Application of DPP4 activity as individual stress marker and immunotherapy synergistic target

Abstract

The invention relates to the field of biomarkers, in particular to application of DPP4 activity as a stress individual marker and an immunotherapy synergistic target. It is found that the activity of DPP4 is obviously related to the stress of an individual, and a DPP4 inhibitor can reverse immunosuppression occurring in the individual of the type, so that the benefit of immunotherapy administered by the DPP4 inhibitor is obviously increased. On the basis, an objective stress marker is provided, and clinical screening and diagnosis of related individuals are facilitated; in addition, a treatment mode which can benefit from DPP4 inhibitor treatment is provided for the type of individuals, and accurate locking of treatment crowds in clinic is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomarkers, and in particular, to the application of DPP4 activity as a stress individual marker and immunotherapy synergistic target. Background Art

[0002] At present, the clinical assessment of individual stress levels and emotional states (anxiety, depression, etc.) mainly relies on a variety of self-assessment and peer-assessment scales, such as the Profile of Mood States Scale (POMS), Patient Health Questionnaire-9 (PHQ-9), Perceived Stress Scale (PSS), Generalized Anxiety Scale (GAD-7), Hamilton Anxiety Rating Scale (HAMA), Montgomery-Asberg Depression Rating Scale (MADRS), etc. Its advantages are simple operation, low cost, suitable for large-scale screening, and standardized scores for easy horizontal comparison. However, the above scales are completely dependent on the subjective perception of patients and assessors, are susceptible to social desirability bias, and cultural differences may lead to decreased scale validity. Therefore, it is urgent to explore reliable and objective biomarkers for stressed individuals. The sampling of peripheral blood (plasma, serum, blood cells) samples is convenient, low-invasive, high in patient compliance, and can achieve continuous observation. Mining and verifying peripheral blood biomarkers of individual stress has better prospects for clinical application and promotion.

[0003] Clinical studies and epidemiological surveys show that about 1 / 3 of cancer patients experience stress and negative emotions (10% have concurrent anxiety, 20% have concurrent mild depression, and 15% have severe depression), which is much higher than normal people and patients with other diseases. Tumor diagnosis, malignant progression, and treatment process (surgery, radiotherapy, chemotherapy, etc.) can induce or aggravate symptoms such as anxiety, depression, and post-traumatic stress. More importantly, stress is closely related to rapid tumor progression, recurrence, metastasis, and poor prognosis. In addition, stress is often closely related to aggravated viral infection, disease progression, aggravated sequelae of infection, and decreased vaccine protection. Recent studies have shown that stress can induce local and systemic immunosuppression in lesions, manifested as a decrease in the proportion of immune effector cells, abnormal differentiation, blocked activation, and weakened anti-tumor and anti-viral efficacy. Research on specific mechanisms and intervention strategies is still in its infancy, and the clinical application prospects of innovative results in this field are broad.

[0004] Dipeptidyl peptidase-4 (DPP4, also known as T cell antigen CD26, adenosine deaminase complexing protein 2) is a multifunctional protein that can catalyze the hydrolysis of the two N-terminal peptide segments of proteins and polypeptides, and can also function as a binding protein or ligand molecule. Membrane-bound DPP4 is widely expressed in various cells throughout the body, but can also be shed from the membrane to become soluble DPP4 that circulates in the plasma. Both forms have dipeptidase activity. The blood glucose-regulating function of DPP4 has been studied most thoroughly, and DPP4 inhibitors (DPP4i) have also been developed as a class of commonly used diabetes treatment drugs. DPP4i can prevent the enzymatic degradation of incretins (glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide), inhibit glucagon release, promote insulin secretion, and slow down gastric emptying, thereby reducing blood glucose levels. The possible cleavage sites of DPP4 are proline, alanine, valine, glycine, serine, threonine, and leucine at the second position from the N-terminus of the protein. Currently, the systematic study of DPP4 substrates is still relatively scarce. There are not many experimentally confirmed DPP4 substrates, only a few chemokines, cytokines, and polypeptide hormones, etc. This has also hindered the exploration of the biological functions and physiological and pathological significance of DPP4, the use of DPP4 for disease diagnosis, and the use of DPP4i for disease treatment strategies.

[0005] Some studies suggest that DPP4 has immunomodulatory functions and may play a dual role in tumors and viral infectious diseases. As an activation antigen and co-stimulatory molecule on the surface of T cells, DPP4 participates in the whole process of T cell maturation, activation, effector function, and memory formation, and can effectively activate anti-tumor immunity. Conversely, DPP4i can prevent the cleavage and inactivation of chemokine CXCL10, which is beneficial for recruiting CXCR3 +Effector T cells to enhance anti-tumor immune responses. DPP4 can also regulate dendritic cell antigen presentation, monocyte migration, hematopoietic stem cell homing, regulatory T cell and Th17 cell differentiation and function. There are also conflicting conclusions regarding whether tumor treatment can benefit from DPP4i. Compared with normal tissues, the expression of DPP4 in different types of tumor tissues may be upregulated or downregulated. A retrospective study of colorectal cancer patients with co-existing diabetes showed that compared with the multi-target diabetes drug metformin, DPP4i can significantly reduce the risk of tumor recurrence. DPP4i can inhibit monocyte-macrophage differentiation and M2 polarization of macrophages, and significantly improve the efficacy of PD-L1 blocking drugs in mice with non-small cell lung cancer. However, the potential risks of DPP4i in specific populations are also worthy of attention. In diabetic patients with female reproductive system malignancies, DPP4i can activate the epithelial-mesenchymal transition pathway, promoting tumor invasion, metastasis and chemotherapy resistance. During viral infections, the level of DPP4 also fluctuates. Compared with normal volunteers, the peripheral blood DPP4 activity of patients with chronic hepatitis C virus infection, hepatitis A virus infection, and Epstein-Barr virus infection is significantly increased, while the plasma DPP4 level of Middle East respiratory syndrome coronavirus patients is significantly decreased, and is correlated with the levels of some cytokines and growth factors. Thus, DPP4 may have a two-way impact on the evolution and outcome of tumors and viral infection diseases. In-depth mechanism exploration and biomarker-driven precision treatment are crucial for achieving the maximum benefit of DPP4i treatment.

[0006] Most existing studies focus on the effects of DPP4i in directly treating tumors or viral infections. Due to the influence of various factors such as cancer types, virus types, disease stages, individual differences, drug dosage and duration, conflicting research conclusions are often obtained, making it difficult to determine the best treatment scenario and drug-using population during clinical treatment.

[0007] Traditional vaccine adjuvant technologies mainly innovate in dimensions such as antigen design, adjuvant development, delivery systems, and combination therapies. Currently, the development of tumor vaccines focuses on: screening and personalized design of tumor neoantigens, precise delivery and stability improvement of antigens, reversing the immunosuppressive tumor microenvironment, etc. The development of viral vaccines focuses on: screening strongly immunogenic epitopes to enhance protective effects, achieving cross-protection through broad-spectrum antigen design, strengthening mucosal immunity, etc. The above ideas are completely based on immunological principles, do not view diseases from an overall perspective and systematic theory, and ignore the close interactive dialogue between the lesion tissue and multiple systemic organs of the body. Macro-environmental factors of the body (especially the neuro-endocrine-immune-metabolism dialogue) are also important determinants of disease evolution and (treatment) outcome. Recently, several important review papers have summarized and prospected this field. There are still huge cognitive gaps and exploration spaces in this field, and a breakthrough in this direction is expected to bring new ideas and strategies for disease diagnosis and treatment. Summary of the Invention

[0008] The present invention focuses on the adverse effects of stress on the body's immune response, and discovers the phenomenon of abnormally increased DPP4 enzyme activity caused by neuroendocrine disorders under stress conditions. Taking this as a breakthrough point, on the one hand, the present invention explores and discovers that DPP4 enzyme activity can be used as a biomarker for diagnosing an individual's stress and emotional state; on the other hand, it is also found that by means of DPP4i, the immune treatment benefit of patients in a stress and negative emotional state can be enhanced.

[0009] Based on the above findings, the present invention first provides the use of a detection reagent in diagnosing or assisting in diagnosing the stress state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity.

[0010] Furthermore, the present invention also provides the use of a detection reagent in preparing a kit for diagnosing or assisting in diagnosing the stress state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity.

[0011] Furthermore, the present invention also provides the use of a detection reagent in diagnosing or assisting in diagnosing the immunosuppressive state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity; the immunosuppression is stress-induced immunosuppression.

[0012] Furthermore, the present invention also provides the use of a detection reagent in preparing a kit for diagnosing or assisting in diagnosing the immunosuppressive state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity; the immunosuppression is stress-induced immunosuppression.

[0013] Furthermore, the present invention also provides the use of a detection reagent in predicting an individual's response to combined DPP4 inhibitor and immunotherapy; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity.

[0014] Furthermore, the present invention also provides the use of a detection reagent in preparing a kit for predicting an individual's response to combined DPP4 inhibitor and immunotherapy; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity.

[0015] Furthermore, the present invention also provides a detection system, which includes: a data processing module, the data processing module is used to obtain biomarker information in a sample to be tested, and then obtain a detection result regarding the stress state of an individual based on the biomarker information; and, a result output module, the result output module is used to obtain and output the detection result; wherein, the biomarker information includes DPP4 activity.

[0016] Furthermore, the present invention also provides the use of a DPP4 inhibitor in preventing, treating or alleviating stress-induced immunosuppression in an individual; wherein, the DPP4 activity in the individual's body is higher than the normal value.

