Use of slamf1 as a biomarker in predicting response to immunotherapy

Abstract

The application proposes the application of SLAMF1 as a biomarker in predicting the response of immunotherapy, belonging to the field of biotechnology. The application proposes to use SLAMF1 as a functional predictive biomarker of the response of immunotherapy, and finds that the prediction of the response of immunotherapy based on the expression amount of SLAMF1 has high accuracy and wide applicability, and can predict the curative effect before or in the early stage of treatment, and reduce the economic burden and adverse reaction risk brought by ineffective treatment.

Description

Technical Field

[0001] This application belongs to the field of biotechnology. Specifically, this application relates to the application of SLAMF1 as a biomarker in predicting immunotherapy response, the application of SLAMF1 in preparing immunotherapy response biomarker reagents, and kits. Background Technology

[0002] In recent years, immunotherapy, especially immune checkpoint inhibitors targeting programmed death receptor-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and lymphocyte activation gene-3 (LAG-3), has become an important treatment for various malignant tumors. This type of therapy, by relieving the immunosuppressive microenvironment and restoring the body's own immune system's ability to kill tumor cells, has significantly prolonged patient survival in various tumors such as melanoma, lung cancer, and liver cancer. However, in clinical practice, only a portion of patients can benefit from this therapy in the long term, with most exhibiting primary or secondary resistance. Therefore, developing biomarkers that can predict immunotherapy response and resistance risk is crucial for achieving personalized precision treatment and avoiding the toxic side effects and economic waste caused by ineffective treatment.

[0003] Currently, commonly used predictive biomarkers in clinical practice include tumor cell PD-L1 expression levels, tumor mutational burden (TMB), and microsatellite instability (MSI). However, these biomarkers have limited predictive efficacy (e.g., PD-L1 expression exhibits spatiotemporal heterogeneity, and some PD-L1-negative patients can still benefit), leading to insufficient accuracy in predicting immunotherapy responses in some patients. Summary of the Invention

[0004] This application is based on the inventor's discovery of the following problems: Although SLAMF1 (CD150) is widely recognized as an important immune costimulatory molecule, it is expressed on the surface of various immune cells such as B cells and T cells and participates in lymphocyte activation and signal transduction. However, related technologies are only at the level of detecting SLAMF1 expression levels. The intrinsic relationship between SLAMF1 and the clinical efficacy and drug resistance risk of solid tumor patients after receiving immunotherapy has not yet been revealed, nor have clear evaluation criteria been formed to guide clinical medication decisions. As a result, the accuracy and practicality of predicting immunotherapy response are significantly limited.

[0005] Based on the above findings, this application proposes the application of SLAMF1 as a biomarker in predicting immunotherapy response, thereby improving the accuracy of immunotherapy response prediction.

[0006] Specifically, the technical solution of this application is as follows: Firstly, this application proposes the use of SLAMF1 as a biomarker in predicting immunotherapy response.

[0007] This application is the first to propose SLAMF1 as a functional predictive biomarker for immunotherapy response, and finds that predicting immunotherapy response based on SLAMF1 expression level is highly accurate and widely applicable. It can predict efficacy before or in the early stages of treatment, reducing the economic burden and adverse reaction risks caused by ineffective treatment.

[0008] Secondly, this application proposes the application of SLAMF1 in the preparation of immunotherapy response biomarker reagents, including: preparing diagnostic reagents for immunotherapy response using SLAMF1 as a target, or preparing prognostic evaluation reagents for immunotherapy response, or preparing reagents for predicting the survival time of target subjects.

[0009] The inventors discovered that the expression status of SLAMF1 can reflect the functional status of immune cells and the degree of remodeling of the immune microenvironment, and it exhibits a stable correlation with the efficacy of immunotherapy and the risk of drug resistance. Therefore, by introducing SLAMF1 into the target system of immunotherapy response-related reagents, the prepared reagents can comprehensively assess the efficacy of immunotherapy, disease progression trends, and survival outcomes in target subjects. Thus, the reagents can not only be used for pre-treatment screening of patients who will benefit from treatment, but also for dynamic monitoring and prognostic stratification during treatment, thereby providing an objective basis for the selection and adjustment of clinical treatment plans and improving the overall clinical benefit rate of immunotherapy.

[0010] Thirdly, this application proposes a kit. According to an embodiment of this application, the kit includes an antibody that specifically recognizes SLAMF1.

[0011] By utilizing this antibody to specifically detect SLAMF1 in target samples, the kit enables accurate identification and quantitative analysis of SLAMF1-positive immune cells. Since the expression level of SLAMF1 and the proportion of positive immune cells are stably correlated with immunotherapy response status and drug resistance risk, the kit can directly use SLAMF1 detection results for immunotherapy response prediction, risk stratification, and prognostic assessment. Therefore, the kit not only improves the specificity and sensitivity of SLAMF1 detection but also gives the detection results clear clinical guidance significance, thereby enhancing the accuracy and operability of personalized immunotherapy decisions.

