Predictive biomarker for treating adverse reaction of tumor patient by using CCL5 as immune checkpoint inhibitor and application of predictive biomarker
By detecting the dynamic changes of CCL5 chemokine in peripheral blood, this study solves the problem of early warning of adverse reactions in cancer patients treated with immune checkpoint inhibitors. It achieves high specificity and early prediction, is applicable to various tumor types and immune checkpoint inhibitors, and has the potential for clinical promotion and translational application.
Patent Information
- Application Number
- CN202511653395.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-02-06
AI Technical Summary
Existing methods for detecting adverse reactions to immune checkpoint inhibitor therapy for tumors suffer from diagnostic lag and insufficient specificity of detection indicators, making it difficult to achieve early warning and accurate prediction.
The dynamic changes in the proportion of CCL5 chemokines were used as a predictive biomarker. By detecting changes in CCL5 in peripheral blood, especially changes in the proportion ≥50% or a sustained increase before and after treatment, adverse reactions could be predicted.
It enables the early identification of high-risk patients before adverse reactions occur, reducing the probability of serious adverse events. It has high specificity and operability, is applicable to various tumor types and immune checkpoint inhibitors, and has the potential for clinical promotion and translational application.
Smart Images
Figure CN121476613A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a CCL5 as a predictive biomarker for adverse reactions of immunological checkpoint inhibitor treatment of tumor patients and application thereof. BACKGROUND
[0002] Although existing immunological checkpoint inhibitors (ICIs) have achieved remarkable effects in tumor treatment, the immune-related adverse reactions (irAEs) accompanying them are still an important problem limiting clinical application. On the one hand, most of the current clinical monitoring methods belong to post-diagnostic type, such as detecting myocardial injury markers (such as cTn-I), performing chest CT or endoscopic examination to find pneumonia or enteritis. These methods can only indicate abnormalities when organ damage has occurred, lack the ability to predict before the occurrence of clinical symptoms, and lead to delayed intervention, which is not conducive to individualized management of patients. On the other hand, some studies attempt to use serum inflammatory factors (such as CRP, IL-6) levels, or peripheral blood total CD8 + T cell numbers and their activation markers (such as HLA-DR, CD38) to predict irAEs risk. However, these indicators have limited specificity and are easily disturbed by factors such as infection, comorbidities or tumor progression, making it difficult to accurately reflect the inflammatory chemotaxis directly related to irAEs in the immune system, thereby limiting their clinical application value. In recent years, high-throughput technologies such as single-cell transcriptome and immune lineage sequencing have revealed that changes in chemotactic factors secreted by immune cells may be an early signal of immune overactivation, but these studies have mostly remained at the laboratory level, and there is still a lack of operable detection methods that can be routinely implemented in the clinic.
[0003] Further studies have shown that CCL5 (also known as RANTES), as an important chemotactic factor, plays a key role in recruiting CD8 + T cells, monocytes and NK cells in immune response. Multiple studies have shown that CCL5 is involved in immune cell migration in the tumor microenvironment. Therefore, simply relying on detection of total CD8 + T cell numbers or general inflammatory factors makes it difficult to capture early chemical signals of immune overactivation.
[0004] In summary, the existing technology mainly has the following defects: first, the diagnosis is delayed, and early warning of irAEs cannot be achieved; second, the detection index has insufficient specificity and cannot effectively reflect immune chemotaxis abnormalities. SUMMARY
[0005] The present application aims to provide a predictive biomarker for adverse reactions of immunological checkpoint inhibitor treatment of tumor patients and application thereof, and aims to solve the above-mentioned problems existing in the detection means of adverse reactions.
[0006] To achieve the above-mentioned application purposes, the technical scheme adopted by the present application is as follows:
[0007] In a first aspect, the present application provides a predictive biomarker of CCL5 as an immune checkpoint inhibitor for treating adverse reactions of tumor patients and an application thereof.
[0008] As a preferred design, the predictive biomarker is a dynamic change in the proportion of chemokine CCL5.