[0017] Furthermore, the present invention also provides the use of a DPP4 inhibitor in the preparation of a drug for preventing, treating or alleviating stress-induced immunosuppression in an individual; wherein, the DPP4 activity in the individual's body is higher than the normal value.

[0018] Furthermore, the present invention also provides the use of a DPP4 inhibitor in improving the immunotherapy effect on a stressed individual; wherein, the DPP4 activity in the individual's body is higher than the normal value.

[0019] Furthermore, the present invention also provides the use of a DPP4 inhibitor in the preparation of a drug for improving the immunotherapy effect on a stressed individual; wherein, the DPP4 activity in the individual's body is higher than the normal value.

[0020] Furthermore, the present invention also provides a combination therapy product, which provides a DPP4 inhibitor and an immunotherapy drug with one or more reagents.

[0021] The present invention discovers that the activity of DPP4 is significantly correlated with the stress of an individual, and the DPP4 inhibitor can reverse the immunosuppression that appears in this type of individual, significantly increasing the benefit of immunotherapy administered to them. Based on this, the present invention not only provides an objective stress biomarker, which is helpful for the clinical screening and diagnosis of related individuals; but also provides a treatment method that can benefit from DPP4i treatment for this type of individual, which is conducive to accurately targeting the treatment population in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1Behavioral changes of stressed mice and test results of various indicators in the embodiments of the present invention; in this figure, A is a schematic diagram of the stress model of repeatedly socially frustrated (SD) mice; B is the result of the light-dark box experiment; C is the result of the open field experiment; D is the result of the social avoidance experiment; E is the ELISA test result of the plasma glucocorticoid (corticosterone) level of mice; F is the blood glucose test result in the body of mice; G is the DPP4 enzyme activity test result in the plasma of mice; H-I are respectively the linear regression analysis results of DPP4 enzyme activity and glucocorticoid (H) or blood glucose level (I).

[0024] Figure 2 Linear regression analysis results of POMS scores and plasma DPP4 enzyme activity of cancer patients in the embodiments of the present invention; in this figure, A and B are respectively the analysis results of two groups of lung cancer patients; C is the analysis result of colorectal cancer patients.

[0025] Figure 3 Prediction results obtained by using the mood binary classification prediction model of cancer patients in the embodiments of the present invention; in this figure, A-C are respectively the prediction model results of lung cancer, colorectal cancer, and the combination of two cancer types.

[0026] Figure 4 Regulation results among stress, glucocorticoid, and DPP4 enzyme activity in the embodiments of the present invention; in this figure, A-C are respectively the glucocorticoid level, DPP4 enzyme activity, and blood glucose test results before and after using mifepristone; D-E are respectively the DPP4 enzyme activity and blood glucose test results before and after using DPP4i; F-H are respectively the changes in the total movement distance, the number of times entering the light box, and the plasma glucocorticoid secretion level of mice before and after stress by DPP4i.

[0027] Figure 5 Effects of injecting whole-cell tumor vaccine and using DPP4i on tumor growth of Ctrl and SD mice in the embodiments of the present invention; in this figure, A is the experimental flow chart of mouse osteosarcoma vaccine and DPP4i intervention; B is the tumor growth curve (upper) and the statistical result of the area under the curve (lower) of Ctrl and SD groups (Ctrl non-Vac, SD non-Vac) of mice that did not receive tumor vaccine injection but were inoculated with subcutaneous tumors; C is the tumor growth curve (upper) and the statistical result of the area under the curve (lower) of Ctrl group mice that received tumor vaccine and subcutaneous tumor inoculation when not using and using DPP4i (Ctrl Vac, Ctrl Vac DPP4i); D is the tumor growth curve (upper) and the statistical result of the area under the curve (lower) of Ctrl group, SD group, and DPP4i-treated SD group mice (CtrlVac, SD Vac, SD Vac DPP4i) that received tumor vaccine and subcutaneous tumor inoculation.

[0028] Figure 6The effects of stress and DPP4i on the protective effect of whole-cell tumor vaccines in lung cancer in the embodiments of the present invention; in this figure, A is the tumor pictures of mice in each group at the experimental endpoint; B to C are the tumor growth curves and the statistical results of the area under the curve of mice in each group respectively.

[0029] Figure 7 The effects of DPP4i on the proportion of immune cells in the tumors of stressed individuals in the embodiments of the present invention; in this figure, A is the flow cytometry analysis strategy; B is the proportion of each immune cell subset calculated based on surface antibody staining.

[0030] Figure 8 The effects of DPP4i on the secretion of effector molecules by immune cells in the tumors of stressed individuals in the embodiments of the present invention; in this figure, A is the flow cytometry analysis strategy; B is the levels of IFN-γ and TNF-α calculated based on intracellular antibody staining.

[0031] Figure 9 The effects of DPP4i on the activation of antigen-specific T cells in the embodiments of the present invention; in this figure, A is the test experimental pattern diagram of the tumor vaccine activating antigen-specific T cells in the draining lymph nodes in vivo; B is the flow cytometry gating strategy diagram for analyzing the in vivo proliferation status of adoptively transferred OT-1 cells; C is the representative results of the tumor vaccine activating antigen-specific T cell proliferation in Ctrl and SD mice in vivo; D is the effects of DPP4i on the proportion, proliferation rate, division index, and proliferation index of OT-1 cells in the lymph nodes.

[0032] Figure 10 The effects of DPP4i on tumor antigen presentation under stress conditions in the embodiments of the present invention; in this figure, A is the DPP4 enzyme activity in the plasma of mice in each group; B is the schematic diagram of co-culturing various types of cells in vitro and the ELISA test results of the IFN-γ concentration in the culture supernatant; C is the reversal effect of different types of DPP4i on the plasma immunosuppressive effect of stressed mice; D is the effects of DPP4i on tumor antigen presentation and antigen-specific T cell IFN-γ secretion in different types of tumor cell models.

[0033] Figure 11 The screening and verification results of highly probable substrates of DPP4 in the embodiments of the present invention; in this figure, A is the flow chart of plasma protein sample pretreatment and LC-MS / MS analysis; B is the PCA analysis of the plasma proteome of mice in each group; C is the differential protein analysis and volcano plot display between different groups; D is the Venn diagram of 210 potential DPP4 substrates circled according to the shown logical idea; E is the clustering analysis result of 210 DPP4 potential substrates; F is 46 highly probable substrate proteins matching the DPP4 structure screened; G is the predicted structure of the complement molecule CFB binding to DPP4; H is the ELISA detection results of CFB in the bodies of mice in each group.

[0034] Figure 12 This figure shows the effect of DPP4i on the antiviral vaccine efficacy under stress in the embodiments of the present invention. In this figure, A and B respectively show the quantitative analysis of the secretion of antigen-specific IgG in mice of the Ctrl and SD groups after 14 days (A) and 35 days (B) of immunization with the antiviral vaccine.

[0035] In each figure, "Ctrl" refers to the group of mice without stress and without tumors; "SD" refers to the group of mice with stress and without tumors; "SD Mife" refers to the group of mice with stress, treated with mifepristone and without tumors; "SD DPP4i" refers to the group of mice with stress, treated with DPP4i and without tumors; "Ctrl non-Vac" refers to the group of mice without stress and with tumors without using the tumor vaccine; "SD non-vac" refers to the group of mice with stress and with tumors without using the tumor vaccine; "CtrlVac" refers to the group of mice without stress and with tumors using the tumor vaccine; "SD Vac" refers to the group of mice with stress and with tumors using the tumor vaccine; "Ctrl Vac DPP4i" refers to the group of mice without stress and with tumors using the tumor vaccine and DPP4i; "SD Vac DPP4i" refers to the group of mice with stress and with tumors using the tumor vaccine and DPP4i; "CtrlRBD Vac" refers to the group of mice without stress using the RBD vaccine; "SD RBD Vac" refers to the group of mice with stress using the RBD vaccine; "Ctrl RBD Vac DPP4i" refers to the group of mice without stress using the RBD vaccine and DPP4i; "SDRBD Vac DPP4i" refers to the group of mice with stress using the RBD vaccine and DPP4i. Detailed Embodiments

[0036] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention. Those skilled in the art can make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce a further embodiment.

[0037] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose the present invention are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains. Through further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] The terms "comprising", "containing" and "including" used in the present invention are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0039] In the present invention, a numerical range represented by endpoints includes all the numerical values and fractions included within the range, as well as the recited endpoints.

[0040] In the present invention, when referring to concentration values, their meanings include fluctuations within a certain range. For example, they can fluctuate within the corresponding precision range. For example, for 2%, a fluctuation within the range of ±0.1% is allowed. For larger numerical values or those that do not require overly precise control, larger fluctuations are also allowed. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. When referring to molecular weight, a fluctuation of ±10% is allowed.

[0041] In the present invention, descriptions such as "a plurality of" and "a variety of", without special limitations, refer to a quantity greater than or equal to 2.

[0042] In the present invention, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the recited features, as well as an open technical solution containing the recited features.

[0043] In the present invention, "preferred", "better", "more preferable", and "preferably" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present invention.

[0044] In the present invention, "optionally", "optional", "option", "optionally", "optional", "option", mean that it can be either present or absent, that is, it refers to any one of the two alternative options of "present" or "absent". If there are multiple "optionally" or "optional" in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optionally" or "optional" is independent.

