapy; when they are lower than the preset thresholds, it indicates that the patient is more likely to continue to benefit from immunotherapy.

5. The method according to claim 4, characterized in that: The biological sample is a tumor tissue sample or a single-cell suspension; in step 2, the expression of CDH4 and HAVCR1 proteins is detected by flow cytometry or immunohistochemistry; when using flow cytometry, the expression of CD45 is detected. + CD31 + CAIX + In the tumor cell population, CDH4 + HAVCR1 + CAIX + The proportion of cells is used to determine this; when the proportion is greater than CAIX + When tumor cells reach 27%, it indicates a high risk of drug resistance.

6. The method according to claim 4, characterized in that: The biological sample is a serum or plasma sample; in step 2, the concentration of HAVCR1 protein is detected by immunological detection methods.

7. A kit for implementing the method according to any one of claims 4-6, characterized in that, The kit contains reagents capable of specifically detecting the expression level or amount of CDH4 and / or HAVCR1.

8. The reagent kit according to claim 7, characterized in that: When used for tissue or cell sample testing, the kit comprises: a first antibody that specifically recognizes CDH4, a second antibody that specifically recognizes HAVCR1, and a corresponding detection system; the first antibody is an anti-CDH4 monoclonal antibody, and the second antibody is an anti-HAVCR1 monoclonal antibody; the detection system comprises a fluorescently labeled or enzyme-labeled secondary antibody and a corresponding substrate; When used for serum or plasma sample testing, the kit is a chemiluminescent immunoassay kit based on a double-antibody sandwich method, comprising: a solid-phase carrier coated with an anti-HAVCR1 capture antibody and an anti-HAVCR1 detection antibody labeled with a chemiluminescent marker; the chemiluminescent marker is selected from acridine ester, alkaline phosphatase, horseradish peroxidase or ruthenium tripyridine derivative.

9. The use of the combination of biomarkers according to any one of claims 1-3 in the preparation of a diagnostic product for monitoring the response of patients with renal cell carcinoma to immune checkpoint inhibitor therapy.

10. A non-volatile computer-readable storage medium storing a computer program, characterized in that, When the computer program is executed by the processor, it implements step 3 of the method according to any one of claims 4-6, namely, outputting a prediction result of the response to immune checkpoint inhibitor treatment based on the input expression data of CDH4 and HAVCR1.

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