Method for evaluating an immune response associated with an immunotherapeutic drug

Abstract

The present application belongs to the technical field of biological medicine, and relates to a method for evaluating immune response related to immunotherapy drugs and application thereof. The method comprises the following steps: obtaining a peripheral blood sample of a subject and separating PBMCs and T cells; respectively co-culturing the PBMCs and / or T cells with HER2-positive tumor cells in vitro; and detecting the levels of immune-related cytokines such as IL-2, IFN-gamma, TNF-alpha and IL-6 in the supernatant of the co-culture. The present application can be used for pharmacodynamic evaluation of immune therapy drugs with complex action mechanisms, mechanism research and / or response state evaluation of subjects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to an in vitro evaluation method for immune responses related to immunotherapy drugs and its application, and more particularly to a detection and analysis method based on the in vitro co-culture of subject-derived immune cells and tumor cells and the detection of immune-related indicators and its application. Background Technology

[0002] Tumor immunotherapy, which activates or modulates the body's immune system to recognize and eliminate tumor cells, has become a crucial strategy in the treatment of malignant tumors. Unlike traditional cytotoxic drugs, the efficacy of immunotherapeutic drugs is typically accompanied by complex immune activation, regulation, and effector processes, and significant heterogeneity in responses often exists among different subjects. Therefore, establishing timely, feasible, and relatively accurate evaluation methods to reflect the characteristics of drug-related immune responses is of great significance for pharmacodynamic studies, mechanism analysis, and the exploration of potential biomarkers for immunotherapeutic drugs.

[0003] On the one hand, imaging changes in tumors cannot directly and promptly indicate the effectiveness of immunotherapy; on the other hand, in studies of combined radiotherapy, chemotherapy, and immunotherapy, it is also necessary to track patients' immune response indicators in a timely manner. Therefore, developing timely and feasible in vitro evaluation methods or pharmacodynamic research methods for different immunotherapy drugs is an urgent problem to be solved in tumor immunotherapy drug research.

[0004] Currently, the evaluation of immune responses related to immunotherapy drugs typically employs methods such as peripheral blood cytokine detection, immune cell phenotypic analysis, in vitro functional experiments, and clinical efficacy follow-up. Among these, peripheral blood cytokine detection offers advantages such as convenient sampling and dynamic monitoring, and can reflect the overall immune status of the subject to some extent. However, these indicators are easily affected by various factors, including the patient's baseline immune status, tumor burden, sampling time, the complexity of cytokine sources, and the in vivo microenvironment. Therefore, relying solely on changes in peripheral blood indicators often fails to directly reflect the true functional status of the patient's immune cells, especially T cells, after exposure to tumor cells. While clinical efficacy information can reflect treatment outcomes, it usually lacks a direct functional correspondence with the specific immune response process.

[0005] For immunotherapies with relatively simple mechanisms of action, such as single-target drugs like PD-1 antibodies, receptor occupancy (RO) is often used as a reference indicator linking pharmacokinetic and pharmacodynamic relationships in clinical studies. In addition, some studies combine changes in PD-1-related phenotypes in peripheral blood to assess the immune status after drug administration. These indicators have some reference value when the specific target or pathway is well-defined. However, for immunotherapies with complex mechanisms of action, establishing an evaluation method that can comprehensively reflect the characteristics of their related immune responses remains a pressing technical problem that needs to be solved in related drug research.

[0006] Existing studies analyze cell activation status and cytokine release through the culture of immune cells alone or in co-culture with tumor cells. However, for immunotherapeutic drugs with complex mechanisms of action, single cell systems or single detection indicators still have certain limitations and are insufficient to comprehensively characterize the related immune response features, especially failing to reflect the dynamic changes in the immune function status of the same subject after in vivo drug exposure at different administration stages. Therefore, it is still necessary to establish an in vitro functional evaluation method based on the co-culture of patient-derived immune cells and tumor cells to more comprehensively reveal the characteristics and heterogeneity of immune responses related to immunotherapeutic drugs. Summary of the Invention

[0007] Given that the existing technology for evaluating immune responses related to immunotherapy drugs still suffers from limited information dimensions and difficulty in timely and comprehensively reflecting the immune function status and individual response differences of subjects at different drug administration stages, the purpose of this invention is to provide an immune response evaluation method and its application based on the in vitro co-culture of subject-derived immune effector cells and target cells.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides an in vitro evaluation method for immune responses, the method comprising the following steps: (1) Collect peripheral blood samples from subjects at different administration stages after receiving the drug to be evaluated; (2) PBMCs and T cell communities were isolated from the peripheral blood sample; (3) Establish in vitro co-culture systems of the immune effector cells from different drug administration stages with target cells respectively; (4) Detect one or more immune-related indicators in each co-culture system; (5) Compare the immune-related indicators of different cells in each co-culture system; Preferably, the different drug administration stages in step (1) include different treatment cycles or different time points before and after drug administration.