[0045] In the present invention, the term "DPP4 (Dipeptidyl Peptidase-4)" refers to dipeptidyl peptidase-4, also known as T cell antigen CD26 or adenosine deaminase complexing protein 2, which is a multifunctional protein that can not only catalyze the hydrolysis of the two peptide segments at the N-terminus of proteins and polypeptides, but also act as a binding protein or ligand molecule.

[0046] In the present invention, the term "stress" refers to the physiological and psychological reactions experienced by an individual when facing external environmental changes or challenges (such as work pressure, interpersonal relationships, diseases, etc.). In terms of physiological stress responses, stressors generally activate the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system, leading to increased levels of hormones such as cortisol and adrenaline, and causing physiological changes such as increased heart rate and blood pressure. In terms of psychological stress responses, after an individual cognitively evaluates a stressor, negative emotional reactions such as anxiety, depression, and fear may occur, as well as cognitive and behavioral changes. Moderate stress can improve alertness and efficiency, but chronic or excessive stress can lead to health problems such as decreased immune system function, cardiovascular diseases, and mental illnesses. In the present invention, stress includes negative emotional reactions, that is, the negative emotional states experienced by an individual, such as anxiety, depression, anger, sadness, etc.

[0047] In the present invention, the term "immunosuppression" refers to a state in which the function of the immune system is weakened or inhibited. Immunosuppression can lead to a decrease in an individual's defense ability against pathogens, an increased risk of infection, a reduced protective effect of immunotherapeutic drugs such as vaccines, an increased risk of certain cancers, and at the same time, it can affect wound healing and tissue repair.

[0048] In the present invention, the term "Normal Values" is also referred to as "Reference Values", which refers to the range of measured values of biomarkers in a specific population. Normal values are considered as indicators of a healthy or disease-free state. In practical applications, normal values are usually not a fixed threshold but a range because there is a certain variability in the measured values of biomarkers among healthy individuals. In specific implementations, those skilled in the art can confirm the normal values of biomarkers in combination with conventional methods. As an example, in some specific embodiments, the normal values can be confirmed through the following steps: (1) Select a reference population: Select a group of healthy individuals as the reference population. These individuals should have no relevant medical history, and their characteristics such as age, gender, and race should match those of the target population. (2) Collect samples: Collect biomarker samples from the reference population, such as blood, urine, etc. (3) Measure and analyze: Use standardized laboratory methods to measure and analyze the samples to obtain the concentration or activity of the biomarkers. (4) Statistical analysis: Conduct statistical analysis on the measurement results to determine the range of normal values. Commonly used statistical methods include calculating the mean and standard deviation (Mean±SD), percentiles (such as the 2.5th percentile and the 97.5th percentile), or confidence intervals (such as the 95% confidence interval). (5) Consider influencing factors: Evaluate the influence of factors such as age, gender, race, and lifestyle habits on the biomarkers, and perform stratified analysis on the data as needed. (6) Establish a reference range: Based on the results of the statistical analysis, establish the range or reference range of normal values. This range usually includes the measured values of most healthy individuals, such as 95% of the reference population. (7) Validate and update: Validate the accuracy and applicability of the reference values by comparing with data from other laboratories and studies. As new data accumulates and analysis methods are improved, the reference values may need to be updated regularly.

[0049] In the present invention, the term "Glucocorticoids" refers to a class of steroid hormones secreted by the adrenal cortex, which have a variety of important physiological functions, especially playing a key role in regulating metabolism, immune response, and stress response. In humans, Cortisol is the main glucocorticoid secreted by the adrenal cortex. In rodents (such as mice and rats), Corticosterone is the main glucocorticoid secreted by the adrenal cortex. Corticosterone plays a role similar to Cortisol in rodents, including regulating metabolism and stress response.

[0050] In the present invention, the term "Dipeptidyl Peptidase-4 Inhibitors", also written as "DPP4i" herein, refers to reagents that can inhibit the activity of DPP4 enzyme.

[0051] In the present invention, the term "individual" refers to an independent living organism capable of performing basic life activities such as metabolism, growth, reproduction, etc. In the present invention, the individual includes humans (usually referring to patients, i.e., independent individuals receiving medical services) and other mammals. In some specific embodiments, the other mammals include, but are not limited to: experimental animals such as mice, rats, rabbits, guinea pigs, hamsters, etc.; domestic animals and pets such as pigs, dogs, cats, etc.; primates such as rhesus monkeys, marmosets, etc.; and so on.

[0052] In the present invention, the term "in vivo" refers to the interior of a living organism. In the present invention, when referring to the DPP4 activity or glucocorticoid level in an individual in vivo, it can be confirmed by detecting blood (or serum), urine, or tissue samples.

[0053] The present invention first provides the use of a detection reagent in diagnosing or assisting in diagnosing the stress state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity. That is, the present invention provides a method for diagnosing or assisting in diagnosing the stress state of an individual, which includes: using the detection reagent to detect a sample to be tested; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity. The following further limitations on the application scheme also apply to the said method.

[0054] Furthermore, the present invention also provides the use of a detection reagent in preparing a kit for diagnosing or assisting in diagnosing the stress state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity.

[0055] In the present invention, the stress includes negative emotional reactions.

[0056] In some embodiments, a DPP4 activity higher than the normal value indicates that the individual is in a stress state.

[0057] Furthermore, the present invention also provides the use of a detection reagent in diagnosing or assisting in diagnosing the immunosuppressive state of an individual; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity; the immunosuppression is immunosuppression caused by stress. That is, the present invention provides a method for diagnosing or assisting in diagnosing the immunosuppressive state of an individual, which includes: using the detection reagent to detect a sample to be tested; wherein, the detection reagent includes one or more reagents for detecting DPP4 activity; the immunosuppression is immunosuppression caused by stress. The following further limitations on the application scheme also apply to the said method.

[0058] Further, the present invention also provides the use of a detection reagent in the preparation of a kit for diagnosing or assisting in the diagnosis of the immunosuppressed state of an individual; wherein, the detection reagent comprises one or more reagents for detecting DPP4 activity; and the immunosuppression is stress-induced immunosuppression.

[0059] In some embodiments, the stress includes negative emotional responses.

[0060] In some embodiments, a DPP4 activity higher than the normal value indicates that the individual is in a stress-induced immunosuppressed state.

[0061] In the present invention, the stress-induced immunosuppressed state includes the immunosuppressed state caused by negative emotional responses.

[0062] In some embodiments, the immunosuppressed state includes a reduced response to immunotherapy.

[0063] Further, the present invention also provides the use of a detection reagent in predicting the response of an individual to combination immunotherapy with a DPP4 inhibitor; wherein, the detection reagent comprises one or more reagents for detecting DPP4 activity. That is, the present invention provides a method for predicting the response of an individual to combination immunotherapy with a DPP4 inhibitor, which comprises: using a detection reagent to detect a test sample; wherein, the detection reagent comprises one or more reagents for detecting DPP4 activity. The following further limitations on the application scheme are equally applicable to the said method.

[0064] Further, the present invention also provides the use of a detection reagent in the preparation of a kit for predicting the response of an individual to combination immunotherapy with a DPP4 inhibitor; wherein, the detection reagent comprises one or more reagents for detecting DPP4 activity.

[0065] In some embodiments, a DPP4 activity higher than the normal value indicates that the individual will benefit more from combination immunotherapy with a DPP4 inhibitor.

[0066] The application of the present invention is based on the general mechanism of immunotherapy, and its scope of application is not limited to specific immunotherapy techniques or methods.

[0067] In some embodiments, the immunotherapy includes at least one of vaccine therapy, immune checkpoint inhibitor therapy, cell immunotherapy, cytokine therapy, oncolytic virus therapy, and immunomodulator therapy.

[0068] In some specific embodiments, the vaccine includes but is not limited to at least one of whole cell tumor vaccines, viral vaccines, nucleic acid vaccines, recombinant protein vaccines, polypeptide vaccines, polysaccharide vaccines, polysaccharide-conjugate vaccines, and toxoid vaccines.

[0069] Pre-treating tumor cells with drugs (mitoxantrone, arsenic trioxide, doxorubicin, oxaliplatin, etc.) that efficiently induce immunogenic cell death (ICD) can prepare whole-cell tumor vaccines. In the past nearly 20 years, series of studies by multiple teams at home and abroad have shown that whole-cell tumor vaccines carry all the antigenic epitopes of tumors, and the released and exposed ICD molecules (adenosine triphosphate, heat shock proteins, high-mobility group protein B1, calreticulin, etc.) can serve as natural adjuvants to promote anti-tumor immune responses by binding to pattern recognition receptors. The above characteristics are the same as the core components and action principles of tumor vaccines "antigen + adjuvant".

[0070] In some specific embodiments, the whole-cell tumor vaccine of the present invention is prepared by the following method: pre-treating tumor cells (such as MCA205 osteosarcoma cells) in vitro with mitoxantrone (MTX) (preferably 1 - 10 μM, 8 - 24 hours). After centrifuging to obtain cell pellets, wash away the residual MTX with PBS solution.

[0071] In some specific embodiments, the whole-cell tumor vaccine of the present invention is prepared by the following method: treating tumor cells (such as TC-1 lung cancer cells) with arsenic trioxide (preferably 5 - 50 μM, 8 - 24 hours), obtaining cell pellets and washing away the drug residues with PBS solution.

[0072] In some specific embodiments, the viral vaccine of the present invention is prepared by the following method: mixing a viral antigen or its receptor-binding fragment (such as the receptor-binding domain RBD epitope of the SARS-CoV-2 spike protein) with an equal volume of MF59 emulsion adjuvant to prepare a viral vaccine (such as a SARS-CoV-2 virus vaccine). Among them, the concentration of the viral antigen or its receptor-binding fragment is preferably 0.1 mg / mL - 2 mg / mL.