[0012] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 A schematic diagram illustrating the process of SLAMF1 in predicting the response to immunotherapy for liver cancer, as provided in an embodiment of this application; Figure 2 CD150 in patient peripheral blood and paired tumor tissue provided in the embodiments of this application + Schematic diagram of cell proportion results; where A is the flow cytometry result; B is the immunohistochemistry result; C is the correlation diagram between flow cytometry and immunohistochemistry results; Figure 3 A schematic diagram illustrating the prediction results of different prediction indicators provided in the embodiments of this application; Figure 4 CD150 provided for embodiments of this application + Schematic diagram showing the expression of B cells in tumor tissue. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0016] 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 non-exclusive inclusion; for example, a process, method, system, product, or server 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 devices.

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] In this application, "SLAMF1," also known as CD150, is a member of the SLAM family and a transmembrane immunoglobulin superfamily molecule. It is mainly expressed on the surface of immune cells such as B cells, T cells, dendritic cells, and monocytes, and participates in immune cell activation, proliferation, intercellular interactions, and signal transduction. In this application, SLAMF1 serves as a functional biomarker for immunotherapy response, and its expression status is used to predict the efficacy of immunotherapy, drug resistance risk, and prognostic outcomes in target subjects.

[0019] In the embodiments of this application, "SLAMF1 positive immune cells" refers to immune cells that are confirmed by detection methods to express SLAMF1 protein or the nucleic acid sequence encoding SLAMF1 on their surface or inside the cells, including but not limited to B cells, T cells and their subsets.

[0020] In the embodiments of this application, "immune cells" include a population of cells that participate in the body's immune response, specifically including but not limited to B cells, T cells, natural killer cells, dendritic cells, and monocytes. In this application, the immune cells are preferably B cells or T cells.

[0021] In the embodiments of this application, "immunotherapy" refers to a treatment method that enhances the anti-tumor immune response by modulating the body's immune system, including but not limited to immune checkpoint inhibitor therapy. The immunotherapy described in this application includes at least one of anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-LAG-3 immunotherapies.

[0022] In the embodiments of this application, "anti-PD-1 immunotherapy" refers to an immune checkpoint inhibitor therapy targeting programmed death receptor 1 (PD-1), which restores the anti-tumor activity of T cells by blocking the PD-1 signaling pathway.

[0023] In the embodiments of this application, "anti-PD-L1 immunotherapy" refers to an immune checkpoint inhibitor therapy targeting programmed death-ligand 1 (PD-L1), which enhances the anti-tumor immune response by blocking PD-L1-mediated immunosuppressive signals.

[0024] In the embodiments of this application, "anti-CTLA-4 immunotherapy" refers to an immune checkpoint inhibitor therapy targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) to relieve immunosuppression during the initial activation phase of T cells.

[0025] In the embodiments of this application, "anti-LAG-3 immunotherapy" refers to an immune checkpoint inhibitor therapy targeting lymphocyte activation gene 3 (LAG-3) to relieve T cell depletion-related immunosuppressive signals.

[0026] In the embodiments of this application, "target subject" refers to a subject who has received or is to receive immunotherapy, including but not limited to patients with solid tumors.

[0027] Application of SLAMF1 as a biomarker in predicting immunotherapy response Firstly, this application provides the application of SLAMF1 as a biomarker in predicting immunotherapy response. Traditional immunotherapy assessment methods often rely on quantitative analysis of immune cells in tumor tissue or predict efficacy using single immune checkpoint molecules such as PD-L1. However, these methods fail to fully consider the functional state of immune cells and their dynamic changes during immunotherapy, thus limiting their ability to predict immunotherapy efficacy and drug resistance risk. This application, by introducing SLAMF1, an important co-stimulatory molecule in the immune microenvironment, fills the gaps in the assessment of immune cell functional state in existing technologies, effectively predicting the efficacy and drug resistance risk of immunotherapy, and providing precise guidance for the development of personalized immunotherapy regimens.

[0028] In an exemplary embodiment, the immunotherapy of this application includes at least one of anti-PD-1 immunotherapy, anti-PD-L1 immunotherapy, anti-CTLA-4 immunotherapy, and anti-LAG-3 immunotherapy.

[0029] It is understood that the aforementioned immunotherapy modalities are merely examples, not an exhaustive list. The protocol described in this application is applicable to predicting the efficacy of monotherapy and combination therapy.

