Use of L-histidine in the preparation of an adjuvant for enhancing the efficacy of immune checkpoint inhibitor therapy for colorectal cancer

Abstract

The application discloses application of L-histidine in preparation of an auxiliary synergist for enhancing the curative effect of an immune checkpoint inhibitor in treatment of colorectal cancer, and belongs to the field of biological medicines. The application combines L-histidine with anti-PD-1 monoclonal antibodies, supplements L-histidine through oral administration, improves the levels of histidine-rich glycoprotein and histamine in plasma, promotes the polarization of tumor-related macrophages to M1 type, increases CD8 + T cell infiltration, reduces regulatory T cell infiltration, up-regulates the expression of anti-tumor effect molecules, thereby synergistically enhances the anti-tumor immune effect, and improves the drug resistance or poor response problem caused by the use of PD-1 monoclonal antibodies alone. In the application, L-histidine can be used as an amino acid adjuvant to synergistically use a first-line clinical immune checkpoint inhibitor for tumor immunotherapy, thereby providing a new solution for the problem of drug resistance of the use of ICIs alone, and having a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of L-histidine in the preparation of adjuvant synergists for enhancing the efficacy of immune checkpoint inhibitors in the treatment of colorectal cancer. Background Technology

[0002] Immune checkpoint inhibitors (ICIs) have made significant progress in anti-tumor immunotherapy, showing good efficacy, especially for "hot tumors" with high immune infiltration. However, primary and acquired resistance are prevalent, limiting the clinically beneficial population. Combination therapy is considered one of the effective strategies to improve the efficacy of ICIs. In colorectal cancer (CRC), only about 5% of patients with DNA mismatch repair deficiency (dMMR) or high microsatellite instability (MSI-H) can benefit from ICI monotherapy; the vast majority of patients respond poorly to ICI treatment due to low tumor mutational burden, insufficient T-cell infiltration, and an immunosuppressive microenvironment. Therefore, there is an urgent need to develop novel combination therapy strategies that can synergistically enhance the efficacy of ICIs to expand the population that benefits from immunotherapy.

[0003] Amino acids, as core nutrients for the body, have recently been found to possess immunomodulatory functions, influencing the phenotype and function of immune cells in the tumor microenvironment through metabolic reprogramming. Targeting the metabolic pathways of amino acids such as tryptophan, arginine, and glutamine has been shown to regulate tumor immune escape and T cell function, thereby enhancing anti-tumor immune responses. Histidine, as an essential amino acid, has metabolites, histamine and histidine-rich glycoprotein (HRG), which have been shown to play complex regulatory roles in innate and adaptive immunity. HRG can alleviate immunosuppression by inhibiting macrophage M2 polarization. However, current research has largely focused on the role of histidine in inflammation, allergies, nervous system diseases, and gastrointestinal diseases. The direct role of histidine metabolism in anti-tumor immunity and its molecular mechanisms remain unclear. Whether histidine can synergistically enhance anti-tumor immune effects through metabolic regulation pathways with intracellular immunoglobulins (ICIs) has not yet been reported.

[0004] To address the aforementioned technical issues, this invention aims to provide a novel application of L-histidine in adjuvant synergistic effects of PD-1 monoclonal antibody immunotherapy for colorectal cancer. Existing technologies lack systematic research on the combined antitumor effects of histidine metabolism and immune checkpoint inhibitors (ICIs). This is primarily due to the complexity of the histidine metabolic network, the unclear regulatory targets of histidine on immune cell subsets within the tumor microenvironment, and the fact that previous studies have largely focused on the independent functions of histamine or HRG, failing to explore the synergistic effects of histidine supplementation on the efficacy of immune checkpoint inhibitors from the perspective of amino acid metabolic reprogramming. These technological gaps have prevented the development and application of histidine as a potential immunomodulator in CRC immunotherapy. Summary of the Invention