[0073] In some embodiments, the individual is a cancer patient.

[0074] The application of the present invention is based on the general action mechanism of immunotherapy, and its scope of application is not limited to specific tumor types or tissue sources.

[0075] In some specific embodiments, the tumors include but are not limited to at least one of lung cancer, colorectal cancer, osteosarcoma, melanoma, head and neck cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, cervical cancer, esophageal cancer, endometrial cancer, lymphoma, multiple myeloma, acute lymphoblastic leukemia.

[0076] In some embodiments, the detection reagent further includes one or more reagents for detecting glucocorticoid levels.

[0077] In some embodiments, DPP4 activity and glucocorticoid levels higher than normal values indicate at least one of the following results: i) the individual is in a stress state; ii) the individual is in an immunosuppressed state caused by stress; iii) the individual benefits more from the combination of DPP4 inhibitor and immunotherapy.

[0078] When used in combination with a reagent for detecting glucocorticoid levels, it is beneficial to further improve the accuracy of diagnosis or prediction.

[0079] In some specific embodiments, the detection reagent further includes other reagents related to the accuracy of diagnosis or prediction. For example, reagents for detecting the levels or activities of other related markers.

[0080] The application of the present invention is based on the fundamental pharmacological mechanism of DPP4 inhibitors, that is, the purpose of its administration is achieved by inhibiting DPP4 enzyme activity, and its scope of application is wide, not limited to specific DPP4 inhibitor molecules or compounds.

[0081] In some embodiments, the DPP4 inhibitor includes one or more of sitagliptin, linagliptin, saxagliptin, alogliptin, vildagliptin, and menispermaceous alkaloid compounds.

[0082] In some embodiments, the detection reagent is suitable for using plasma or serum as the sample to be tested.

[0083] In specific implementation, those skilled in the art can confirm the detection methods and detection reagents for DPP4 activity and glucocorticoid levels in combination with common knowledge.

[0084] In some specific embodiments, the DPP4 activity can be detected by the following methods: (i) Fluorescence method: Utilize the hydrolysis reaction of a fluorescent substrate (such as H-Gly-Pro-AMC) under the action of DPP4, and determine the enzyme activity by detecting the intensity of the fluorescent product (AMC); (ii) Colorimetric method: Use a substrate (such as Gly-Pro-pNA) to generate a colored product (p-nitroaniline) under the catalysis of DPP4, and quantify the enzyme activity through the change in absorbance; (iii) Chemiluminescence method: Based on the reaction of a chemiluminescent substrate (such as Z-Gly-Pro-aminoluciferin), it has high sensitivity and low background interference; (iv) ELISA method: Detect the DPP4 protein level through specific antibodies to indirectly reflect its activity, and the commonly used reagent is a secondary antibody labeled with HRP or AP.

[0085] In some specific embodiments, the glucocorticoid level can be detected by the following methods: (i) ELISA method: Specific antibodies (such as anti-cortisol or corticosterone antibodies) are used to detect glucocorticoids (such as cortisol or corticosterone) in serum or urine, and the commonly used reagent is a secondary antibody labeled with HRP or AP; (ii) Liquid chromatography-tandem mass spectrometry (LC-MS / MS): Glucocorticoid molecules are directly detected by high-resolution mass spectrometry, which has high sensitivity and specificity; (iii) Radioimmunoassay (RIA): Radioactively labeled glucocorticoids are used to compete with the sample for binding to antibodies, and the hormone level is quantified by the radioactivity intensity; (iv) Chemiluminescence immunoassay (CLIA): Glucocorticoids are detected based on chemiluminescence reaction, which has high sensitivity and a wide linear range.

[0086] In specific implementation, those skilled in the art can combine the above embodiments to obtain better embodiments regarding the application or method of the present invention.

[0087] Furthermore, the present invention also provides a detection system, which includes: a data processing module, the data processing module is used to obtain biomarker information in a sample to be tested, and then obtain a detection result regarding the stress state of an individual based on the biomarker information; and, a result output module, the result output module is used to obtain and output the detection result; wherein, the biomarker information includes DPP4 activity.

[0088] In some embodiments, the stress includes negative emotional reactions.

[0089] In some embodiments, the data processing module obtains a detection result that the individual is in a stress state when the DPP4 activity is higher than the normal value.

[0090] In some embodiments, when the DPP4 activity is higher than the normal value, the data processing module also obtains at least one of the following detection results 1) to 2): 1) The individual is in an immunosuppressed state caused by stress; 2) The individual benefits more from the combination immunotherapy of DPP4 inhibitors.

[0091] In some embodiments, the biomarker information includes DPP4 activity and glucocorticoid level.

[0092] In some embodiments, when the DPP4 activity and the glucocorticoid level are higher than the normal value, the data processing module obtains at least one of the following results i) to iii): i) The individual is in a stress state; ii) The individual is in an immunosuppressed state caused by stress; iii) The individual benefits more from the combination immunotherapy of DPP4 inhibitors.

[0093] In some embodiments, the detection system further includes: a detection end, which is used to detect biomarker information in a sample to be tested; the detection end is communicatively connected to the data processing module.

[0094] In some specific embodiments, the detection end is used to detect the activity of DPP4 in a sample to be tested.

[0095] In some specific embodiments, the detection end is used to detect the activity of DPP4 and the glucocorticoid level in a sample to be tested.

[0096] In specific implementation, those skilled in the art can confirm the aforementioned detection end in combination with common knowledge. In some specific embodiments, the detection end for detecting the activity of DPP4 in a sample to be tested can be a fluorescence detector (such as a fluorescence microplate reader), an ultraviolet-visible spectrophotometer, a chemiluminescence detector, an ELISA detector (such as an enzyme-linked immunosorbent assay instrument), etc. In some specific embodiments, the detection end for detecting the glucocorticoid level in a sample to be tested can be an ELISA detector (such as an enzyme-linked immunosorbent assay instrument), a liquid chromatography-mass spectrometry system, a gamma counter or a liquid scintillation counter, a chemiluminescence immunoassay analyzer, etc. In some specific embodiments, the detection end can also be a microfluidic detection system, which can simultaneously detect multiple indicators including the activity of DPP4 and the glucocorticoid level.

[0097] In specific implementation, those skilled in the art can combine the foregoing embodiments in combination with common knowledge to obtain more embodiments of the detection system of the present invention.

[0098] Furthermore, the present invention also provides the use of a DPP4 inhibitor in preventing, treating or alleviating immune suppression caused by stress in an individual; wherein, the DPP4 activity in the individual's body is higher than the normal value. That is, the present invention provides a method for preventing, treating or alleviating immune suppression caused by stress in an individual, wherein the DPP4 activity in the individual's body is higher than the normal value, and the method includes: administering a DPP4 inhibitor to the individual. The following further limitations on the application scheme also apply to the aforementioned method.

[0099] Furthermore, the present invention also provides the use of a DPP4 inhibitor in the preparation of a drug for preventing, treating or alleviating immune suppression caused by stress in an individual; wherein, the DPP4 activity in the individual's body is higher than the normal value.

[0100] In some embodiments, the stress includes negative emotional reactions.

[0101] Furthermore, the present invention also provides the use of a DPP4 inhibitor in improving the immunotherapeutic effect on a stressed individual; wherein, the DPP4 activity in the individual's body is higher than the normal value. That is, the present invention provides a method for improving the immunotherapeutic effect on a stressed individual, wherein the DPP4 activity in the individual's body is higher than the normal value, and the method includes: administering a DPP4 inhibitor to the individual. The following further limitations on the application scheme are equally applicable to the said method.

[0102] Furthermore, the present invention also provides the use of a DPP4 inhibitor in the preparation of a drug for improving the immunotherapeutic effect on a stressed individual; wherein, the DPP4 activity in the individual's body is higher than the normal value.

[0103] In some embodiments, the stress includes negative emotional responses.

[0104] In some specific embodiments, the fact that the DPP4 activity in the individual's body is higher than the normal value specifically means that the DPP4 activity in the plasma or serum of the individual is higher than the normal value.

[0105] The application of the present invention is based on the general mechanism of immunotherapy, and its scope of application is not limited to specific immunotherapy techniques or methods.

[0106] In some embodiments, the immunotherapy includes at least one of vaccine therapy, immune checkpoint inhibitor therapy, cellular immunotherapy, cytokine therapy, oncolytic virus therapy, and immunomodulator therapy.

[0107] In some embodiments, the vaccine includes at least one of whole cell tumor vaccine, viral vaccine, nucleic acid vaccine, recombinant protein vaccine, polypeptide vaccine, polysaccharide vaccine, polysaccharide-conjugated protein vaccine, and toxoid vaccine.

[0108] In some embodiments, the individual is a cancer patient.

[0109] The application of the present invention is based on the general mechanism of action of immunotherapy, and its scope of application is not limited to specific cancer types or tissue sources.

[0110] In some embodiments, the cancer includes at least one of, but is not limited to, lung cancer, colorectal cancer, osteosarcoma, melanoma, head and neck cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, cervical cancer, esophageal cancer, endometrial cancer, lymphoma, multiple myeloma, and acute lymphoblastic leukemia.

[0111] In some embodiments, the DPP4 activity and glucocorticoid level in the individual's body are higher than the normal value.

[0112] In some specific embodiments, the DPP4 activity and glucocorticoid level in the individual being higher than the normal value specifically means that the DPP4 activity and glucocorticoid level in the plasma or serum of the individual are higher than the normal value.

