Application of two metabolites in preparation of immune checkpoint inhibitor sensitizer based on body mass index layering

Abstract

The invention discloses application of two metabolites in preparation of an immune checkpoint inhibitor (ICIs) sensitizer based on body mass index (BMI) stratification. The metabolites are flavin adenine dinucleotide and docosahexaenoic acid. Experiments prove that: when a cancer patient has BMILlt; when the time is 25, the treatment effect of the tumor immune checkpoint inhibitor is better according to the docosahexaenoic acid (DHA) and ICIs treatment scheme; when the BMI of a cancer patient is greater than or equal to 25, the curative effect of the tumor immune checkpoint inhibitor is better according to a flavin adenine dinucleotide (FAD) combined ICIs treatment scheme. Therefore, the invention provides a BMI difference-based personalized metabolite combined immune checkpoint inhibitor (ICIs) treatment scheme, and different metabolites are respectively screened and applied as sensitizers of ICIs for obese (BMI is greater than or equal to 25) and non-obese (BMILt, 25) cancer patients, so that the accurate improvement of the curative effect of the immune checkpoint inhibitor is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular to the use of two metabolites in the preparation of an immune checkpoint inhibitor sensitizer based on body mass index stratification. BACKGROUND

[0002] Immune checkpoint inhibitors (ICIs) can activate anti-tumor immune responses by blocking the immune inhibitory pathways such as programmed death 1 (PD-1) and its ligand PD-L1, or cytotoxic T lymphocyte antigen 4 (CTLA-4), thus relieving the inhibition of T cells in the tumor microenvironment. Although such drugs have shown breakthrough efficacy in a variety of malignancies, overall only a portion of patients can obtain durable clinical benefit, suggesting that immune checkpoint blockade itself is not enough to restore effective anti-tumor immunity in all patients.

[0003] Previous studies have gradually recognized that the occurrence and maintenance of tumor immune responses are highly dependent on the metabolic state of immune cells. The availability of energy substrates, redox state, and lipid and amino acid metabolism characteristics in the tumor microenvironment can profoundly affect the proliferation, differentiation, and killing function of effector T cells. Therefore, improving the functional state of immune cells through metabolic intervention is considered an important strategy to enhance the efficacy of ICIs.

[0004] However, the existing metabolic-related combination therapy schemes generally have a key defect, that is, the systematic differences in the overall metabolic state of patients are not taken into account in the design of the treatment strategy. In clinical practice, cancer patients have significant individual differences in body composition, fat distribution, basal energy metabolism, and chronic inflammation levels, among others. Body mass index (BMI) as an important indicator reflecting the overall metabolic characteristics shows stable and quantifiable stratification characteristics in different patient populations. Obese or overweight patients and normal weight or cachectic patients have systematic differences in fat factor secretion, lipid metabolism, mitochondrial function, and immune metabolic regulation pathways, which can lead to different and even opposite regulatory effects of the same metabolite on immunotherapy in different patients.

[0005] There is currently a lack of a systematic technical solution based on patient metabolic phenotype stratification and the selection and application of specific metabolites to enhance the efficacy of immune checkpoint inhibitors. Therefore, it is of great theoretical value and clinical application prospect to establish a personalized immune metabolic regulation strategy that combines patient metabolic state and metabolite selection. SUMMARY

[0006] In view of the existing technical problems, the purpose of the present application is to provide the use of two metabolites in the preparation of an immune checkpoint inhibitor sensitizer based on body mass index stratification.

[0007] The purpose of the present application is achieved by the following technical solutions: In the first aspect, the present application provides the use of two metabolites in the preparation of an immune checkpoint inhibitor sensitizer based on body mass index stratification, wherein the metabolites are flavin adenine dinucleotide (FAD) and docosahexaenoic acid (DHA).

[0008] As a preferred solution, the sensitizer can improve the therapeutic effect of the immune checkpoint inhibitor for the cancer patient.

[0009] As a preferred solution, when the cancer patient is an obese cancer patient with a BMI of ≥25, the metabolite is flavin adenine dinucleotide.

[0010] As a preferred solution, when the cancer patient is a cachectic cancer patient with a BMI of <25, the metabolite is docosahexaenoic acid.

[0011] As a preferred solution, the cancer is colorectal cancer or melanoma.

[0012] As a preferred solution, the immune checkpoint inhibitor includes at least one of an anti-PD-1 inhibitor, an anti-PD-L1 inhibitor, and an anti-CTLA-4 inhibitor.

[0013] As a preferred solution, the immune checkpoint inhibitor is an anti-PD-1 inhibitor.