[0005] The purpose of this invention is to provide the application of L-histidine in the preparation of adjuvant potentiators for enhancing the efficacy of immune checkpoint inhibitors in the treatment of colorectal cancer, thereby addressing the problems existing in the prior art. This invention demonstrates that the combination of L-histidine and anti-PD-1 monoclonal antibodies has a synergistic anti-colorectal cancer effect, promoting macrophage M1 polarization and increasing CD8+. + T-cell infiltration, reduction of Tregs, and upregulation of antitumor effector molecules improve the immunosuppressive microenvironment. L-histidine has good safety and low cost, and can be used as an adjuvant to enhance the efficacy of ICIs, providing a new solution to the problem of drug resistance.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of L-histidine in the preparation of adjuvant synergists for enhancing the efficacy of immune checkpoint inhibitors in the treatment of colorectal cancer.

[0007] Optionally, the adjuvant synergist may be administered in combination with the PD-1 monoclonal antibody.

[0008] Optionally, the immune checkpoint inhibitor is a PD-1 antibody.

[0009] The present invention also provides the use of L-histidine in the preparation of pharmaceutical compositions for the treatment of colorectal cancer.

[0010] The present invention also provides a pharmaceutical composition for treating colorectal cancer, the pharmaceutical composition comprising L-histidine and an immune checkpoint inhibitor.

[0011] Optionally, the immune checkpoint inhibitor is a PD-1 antibody.

[0012] Optionally, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

[0013] Optionally, the pharmaceutically acceptable carrier includes at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, or buffer.

[0014] Optionally, the dosage form of the pharmaceutical composition includes at least one of suspension, granules, capsules, powder, tablets, pills, injections, suppositories, or drops.

[0015] This invention also provides the use of L-histidine in the preparation of drugs for regulating the tumor immune microenvironment of colorectal cancer, wherein the regulation includes at least one of the following: a) Increase the levels of histidine-rich glycoproteins and / or histamine in plasma; b) Promotes the polarization of tumor-associated macrophages towards the M1 type; c) Increases the infiltration of CD8+ T cells in the tumor microenvironment; d) Reduce the infiltration of regulatory T cells in the tumor microenvironment; e) Increase the expression of antitumor effector molecules IFN-γ, TNF-α, Perforin and / or Granzyme B.

[0016] The present invention discloses the following technical effects: This invention experimentally demonstrates that the antitumor effect of the combination of L-histidine and anti-PD-1 monoclonal antibody is significantly superior to that of PD-1 monoclonal antibody alone or L-histidine alone. Evaluation using the Bliss independent model showed that the actual tumor inhibition rate of the combination therapy was significantly greater than the expected tumor inhibition rate, indicating a synergistic effect between the two drugs, which can significantly improve the drug resistance or poor response problems caused by PD-1 monoclonal antibody alone. Simultaneously, L-histidine supplementation can effectively increase the levels of histidine-rich glycoprotein (HRG) and histamine in plasma, promote the polarization of tumor-associated macrophages towards the M1 type, and increase CD8+. + This approach reduces T cell infiltration, decreases regulatory T cell (Treg) infiltration, and upregulates the expression of anti-tumor effector molecules such as IFN-γ, TNF-α, Perforin, and Granzyme B, thereby systematically alleviating the inhibitory state of the tumor immune microenvironment and providing a new technical solution for expanding the beneficiary population of colorectal cancer immunotherapy.