[0009] Preferably, the drug to be evaluated is an immunotherapy drug; more preferably, the drug to be evaluated is an immunotherapy drug with a combined mechanism of action.

[0010] Preferably, the immune effector cells in step (2) include peripheral blood mononuclear cells (PBMCs) and T cells obtained by further separation from PBMCs.

[0011] Preferably, the target cells in step (3) can be selected from Her2-expressing tumor cells, such as NCI-N87 cells and MCF-7 cells.

[0012] Preferably, the in vitro co-culture system in step (3) can be set with different effective-to-target ratios and / or different culture times.

[0013] Preferably, the immune-related indicators in step (4) include one or more of IL-2, IFN-γ, TNF-α, IL-6, and perforin.

[0014] A second aspect of this invention provides the application of the above-mentioned in vitro evaluation method in pharmacodynamic studies of drugs to be evaluated, analysis of immune response characteristics, evaluation of individual differences in immune response of subjects, and / or research on potential immune-related biomarkers.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides an in vitro co-culture evaluation system based on subject-derived immune effector cells and target cells. This system can reflect changes in the functional status of immune effector cells after in vivo exposure to the drug being evaluated under relatively controlled conditions, thereby facilitating the analysis of the drug-induced immune response characteristics. This method can be used for pharmacodynamic studies, immune response characteristic analysis, evaluation of individual response differences, and potential biomarker research of the drugs being evaluated, particularly immunotherapeutic drugs with combined mechanisms of action. Attached Figure Description

[0016] Figure 1 These are the results of cytokine release assays performed on PBMCs derived from the subjects under different co-culture conditions. Figure 2 These are the results of cytokine release assays performed on subject-derived T cells under different co-culture conditions. Figure 3 These are the cytokine release detection results of the PBMC-T cell co-culture system in samples 003 and 004. Figure 4 The results of cytokine release detection in the T cell co-culture system of sample 003. Figure 5 This is the result of cytokine release detection in the T cell co-culture system of sample 001. Figure 6 This is the result of cytokine release detection in the T cell co-culture system of sample 004. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Experimental methods not specifically described in the embodiments were performed according to conventional methods in the art or the conditions recommended by relevant reagent and instrument manufacturers.

[0018] The data provided in the embodiments of the present invention are all from clinical research subjects. Peripheral blood samples were collected and used for subsequent PBMC and / or T cell isolation, in vitro co-culture and detection of immune-related indicators. Example

[0019] An in vitro evaluation model for related immune responses based on subject-derived PBMCs and the co-incubation of isolated T cells with tumor cells. In this embodiment, peripheral blood mononuclear cells (PBMCs) derived from the subject and T cells further isolated from the PBMCs were used to establish an in vitro co-culture system with HER2-positive tumor cells. One or more immune response-related indicators in the co-culture system were detected to analyze and evaluate the immune response related to immunotherapy drugs.

[0020] (1) Culture and plating of HER2-positive tumor cells: After revival, HER2-positive tumor cells were seeded into complete culture medium and cultured and passaged at 37°C and 5% CO2. When the cells were in good growth condition, they were digested, counted, and the cell density was adjusted to 1×10^5 cells / mL. 200 μL of cell suspension was seeded into 96-well flat-bottom culture plates, with 2×10^4 tumor cells seeded into each well, and placed in an incubator for later use.

[0021] (2) Isolation of PBMCs: Peripheral blood samples were collected, and whole blood was diluted with PBS at a 1:1 volume ratio. The diluted whole blood was then added to the upper layer of Ficoll separation solution and centrifuged at room temperature. The white membrane cells were aspirated, washed, and resuspended to obtain a suspension of subject-derived PBMCs for later use.

[0022] (3) Isolation of T cells T cells were further isolated from the PBMCs. The T cells could be isolated using methods commonly used in the art, such as magnetic bead sorting. The obtained PBMCs and T cells were counted separately and then used for later use.

[0023] (4) Establishment of in vitro co-culture system PBMCs and / or T cells were resuspended in complete culture medium and adjusted to the predetermined cell density. PBMCs and / or T cells were added to culture plates pre-coated with HER2-positive tumor cells at the set effector-to-target ratio (E:T) to establish an in vitro co-culture system.