[0030] It is understandable that the technical solution of this application, in addition to its application in cancer immunotherapy, can also be extended to the treatment of other immune-related diseases, especially those involving immune resistance. By assessing the proportion of SLAMF1-positive immune cells, a new biomarker can be provided for predicting the efficacy of immunotherapy, determining drug resistance, and assessing prognostic survival, thus offering patients more precise and personalized treatment plans. Compared with existing technologies, this solution, by introducing SLAMF1, a dynamic regulator of the immune microenvironment, overcomes the limitation of traditional biomarkers that only reflect static immune status, significantly improving the accuracy and reliability of predicting immunotherapy responses.

[0031] In exemplary embodiments, the proportion of SLAMF1-positive immune cells in immune cells is determined by detecting the protein or nucleic acid levels of SLAMF1 in the target sample. This proportion reflects the enrichment of SLAMF1-positive immune cells in the immune microenvironment, thus providing a basis for prognostic assessment of immunotherapy. In the embodiments of this application, the detection results of the proportion of SLAMF1-positive immune cells not only have high sensitivity and specificity, but also dynamically reflect the possible remodeling and functional changes of immune cells during immunotherapy. Unlike traditional static phenotypic analysis, this approach provides clinicians with a more guiding biomarker by quantifying the proportion of SLAMF1-positive immune cells, assisting in determining whether the target subject has a good capacity for immunotherapy response.

[0032] The target sample sources mentioned above include, but are not limited to, the target object's blood or tumor tissue. In an exemplary preferred embodiment, the target object's blood is selected as the sample source to avoid the problems of difficulty in obtaining tumor tissue samples or the existence of heterogeneity.

[0033] The determination of SLAMF1 protein levels in the above-mentioned target samples includes, but is not limited to: flow cytometry, immunohistochemistry, Western blotting, or liquid microarray.

[0034] For the detection of SLAMF1 nucleic acid levels, quantitative analysis of the mRNA encoding SLAMF1 can be performed, such as quantitative PCR (qPCR) and RNA sequencing (RNA-seq).

[0035] In an exemplary embodiment, this application also proposes an immunotherapy resistance risk stratification scheme. When the proportion of SLAMF1-positive immune cells in immune cells is not lower than a certain predetermined threshold, it indicates that the target subject may have a high risk of immunotherapy resistance; conversely, when the proportion is lower than the predetermined threshold, it indicates that the target subject has a low risk of immunotherapy response. This proportion-based criterion helps clinicians accurately assess the potential efficacy of immunotherapy, thereby developing personalized treatment plans and avoiding blind treatment or treatment failure. In practical applications, the selected predetermined threshold is typically between 35% and 45%. Clinical validation has shown that this threshold range can effectively distinguish between patients with good immunotherapy response and those with a high risk of resistance.

[0036] Quantitative analysis of the proportion of SLAMF1-positive immune cells enables accurate prediction of immunotherapy response and drug resistance risk. This not only provides a scientific basis for the development of individualized immunotherapy regimens but also plays an important role in clinical applications, improving the clinical benefits of immunotherapy and reducing unnecessary treatment side effects.

[0037] To facilitate understanding, we will use the example of predicting the response to anti-PD-1 / PD-L1 immunotherapy in cancer patients for a detailed explanation. By detecting the proportion of SLAMF1-positive immune cells and combining it with a pre-set threshold, we can predict the immunotherapy response, thus providing a strong basis for clinical decision-making.

[0038] 1) Sample Acquisition and Processing Peripheral blood samples were obtained from patients with tumors to be tested. To ensure sufficient immune cells were obtained and unnecessary cellular components were removed, the peripheral blood samples were separated using density gradient centrifugation, and peripheral blood mononuclear cells (PBMCs) were extracted.

[0039] 2) Marker detection After obtaining PBMCs, cell staining and detection were performed using multicolor flow cytometry with antibodies that specifically bind to CD150 (e.g., fluorescently labeled anti-human CD150 antibody) and antibodies that specifically bind to B cell markers (e.g., CD19 antibody). Flow cytometry allows for the simultaneous detection of multiple immune cell markers, precisely quantifying CD150-positive B cells (CD20... + CD150 + ) in total B cells (CD19) + The proportion in ).

[0040] 3) Threshold comparison and judgment The percentage of CD150-positive B cells obtained in step 2) is compared with a pre-defined clinical threshold. Based on clinical studies, a predetermined threshold (e.g., 40%) is set to assess the patient's immunotherapy response. If the measured percentage of CD150-positive B cells is not lower than (i.e., higher than or equal to) the predetermined threshold, it indicates a higher risk of resistance to anti-PD-1 / PD-L1 monotherapy. Therefore, in this case, it is recommended that the patient not use anti-PD-1 / PD-L1 therapy alone, but rather consider alternative therapies, such as a combination of anti-CD150 antibody and anti-PD-1 / PD-L1 therapy, or other non-PD-1 / PD-L1 inhibitor therapies.

