Application of L-histidine as PD-1 antibody for treating microsatellite stable colon cancer sensitizer
By adding L-histidine as a sensitizer to PD-1 antibodies, the metabolism and immune microenvironment of colon cancer cells were regulated, which solved the problem of poor efficacy of PD-1 antibodies in the treatment of microsatellite stable colon cancer and achieved a significant tumor suppression effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-03
- Publication Date
- 2026-03-24
AI Technical Summary
Existing PD-1 antibody drugs are not effective in treating microsatellite stable colorectal cancer, and about 85% of colorectal cancer patients are not sensitive to current PD-1/PD-L1 inhibitors, making it difficult to improve the tumor immune microenvironment to enhance the activity of cytotoxic T cells.
Using L-histidine as a sensitizer for PD-1 antibodies, the combination of L-histidine and PD-1 antibodies in a specific ratio is administered via injection or oral administration to regulate the oxidative phosphorylation of colon cancer cells and the metabolism of energy and one-carbon units such as glycine and serine, thereby affecting the cell cycle and apoptosis pathways, relieving the immunosuppressive microenvironment, and enhancing the therapeutic effect of PD-1 antibodies.
L-histidine significantly enhanced the therapeutic effect of PD-1 antibody in microsatellite stable colorectal cancer, inhibited the proliferation of colorectal cancer cells, promoted cell death, improved the sensitivity to PD-1 antibody treatment, and significantly reduced tumor volume and weight.
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Figure CN121714565A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medicine, in particular, relates to the application of L-histidine as a PD-1 antibody treatment microsatellite stable colon cancer sensitizer. BACKGROUND
[0002] Targeted PD-1 monoclonal antibody drugs have been approved for first-line treatment of microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer patients, however, about 50% of advanced colorectal cancer patients will have primary or secondary drug resistance problems. More seriously, more than 85% of colorectal cancer patients are microsatellite stable (MSS), and are generally not sensitive to current PD-1 / PD-L1 inhibitors, and are not suitable for immune checkpoint blockade therapy. Improvement of the tumor immune microenvironment of colorectal cancer can improve the activity of killer T cells and enhance the efficacy of immunotherapy, thereby improving the therapeutic effect of targeted PD-1 monoclonal antibody drugs on microsatellite stable colon cancer.
[0003] Therefore, how to improve the tumor immune microenvironment of colorectal cancer, transform "cold" tumors into "hot" tumors, and improve the activity of killer T cells is urgent and challenging, and is also a problem to be solved at present. SUMMARY
[0004] The present application proposes the application of L-histidine as a PD-1 antibody treatment microsatellite stable colon cancer sensitizer, which solves the problem of poor effect of PD-1 antibody drug treatment of microsatellite stable colon cancer in the related art.
[0005] The technical solution of the present application is as follows: The present application proposes the application of L-histidine as a PD-1 antibody treatment microsatellite stable colon cancer sensitizer.
[0006] As a further technical solution, the mass ratio of the PD-1 antibody and L-histidine is 1:31.
[0007] As a further technical solution, the PD-1 antibody is applied in the form of injection.
[0008] As a further technical solution, the L-histidine is applied in the form of oral administration.
[0009] As a further technical solution, the L-histidine is formulated into an L-histidine-phosphate buffer solution for application.
[0010] As a further technical solution, the PD-1 antibody is applied in the form of a PD-1 antibody solution.
[0011] As a further technical solution, the concentration of L-histidine in the L-histidine-phosphate buffer solution is 15.5 mg / mL.
[0012] As a further technical solution, the concentration of PD-1 antibody in the PD-1 antibody solution is 1 mg / mL.
[0013] As a further technical solution, the L-histidine-phosphate buffer solution is obtained by mixing L-histidine and a phosphate buffer solution.
[0014] As a further technical solution, the pH of the phosphate buffer solution is 7.5-7.8.
[0015] As a further technical solution, the PD-1 antibody solution comprises PD-1 antibody, a phosphate buffer solution and L-histidine.
[0016] As a further technical solution, the mass ratio of PD-1 antibody to L-histidine in the PD-1 antibody solution is 16-16.5:1.