[0017] Furthermore, L-histidine, as an essential amino acid for the human body, has been widely used in the pharmaceutical and food fields, possesses a sound safety profile, and can be developed and applied in various forms such as pharmaceutical formulations, functional food ingredients, or dietary supplements. Compared with some combination therapy regimens, its production and usage costs are relatively low, demonstrating good application feasibility. In this invention, L-histidine can be used as an amino acid adjuvant in conjunction with first-line clinical immune checkpoint inhibitors for tumor immunotherapy, providing a new solution to the problem of resistance to ICIs alone, and has broad clinical application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows the effect of L-histidine combined with anti-PD-1 monoclonal antibody in the treatment of colorectal cancer. Among them, A is the experimental design of the MC38 tumor model; B is the representative tumor phenotype image from four different treatment groups; C is the final volume statistics of tumors from four different treatment groups; D is the final weight statistics of tumors from four different treatment groups; and E is the MC38 tumor volume growth curve of four different treatment groups. Figure 2 The image shows the results of the tumor immune microenvironment detection; where A represents the CD8+ detection by flow cytometry. + CD3 + The proportion of T cells; B is CD25 + FOXP3 + CD4 + The proportion of T cells; C represents CD86. + F4 / 80 + CD11b + The proportion of macrophages of type M1; D represents the expression of tumor-killing cytokine IFN-γ detected by RT-qPCR; E represents the expression of tumor-killing cytokine Granzyme B detected by RT-qPCR; F represents the expression of tumor-killing cytokine TNF-α detected by RT-qPCR; G represents the expression of tumor-killing cytokine Perforin detected by RT-qPCR. Figure 3This study aims to detect histidine and its metabolites and their effects on macrophage polarization. A represents products of the histidine metabolic pathway; B and C represent plasma HRG and histamine levels in mice, detected by enzyme-linked immunosorbent assay (ELISA); D and G represent plasma glutamate, carnosine, 1-methyl-L-histidine, and 3-methyl-L-histidine levels in mice, detected by LC-MS targeting amino acids; H represents the mRNA expression level of macrophage M1 / M2 polarization markers, detected by RT-qPCR; and I represents the mRNA expression level of histamine receptor 1 (HRH1) and histamine receptor 2 (HRH2), detected by RT-qPCR. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: The effect of L-histidine on enhancing the anti-colorectal cancer growth of PD-1 monoclonal antibody MC38 colorectal cancer cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM medium at 37°C with 5% CO2. Regular checks for mycoplasma contamination and cell line identification were performed. Cell passages used for modeling and in vitro experiments were limited to no more than five generations. Five-week-old male and female C57BL / 6 mice were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. All mice were housed in a specific pathogen-free (SPF) environment with 12 hours of light and dark daily, and had free access to food and water. PD-1 monoclonal antibody (RMPI-14) was purchased from BioXCell, and L-histidine (HY-N0832) was purchased from MCE.

[0026] I. Experimental Methods 1. Establishment of animal models MC38 cells in logarithmic growth phase were resuspended in sterile PBS, and the cell concentration was adjusted to 2.5 × 10⁻⁶. 6 Cells / mL. Take 0.2 mL of the above cell suspension (containing 5 × 10⁶ cells / mL). 5 (100 cells) were subcutaneously injected into the right axilla of C57BL / 6 mice. The general condition of the mice and tumor growth were observed daily.

[0027] 2. Grouping and administration When the tumor volume reaches 50-100 mm 3 At that time, the tumor-bearing mice were randomly divided into four groups (no fewer than 6 mice in each group): Anti-PD-1 monoclonal antibody monotherapy group: Intraperitoneal injection of anti-mouse PD-1 monoclonal antibody at a dose of 10 mg / kg, once every 3 days, for a total of 4 times.

[0028] L-histidine monotherapy group: L-histidine was administered by gavage at a dose of 1 mg / kg mouse body weight, prepared as a suspension with 0.5% CMC-Na, with an administration volume of 200 μL per mouse. Administration was once daily.

[0029] The combination therapy group received an intraperitoneal injection of anti-mouse PD-1 monoclonal antibody at a dose of 10 mg / kg, administered once every 3 days for a total of 4 times. Simultaneously, L-histidine was administered daily by gavage at a dose of 1 mg / kg body weight, prepared as a suspension in 0.5% CMC-Na, with an administration volume of 200 μL per mouse. This was done once daily.

[0030] The control group received the same dose of saline via gavage and intraperitoneal injection.

[0031] Each group was designated as day 0 from the first day of administration, and tumor volume was measured every 2 days. The tumor volume was calculated using the formula: V = (major axis × minor axis) 2) / 2.

[0032] 3. Experimental endpoint and sample collection The day after the fourth dose of anti-PD-1 monoclonal antibody, or when the tumor volume of any experimental mouse reaches the ethical endpoint (e.g., ≥2000 mm). 3 At this point, all mice were euthanized. The tumor tissue was completely dissected, the tumor weight was measured, and the final tumor volume was determined.