[0041] If the measured proportion of CD150-positive B cells is below a preset threshold, it indicates that the patient may have good potential to respond to anti-PD-1 / PD-L1 monotherapy. Therefore, it is recommended to consider continuing anti-PD-1 / PD-L1 monotherapy to maximize the clinical efficacy of immunotherapy.

[0042] This embodiment assesses the dynamic changes of SLAMF1-positive B cells in the immune microenvironment, which can accurately predict the response rate of immunotherapy, effectively avoid ineffective treatment for drug-resistant patients, thereby reducing unnecessary treatment side effects and costs, and improving clinical treatment outcomes.

[0043] Application of SLAMF1 in the preparation of immunotherapy response biomarker reagents Secondly, this application provides the application of SLAMF1 in the preparation of immunotherapy response biomarker reagents. As a key molecule in the immune microenvironment, SLAMF1 is significantly correlated with the response to immunotherapy, especially anti-PD-1 / PD-L1 immunotherapy. By detecting the expression level of SLAMF1 or the proportion of SLAMF1-positive immune cells, the immunotherapy response of patients can be predicted. Using SLAMF1 as a target, a series of reagents can be prepared, including diagnostic reagents, prognostic evaluation reagents, and survival prediction reagents, for application in different clinical scenarios. For predicting immunotherapy response, these reagents can be used to effectively stratify patients before treatment, identify patients at risk of drug resistance in advance, and thus avoid unnecessary immunotherapy.

[0044] In this application, the biomarker reagent may be an antibody that specifically recognizes SLAMF1; and / or, immune cells, proteins and / or small molecules that target SLAMF1.

[0045] Antibody acquisition methods include using SLAMF1 as an immunogen to immunize animals and extracting specific antibodies from the immunized animals. These antibodies can be monoclonal or polyclonal, depending on the needs of the clinical application.

[0046] Immune cells can be obtained through genetic engineering. For example, T cells and B cells can be combined with SLAMF1 through genetic engineering technology to form immune cells that specifically recognize SLAMF1.

[0047] When preparing these reagents, their form can be adjusted according to different needs. For example, for SLAMF1-related diagnostic reagents, antibodies can be labeled with fluorescence, enzymes, or other detection tags, and detected using techniques such as flow cytometry, ELISA, or immunohistochemistry, thereby achieving accurate detection of SLAMF1-positive immune cells in the patient's immune microenvironment. For prognostic evaluation reagents, in addition to using antibodies, small molecules that interact with SLAMF1 can be prepared, or SLAMF1-related proteins and other biomacromolecules can be used to assess the prognosis of cancer patients after immunotherapy.

[0048] This proposed protocol has broad applicability in various clinical scenarios, particularly in tumor immunotherapy. It enables the assessment of immunotherapy efficacy, resistance risk, and survival time by detecting SLAMF1-related biomarkers, supporting personalized treatment. This not only improves the success rate of immunotherapy but also reduces potential side effects and enhances patients' quality of life.

[0049] Reagent test kit Thirdly, this application provides a kit comprising an antibody that specifically recognizes SLAMF1, used to accurately detect the expression level of SLAMF1 in immune cells, providing strong support for predicting immunotherapy responses. This kit is simple in design, easy to operate, and suitable for clinical laboratories and research environments. It is used to detect SLAMF1-positive cells in the patient's immune microenvironment, thereby assessing their ability to respond to immunotherapy. The kit is not limited to using a single antibody; it may also include multiple enhancing antibodies and controls to improve the reliability and accuracy of the detection results.

[0050] The key component of this kit is an antibody that specifically recognizes SLAMF1. This antibody can be a monoclonal or polyclonal antibody that specifically binds to the SLAMF1 molecule and can be detected by markers (such as fluorescence, enzyme-linked immunosorbent assay, or gold labeling). Using conventional detection methods such as flow cytometry, immunohistochemistry, and ELISA, this antibody can accurately identify and quantify SLAMF1-positive immune cells.

[0051] To further enhance the specificity and sensitivity of the detection, this antibody can be used in combination with other specific immune cell marker antibodies, such as antibodies that recognize B cells, T cells, etc. (e.g., CD19 antibody for B cells and CD3 antibody for T cells). This multi-marker combined detection method can significantly improve the ability to distinguish immune cell subsets, thereby accurately calculating the proportion of SLAMF1-positive immune cells and providing more accurate information for predicting immunotherapy responses.

[0052] It is understandable that the antibodies used to identify B cells, in addition to CD19 antibodies, may also be selected from one or more antibodies of CD20, CD79a and PAX5.