[0113] In some embodiments, the DPP4 inhibitor is used in at least one of the following aspects I) - IV): I) increasing the proportion of immune cells in the tumor of an individual; optionally, the immune cells include at least one of CD8 + T cells, dendritic cells, and macrophages; II) promoting the secretion of effector cytokines by immune cells in the tumor of an individual; optionally, the effector cytokines include at least one of IFN-γ and TNF-α; III) promoting the proliferation of antigen-specific T cells in the individual; IV) enhancing tumor antigen presentation in the individual.

[0114] In some embodiments, the DPP4 inhibitor is used to increase the level of DPP4 substrates in an individual.

[0115] In some embodiments, the DPP4 substrates include at least one of the following: AFM, GHR, SERPINA7, CTSA, CFD, AQP9, SERPINA3K, CANT1, CFB, YIPF3, HGFAC, RNF7, CA1, CACNA2D2, ITIH1, SOD1, OSBPL9, PRKAA2, P4HB, QDPR, MOGS, UBE2Z, ATP6V1B2, SOD3, TDP1, FLT4, CA2, MUG1, TRAPPC3, OPTN, CELA2A, PDIA2, CYCS, MBL1, MERTK, GCSAM, PDE9A, ANK1, GSN, ST3GAL4, PSTPIP2, SPTB, TNNC2, APOA2, ABI3BP, ALDH18A1.

[0116] The application of the present invention is based on the fundamental pharmacological mechanism of DPP4 inhibitors, that is, its application purpose is achieved by inhibiting DPP4 enzyme activity, and its scope of application is wide, not limited to specific DPP4 inhibitor molecules or compounds.

[0117] In some embodiments, the DPP4 inhibitor includes one or more of sitagliptin, linagliptin, saxagliptin, alogliptin, vildagliptin, menispermaceous alkaloid compounds.

[0118] In some embodiments, the DPP4 inhibitor is administered to the individual by one or several of the following administration methods: intravenous injection, intraperitoneal injection, oral administration, subcutaneous injection, intramuscular injection, local administration.

[0119] In some specific embodiments, the DPP4 inhibitor and the immunotherapy can be administered to the individual simultaneously. In some specific embodiments, the DPP4 inhibitor and the immunotherapy can be administered to the individual successively (without limiting the specific order).

[0120] In specific implementation, those skilled in the art can combine the above embodiments to obtain more optimal embodiments of the application or method of the present invention.

[0121] Furthermore, the present invention also provides a combination therapy product, which provides a DPP4 inhibitor and an immunotherapy drug with one or more reagents.

[0122] In some specific embodiments, the combination therapy product is a composition.

[0123] In some specific embodiments, the combination therapy product is a combination kit of drugs in the same or different dosage forms.

[0124] In some embodiments, the DPP4 inhibitor includes one or more of sitagliptin, linagliptin, saxagliptin, alogliptin, vildagliptin, and menispermum alkaloid compounds.

[0125] In some embodiments, the immunotherapy drug includes at least one of vaccines, immune checkpoint inhibitors, cellular immunotherapy drugs, cytokines, oncolytic viruses, and immunomodulators.

[0126] In some embodiments, the vaccine includes at least one of whole cell tumor vaccines, viral vaccines, nucleic acid vaccines, recombinant protein vaccines, polypeptide vaccines, polysaccharide vaccines, polysaccharide-conjugate vaccines, and toxoid vaccines.

[0127] In some embodiments, the dosage form of the DPP4 inhibitor is selected from any one of intravenous injection preparations, intraperitoneal injection preparations, oral preparations, subcutaneous injection preparations, intramuscular injection preparations, and topical administration preparations.

[0128] In some embodiments, the combination therapy product further contains other active ingredients and / or adjuvants.

[0129] In specific implementation, those skilled in the art can combine the foregoing embodiments with common knowledge to obtain more embodiments of the combination therapy product of the present invention.

[0130] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present invention is preferably referred to. It can also be carried out according to the experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0131] In the following specific embodiments, regarding the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0132] I. Experimental Materials and Methods

[0133] 1. Mouse repeated social defeat (SD) model: Order male C57BL / 6N mice at 7 - 8 weeks old and male ICR (CD - 1) retired breeding mice at 6 - 7 months old. Aggressive male CD - 1 mice are first acclimated in separate cages for 7 days. Before contacting C57BL / 6N mice every day, CD - 1 mice are exposed to red light in a dark room for 30 minutes to enhance their aggressiveness. Each C57BL / 6N mouse is placed in a new CD - 1 mouse cage every day, and the two mice are in direct contact for 10 minutes. Then, the two mice are physically separated by a plexiglass partition with small holes, so that they cannot have direct physical contact, but they experience 24 - hour visual, olfactory, and auditory contact. Repeat the above steps once a day for 10 consecutive days, or twice a day for 5 consecutive days to induce stress - group (SD) mice ( Figure 1 A in). Control - group (Ctrl) mice are raised alone without contact with CD - 1 mice.

[0134] 2. Mouse behavioral tests: All behavioral tests were arranged within 48 hours after the last social defeat. The inner and outer surfaces of the instruments were pre-cleaned with an alcohol solution and water to remove any residual mouse odor. The light-dark box experiment was conducted in an apparatus consisting of a dark box (18×27×30 cm) and a bright box (illuminance of approximately 200 lux, 27×27×30 cm). The two chambers were connected by a small door (6.5×6.5 cm) through which the mouse could freely pass. The mouse was placed in the dark chamber with its back to the door and allowed to freely explore for 10 minutes before the test was completed. The open field experiment was carried out in an open box apparatus measuring 50 cm × 50 cm, with a central area (20 cm × 20 cm) set artificially. Each mouse was allowed to freely explore for 30 minutes under low light and quiet conditions before the test was completed. The social avoidance test was conducted in an open box apparatus under low light conditions (such as an illuminance of 5 lux). During the first 150 seconds of the test, the mouse was allowed to freely explore the open field, and a hollow circular wire cage (18 cm × 9 cm) was placed in the center of one side of the open box. During the second 150 seconds of the test, the empty wire cage was replaced with a cage containing a CD-1 mouse (Target, the target mouse). The social interaction between the test mouse and the CD-1 mouse was quantified by the time spent entering and staying in the "virtual interaction area (the projected area 8 cm around the wire cage)". The calculation formula for the interaction rate was: (time spent in the interaction area in the presence of the target mouse / time spent in the interaction area in the absence of the target mouse) × 100%. The above experiments all used a video tracking system and SuperMaze V2.0 software to record and analyze the spontaneous movement and real-time position of each mouse, and then analyze parameters such as the total movement distance, the number of entries into the central area, the total movement distance in the central area, and the time spent staying in the interaction area, based on which it was determined whether the mouse had an anxious or depressive-like behavioral phenotype.

[0135] 3. Analysis of mouse plasma DPP4 enzyme activity, blood glucose, and plasma corticosterone levels: Mouse blood samples were collected, and the blood glucose level of the mouse was directly measured using a blood glucose test strip and a blood glucose meter (Yuwell 580, China). The blood samples treated with EDTA anticoagulant were centrifuged at 4000×g for 15 minutes to obtain plasma. The DPP4 enzyme activity was measured using a DPPIV-Glo TM Protease Assay Kit (Promega), and the corticosterone concentration was detected using a corticosterone ELISA kit (ENZO Life Sciences, ADI-901-097) as a reference index for glucocorticoid levels. As an antagonist of the glucocorticoid receptor, mifepristone (Mife) can be used to detect whether the increase in plasma DPP4 enzyme activity after stress is regulated by the glucocorticoid receptor signaling pathway.

[0136] 4. Tumor vaccine model: MCA205 osteosarcoma cells were pretreated with 2 μM mitoxantrone (MTX) in vitro for 16 hours. After centrifugation to obtain cell pellets, the residual MTX was removed by washing with PBS solution. TC-1 lung cancer cells were treated with 25 μM arsenic trioxide for 16 hours, and cell pellets were obtained and washed with PBS solution to remove drug residues. PBS solution or whole-cell tumor vaccine prepared by the above protocol was injected subcutaneously into Ctrl and SD mice at 1×10 6 cells / mouse. After the immune response was initiated (8 days later), tumor cells without drug treatment were subcutaneously inoculated into the above mice at 1×10 6 cells / mouse (tumor rechallenge experiment). In this experiment, Ctrl and SD mice were intraperitoneally injected with PBS solution or DPP4i (sitagliptin) at a dose of 100 mg / Kg each time, and each mouse received a total of 6 administrations (once a day). Thereafter, tumor growth was monitored every 2 - 3 days, and when the tumor reached 300 mm 2 , the mice were euthanized (A in Figure 5 ).

[0137] 5. Viral vaccine model: The receptor-binding domain (RBD) epitope of the SARS-CoV-2 spike protein at a concentration of 0.5 mg / mL was mixed with MF59 emulsion adjuvant in equal volume to prepare the SARS-CoV-2 virus vaccine. 100 μL of the vaccine solution was subcutaneously inoculated at multiple points into Ctrl and SD group mice. From the day of vaccine inoculation, Ctrl and SD group mice were injected with PBS control or DPP4i (sitagliptin) at a dose of 100 mg / Kg each time, once a day, and the injection duration covered the critical stage of immune response initiation, for a total of 8 days. Mouse peripheral blood was collected at different time points, and the IgG level against the RBD antigen epitope was detected by ELISA. Mouse plasma was collected 14 days and 35 days after vaccine inoculation, and the mice were sacrificed 35 days later to end the experiment.