[0033] Experimental design scheme as follows Figure 1 As shown in A.

[0034] 4. Methods for assessing combined effects The combined effect of L-histidine and anti-PD-1 monoclonal antibody was evaluated using the Bliss independent model method. The expected combined effect was calculated using the following formula: E(a+b) expected = Ea + Eb − (Ea × Eb), E(a+b) observed =E(a+b), where: Ea represents the tumor inhibition rate (%) in the anti-PD-1 monoclonal antibody monotherapy group. Eb represents the tumor inhibition rate (%) of the L-histidine monotherapy group. E(a+b) expected represents the expected combined tumor suppression rate (%). E(a+b) observed represents the actual combined tumor suppression rate (%).

[0035] If E(a+b) observed > E(a+b) expected, then the two drugs are considered to have a synergistic effect.

[0036] II. Experimental Results During the drug administration period, the mice in all groups were generally in good condition, with no significant weight loss or behavioral abnormalities. Figure 1 As shown in the BE (Brain-Earnings Interaction) results, compared with the anti-PD-1 monoclonal antibody monotherapy group, the L-histidine combination therapy group showed a significant reduction in tumor volume and tumor weight at the experimental endpoint. The tumor inhibition rates and combined effects of the four treatment groups are evaluated in Table 1. Calculations using the Bliss independent model showed that the actual tumor inhibition rate of the combination therapy group was significantly greater than the expected tumor inhibition rate, indicating that L-histidine and anti-PD-1 monoclonal antibody have a synergistic anti-tumor effect in a mouse model of colorectal cancer.

[0037] Table 1. Tumor suppression rate and combined effect assessment of the four treatment groups Example 2: Regulation of the tumor immune microenvironment by L-histidine combined with PD-1 monoclonal antibody The experimental animals and cells are the same as in Example 1.

[0038] The flow cytometry antibodies used in the experiment are as follows: Anti-CD45 (30-F11, 1:100 dilution, purchased from BD), anti-CD3 (17A2, 1:100 dilution, purchased from Biolegend), anti-CD4 (RM4-5, 1:100 dilution, purchased from BD), anti-CD8 (53-6.7, 1:100 dilution, purchased from BD), anti-CD25 (PC61.5, 1:100 dilution, purchased from Invitrogen), anti-FO XP3 (FJK-16s, 1:100 dilution, purchased from Invitrogen), anti-CD11b (M1 / 70, 1:100 dilution, purchased from BD), anti-F4 / 80 (BMB, 1:100 dilution, purchased from Invitrogen), anti-CD86 (GL-1, 1:100 dilution, purchased from Biolegend), and anti-CD16 / 32 (2.4G2, 1:100 dilution, purchased from BD). ZombieAqua™ dye was used as the live / dead cell dye, diluted 1:100.

[0039] The RT-qPCR reagents are as follows: RNA was extracted from tumor samples using RNAiso Plus reagent (Takara Bio, Japan), chloroform (SCR, China), isopropanol (SCR, China), and 75% ethanol (SCR, China). cDNA synthesis was performed using the Prime Script™ RT kit (Takara Bio, Japan). PCR amplification was performed using TB Green Premix Ex Taq™ (Takara Bio, Japan).

[0040] The primers used for RT-qPCR are shown in Table 2: Table 2 Primers used for RT-qPCR I. Experimental Methods 1. Animal model establishment and dosing regimen The animal model establishment and administration regimen were the same as in Example 1. The experiment was divided into four groups: control group, anti-PD-1 monoclonal antibody monotherapy group, L-histidine monotherapy group, and combination therapy group. Mice were sacrificed the day after the fourth dose of anti-PD-1 monoclonal antibody, and tumor tissue was excised for the following tests.

[0041] 2. Detection of tumor-infiltrating immune cells by multicolor flow cytometry 2.1 Preparation of single-cell suspension Mice were euthanized at the experimental endpoint, and tumor tissue was completely dissected and immediately immersed in pre-chilled PBS. The tumor tissue was transferred to a petri dish containing an appropriate amount of pre-chilled PBS, minced with sterile ophthalmic scissors, and then physically ground and passed through a 70 μm cell filter to prepare a concentration of 1×10⁻⁶. 6 -1×10 7 A single-cell suspension of cells per mL.