[0053] To further enhance the effectiveness and accuracy of the kit, reference controls can be added. These controls may include isotype control antibodies, control cells known to be SLAMF1 positive, and control cells known to be SLAMF1 negative. These controls serve as reference standards during the assay, helping to verify the accuracy and reliability of the experiment. Isotype control antibodies can eliminate the influence of non-specific binding, while control cells known to be SLAMF1 positive provide a positive control, ensuring that the detected signal truly originates from specific SLAMF1 binding. Control cells known to be SLAMF1 negative eliminate potential background noise, ensuring the clarity and accuracy of the test results. The use of controls enables quality control of the experimental process, guaranteeing the reproducibility and reliability of the kit in various experimental environments.

[0054] The kit described in this embodiment is not only suitable for basic research and preclinical studies, but can also be widely used in the clinical monitoring of immunotherapy, especially for predicting immunotherapy responses in cancer patients. In tumor immunotherapy, by detecting the proportion of SLAMF1-positive immune cells, the patient's immunotherapy response can be predicted, providing a basis for clinical practice, optimizing individualized treatment plans, and reducing the occurrence of ineffective treatment. Simultaneously, due to its high sensitivity and specificity, the kit can be used to monitor early immune responses, promptly identify the risk of immunotherapy resistance, and assist clinicians in adjusting treatment strategies in a timely manner.

[0055] The embodiments of this application will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0056] Example 1: Application of SLAMF1 as a biomarker in predicting immunotherapy response This embodiment predicts the response to anti-PD-1 immunotherapy in hepatocellular carcinoma (HCC) patients and guides treatment decisions based on the expression level of SLAMF1 in peripheral blood B cells. By detecting immune cell markers in peripheral blood samples, it achieves indirect assessment of the tumor immune microenvironment, thereby enabling risk stratification of patients and the development of corresponding individualized treatment plans before treatment.

[0057] 1) Sample collection and processing In this embodiment, the study subjects were patients with hepatocellular carcinoma diagnosed by pathological examination. Peripheral blood samples and tumor tissue samples were collected simultaneously before the patients received immunotherapy to ensure that the test results accurately reflected their pre-treatment immune status. Peripheral blood samples were collected using heparin-anticoagulated tubes, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation (Ficoll). After washing, the samples were cryopreserved in liquid nitrogen using a cryopreservation solution containing 10% fetal bovine serum. Tumor tissue samples were obtained from surgically removed tissue, a portion of which was fixed in 4% paraformaldehyde, embedded in paraffin, and used for immunohistochemical detection.

[0058] 2) Flow cytometry detection of SLAMF1 expression in peripheral blood B cells Resuscitated PBMCs: Incubate overnight with DMEM + 10% FBS before testing, and wash twice with PBS.

[0059] Labeling for live and dead cells: First, dilute FSV700 live and dead dye 1:100 with PBS. Resuspend each tube of cells in 100ul of dilution buffer and label for live and dead cells at 4 degrees Celsius for 15 minutes in the dark. Then add 100ul of FBS at room temperature for 5 minutes to block the cell cycle. Finally, wash twice with PBS.

[0060] Labeling flow cytometry external standard: First, block solution was prepared using 10% mouse serum + 90% PBS, and the mixture was blocked at 4°C for 15 minutes. Then, surface staining was performed using a combination of CD45, CD19, and SLAMF1 antibodies (protected from light, 4°C, 30 minutes). Data were acquired via flow cytometry. Subsequent analysis recorded the percentage of CD19+SLAMF1+ cells among CD19+ B cells for each patient.

[0061] 3) Immunohistochemical (IHC) detection of SLAMF1 expression in the tumor microenvironment Sectioning and baking: Continuously section the paraffin tissue block to a thickness of 4 μm. Float the sections in 40℃ warm water to spread them, then remove them and place them on glass slides. Bake the slides in a 60℃ oven for 1-2 hours to ensure tight adhesion of the tissue.

[0062] Dewaxing and hydration: Immerse the sections in xylene I and II for 10 minutes each. Then immerse them in a gradient of 100%, 95%, 80%, and 70% ethanol for 5 minutes each, and finally wash with running distilled water for 5 minutes.

[0063] Antigen retrieval: Immerse the slides in a container of EDTA alkaline antigen retrieval solution and autoclave for 25 minutes. Then remove and allow to cool naturally to room temperature for 30 minutes. Post-retrieval: Rinse the slides three times with PBST (PBS + 0.05% Tween), 5 minutes each time.

[0064] Endogenous peroxidase inhibition: Cover the tissue with sufficient 3% H2O2 solution and incubate at room temperature in the dark for 10-15 minutes to block endogenous peroxidase activity. Rinse three times with PBST, 5 minutes each time.