[0024] (5) Detection of immune response-related indicators in the co-culture system After co-culture, one or more immune response-related indicators in the co-culture system were detected.

[0025] Supernatants from co-culture studies were collected from each group, and cytokines were detected using flow cytometry microsphere assay. These cytokines included, but were not limited to, one or more of IFN-γ, TNF-α, IL-2, and IL-6. Perforin was detected using ELISA to evaluate cytotoxic effects in immunotherapy-related immune responses. These assays could be performed using standard kits in the art, following the manufacturer's instructions.

[0026] In one specific implementation, subject-derived PBMCs were co-cultured with MCF-7 cells or NCI-N87 cells at effector-to-target ratios (E:T) of 1:1, 10:1, and 50:1, with two detection time points set at 2 h and 16 h, respectively; simultaneously, T cells were co-cultured with MCF-7 cells or NCI-N87 cells at effector-to-target ratios (E:T) of 1:1 and 10:1 for 8 h.

[0027] After the culture was completed, samples from each group were collected, and the levels of IFN-γ, TNF-α, IL-2 and IL-6 were detected by flow cytometry microsphere detection method, and the perforin level was detected by ELISA method.

[0028] Relevant test results are as follows Figure 1 and Figure 2 As shown. Example

[0029] Cytokine release assays in PBMC and T cell systems Following the method in Example 1, co-culture systems were established for subject-derived PBMCs and HER2-positive tumor cells, as well as for subject-derived isolated T cells and HER2-positive tumor cells. The differences in cytokine release levels between the two systems under HF50 treatment were compared. Specifically, subject-derived PBMCs or T cells further isolated from PBMCs were used as effector cells and co-cultured with HER2-positive tumor cells; the HER2-positive tumor cells could be MCF-7 cells and / or NCI-N87 cells. In some embodiments, the effector-to-target ratio (E:T) could be set to 1:1, 10:1, or 50:1, and the co-culture time could be set to 2 h, 16 h, or 20 h. After co-culture, the culture supernatant was collected, and the levels of TNF-α, IL-6, IL-2, and IFN-γ were detected.

[0030] The cytokine release patterns under HF50 treatment differed between the PBMC system and the isolated T cell system. Elevated levels of cytokines such as IL-6 and TNF-α were more readily observed in the PBMC system compared to the isolated T cell system. The PBMC co-incubation system was more likely to reflect a comprehensive immune activation state, while the isolated T cell system was better suited to reflecting directly mediated functional output by T cells. Related detection results are as follows: Figure 3 As shown. Example

[0031] Dynamically monitor changes in cytokine release patterns in an in vitro co-culture model during immunotherapy. This example illustrates that, at different stages of treatment, the levels of immune-related cytokines such as TNF-α, IL-6, IL-2, and IFN-γ in the supernatant of a co-culture model of PBMCs, T cells, and HER2-positive tumor cells in the same subject showed significant changes. Relevant detection results are as follows: Figure 4 As shown, Example

[0032] Cytokine release in in vitro co-culture models suggests an immune response In an in vitro co-culture system, the release of IFN-γ, TNF-α, IL-6, and IL-2 from a subject's sample was detected at different time points. This suggests that the subject possesses a relatively stable ability to release effector and inflammation-related cytokines. Related detection results are as follows: Figure 5 As shown.

[0033] The perforin level was further detected. In the in vitro co-culture system, perforin was detected after co-incubation with N87 cells for 3 h, with a detection value of 5371.25 pg / mL. The relevant detection results are detailed in Table 1. Example

[0034] Changes in the expression of different cytokines in the co-culture model suggest changes in the immune response status. In an in vitro co-culture system of T cells derived from a subject and HER2-positive tumor cells, the release of IFN-γ, TNF-α, and IL-2 was detected under different effector-to-target ratios and incubation times, but the changes in different cytokines varied. This suggests that the subject's immune response exhibited a dynamic characteristic of rapid activation in the early stages of drug administration, followed by subsequent functional adjustments.

Claims

1. A method for evaluating immunotherapy-related immune responses, wherein peripheral blood of a subject is obtained, PBMCs and T cells are separated, co-cultured with tumor cells, and cytokine secretion levels are detected.

2. The application of the method as described in claim 1, by comparing the differences in the levels of cytokines secreted by PBMCs and T cells in the same sample, evaluates the mechanism by which the drug acts on different subtypes of immune cells.

3. The application of the method as described in claim 1, by comparing changes in cytokine secretion at different treatment stages in the same subject, evaluates changes in the immune response effect produced by the drug.

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