[0042] 2.2 Fc receptor blocking Transfer 1 mL of single-cell suspension to a 1.5 mL EP tube and centrifuge at 450 × g for 5 min at 4°C. Discard the supernatant, retaining approximately 100-200 μL of liquid. Add 0.5 μL of anti-CD16 / 32 antibody (Fc receptor blocking antibody), vortex to mix, and incubate at room temperature for 15 min. Add 1 mL of PBS to terminate the blocking reaction, centrifuge at 450 × g for 5 min at 4°C, and discard the supernatant.

[0043] 2.3 Staining of live and dead cells Add 0.3 μL of Zombie Aqua™ live / dead dye to each tube, vortex to mix, and incubate at room temperature in the dark for 10 min. Add 1 mL of PBS to stop staining, centrifuge at 450 × g for 5 min at 4 °C, and discard the supernatant.

[0044] 2.4 Surface marker staining Add premixed surface antibody working solution (containing anti-CD45, anti-CD3, anti-CD8, anti-CD4, anti-CD25, anti-CD11b, and anti-F4 / 80, each antibody diluted 1:100) to each tube, vortex to mix, and incubate at 4°C in the dark for 30 min. Add 1 mL of PBS to stop staining, centrifuge at 450×g for 5 min at 4°C, and discard the supernatant.

[0045] 2.5 Fixation, membrane rupture, and intranuclear staining Add 500 μL of fixation and perforation buffer to each tube, vortex to mix, and incubate at room temperature for 50 min. Add 1 mL of washing buffer to each tube, centrifuge at 800×g for 10 min at 4°C, discard the supernatant, and repeat the washing process once. Add premixed nuclear antibody working solution (containing anti-FOXP3, diluted 1:100) to each tube, vortex to mix, and incubate at 4°C in the dark for 60 min. After incubation, add 1 mL of PBS to stop staining, centrifuge at 800×g for 10 min at 4°C. Discard the supernatant, retain 100-200 μL of the liquid, resuspend the cells, and then perform the analysis.

[0046] 2.6 Flow Cytometry Data Analysis Data were acquired using flow cytometry and processed using appropriate analysis software. Gating analysis of various immune cell subsets was performed based on the following biomarker combinations: CD8 + T cells: CD45 + CD3 + CD8 + ; Treg cells (regulatory T cells): CD45 + CD3 + CD4 + CD25 + FOXP3 + ; M1 macrophages: CD45 + CD11b + F4 / 80 + CD86 + .

[0047] 3. RT-qPCR detection of antitumor effector molecule expression Take about 50-100 mg of tumor tissue, extract total RNA using an RNA extraction kit, reverse transcribe to obtain cDNA, and store at -20℃ for real-time quantitative PCR.

[0048] Using TB Green Premix Ex Taq TM PCR amplification was performed. The reaction mixture (20 μL) consisted of: 10 μL TB GreenPremix Ex Taq, 0.8 μL each of forward and reverse primers (10 μmol / L), 0.4 μL ROX Reference Dye, 2 μL cDNA template, and ddH2O to a final volume of 20 μL. Reaction conditions: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 5 sec, 60℃ annealing and extension for 34 sec, for a total of 40 cycles. GAPDH was used as an internal control gene, and 2... -ΔΔCt The relative expression levels of each target gene were calculated. The target genes detected included IFN-γ, TNF-α, Perforin, and Granzyme B.

[0049] II. Experimental Results 1. Effects of L-histidine combined with anti-PD-1 monoclonal antibody on tumor-infiltrating lymphocytes Flow cytometry results showed ( Figure 2 A- Figure 2 (C): Compared with the anti-PD-1 monoclonal antibody monotherapy group and the L-histidine monotherapy group, the combination therapy group showed higher CD8 concentrations in the tumor microenvironment. + The proportion of T cell infiltration increased significantly ( Figure 2The proportion of regulatory T cells (Tregs) infiltrating was significantly reduced (A). Figure 2 B), M1 macrophages (CD86) + The infiltration ratio increased significantly. Figure 2 (C). The results showed that the combination of L-histidine and anti-PD-1 monoclonal antibody could remodel the tumor immune microenvironment, promote the infiltration of anti-tumor immune cells, and inhibit the aggregation of immunosuppressive cells.