[0065] Serum blocking: Blot dry the PBS around the tissue with absorbent paper, add antigen blocking solution, and block at room temperature for 30 minutes to reduce non-specific background staining.

[0066] Primary antibody incubation: Discard the blocking solution. Directly add 1:100 diluted anti-SLAMF1 primary antibody working solution, ensuring complete tissue coverage. Place the sections in a humidified chamber and incubate overnight at 4°C (approximately 16-18 hours) to obtain the optimal signal-to-noise ratio.

[0067] Secondary antibody incubation: The next day, bring the humidified chamber to room temperature. Discard the primary antibody and wash three times with PBST, 5 minutes each time. Add HRP-labeled polymeric secondary antibody working solution and incubate at room temperature for 30-60 minutes. Wash three times with PBS, 5 minutes each time.

[0068] Developing color: Add freshly prepared DAB chromogenic solution and closely monitor the chromogenic process under a microscope (usually 30 seconds to 2 minutes). When a clear and specific brownish-yellow signal appears in the target area (cell membrane / cytoplasm) and the background is not stained, immediately immerse the section in distilled water to stop the chromogenic process.

[0069] Counterstaining, dehydration, and mounting: Counterstain cell nuclei with hematoxylin for 30 seconds, rinse with running water for 11 minutes to reverse blue staining. Dehydrate sequentially by immersing in 70%, 80%, 95%, and 100% ethanol solutions for 10 seconds each. Clear the slides by immersing in xylene I and II solutions for 10 minutes each. Remove the slides from the xylene solution, wipe away excess liquid, add a suitable amount of neutral resin, and mount with a coverslip.

[0070] Digital and quantitative analysis: High-resolution digital images were acquired using a full-slide scanner. Professional image analysis software (QuPath) was employed to quantitatively calculate the proportion of SLAMF1-positive immune cells among all infiltrating immune cells through nuclear identification and cell membrane / cytoplasmic staining intensity analysis.

[0071] like Figure 2 As shown in Figure AC, a comparative analysis of peripheral blood flow cytometry results and tumor tissue immunohistochemical results from the same patient revealed a positive correlation between the proportion of SLAMF1-positive B cells in peripheral blood and the proportion of SLAMF1-positive immune cells in tumor tissue (r=0.8208). The expression status of SLAMF1 in peripheral blood B cells can reflect SLAMF1-related immune characteristics in the tumor microenvironment to some extent. The results of this embodiment indicate that the proportion of SLAMF1+ B cells in peripheral blood can predict the proportion of SLAMF1+ immune cells in tissues.

[0072] Based on this, this embodiment constructs an immunotherapy decision-making process based on SLAMF1 expression levels, the overall process of which is as follows: Figure 1 As shown in the diagram, the process sequentially includes diagnosis and confirmation of hepatocellular carcinoma, quantitative detection of SLAMF1 expression in peripheral blood B cells, risk stratification based on preset thresholds, and selection of corresponding treatment regimens. Specifically, when the proportion of SLAMF1 expression in peripheral blood B cells is detected to be not lower than a preset threshold (e.g., 40%), the patient is classified as a high-expressing SLAMF1 population. This population indicates a higher risk of resistance to PD-1 inhibitor monotherapy, therefore, PD-1 inhibitor combined with SLAMF1 targeted therapy is the preferred treatment decision. When the proportion of SLAMF1 expression is detected to be lower than this threshold, the patient is classified as a normal-expressing SLAMF1 population. This population has a lower risk of resistance to PD-1 inhibitor monotherapy, and PD-1 inhibitor monotherapy is the preferred treatment regimen.

[0073] This embodiment achieves the goal of directly incorporating peripheral blood immune marker detection results into the immunotherapy decision-making process. This allows treatment selection to move beyond reliance solely on tumor tissue sampling or empirical judgment, and instead enables stratified decision-making based on quantifiable and repeatable immunological indicators. This method improves the accuracy of immunotherapy regimen matching without increasing the patient's invasiveness, contributing to higher treatment response rates and reducing the risk of ineffective treatment.

[0074] Example 2: A method for predicting overall survival in hepatocellular carcinoma patients based on SLAMF1 expression This embodiment is used to statistically analyze the association between multiple factors and overall survival (OS) in patients with hepatocellular carcinoma.

[0075] Specifically, the following statistical analysis procedure was used to assess patient survival outcomes: 1) Survival analysis and intergroup comparisons were performed. The Kaplan-Meier method was used to estimate overall survival and plot survival curves. Patients were divided into high-expression and low-expression groups based on the expression level of the target biomarker SLAMF1. Peripheral blood CD19... + SLAMF1 + Cell percentage was set at 40% as the grouping threshold. The log-rank test was used to compare the differences in survival distribution between different groups to assess the statistical significance of survival differences.