[0009] As a preferred design, the dynamic change refers to a change in the proportion of chemokine CCL5 before and after the use of an immune checkpoint inhibitor; if the dynamic change is ≥ 50% or continuously increases, it indicates that the immune checkpoint inhibitor treatment of the tumor patient causes an adverse reaction.
[0010] As a possible design, the tumor patient includes non-small cell lung cancer, melanoma, renal cancer, pancreatic cancer, or liver cancer.
[0011] In a second aspect, the present application provides an application of a detection reagent of a predictive biomarker in the preparation of an adverse reaction detection kit, and the detection kit is used for detecting the proportion of chemokine CCL5.
[0012] As a possible design, the chemokine CCL5 is derived from the peripheral blood of a tumor patient.
[0013] As a possible design, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
[0014] The detection and determination method proposed in the present application is suitable for a variety of immune checkpoint inhibitors, including but not limited to PD-1 inhibitors (such as nivolumab, pembrolizumab), PD-L1 inhibitors (such as atezolizumab, durvalumab), and CTLA-4 inhibitors (such as ipilimumab). This ensures the wide applicability of the present application in clinical practice.
[0015] As a possible design, the tumor patient includes non-small cell lung cancer, melanoma, renal cancer, pancreatic cancer, or liver cancer.
[0016] The present application is not limited to a single cancer, but is suitable for a variety of tumor patients receiving immunotherapy, including non-small cell lung cancer, melanoma, renal cancer, pancreatic cancer, liver cancer, etc. Since the mechanism of irAEs has commonality, the kit of the present application can be used as a universal prediction tool across cancer types.
[0017] As a possible design, the detection kit is an enzyme-linked immunosorbent assay kit, a chemiluminescence immunoassay kit, a multiplex fluorescent immunoassay kit, a kit based on a microfluidic chip high-sensitivity detection system, or a kit based on an immunosensor high-sensitivity detection system.
[0018] The present application not only limits to the conventional method of detecting the concentration of CCL5 in peripheral blood plasma by enzyme-linked immunosorbent assay (ELISA), but also covers various equivalent detection technologies, including chemiluminescence immunoassay (CLIA), multiplex fluorescent immunoassay (such as Luminex or Cytometric Bead Array), and kits based on microfluidic chip or immunosensor high-sensitivity detection systems. Among them, the ELISA method is simple to operate, high in sensitivity, and good in repeatability, and is suitable for clinical routine laboratory; chemiluminescence immunoassay can realize rapid quantitative detection on an automated platform, and is suitable for large-scale sample screening; multiplex fluorescent immunoassay can simultaneously quantify multiple cytokines (such as CCL5, IL-6, CXCL10, etc.), which helps to establish a multi-index joint prediction model; the detection system based on microfluidic chip has the advantages of small sample size, high detection sensitivity, and high integration level, and is suitable for future rapid diagnosis and point-of-care testing (POCT) applications. By covering the above-mentioned various detection approaches, the protection scope of the present application is not limited to a specific platform or reagent system. Any method that can realize accurate and repeatable detection of the change of the concentration of CCL5 in plasma, including traditional immunoassay, automated chemiluminescence system, chip-based biosensor, or other equivalent detection technologies, belongs to the protection scope of the present application.
[0019] In a third aspect, the present application provides a predictive biomarker or a detection kit involving the detection reagent for use in a clinical follow-up system for monitoring adverse reactions.
[0020] The present application not only proposes a predictive biomarker for adverse reactions of immune checkpoint inhibitors, but also reserves the conversion space to in vitro diagnostic kits or standardized clinical detection processes. For example, commercial kits of enzyme-linked immunosorbent assay, chemiluminescence immunoassay, or multiplex fluorescent immunoassay can be developed for direct use by hospital laboratories; or they can be embedded in a clinical follow-up system to realize dynamic risk monitoring during treatment. Through this expansion, the present application has the convertibility and generalizability from research achievements to clinical applications.