[0014] In the second aspect, the present application provides a pharmaceutical composition for treating cancer, which includes an immune checkpoint inhibitor and a sensitizer; the sensitizer includes a metabolite; The metabolite is selected from flavin adenine dinucleotide or docosahexaenoic acid.

[0015] As a preferred solution, the cancer is colorectal cancer or melanoma.

[0016] As a preferred solution, when the cancer patient is an obese cancer patient with a BMI of ≥25, the metabolite is flavin adenine dinucleotide; When the cancer patient is a cachectic cancer patient with a BMI of <25, the metabolite is docosahexaenoic acid.

[0017] As a preferred solution, the effective dose of flavin adenine dinucleotide is 0.5-50 mg / kg; more preferably, the effective dose is 2 mg / kg.

[0018] As a preferred solution, the effective dose of the docosahexaenoic acid is 10-100 mg / kg; more preferably, the effective dose is 30 mg / kg.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application proposes a new personalized metabolite combined ICIs treatment scheme for obese (BMI≥25) and lean (BMI<25) cancer patients, and finds that different metabolites are provided as sensitizers of immune checkpoint inhibitors for obese or lean patients according to BMI difference, so as to realize precise enhancement of tumor immune checkpoint inhibitor efficacy.

[0020] The present application has been verified to find that when the BMI of a cancer patient is less than 25, the docosahexaenoic acid (DHA) combined ICIs treatment scheme has better tumor immune checkpoint inhibitor efficacy, and when the BMI of a cancer patient is greater than or equal to 25, the flavin adenine dinucleotide (FAD) combined ICIs treatment scheme has better tumor immune checkpoint inhibitor efficacy, indicating that the personalized immune enhancement strategy based on BMI difference is more precise and targeted than the existing immune checkpoint inhibitor monotherapy treatment scheme. BRIEF DESCRIPTION OF DRAWINGS

[0021] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the accompanying drawings: Fig. 1 is the evaluation results of the personalized metabolites obtained based on the treatment response of different BMI populations and their role in enhancing the efficacy of tumor immune checkpoint inhibitors; wherein Fig. 1A is the characteristic metabolite FAD for sensitizing anti-PD-1 / PD-L1 treatment response of cancer patients with BMI≥25 obtained by screening; Figure 1 Fig. 1B is the characteristic metabolite DHA for sensitizing anti-PD-1 / PD-L1 treatment response of cancer patients with BMI<25 obtained by screening; Fig. 1C is the evaluation results of FAD in enhancing the efficacy of tumor immune checkpoint inhibitors in the MC38 tumor model; Fig. 1D is the evaluation results of FAD in enhancing the efficacy of tumor immune checkpoint inhibitors in the B16-F0 tumor model; Fig. 1E is the evaluation results of DHA in enhancing the efficacy of tumor immune checkpoint inhibitors in the MC38 tumor model, Figure 1 Fig. 1F is the evaluation results of DHA in enhancing the efficacy of tumor immune checkpoint inhibitors in the B16-F0 tumor model; Fig. 1G is the left graph of the experimental process, the middle graph is the change results of the tumor volume of mice in each group during treatment, and the right graph is the comparison of the tumor volume of mice in each group after 19 days of treatment. DETAILED DESCRIPTION

[0022] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1 1. Patient recruitment and sample collection A total of 196 cancer patients receiving anti-PD-1 / PD-L1 therapy were recruited from our collaborating institutions. Cohort details are shown in Table 1. Patient recruitment and sample collection protocols were approved by the Medical Ethics Committee of Renji Hospital affiliated with Shanghai Jiao Tong University School of Medicine (ID: LY2020-067-B), Shanghai Cancer Center of Fudan University, Shanghai Medical College of Fudan University (ID: 050432-4-1911D), and Shanghai Chest Hospital affiliated with Shanghai Jiao Tong University School of Medicine (ID: KS23026). Enrolled patients were individuals with pathologically confirmed cancer and available BMI data. Sample collection was conducted before the start of immunotherapy. All patients had no history of autoimmune diseases or previous cancer. All enrolled patients were treatment-naïve and had not received chemotherapy, radiotherapy, or other anti-tumor treatments prior to surgery. Furthermore, enrolled patients were required to be about to begin anti-cancer treatment, be able and willing to comply with the study procedures, and have social security or an equivalent protection plan. Informed consent must be obtained before any research procedure is performed. Exclusion criteria include pregnant or breastfeeding women, patients under legal guardianship or judicial / administrative detention, or patients who cannot provide informed consent.