[0076] 2) Screening and modeling analysis of prognostic factors. First, the target biomarker (peripheral blood CD19) was selected. + SLAMF1 + The cell proportion and multiple clinicopathological and biochemical indicators were incorporated into a univariate Cox proportional hazards regression model to calculate the hazard ratio (HR) and its 95% confidence interval for each variable. The clinicopathological indicators in this embodiment include, but are not limited to, pathological stage, maximum tumor diameter, and TNM stage.

[0077] In univariate analysis, variables that were significantly associated with overall patient survival (with a significance criterion of P<0.05) were selected and used as candidate variables in subsequent multivariate analysis to reduce the impact of potential confounding factors on the analysis results.

[0078] 3) The significant variables selected above are simultaneously included in a multivariate Cox proportional hazards regression model. By conducting joint analysis on multiple variables, the independent prognostic effect of each factor after adjusting for the influence of other variables is evaluated, thereby identifying prognostic factors that have an independent impact on the overall survival of hepatocellular carcinoma patients, especially for assessing the independent association between SLAMF1 expression status and patient survival outcomes.

[0079] 4) To verify the stability and reliability of SLAMF1 as a prognostic indicator, patients were stratified according to key clinical characteristics such as maximum tumor diameter and TNM stage to form multiple subgroups. Survival analysis and Cox regression analysis were repeated within each subgroup to examine whether the association between SLAMF1 and overall survival remained consistent under different clinical backgrounds.

[0080] The Cox proportional hazards regression analysis in this embodiment was performed using SPSS statistical software, and the results are shown in Figure 3. The above analysis shows that, compared with commonly used pathological staging, maximum tumor diameter, and TNM staging, the SLAMF1-based method... +The risk regression model of cell proportions showed higher correlation accuracy in predicting overall survival in patients with hepatocellular carcinoma, indicating that SLAMF1 can serve as an effective biomarker for prognostic assessment of hepatocellular carcinoma.

[0081] Example 3: Detection of CD45 in hepatocellular carcinoma tissue by multiplex immunofluorescence staining + SLAMF1 + cell This embodiment uses multiplex immunofluorescence staining technology to detect CD45 in hepatocellular carcinoma tissue. + SLAMF1 + Cells were used to analyze the differences in cellular composition in the tumor immune microenvironment of patients with PD-1-resistant and non-resistant hepatocellular carcinoma.

[0082] I. Experimental Materials and Equipment In this embodiment, the tissue sample used was a section of hepatocellular carcinoma tissue fixed with 4% paraformaldehyde and embedded in paraffin. The section thickness was 4 μm. The section was attached to a glass slide to prevent detachment and baked overnight at 60 °C for later use.

[0083] Key reagents used include: a fluorescence signal amplification kit (including HRP polymeric secondary antibody, fluorescently labeled tyrosine, and antibody elution / retrieval solution), a series of primary antibodies targeting different targets (including targets such as SLAMF1 and CD20, all of which have been pre-validated), 10% neutral formalin, antigen retrieval solution, DAPI nuclear staining reagent, anti-fluorescence quenching mounting medium, blocking solution, and TBST buffer.

[0084] The main experimental equipment includes: microwave oven, constant temperature shaker, histochemistry pen, humidification chamber, and fluorescence microscopy imaging system.

[0085] II. Multiplex Immunofluorescence Staining 1) Dewaxing, hydration and post-fixation treatment Paraffin sections were dewaxed sequentially in fresh xylene I, II, and III for 10 minutes each time; then hydrated sequentially in a gradient of 100%, 95%, and 70% ethanol for 5 minutes each; finally, the sections were washed three times with sterile deionized water for 1 minute each time.

[0086] After hydration, the sections were immersed in 10% neutral formalin for 10 minutes to fix them and further stabilize the tissue antigen structure. They were then washed three times with sterile water for one minute each time.

[0087] 2) Antigen retrieval Place the slides in a retrieval chamber containing preheated antigen retrieval solution (pH 9.0 EDTA buffer). Microwave on high until boiling, then reduce to low heat and maintain a gentle boil for 15 minutes to ensure effective exposure of the antigen epitopes. After retrieval, allow the slides to cool naturally to room temperature.

[0088] 3) Sealing treatment After aspirating excess liquid around the slide, draw a hydrophobic zone around the tissue using a histochemical pen, and add blocking solution within the zone to completely cover the tissue. The blocking solution is a TBST solution containing 5% BSA or normal serum homologous to the secondary antibody. Place the slide in a humidified chamber and incubate at room temperature for 20 minutes to block nonspecific binding sites.