[0138] 6. Detection of the efficacy of tumor-reactive T cells: Ctrl and SD mice received intraperitoneal injection of PBS control or DPP4i (sitagliptin) at a dose of 100 mg / kg every day. Two days after the first injection of DPP4i or PBS solution, all mice received 1 subcutaneous vaccine injection at the footpad. The vaccine was prepared from MTX-pretreated MCA205 tumor cells expressing ovalbumin (OVA) (the method was the same as above). OT-1 cells were purified from the spleen cells of OT-1 mice using magnetic beads (130 - 104 - 075, Miltenyi Biotec), and cell proliferation dye eFluor TM 670 (65 - 0840 - 90, eBioscience TM)Perform cell pre-labeling. Transfer OT-1 cells to the above mice by intravenous injection. Three days later, collect the popliteal lymph nodes on the vaccination side and analyze the proliferation status of OT-1 cells by flow cytometry. The more cell proliferation generations there are, the weaker the fluorescence dye signal they carry. Calculate the cell proliferation index and division index with the help of Flowjo software ( Figure 9 A in). To simulate tumor antigen presentation, stimulate mouse bone marrow cells with 10 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in vitro for 7 days to induce bone marrow-derived dendritic cells (BMDCs). Mix them with MTX-pretreated MCA205-OVA cells (or other mouse solid tumor cells expressing OVA) and OT-1 cells, and place them in DMEM medium containing 25% mouse plasma (derived from the Ctrl group or SD group). In each of the above culture systems, add PBS solution (control) and various DPP4is: 2 μg / mL sitagliptin, 0.1 μg / mL linagliptin, 0.1 μg / mL saxagliptin, 0.4 μg / mL alogliptin, 1 μg / mL vildagliptin. Collect the supernatant after 48 hours and compare the IFN-γ secretion levels in each group using an ELISA kit (BioLegend, 430801).

[0139] 7. Flow cytometry analysis of the immune microenvironment: After cutting the freshly collected tumor tissue into small pieces, place it in a digestive solution containing 0.4 Wünsch U / mL Liberase TL (Roche) and 200 U / mL DNase I (Calbiochem) prepared with serum-free DMEM medium. After digesting at 37 °C for 30 minutes, obtain a single-cell suspension using a 70 μm cell strainer, and block non-specific antibody binding using CD16 / 32 antibody (clone number 2.4G2). Use a dead cell staining kit (LIVE / DEAD TM fixable yellow dead cell stain kit, L34959, Thermo Fisher Scientific) for labeling to exclude dead cells in subsequent analysis. Mix the following antibodies labeled with fluorescent dyes and complete surface marker staining at 4 °C for 30 minutes. To detect the intracellular cytokine secretion level, use GolgiPlug TM and GolgiStop TM (product numbers 555029 and 554724 respectively, purchased from BD Bioscience) to block factor release, and at the same time stimulate the cells with PMA (100 ng / mL, S1819, Beyotime) and ionomycin (5 μM, S1672, Beyotime) at 37 °C for 4 hours. Use BD Cytofix / CytopermTM Cells were treated with a fixation / permeabilization kit, and then mixed fluorescent antibodies were added for staining. The antibodies used included: CD3 (17A2), CD4 (GK1.5), CD8a (53-6.7), CD11b (M1 / 70), Ly6G (1A8), Ly6C (HK1.4), I-A / I-E (M5 / 114.15.2), CD11c (HL3), F4 / 80 (BM8), CD19 (6D5), NK1.1 (PK136), IFN-γ (XMG1.2) and TNF-α (MP6-XT22), all purchased from BioLegend or BD Biosciences. Samples were analyzed using an LSR Fortessa TM flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

[0140] 8. Plasma protein enrichment and profiling: 3 mg of nanoscale magnetic particles (NMPs) were washed and resuspended in 1 mL of washing buffer (10 mM Tris, pH = 7.4, 150 mM KCl, 0.05% CHAPS). Every 40 μg of NMPs was resuspended in 200 μL of washing buffer, mixed with 40 μL of plasma sample, and incubated with shaking at 37 °C for 1 hour. The incubated NMPs were collected by a magnet and washed 3 times with 200 μL of washing buffer. 30 μL of freshly prepared lysis buffer (0.5% SDC (w / v), 10 mM TCEP, 40 mM CAA, 50 mM TEAB) was added to the above NMPs, followed by 10 minutes of sonication and heating at 95 °C for 10 minutes. After the sample was cooled to room temperature naturally, trypsin digestion buffer (40 μL of 50 mM TEAB solution containing 0.5 μg of trypsin) was added, and the mixture was incubated with shaking at 37 °C for 12 hours. The solution was acidified with trifluoroacetic acid (TFA) at a final concentration of 1%, and centrifuged at 16000×g for 10 minutes. The precipitate was further acidified with 100 μL of 1% TFA, sonicated for 5 minutes and then centrifuged at 16000×g for 10 minutes. After the supernatants were combined, desalting was completed using a MSC18 96-well plate. The MSC18 96-well plate was pre-activated with 3 volumes of methanol, equilibrated with 3 volumes of 0.1% formic acid (FA) before adding the peptide sample, and then desalted with 3 volumes of 0.1% FA. The peptides bound to the column were eluted with 40% acetonitrile (ACN) / 0.1% FA and 60% ACN / 0.1% FA respectively. The eluted peptides were collected, dried using a vacuum concentrator and stored at -80 °C in a refrigerator. With the help of Vanquish TMNeo ultra-high performance liquid chromatography system and Orbitrap Astral mass spectrometer (Thermo Fisher Scientific TM ), liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed in data-independent acquisition mode. With the support of the EASYSpray TM online ion source, data acquisition was carried out using an Aurora EliteTS analytical column. Full-scan mass spectrometry analysis was performed in the mass-to-charge ratio range of 380 - 980 m / z, and the resolution was set to 240,000. The automatic gain control of the full-scan mass spectrometry was adjusted to 500%. The isolation window for tandem mass spectrometry scanning was set to 2 Th, and the maximum ion injection time was 3 ms. The mass-to-charge ratio range of tandem mass spectrometry scanning was also set at 380 - 980 m / z. High-energy collision dissociation (HCD) was used to fragment the separated ions, and the normalized collision energy (NCE) was set to 27%. The raw data was analyzed using Spectronaut v19 (Biognosys) software, and the PG.Quantity value was exported as protein abundance data. Student's t-test was performed between different groups. Proteomics analysis used R software and the following software packages: ggord for principal component analysis, ggplot2 for drawing volcano plots, clusterProfiler for enrichment analysis, and aPEAR for gene ontology network analysis.

[0141] 9. Mood state assessment and plasma index detection in cancer patients: After excluding the history of glucocorticoid treatment, newly diagnosed lung cancer and colorectal cancer patients were included in the study. The plasma of patients was used to detect DPP4 enzyme activity using a DPPIV-Glo TM protease assay kit (Promega), and plasma cortisol was detected using an ELISA kit (ENZO Life Sciences, ADI-900-071). Before receiving any treatment regimen and within 7 days after blood collection, the Profile of Mood States (POMS) was used to assess the mood state of inpatients with cancer. The POMS questionnaire assesses five negative mood states (tension, anger, fatigue, depression, and panic) and two positive mood states (vigorous and confident), and each state was measured using a 5-point scale (0 = none, 1 = mild, 2 = moderate, 3 = more, 4 = severe). The POMS score of each patient was calculated using the following formula: 100 + (tension × 0.72) + (anger × 0.82) + (fatigue × 0.71) + (depression × 0.66) + (panic × 0.63) - (vigorous × 0.62) - (confident × 0.63). With the help of linear regression, the correlation between the POMS score and plasma DPP4 enzyme activity was analyzed.

[0142] 10. According to the POMS score, the patients were divided into a stress group (POMS>110) and a normal group (POMS≤110), and a binary classification label was assigned to them. The patient's plasma cortisol level and DPP4 enzyme activity were used as input features to construct a binary classification prediction model. To ensure the reliability of the model and the robustness of the evaluation results, the five-fold cross-validation method was used for model training and evaluation. Specifically, the complete data set was randomly divided into five subsets of similar size, four of which were used for model training each time, and the remaining subset was used for validation. The cycle was repeated five times to ensure that each sample was used for validation once. In the validation stage, the area under the receiver operating characteristic curve (AUC) was calculated as a model performance evaluation indicator. The final prediction model was further constructed by an ensemble learning method. Specifically, the five models trained in the five-fold cross-validation were combined through bagging technology to obtain a comprehensive model to improve the prediction stability and accuracy. Subsequently, the final model was tested with the entire data set, lung cancer data set, and colorectal cancer data set, and the AUC value was calculated to comprehensively evaluate the predictive ability of the model and its applicability in patients with different cancer types.