[0025] Standard treatment for patients will continue until disease progression or unacceptable adverse events occur. Baseline clinical characteristics should be recorded, including sex, age, BMI, type of immune checkpoint inhibitor (ICIs), cancer type, and antibiotic use. A detailed list of concomitant medications used in the three months prior to initiating ICIs, follow-up duration, and treatment outcomes should also be provided.

[0026] All participants signed written informed consent forms for sample collection and subsequent analysis, some of which had been previously reported by our team. Patient treatment response was assessed according to the RECIST v1.1 criteria for evaluating the efficacy of treatment in solid tumors. The primary endpoint was the investigator-assessed objective response rate (ORR), defined as the number and proportion of patients achieving a confirmatory complete response (CR) or partial response (PR). Best overall response (BOR) was defined as the best response from initial treatment to the onset of tumor progression or initiation of subsequent treatment according to RECIST v1.1 criteria (whichever occurred first). BOR was primarily assessed at 6-month intervals. Complete response (CR), partial response (PR), or stable disease (SD) were considered responders (R), while disease progression (PD) was considered a non-responder (NR). For patients who did not experience recorded disease progression or initiation of subsequent treatment, all available response data were used for BOR assessment. For patients still alive at the time the database was locked, follow-up ended at the last recorded contact date.

[0027] Table 1 Summary of cohort clinical information 2. Screening for characteristic metabolites in cancer patients with BMI ≥ 25 that enhance response to anti-PD-1 / PD-L1 therapy The study was commissioned to Zhongke New Life Biotechnology Co., Ltd. Fecal samples were thawed at room temperature after being removed from -80℃, and then rapidly subjected to quantitative suspension preparation and targeted mass spectrometry analysis (specifically, ultra-high performance liquid chromatography-quadrupole mass spectrometry (UHPLC-QTRAP MS) was used to detect metabolites in the samples). MultiQuant or Analyst software was used to extract peaks from the raw MRM data, obtaining the ratio of peak area to internal standard peak area for each substance, and calculating the content based on the standard curve. Data analysis included univariate statistical analysis, multidimensional statistical analysis, differential metabolite screening, differential metabolite correlation analysis, and KEGG pathway analysis. Based on the cohort analysis data, differential analysis was performed, ultimately identifying a characteristic metabolite of flavin adenine dinucleotide (FAD) in cancer patients with a BMI ≥ 25 who showed sensitized response to anti-PD-1 / PD-L1 therapy. Specific information is as follows... Figure 1 As shown in Figure A. Figure 1 Results A showed that there was a significant difference in FAD levels between anti-PD-1 / PD-L1 treatment responders (R) and non-responders (NR) with a BMI ≥ 25; however, there was no significant difference in FAD levels between anti-PD-1 / PD-L1 treatment responders (R) and non-responders (NR) with a BMI < 25.

[0028] 3. Screening of characteristic metabolites for sensitizing anti-PD-1 / PD-L1 treatment response in cancer patients with BMI < 25 The serum samples were thawed from -80°C to room temperature, and then quickly prepared into quantitative suspensions and separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) HILIC and C18 chromatographic column. Mass spectrometry was performed using AB 6500+ QTRAP mass spectrometer (AB SCIEX). The MRM raw data were extracted using MultiQuant or Analyst software to obtain the peak area ratio of each substance and internal standard peak area, and the content was calculated according to the standard curve. The data analysis includes univariate statistical analysis, multidimensional statistical analysis, differential metabolite screening, differential metabolite correlation analysis, KEGG pathway analysis, etc. Based on the analysis data of the cohort, correlation analysis was performed, and finally one characteristic metabolite most closely related to the sensitization of anti-tumor immune response in cancer patients with BMI < 25 was obtained: docosahexaenoic acid (DHA), specific information as shown in Table B. Figure 1 The results of B show that the content of docosahexaenoic acid in the blood of 27 cancer patients is significantly positively correlated with the relative proportion of CD8-positive T cell infiltration in the primary tumor. Figure 1

[0029] Example 2 This example carried out a personalized metabolite function verification of tumor immune checkpoint inhibitor efficacy for cancer patients with BMI ≥ 25. The specific steps are as follows: ​1. All DIO mice were randomly grouped and housed in a laminar flow biosafety cabinet under specific pathogen-free (SPF) conditions at 20-25 °C, 30-70% humidity, 12 hours light / 12 hours dark cycle. Mice were housed in ventilated cages with up to 5 mice per cage and had free access to food and water. High-fat diet-induced C57BL / 6J mice were used as the obese mouse model (DIO mice). Six-week-old male C57BL / 6J mice (purchased from GemPharmatech / Jiangsu Jiesu Pharmaceutical Biotechnology Co., Ltd.) were randomly housed (5 mice per cage) and given either a 60% fat diet (Research Diets, D12492) or a 10% fat, sucrose-matched control diet (Research Diets, D12492J). Mice were continuously given the corresponding diet for 20 weeks before starting the subcutaneous injection of tumor experiment to obtain DIO experimental mice. Metabolic parameters were evaluated at weeks 19-20 of dietary intervention. All mouse experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures in this study were approved by the Committee on Animal Care and Use of Experimental Animals of Renji Hospital, School of Medicine, Shanghai Jiao Tong University.