[0021] The present application has the following beneficial effects:
[0022] 1. Early prediction can be realized
[0023] Unlike existing methods that rely on clinical symptoms or organ damage to indicate abnormalities, the present application can identify high-risk patients through peripheral blood testing before irAEs have obvious clinical manifestations, providing clinicians with the basis for early intervention and reducing the probability of serious adverse events.
[0024] 2. Higher specificity
[0025] Compared with traditional inflammatory markers (such as CRP, IL-6), the detection of cytokine CCL5 has higher specificity in predicting immune-related adverse reactions (irAEs). CRP and IL-6 are acute phase response proteins or broad-spectrum inflammatory factors that can be elevated by almost all types of inflammation (infection, surgery, tumor necrosis, etc.). They reflect systemic inflammation or "passive immune response", rather than the active attack of immune cells specific to irAEs. CCL5 (also known as RANTES) is a chemokine secreted by activated effector CD8 + T cells, Th1 helper T cells, and monocytes, which mediate the directional migration of immune cells to specific tissues, are the main effector populations that cause irAEs after the activation of immune checkpoint inhibitors (ICIs). When the immune system is overactivated, CCL5 will rise early, reflecting the abnormal initiation of inflammatory chemotaxis. Therefore, the dynamic changes in CCL5 levels in peripheral blood can serve as an early and more specific predictor, helping to identify high-risk patients before clinical symptoms appear and enabling early warning and intervention of immune side effects.
[0026] 3. Stronger operability and generalizability
[0027] Peripheral blood can be collected for detection, and sampling is convenient. It can be achieved based on conventional immunological detection methods such as enzyme-linked immunosorbent assay (ELISA), and the detection process is mature and laboratories generally have the relevant equipment. Compared with high-cost methods such as single-cell sequencing, it is easier to promote in clinical practice.
[0028] 4. Dynamic monitoring capability
[0029] The present application emphasizes the dynamic change trend (relative baseline proportion increase) of individual patients to determine the risk, rather than relying on a single time point or absolute value. This not only enhances the applicability of the determination, but also can indicate potential risks earlier.
[0030] 5. Wide clinical application range
[0031] The present application method can be applied to various immune checkpoint inhibitors (such as PD-1, PD-L1, CTLA-4 inhibitors) and various tumor types (including lung cancer, melanoma, pancreatic cancer, etc.), and has strong universality, not limited to specific drugs or cancer types.
[0032] 6. Potential for translational applications
[0033] The present application is not limited to scientific research detection, but can be further developed into an in vitro diagnostic kit or a clinical standardized detection process, which helps to be integrated into the patient treatment follow-up system and form an operable early warning tool. BRIEF DESCRIPTION OF DRAWINGS
[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.
[0035] Figure 1 is a bubble chart of animal models in Example 1 of the present application to reproduce the clinical side effects during tumor immune checkpoint blockade (ICI) treatment;
[0036] Figure 2 is a CT scan result chart of 6 patients with irae induced by PD-1 inhibitor blockade receiving ICI treatment in Example 1 of the present application;
[0037] Figure 3 is a result chart of the proportion of CCL5 concentration in the plasma of the four stages of 6 patients with irae induced by PD-1 inhibitor blockade in Example 1 of the present application. DETAILED DESCRIPTION
[0038] In order to make the technical problems, technical solutions and beneficial effects of the present application more clear, the present application will be further described in detail in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.
[0039] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can represent the following cases: A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.
[0040] It should be understood that in various embodiments of the present application, the size of the sequence number of the above processes does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
[0041] The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in the embodiments of the present application and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
[0042] The weight of the related components mentioned in the embodiments of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component. Therefore, as long as the content of the related components in the embodiments of the present application is proportionally enlarged or reduced, it is within the scope disclosed in the embodiments of the present application. Specifically, the mass mentioned in the embodiments of the present application can be µg, mg, g, kg, and other mass units commonly known in the chemical field.
[0043] The term "ICIs" is an abbreviation of "Immune Checkpoint Inhibitors", which means immune checkpoint inhibitors; the term "irAEs" is an abbreviation of "Immune-Related Adverse Events", which means adverse reactions.