[0050] 2. Effects of L-histidine combined with anti-PD-1 monoclonal antibody on the expression of anti-tumor effector molecules RT-qPCR test results showed ( Figure 2 D- Figure 2 (G): Compared with the anti-PD-1 monoclonal antibody group and the L-histidine monoclonal antibody group, the mRNA expression levels of anti-tumor effector molecules IFN-γ, TNF-α, perforin, and Granzyme B in tumor tissues were significantly upregulated in the combination therapy group. This result further confirms that the combination of L-histidine and anti-PD-1 monoclonal antibody can enhance the effector function of cytotoxic T lymphocytes.

[0051] Example 3: Detection of L-histidine metabolites after gavage and their regulatory effect on macrophage polarization I. Experimental Methods 1. Animal model establishment and dosing regimen The animal model establishment and administration regimen were the same as in Example 1. The experiment was divided into four groups: control group, anti-PD-1 monoclonal antibody monotherapy group, L-histidine monotherapy group, and combination therapy group. Mice were sacrificed the day after the fourth dose of anti-PD-1 monoclonal antibody. Tumors were removed and blood was collected from the orbits of the mice. Whole blood was collected and centrifuged at 2000×g for 15 min at 4°C. The supernatant plasma was collected, aliquoted, and stored at -80°C for later use.

[0052] The primer sequences used to detect macrophage polarization marker genes are shown in Table 3.

[0053] Table 3 Primer sequences used to detect macrophage polarization marker genes 2. Targeted detection of amino acid metabolites 2.1 Sample Pretreatment Take 10 μL of plasma, dilute it 10-fold with DEPC water, add 60 μL of methanol containing the internal standard, shake for 5 min, and then centrifuge at 14000 r / min for 10 min. Take 10 μL of the supernatant, add 70 μL of buffer and 20 μL of derivatization reagent, shake and let stand for 1 min, then react at 55℃ for 10 min. Collect the supernatant of each sample and dilute it with pure water. After dilution, centrifuge the sample again, and transfer 80 μL of the supernatant for injection analysis.

[0054] 2.2 Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) analysis Amino acids and their metabolites were detected using a UHPLC-MS / MS system. Mobile phase A was water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid). The gradient elution program was as follows: 0–4 min, 2%–22% B; 4–4.1 min, 22%–95% B; 4.1–4.4 min, 95%–100% B; 4.4–4.6 min, 100%–2% B; 4.6–6.0 min, hold at 2% B. The column temperature was maintained at 60℃. The mass spectrometer capillary voltage was set to +5.5 kV. Data acquisition and analysis were performed using appropriate analytical software, including peak data integration, calibration curve establishment, and quantitative analysis of individual amino acids. The detected metabolites included histidine, histamine, histidine-rich glycoprotein (HRG), glutamate, carnosine, and methylated decarboxylation modified products.

[0055] 3. ELISA method for detecting plasma HRG and histamine levels Frozen plasma samples were thawed at 4°C and then processed according to the ELISA kit instructions. Samples and standards were added to pre-coated microplates, incubated, washed, and then enzyme-labeled antibody was added. The plates were incubated and washed again, followed by the addition of chromogenic substrate for incubation in the dark, and finally, stop solution was added. The absorbance of each well was measured at 450 nm using a microplate reader. The concentrations of HRG and histamine in each sample were calculated based on a standard curve generated using four-parameter logistic regression. All samples were analyzed in duplicate.

[0056] 4. Detection of macrophage polarization marker genes in tumor tissue 4.1 Real-time quantitative PCR Total RNA extraction and cDNA synthesis were performed as in Example 2. TB Green Premix Ex Taq was used. TM PCR amplification was performed, using the same reaction system and conditions as in Example 2. GAPDH was used as an internal reference gene, and 2... -ΔΔCt The relative expression levels of M1 macrophage marker genes (NOS2, IL-1β) and M2 macrophage marker genes (Arg1, IL-10) were calculated.