[0089] 4) First round of immunostaining and signal amplification After discarding the blocking solution, add the working solution of the first target primary antibody (e.g., anti-SLAMF1 antibody) directly without washing, ensuring complete coverage of the tissue. Incubate the sections in a 37°C shaker with low-speed shaking for 1 hour. After incubation, wash the sections three times with TBST buffer for 5 minutes each time.

[0090] Subsequently, an HRP-labeled polymeric secondary antibody matching the primary antibody species was added, and the mixture was incubated at room temperature for 30 minutes, followed by three washes with TBST. Next, a first fluorescein-labeled tyrosine working solution was added, and the mixture was incubated at room temperature in the dark for 10 minutes to allow the fluorescent signal to deposit at the target antigen site. After incubation, the mixture was washed three times with TBST in the dark.

[0091] 5) Antibody elution and cyclic staining After signal amplification, the slides were immersed in antibody elution / repair solution and heated to boiling in a microwave oven on high power, then reduced to low power and maintained for 10-15 minutes to dissociate the primary and secondary antibody complexes bound in the previous round and inactivate residual HRP activity. The slides were then allowed to cool naturally to room temperature and washed sequentially with TBST and sterile water.

[0092] After completing the above elution steps, repeat the blocking, primary antibody incubation, secondary antibody incubation, fluorescence signal amplification and elution steps, changing the primary antibody to different targets and the corresponding fluorescence channels, until all target markers are stained.

[0093] 6) Nuclear staining and sealing After the final round of immunostaining, DAPI working solution was added to counterstain the cell nuclei, and the sections were incubated at room temperature in the dark for 5 minutes. The sections were then washed three times with TBST and briefly rinsed with sterile water. Excess water was aspirated, and anti-fluorescence quenching mounting medium was added to the tissue, covered with coverslips and sealed. The resulting sections were stored at 4 °C in the dark.

[0094] III. Image Acquisition and Result Analysis The AKOYA PhenoImager Fusion system was used to acquire multi-channel fluorescence images of stained sections, and the Qupath software was used for multi-channel overlay, fluorescence signal co-localization analysis, and quantitative analysis of cell phenotype.

[0095] The results are as follows Figure 4 As shown, in samples of hepatocellular carcinoma patients resistant to PD-1 therapy (PD), CD45 infiltrates within the tumor tissue. + SLAMF1 + The number of cells was significantly higher in the PD-1 treatment non-resistant group (PR), suggesting CD45 + SLAMF1 + Cellular resistance is associated with PD-1 therapy.

[0096] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0097] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0098] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Application of SLAMF1 as a biomarker in predicting immunotherapy response.

2. The application according to claim 1, characterized in that, The proportion of SLAMF1-positive immune cells in immune cells is determined based on the protein or nucleic acid levels of SLAMF1.

3. The application according to claim 2, characterized in that, The proportion of SLAMF1-positive immune cells in immune cells is not lower than a predetermined threshold, indicating that the target subject has a high risk of drug resistance. The proportion of SLAMF1-positive immune cells in immune cells is lower than a predetermined threshold, indicating that the target subject has a low risk of drug resistance.

4. The application according to claim 3, characterized in that, The predetermined threshold is selected from 35%-45%.

5. The application according to any one of claims 2-4, characterized in that, The immune cells are selected from B cells or T cells.

6. The application according to any one of claims 2-4, characterized in that, The samples used to determine the protein or nucleic acid levels of SLAMF1 were selected from the target subject's blood or tumor tissue.

7. The application according to any one of claims 2-4, characterized in that, The immunotherapy includes: At least one of anti-PD-1 immunotherapy, anti-PD-L1 immunotherapy, anti-CTLA-4 immunotherapy, and anti-LAG-3 immunotherapy; Optionally, the protein level of the SLAMF1 is determined based on any of the following methods: Flow cytometry, immunohistochemistry, Western blotting, or liquid microarray; The nucleic acid level of SLAMF1 was determined by quantifying the mRNA encoding SLAMF1.

8. Applications of SLAMF1 in the preparation of immunotherapy response biomarker reagents, including: Diagnostic reagents for immunotherapy response can be prepared using SLAMF1 as a target, or prognostic reagents for immunotherapy response can be prepared, or reagents for predicting the survival time of target subjects can be prepared.

9. The application according to claim 8, characterized in that, The reagent is used to prepare antibodies by immunizing animals with SLAMF1 as an immunogen; and / or to prepare immune cells, proteins and / or small molecules with SLAMF1 as a target.

10. A reagent kit, characterized in that, include: Antibodies that specifically recognize SLAMF1; Optionally, it further includes: antibodies that specifically recognize immune cell markers; Optionally, it further includes: a reference control, said reference control comprising at least one of an isotype control antibody, control cells known to be positive for SLAMF1 expression, and control cells known to be negative for SLAMF1 expression.

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