[0044] The following will be described in conjunction with specific embodiments.
[0045] Embodiment 1
[0046] The present embodiment discloses an immune therapy adverse reaction prediction method based on the proportion change of peripheral blood chemokine CCL5 as a predictive biomarker. The method has clear process and operable steps, and specifically includes the following steps:
[0047] 1. Sample collection
[0048] During the treatment of the patient with immune checkpoint inhibitors (such as PD-1, PD-L1 or CTLA-4 inhibitors), peripheral blood samples are collected according to the time point rules: (1) baseline sample: collected before the first administration; (2) dynamic follow-up sample: collected before each administration and 1-2 days after administration.
[0049] Sample volume: generally 2-5 mL, stored in a vacuum blood collection tube containing an anticoagulant (such as EDTA or sodium citrate).
[0050] 2. Plasma separation
[0051] Centrifuge at 1000 G for 15 minutes at 4°C, and aspirate the upper plasma in the peripheral blood.
[0052] 3. CCL5 cytokine detection in plasma
[0053] CCL5 concentration changes were detected by enzyme-linked immunosorbent assay (ELISA) kit (kit brand: genie, item number: HUFI00231).
[0054] 4. Risk determination
[0055] The dynamic detection results were compared with the baseline level:
[0056] No risk / low risk: no significant change in CCL5 cytokine concentration in peripheral blood plasma compared with baseline;
[0057] High risk warning: the proportion increased by ≥50% or continued to rise compared with baseline;
[0058] Clinical application: once high risk is prompted, the clinician should be advised to perform function tests of key organs such as heart, lung, and intestine, and assess whether to adjust the drug regimen or intervene in advance.
[0059] 5. Clinical application scenarios
[0060] Individualized treatment monitoring: through the detection results of peripheral blood, the risk of patients developing irAEs is dynamically evaluated to guide whether to continue, reduce or suspend ICI treatment;
[0061] Early warning mechanism: when the patient has not yet shown clinical symptoms, potential high-risk individuals are prompted to reduce the incidence of serious irAEs;
[0062] Auxiliary drug selection: in high-risk patients, the use of immunosuppressants (such as selective JAK1 inhibitors) or enhanced monitoring is provided as a reference.
[0063] The present embodiment also discloses a model to reproduce clinical side effects during tumor immune checkpoint blockade (ICI) treatment, as follows:
[0064] 1. Subcutaneously transplant melanoma cells into C57 mice and intravenously inject PD-1 blocking antibodies, and the results are shown in Figure 1 As can be seen from Figure 1 , compared with the blank control group, the expression of T cells, especially the CD8+ T cell population, was significantly up-regulated in the tumor and organs that developed side effects that received PD-1 blocking antibody treatment. The visualization analysis of immune cell intercellular ligand-receptor interactions in different tissues (tumor, peripheral blood, side effect organ-lung) was analyzed. Bubble chart shows the average expression level (color depth) and statistical significance (bubble size) of various immune-related ligand-receptor pairs between different cell types. Chemokine CCL5 and its receptor CCR1 / CCR5 showed significant interaction signals in multiple tissues, especially in CD8 +The high-intensity pairing between effector T cells (CD8 EFF) and monocytes / macrophages suggests that the CCL5 signaling axis may be involved in immune cell recruitment and immune-related inflammatory responses.
[0065] 2. This embodiment also discloses the collection of information of 6 patients with PD-1 blockade-induced irae, CT scans of cancer patients receiving ICI treatment, and the results are shown in Figure 2 As can be seen from Figure 2 , the CT images before and after four doses of ICI treatment show that the lesions in the right lung (red arrow) of 3 patients are uniform consolidation with peripheral ground glass opacities (GGO), which is consistent with irae-related pneumonia. The other 3 patients received four doses of ICI treatment, and the CT images showed no signs of pneumonia, and the lung parenchyma was normal without consolidation or GGO.