[0143] 2. Experimental Results

[0144] 1. Stress leads to anxiety-depression-like phenotypes, increases plasma corticosterone, blood glucose and DPP4 activity. The light-dark box experiment suggests that SD mice have anxiety-like behavioral changes and a decrease in total movement distance ( Figure 1 B in the figure). The open field test showed that SD mice showed an anxiety-like phenotype, with a significant decrease in the distance traveled in the central area and the number of times they entered the central area ( Figure 1 C in the figure). The social avoidance experiment suggests that mice show a depressive-like phenotype. When there is no target mouse, there is no difference in the length of time that Ctrl and SD mice stay in the interaction zone. When the target mouse appears, the length of time that SD mice stay in the interaction zone is much shorter than that of Ctrl mice ( Figure 1 D in the figure). ELISA showed that the plasma glucocorticoid (corticosterone) level of SD mice was much higher than that of Ctrl mice. The significant increase may indicate overactivation of the hypothalamus-pituitary-adrenal axis and increased individual stress levels. Importantly, there was no difference in the background level of plasma corticosterone between the two groups of mice before the start of the experiment ( Figure 1 E). Compared with the Ctrl group mice, the blood glucose level of SD mice was significantly increased ( Figure 1 F), and plasma DPP4 enzyme activity increased ( Figure 1 The relationship between DPP4 enzyme activity and glucocorticoids ( Figure 1 H) or blood sugar level ( Figure 1 The correlation between DPP4 enzyme activity and glucocorticoids and blood glucose levels was shown in Figure 1.

[0145] 2. The POMS score of tumor patients is positively correlated with plasma DPP4 enzyme activity. Lung cancer patient cohorts ( Figure 2 A and B therein) and colorectal cancer patient cohorts ( Figure 2 C therein) were included in the analysis. Linear regression analysis indicated that there was a significant positive correlation between DPP4 enzyme activity and the POMS score.

[0146] 3. Using plasma glucocorticoid and DPP4 enzyme activity as input features, a binary classification prediction model for the mood of tumor patients was constructed. The prediction model results of lung cancer ( Figure 3 A therein), colorectal cancer ( Figure 3 B therein), and the combination of the two cancer types ( Figure 3 C therein) are as shown in Figure 3 , showing that plasma glucocorticoid and DPP4 enzyme activity can effectively infer the mood state of patients.

[0147] 4. Regarding the regulatory relationship among stress, glucocorticoid, and DPP4 enzyme activity, the results showed that by means of the glucocorticoid receptor antagonist mifepristone (Mife), the excessive secretion of glucocorticoid induced by stress ( Figure 4 A therein), the increase in DPP4 enzyme activity ( Figure 4 B therein), and the increase in blood glucose ( Figure 4 C therein) could be reversed. While DPP4i could effectively inhibit the plasma DPP4 enzyme activity ( Figure 4 D therein), reverse the increase in blood glucose in stressed mice ( Figure 4 E therein), but could not normalize the behavioral characteristics ( Figure 4 F and G therein) and the glucocorticoid secretion level ( Figure 4 H therein) of stressed mice.

[0148] 5. Stress and DPP4i affect the protective effect of whole-cell tumor vaccine against osteosarcoma. Figure 5 Statistics of the tumor growth curves and areas under the curves of each group in B–D therein showed that when the vaccine was not inoculated, the tumors of mice all grew rapidly, and there was no obvious difference between the control group and the stress group (Ctrl non-Vac group and SD non-Vac group, Figure 5 B therein). After inoculating the tumor vaccine, the tumor growth of mice in the control group (Ctrl Vac) was significantly delayed, while using DPP4i (Ctrl Vac DPP4i) did not affect the tumor growth in the control group ( Figure 5 C therein). Compared with the control group (Ctrl Vac), the protective effect of the tumor vaccine in the stress group (SD Vac) was significantly reduced, while DPP4i could significantly improve the vaccine benefit of mice in the SD group (SD Vac DPP4i) ( Figure 5 D therein).

[0149] 6. Stress and DPP4i affect the protective effect of whole-cell tumor vaccine against lung cancer. Figure 6 In A of Figure 6 , the tumor pictures of mice in each group at the experimental endpoint are shown. From top to bottom, they correspond to Ctrl Vac, Ctrl Vac DPP4i, SD Vac, and SD Vac DPP4i respectively. The tumor growth curves ( Figure 6 in B of Figure 6 ) and the statistical results of the area under the curve ( Figure 6 in C of Figure 6 ) show that stress significantly hinders the protective effect of the vaccine. DPP4i can significantly improve the vaccine benefit in SD group mice, but does not affect the vaccine effect in Ctrl group.

[0150] 7. DPP4i remodels the infiltration of immune cells in tumors of stressed individuals. Figure 7 In A of Figure 7 is the analysis strategy of flow cytometry. Figure 7 In B of Figure 7 , the proportions of each immune cell subset calculated based on surface antibody staining are shown. The results suggest that DPP4i can significantly increase the proportions of CD8 + T, dendritic cells (DC), and macrophages in the tumors of stressed mice.

[0151] 8. DPP4i can promote the secretion of effector molecules by immune cells in tumors of stressed individuals. Figure 8 In A of Figure 8 is the analysis strategy of flow cytometry. Figure 8 In B of Figure 8 , the proportional changes of CD8 + T and NK cells secreting effector cytokines are calculated. The results suggest that the levels of IFN-γ and TNF-α are enhanced.

[0152] 9. DPP4i can promote the proliferation of antigen-specific T cells and enhance anti-tumor immunity. Figure 9 In B of Figure 9 is the flow cytometry gating strategy diagram for analyzing the in vivo proliferation status of adoptive OT-1 cells. Figure 9 In C of Figure 9 are the representative results of the activation of antigen-specific T cell proliferation by tumor vaccine in Ctrl and SD mice. DPP4i can significantly increase the proportion, proliferation rate, division index, and proliferation index of OT-1 cells in the lymph nodes ( Figure 9 in D of Figure 9 ).

[0153] 10. DPP4i can enhance tumor antigen presentation under stress conditions. Plasma samples from Ctrl and SD mice were obtained, and the DPP4 enzyme activity in these plasma samples was detected with or without the addition of DPP4i. The results showed that DPP4i could effectively reverse the increase in plasma DPP4 enzyme activity induced by stress in vitro ( Figure 10A) in the above. Prepare DMEM medium with the above plasma samples to simulate the in - vivo environment of stressed mice. Culture bone - marrow - derived DC cells, MTX - pretreated tumor cells, and OT - 1 cells in vitro. The IFN - γ concentration in the culture supernatant indicates the ability of tumor antigen presentation and activation of T cells. Serum culture of SD mice significantly inhibits tumor antigen presentation, and this inhibitory effect can be effectively reversed by DPP4i ( Figure 10 B) in the above. Replace the type of DPP4i in the same experimental system, changing from sitagliptin to linagliptin, saxagliptin, alogliptin, or vildagliptin. Both tumor antigen presentation and antigen - specific T - cell activation can be significantly increased ( Figure 10 C) in the above. Replace the type of tumor cells expressing OVA antigen in the same experimental system, changing from TC - 1 lung cancer cells to B16 melanoma, MC38 colorectal cancer, MCA205 osteosarcoma, 4T1 breast cancer, HEPA1 - 6 liver cancer, AKR esophageal cancer, MFC gastric cancer. DPP4i can effectively enhance tumor antigen presentation and antigen - specific T - cell activation in the above systems ( Figure 10 D) in the above.

[0154] 11. Screening and verification of highly probable DPP4 substrates. Figure 11 A in the above is the flow chart of plasma protein sample pretreatment and LC - MS / MS analysis, Figure 11 B in the above is the PCA analysis of plasma proteome of each group of mice, Figure 11 C in the above is the differential protein analysis and volcano plot display between different groups, Figure 11 D in the above is a Venn diagram showing 210 potential DPP4 substrates circled according to the indicated logical idea. Cluster analysis of the 210 potential DPP4 substrates reveals that they are closely related to immune responses, suggesting that DPP4 may participate in multi - level immune regulation through its substrate molecules ( Figure 11 E) in the above. Screen 97 proteins containing potential DPP4 cleavage sites from 210 candidate proteins. According to AlphaFold3 prediction of the molecular docking of DPP4 and substrate proteins, screen 46 highly probable substrate proteins that match the structure of DPP4. According to the list of the second amino acid category at the N - terminus of the cleavage site ( Figure 11 F) in the above. Select one protein - - complement molecule CFB, Figure 11 G in the above shows its predicted structure binding to DPP4. ELISA was used to confirm that the abundance of CFB indeed decreased after stress (consistent with the increase in DPP4 enzyme activity), and the abundance of CFB in the stressed group of mice increased significantly after DPP4i treatment ( Figure 11 H) in the above.

[0155] 12. DPP4i can enhance the secretion of IgG antibodies induced by antiviral vaccines under stress. The receptor-binding domain (RBD) epitope of the SARS-CoV-2 spike protein was mixed with an equal volume of MF59 emulsion adjuvant to prepare an antiviral vaccine, which was inoculated into control and stressed mice with or without DPP4i treatment. Plasma was collected on the 14th and 35th days after vaccination. DPP4i significantly increased the secretion level of IgG in stressed mice ( Figure 12 A and B in

[0156] In summary, the present invention found that stress can lead to a surge in glucocorticoid secretion and abnormal elevation of blood glucose, accompanied by a significant increase in the activity of dipeptidyl peptidase 4 (DPP4). DPP4 inhibitor (DPP4i) can not only normalize the blood glucose level in stressed mice but also reverse stress-induced immunosuppression, significantly enhancing the protective efficacy of tumor vaccines and viral vaccines in stressed mice. A cohort study of lung cancer and colorectal cancer patients without hormone treatment showed that the scores of the Profile of Mood States (POMS) were significantly positively correlated with the plasma DPP4 enzyme activity. Based on the plasma glucocorticoid level and DPP4 enzyme activity of patients, it is possible to effectively infer the mood state and stress level of patients, which is expected to provide objective stress markers to assist clinical screening and diagnosis. In addition, this also suggests that immunosuppression in stressed individuals may benefit from DPP4i treatment, which is conducive to accurately identifying the treatment population. Considering that about one-third of tumor patients and patients with chronic or recurrent infections have poor mood and emotional disorders, the number of patients who are expected to benefit from the application of this invention is huge. In individuals with mental and emotional disorders, DPP4i can enhance the immune activation efficacy of tumor vaccines and viral vaccines, which has potential clinical value for vaccine potentiation. At the same time, the plasma DPP4 activity level and glucocorticoid level can be jointly used as biomarkers to predict the stress level of individuals and whether they will benefit from DPP4i combined with vaccines.