[0057] 4.2 Histamine receptor expression detection The mRNA expression levels of histamine receptors HRH1 and HRH2 in tumor tissues were detected by RT-qPCR.

[0058] II. Experimental Results 1. Detection of L-histidine metabolites in vivo after gavage Detection of targeted amino acid metabolomics products in the plasma of colorectal cancer-bearing mice, such as Figure 3 As shown in Figure A. Targeted amino acid metabolomics analysis of plasma from colorectal cancer-bearing mice revealed changes in the levels of major histidine metabolites in the L-histidine monotherapy group and the combination therapy group compared to the control group. Specifically, the levels of histidine-rich glycoprotein (HRG) and histamine were significantly increased. Figure 3 B- Figure 3 The levels of glutamate, carnosine, and methylated decarboxylation products did not change significantly (C), while the levels of glutamate, carnosine, and methylated decarboxylation products did not change significantly (C). Figure 3 D- Figure 3 (G). This result indicates that exogenously supplemented L-histidine is mainly converted in vivo via the HRG and histamine metabolic pathways.

[0059] 2. The regulatory role of L-histidine in macrophage polarization RT-qPCR test results showed ( Figure 3 Compared with the control group and the anti-PD-1 monoclonal antibody group, the expression levels of M1 macrophage marker genes NOS2 and IL-1β in tumor tissues of the L-histidine monotherapy group and the combination therapy group were significantly increased, while the expression levels of M2 macrophage marker genes Arg1 and IL-10 were significantly decreased. These results indicate that L-histidine supplementation can promote the polarization of tumor-associated macrophages towards the M1 type and reverse the immunosuppressive state.

[0060] 3. Effects of L-histidine on histamine receptor expression To investigate the role of histamine in macrophage polarization regulation, the expression levels of histamine receptors HRH1 and HRH2 in tumor tissue were examined. The results showed ( Figure 3 In the colorectal cancer-bearing mouse model constructed in this invention, L-histidine supplementation downregulated HRH1 expression while upregulating HRH2 expression. This result indicates that exogenously supplemented L-histidine-mediated histamine metabolism tends to bind to the HRH2 receptor. This selective receptor binding pattern may promote macrophages to shift to an M1 polarization state, thereby enhancing anti-tumor immune responses and the efficacy of immune checkpoint inhibitors.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of L-histidine in the preparation of adjuvant synergists for enhancing the efficacy of immune checkpoint inhibitors in the treatment of colorectal cancer.

2. Use according to claim 1, characterized in that, The adjuvant was administered in combination with the PD-1 monoclonal antibody.

3. Use according to claim 1, characterized in that, The immune checkpoint inhibitor is a PD-1 antibody.

4. The use of L-histidine in the preparation of pharmaceutical compositions for the treatment of colorectal cancer.

5. A pharmaceutical composition for treating colorectal cancer, characterized by, The pharmaceutical composition contains L-histidine and an immune checkpoint inhibitor.

6. The pharmaceutical composition of claim 5, wherein, The immune checkpoint inhibitor is a PD-1 antibody.

7. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 5, wherein, The pharmaceutically acceptable carrier includes at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, or buffer.

9. Use according to claim 5, characterized in that, The dosage form of the pharmaceutical composition includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, or drops.

10. Use of L-histidine for the preparation of a medicament for modulating the immune microenvironment of a colorectal cancer tumor, characterized in that, The regulation includes at least one of the following: a) Increase the levels of histidine-rich glycoproteins and / or histamine in plasma; b) Promotes the polarization of tumor-associated macrophages towards the M1 type; c) increasing infiltration of CD8 + T cells into the tumor microenvironment; d) Reduce the infiltration of regulatory T cells in the tumor microenvironment; e) Increase the expression of antitumor effector molecules IFN-γ, TNF-α, Perforin and / or Granzyme B.

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