[0066] 3. This embodiment also discloses the verification of the immune therapy adverse reaction prediction method disclosed in this embodiment based on the change in the proportion of peripheral blood chemokine CCL5 as a predictive biomarker in the above-mentioned 6 patients with PD-1 blockade-induced irae. Specifically, blood plasma components are collected for detection, including baseline and samples after each administration. ELISA is used to detect the dynamic changes of cytokine CCL5 in the blood samples of patients during ICI treatment (n=6 cases). The abscissa represents the administration cycle (“0” represents before administration, “1” represents after the first ICI treatment, and so on). The results are shown in Figure 3 As can be seen from Figure 3 , before the irAEs outbreak, the concentration of CCL5 in the blood of the patients showed a gradually increasing trend. Before the irae outbreak, the concentration of CCL5 in the plasma reached a higher platform compared with each corresponding baseline (the proportion increased by ≥50% or continuously increased, and the triangular star in the figure indicates that the patient developed immune-related pneumonia after this treatment). In contrast, the concentration of CCL5 in the plasma of non-irae patients did not change significantly. Therefore, our research results show that the concentration of CCL5 in the plasma can be used as a diagnostic marker for monitoring and even predicting irae during immunotherapy, specifically: no significant change in the concentration of CCL5 cytokine in peripheral blood plasma compared with the baseline, which is no risk / low risk; an increase of ≥50% or continuous increase in the concentration of CCL5 cytokine in peripheral blood plasma compared with the baseline, which is high risk.
[0067] This embodiment discloses that the change in the concentration of CCL5 cytokine compared with the baseline level can be used as a predictive biomarker for adverse reactions in tumor patients treated with immune checkpoint inhibitors. The patients treated with immune checkpoint inhibitors include but are not limited to PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors, and the tumor patients include but are not limited to non-small cell lung cancer, melanoma, renal cancer, pancreatic cancer, or liver cancer.
[0068] The embodiment also discloses application of the detection reagent of the predictive biomarker in preparation of an adverse reaction detection kit, which is an enzyme-linked immunosorbent assay kit, a chemiluminescence immunoassay kit, a multiplex fluorescence immunoassay kit, a kit based on a microfluidic chip high-sensitivity detection system or a kit based on an immunosensor high-sensitivity detection system.
[0069] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims
1. A predictive biomarker for adverse reactions of CCL5 as an immune checkpoint inhibitor in cancer patients.
2. The predictive biomarker according to claim 1, characterized in that, The predictive biomarker is the dynamic change in the proportion of the chemokine CCL5.
3. The predictive biomarker according to claim 2, characterized in that, The dynamic change refers to the change in the proportion of chemokine CCL5 before and after the use of immune checkpoint inhibitors; if the dynamic change is ≥50% or continues to increase, it indicates that the immune checkpoint inhibitor treatment has caused adverse reactions in cancer patients.
4. The predictive biomarker according to claim 1, characterized in that, The cancer patients include those with non-small cell lung cancer, melanoma, kidney cancer, pancreatic cancer, or liver cancer.
5. The use of a detection reagent for a predictive biomarker according to any one of claims 1-4 in the preparation of an adverse reaction detection kit, wherein the detection kit is used to detect the proportion of chemokine CCL5.
6. The application according to claim 5, characterized in that, The chemokine CCL5 was derived from the peripheral blood of cancer patients.
7. The application according to claim 5, characterized in that, The immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
8. The application according to claim 5, characterized in that, The cancer patients include those with non-small cell lung cancer, melanoma, kidney cancer, pancreatic cancer, or liver cancer.
9. The application according to claim 5, characterized in that, The detection kit is an enzyme-linked immunosorbent assay (ELISA) kit, a chemiluminescent immunoassay kit, a multiplex fluorescent immunoassay kit, a kit based on a microfluidic chip-based high-sensitivity detection system, or a kit based on an immunosensor-based high-sensitivity detection system.
10. The application of a detection reagent for a predictive biomarker as described in any one of claims 1-4 or a detection kit related to the application described in any one of claims 5-9 in a clinical follow-up system for monitoring adverse reactions.