[0030] 2. DIO mice were given a 3-day antibiotic (ABX) pretreatment. The antibiotic cocktail used contained vancomycin (0.5 g / L), ampicillin (1 g / L), metronidazole (1 g / L), and neomycin (1 g / L) added to sterile drinking water.

[0031] 3. The pretreated DIO mice were inoculated with tumor cells MC38 (purchased from Beijing Medical Cell Resource Bank (BMCR)) or B16-F0 (purchased from North China Cell Bank (BNCC)) to obtain MC38 tumor model DIO mice and B16-F0 tumor model DIO mice after inoculation.

[0032] 4. After grouping the MC38 tumor model DIO mice and B16-F0 tumor model DIO mice, experimental group 1 ((ABX)lgG+FAD group) was given FAD (2 mg / kg, once a day) treatment alone; experimental group 2 ((ABX)lgG+PD-1 group) was given anti-mouse PD-1 monoclonal antibody (RMP1-14, BioXCell) treatment alone; experimental group 3 ((ABX)lgG+PD-1+FAD group) was given FAD (2 mg / kg, once a day) and anti-mouse PD-1 monoclonal antibody (RMP1-14, BioXCell) treatment in combination; the control group ((ABX)lgG) was given sterile water only.

[0033] 5. The DIO mice of each group of MC38 tumor model were measured every other day for 19 days during the treatment for the mice weight, tumor length (A) and tumor width (B). The tumor volume was calculated according to the formula (A × B²) / 2, and the tumor volume (average value) of each experimental group and control group was obtained, and the results are shown in Figure 1 Compared with the control group, the tumor volume of the experimental group 1 did not decrease significantly, indicating that FAD alone had no therapeutic effect on MC38 tumor; while the tumor volume of the experimental groups 2 and 3 decreased significantly compared with the control group. Further comparison of the experimental groups 2 and 3 showed that the administration of FAD at the same time as the anti-PD-1 inhibitor treatment could significantly reduce the tumor volume. This indicated that FAD could act as a sensitizer for anti-PD-1 inhibitors, enhancing the tumor treatment effect of anti-PD-1 inhibitors.

[0034] 6. The DIO mice of each group of B16-F0 tumor model were measured every other day for 16 days after treatment for the mice weight, tumor length (A) and tumor width (B). The tumor volume was calculated according to the formula (A × B²) / 2, and the tumor volume (average value) of each experimental group and control group was obtained, and the results are shown in Figure 1 Compared with the control group, the tumor volume of the experimental group 1 did not decrease significantly, indicating that FAD alone had no therapeutic effect on B16-F0 tumor; while the tumor volume of the experimental groups 2 and 3 decreased significantly compared with the control group. Further comparison of the experimental groups 2 and 3 showed that the administration of FAD at the same time as the anti-PD-1 inhibitor treatment could significantly reduce the tumor volume. This indicated that FAD could act as a sensitizer for anti-PD-1 inhibitors, enhancing the tumor treatment effect of anti-PD-1 inhibitors.

[0035] Based on the results of the above verification, it can be seen that the metabolite FAD can act as a sensitizer for enhancing the efficacy of immune checkpoint inhibitors. The supplementation of FAD is a feasible potential strategy, and the combination of FAD and anti-PD-1 can enhance the responsiveness of obese cancer patients to immune checkpoint inhibitors.

[0036] Example 3 This embodiment carried out personalized metabolite promotion of tumor immune checkpoint inhibitor efficacy for BMI < 25 cancer patients. The specific steps are as follows: 1. All lean mice (LFD mice) were randomly grouped and housed in a laminar flow biosafety cabinet under specific pathogen-free (SPF) conditions at 20-25 °C, 30-70% humidity, 12 hours light / 12 hours dark cycle. Mice were housed in ventilated cages, up to 5 mice per cage, with free access to food and water. LFD mice were induced using low-fat fed C57BL / 6J mice. Specifically, 10% fat, sucrose content-matched control diet (Research Diets, D12492J). Mice were continuously treated with corresponding diet for 20 weeks before starting subcutaneous injection of tumor experiment. All mouse experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, National Institutes of Health. All experimental procedures in this study were approved by the Committee on Animal Care and Use of Experimental Animals of Renji Hospital, School of Medicine, Shanghai Jiao Tong University.