[0157] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Application of the detection reagent in any of the following aspects a1) to a2): a1) Diagnose or assist in diagnosing an individual’s stress state; a2) preparing a kit for diagnosing or assisting in diagnosing a stress state of an individual; in, The detection reagents include one or more reagents for detecting DPP4 activity; Optionally, the stress comprises a negative emotional response.

2. The use according to claim 1, wherein: Higher than normal DPP4 activity indicates that the individual is under stress.

3. Application of the detection reagent in any of the following aspects b1) to b2): b1) diagnose or assist in diagnosing an individual’s immunosuppressive state; b2) preparing a kit for diagnosing or assisting in the diagnosis of an individual's immunosuppressive state; in, The detection reagents include one or more reagents for detecting DPP4 activity; The immunosuppression is immunosuppression caused by stress; Optionally, the stress comprises a negative emotional response.

4. The use according to claim 3, wherein: Higher than normal DPP4 activity indicates that the individual is in a state of stress-induced immunosuppression.

5. The use according to claim 3 or 4, wherein: The immunosuppressive state includes a reduced response to immunotherapy.

6. Application of the detection reagent in any of the following aspects c1) to c2): c1) predict individual response to DPP4 inhibitor combined with immunotherapy; c2) preparing a kit for predicting an individual's response to a combination of a DPP4 inhibitor and immunotherapy; in, The detection reagents include one or more reagents for detecting DPP4 activity.

7. The use according to claim 6, wherein: Higher-than-normal DPP4 activity suggests that individuals may benefit more from combined DPP4 inhibitor and immunotherapy.

8. The use according to any one of claims 5 to 7, wherein: The immunotherapy includes at least one of vaccine therapy, immune checkpoint inhibitor therapy, cellular immunotherapy, cytokine therapy, oncolytic virus therapy, and immunomodulator therapy; optionally, the vaccine includes at least one of whole cell tumor vaccine, viral vaccine, nucleic acid vaccine, recombinant protein vaccine, polypeptide vaccine, polysaccharide vaccine, polysaccharide and carrier protein combined vaccine, and toxoid vaccine.

9. The use according to any one of claims 1 to 8, wherein: The individual is a tumor patient; optionally, the tumor includes at least one of lung cancer, intestinal cancer, osteosarcoma, melanoma, head and neck cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, cervical cancer, esophageal cancer, endometrial cancer, lymphoma, multiple myeloma, and acute lymphocytic leukemia.

10. The use according to any one of claims 1 to 9, wherein: The detection reagents also include one or more reagents for detecting glucocorticoid levels; Optionally, DPP4 activity and glucocorticoid levels above normal values ​​indicate at least one of the following results i) to iii): i) The individual is under stress; ii) the individual is in a state of immunosuppression caused by stress; iii) Individuals benefit more from DPP4 inhibitors combined with immunotherapy.

11. The use according to any one of claims 6 to 10, wherein: The DPP4 inhibitor includes one or more of sitagliptin, linagliptin, saxagliptin, alogliptin, vildagliptin, and dauricum alkaloid compounds.

12. The use according to any one of claims 1 to 11, wherein: The detection reagent is suitable for taking plasma or serum as the sample to be tested.

13. A detection system comprising: A data processing module, the data processing module is used to obtain biomarker information in the sample to be tested, and then obtain a test result about the stress state of the individual based on the biomarker information; and, a result output module, the result output module being used to obtain and output the detection result; Wherein, the biomarker information includes DPP4 activity; Optionally, the stress comprises a negative emotional response.

14. The detection system according to claim 13, wherein: The data processing module obtains a detection result indicating that the individual is in a stress state when the DPP4 activity is higher than a normal value.

15. The detection system according to claim 13 or 14, wherein: When the DPP4 activity is higher than the normal value, the data processing module also obtains at least one of the following test results 1) to 2): 1) the individual is in a state of immunosuppression caused by stress; 2) the individual benefits more from the combined immunotherapy of DPP4 inhibitors.

16. The detection system according to any one of claims 13 to 15, wherein: The biomarker information includes DPP4 activity and glucocorticoid levels; Optionally, the data processing module obtains at least one of the following results i) to iii) when the DPP4 activity and glucocorticoid level are higher than normal values: i) The individual is in a state of stress; ii) the individual is in a state of immunosuppression caused by stress; iii) Individuals benefit more from DPP4 inhibitors combined with immunotherapy.

17. The detection system according to any one of claims 13 to 16, further comprising: A detection end, which is used to detect biomarker information in a sample to be tested; The detection end is connected to the data processing module through communication.

18. Use of DPP4 inhibitors in any of the following aspects A1) to A2): A1) preventing, treating or alleviating stress-induced immunosuppression in individuals; A2) preparing a medicament for preventing, treating or alleviating stress-induced immunosuppression in an individual; in, The DPP4 activity in the individual is higher than normal; Optionally, the stress comprises a negative emotional response.

19. Use of DPP4 inhibitors in any of the following aspects B1) to B2): B1) Improve the efficacy of immunotherapy in stressed individuals; B2) preparing a medicament for improving the effect of immunotherapy on stressed individuals; in, The DPP4 activity in the individual is higher than normal; Optionally, the stress comprises a negative emotional response.

20. The use according to claim 19, wherein: The immunotherapy includes at least one of vaccine therapy, immune checkpoint inhibitor therapy, cellular immunotherapy, cytokine therapy, oncolytic virus therapy, and immunomodulator therapy; optionally, the vaccine includes at least one of whole cell tumor vaccine, viral vaccine, nucleic acid vaccine, recombinant protein vaccine, polypeptide vaccine, polysaccharide vaccine, polysaccharide and carrier protein combined vaccine, and toxoid vaccine.

21. The use according to any one of claims 18 to 20, wherein: The individual is a tumor patient; optionally, the tumor includes at least one of lung cancer, intestinal cancer, osteosarcoma, melanoma, head and neck cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, cervical cancer, esophageal cancer, endometrial cancer, lymphoma, multiple myeloma, and acute lymphocytic leukemia.

22. The use according to any one of claims 18 to 21, wherein: The individual has higher than normal DPP4 activity and glucocorticoid levels.

23. The use according to any one of claims 18 to 22, wherein: The DPP4 inhibitor is used for at least one of the following I) to IV): I) increasing the proportion of immune cells in the individual tumor; optionally, the immune cells include CD8 + at least one of a T cell, a dendritic cell, and a macrophage; II) promoting the secretion of effector cytokines by immune cells in the individual tumor; optionally, the effector cytokines include at least one of IFN-γ and TNF-α; III) promoting the proliferation of antigen-specific T cells in an individual; IV) enhancing tumor antigen presentation in a subject.

24. The use according to any one of claims 18 to 23, wherein: The DPP4 inhibitor is used to increase the level of a DPP4 substrate in a subject; Optionally, the DPP4 substrate includes at least one of the following: AFM, GHR, SERPINA7, CTSA, CFD, AQP9, SERPINA3K, CANT1, CFB, YIPF3, HGFAC, RNF7, CA1, CACNA2D2, ITIH1, SOD1, OSBPL9, PRKAA2, P4HB, QDPR, MOGS, UBE2Z, ATP6V1B2, SOD3, TDP1, FLT4, CA2, MUG1, TRAPPC3, OPTN, CELA2A, PDIA2, CYCS, MBL1, MERTK, GCSAM, PDE9A, ANK1, GSN, ST3GAL4, PSTPIP2, SPTB, TNNC2, APOA2, ABI3BP, ALDH18A1.

25. The use according to any one of claims 18 to 24, wherein: The DPP4 inhibitor includes one or more of sitagliptin, linagliptin, saxagliptin, alogliptin, vildagliptin, and dauricum alkaloid compounds.

26. The use according to any one of claims 18 to 25, wherein: The DPP4 inhibitor is administered to the individual by one or more of intravenous injection, intraperitoneal injection, oral administration, subcutaneous injection, intramuscular injection, and local administration.

27. A combination therapeutic product providing a DPP4 inhibitor and an immunotherapeutic drug in one or more agents.

28. The combination therapy product according to claim 27, wherein The DPP4 inhibitor includes one or more of sitagliptin, linagliptin, saxagliptin, alogliptin, vildagliptin, and dauricum alkaloid compounds.

29. The combination therapy product according to claim 27 or 28, wherein The immunotherapy drug includes at least one of a vaccine, an immune checkpoint inhibitor, a cellular immunotherapy drug, a cytokine, an oncolytic virus, and an immunomodulator; optionally, the vaccine includes at least one of a whole cell tumor vaccine, a viral vaccine, a nucleic acid vaccine, a recombinant protein vaccine, a polypeptide vaccine, a polysaccharide vaccine, a polysaccharide and carrier protein combined vaccine, and a toxoid vaccine.

30. The combination therapy product according to any one of claims 27 to 29, wherein The dosage form of the DPP4 inhibitor is selected from any one of intravenous injection preparations, intraperitoneal injection preparations, oral preparations, subcutaneous injection preparations, intramuscular injection preparations, and local administration preparations.

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