[0037] 2. LFD mice were subjected to a 3-day antibiotic (ABX) pretreatment. The antibiotic cocktail used contained vancomycin (0.5 g / L), ampicillin (1 g / L), metronidazole (1 g / L), and neomycin (1 g / L) added to sterile drinking water.

[0038] 3. The pretreated LFD mice were inoculated with tumor cells MC38, and MC38 tumor model LFD mice were obtained after inoculation.

[0039] 4. After grouping the MC38 tumor model LFD mice, experimental group 1 ((ABX)lgG+DHA group) was treated with DHA (30 mg / kg, once a day) alone; experimental group 2 ((ABX)lgG+PD-1 group) was treated with anti-mouse PD-1 monoclonal antibody (RMP1-14, BioXCell) alone; experimental group 3 ((ABX)lgG+PD-1+DHA group) was treated with DHA (30 mg / kg, once a day) and anti-mouse PD-1 monoclonal antibody (RMP1-14, BioXCell) in combination; the control group ((ABX)lgG) was treated with sterile water only.

[0040] 5. The body weight, tumor length (A), and tumor width (B) of each group of MC38 tumor model LFD mice were measured every other day during the 19-day treatment period. The tumor volume was calculated according to the formula (A × B²) / 2, and the tumor volumes of each experimental group and the control group were obtained, as shown in Figure 1As shown in Figure E, compared with the control group, the tumor volume of experimental group 1 decreased, but there was no significant difference, indicating that DHA alone had no obvious therapeutic effect on MC38 tumor; while experimental groups 2 and 3 had significantly decreased tumor volume compared with the control group. Further comparison between experimental groups 2 and 3 showed that DHA could significantly reduce tumor volume when used in combination with anti-PD-1 inhibitor. It was indicated that DHA could be used as a sensitizer to enhance the therapeutic effect of anti-PD-1 inhibitor.

[0041] Based on the results of the above verification, it can be seen that metabolite DHA can be used as a sensitizer to enhance the therapeutic effect of immune checkpoint inhibitors. The supplementation of DHA is a feasible potential strategy, and the combination of DHA and anti-PD-1 can enhance the responsiveness of patients with cachexia to immune checkpoint inhibitors.

[0042] The present application has many specific application approaches, and the above description is only the preferred embodiment of the present application. It should be noted that the above examples are only used to illustrate the present application, and are not used to limit the protection scope of the present application. For ordinary skilled persons in the art, several improvements can be made without departing from the principles of the present application, and these improvements should also be considered as the protection scope of the present application.

Claims

1. The use of two metabolites in the preparation of immune checkpoint inhibitor sensitizers based on body mass index stratification, characterized in that, The metabolites are flavin adenine dinucleotide and docosahexaenoic acid.

2. The use according to claim 1, characterized in that, The sensitizer can enhance the therapeutic efficacy of immune checkpoint inhibitors in cancer patients.

3. The use according to claim 2, characterized in that, When the cancer patient is an obese cancer patient with a BMI ≥ 25, the metabolite is flavin adenine dinucleotide.

4. The use according to claim 2, characterized in that, When the cancer patient is a lean cancer patient with a BMI < 25, the metabolite is docosahexaenoic acid (DHA).

5. The use according to claim 2, characterized in that, The cancer in question is either colorectal cancer or melanoma.

6. The use according to claim 1 or 2, characterized in that, The immune checkpoint inhibitors include at least one of anti-PD-1 inhibitors, anti-PD-L1 inhibitors, and anti-CTLA-4 inhibitors.

7. The use according to claim 6, characterized in that, The immune checkpoint inhibitor is an anti-PD-1 inhibitor.

8. A pharmaceutical composition for treating cancer, characterized in that, This includes immune checkpoint inhibitors and sensitizers; the sensitizers include metabolites; The metabolite is selected from flavin adenine dinucleotide or docosahexaenoic acid.

9. The pharmaceutical composition for treating cancer according to claim 8, characterized in that, The cancer in question is either colorectal cancer or melanoma.

10. The pharmaceutical composition for treating cancer according to claim 8, characterized in that, When the cancer patient is an obese cancer patient with a BMI ≥ 25, the metabolite is flavin adenine dinucleotide; When the cancer patient is a lean cancer patient with a BMI < 25, the metabolite is docosahexaenoic acid (DHA).

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