[0017] As a further technical solution, the mass ratio of PD-1 antibody to L-histidine in the PD-1 antibody solution is 16.49:1.
[0018] The application further provides a composition of L-histidine and PD-1 antibody, comprising the L-histidine and PD-1 antibody, and the composition comprises an L-histidine-phosphate buffer solution and a PD-1 antibody solution.
[0019] The working principle and beneficial effects of the application are as follows: (1) The application first discovers that L-histidine treatment can significantly increase the therapeutic effect of PD-1 antibody on microsatellite stable colon cancer, indicating that L-histidine can increase the immunotherapy of PD-1 antibody on colon cancer.
[0020] (2) The existing L-histidine in PD-1 antibody is a stabilizer of PD-1 antibody, and the addition amount of L-histidine is much lower than that of the application. Through in vivo, in vitro cell and animal experiments, it is confirmed that under the limited amount of L-histidine in the application, L-histidine can inhibit the effective proliferation of colon cancer cells and subcutaneous tumors, improve the treatment sensitivity of microsatellite stable colon cancer to PD-1 antibody, specifically: on the one hand, it can affect the oxidative phosphorylation of colon cancer and the metabolism of glycine, serine and other energy and one-carbon units, and can inhibit the proliferation and promote the death of colon cancer cells by affecting the cell cycle, apoptosis and classical growth signal pathway PI3K-Akt / mTOR; on the other hand, it can relieve the immune suppression microenvironment of microsatellite stable colon cancer and increase the therapeutic effect of PD-1 antibody. BRIEF DESCRIPTION OF DRAWINGS
[0021] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0022] Figure 1 This is a volcano diagram of differential gene analysis in Example 2 of the present invention; Figure 2 This is a KEGG enrichment pathway analysis diagram from Example 2 of the present invention. Detailed Implementation
[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0024] The phosphate buffer solution with a pH of 7.5 used in the following examples has a concentration of 0.15 mol / L.
[0025] Example 1: Effects of L-histidine on the function of colorectal cancer cells 1.1 Cell proliferation experiment Human colon cancer cell line HT29 was seeded in 96-well plates, with approximately 1000 cells per well. Cells were divided into four groups based on different concentrations of L-histidine treatment: a control group (phosphate-buffered saline at pH 7.5), a 1 mM L-histidine treatment group, a 5 mM L-histidine treatment group, and a 10 mM L-histidine treatment group. Each group had six replicates. The L-histidine treatment groups were prepared using L-histidine and phosphate-buffered saline at pH 7.5.
[0026] Cells were cultured at 37°C in a 5% CO2 incubator for 0 h, 24 h, 48 h, and 72 h. 10 μL of CCK-8 solution was added to each well. After adding the CCK-8 solution, the 96-well plate was returned to the 37°C, 5% CO2 incubator for 2 h. The absorbance (OD value) of each well was then measured at 450 nm using a microplate reader.
[0027] 1.2 Cloning Experiment Human colon cancer cell line HT29 was seeded in 6-well plates, with approximately 1000 cells per well. Cells were divided into control group (phosphate-buffered saline, pH 7.5), 1 mM L-histidine treatment group, 5 mM L-histidine treatment group, and 10 mM L-histidine treatment group based on different concentrations of L-histidine. Each group had three replicates. All L-histidine treatments were prepared using L-histidine and phosphate-buffered saline at pH 7.5.
[0028] Cells were cultured at 37°C in a cell culture incubator containing 5% CO2. After 10 days of culture, cells were fixed with 4% paraformaldehyde for 30 min, and then stained with 0.1% crystal violet at room temperature in the dark for 20 min. Visible colonies (diameter greater than 0.1 mm) were photographed and counted.
[0029] 1.3 Apoptosis Experiment Human colon cancer cell line HT29 cells were seeded in 6-well plates, approximately 1 × 10⁶ cells per well. 6 Cells were divided into control group (phosphate buffer at pH 7.5), 1 mM L-histidine treatment group, 5 mM L-histidine treatment group, and 10 mM L-histidine treatment group according to different concentrations of L-histidine treatment. Each group had 3 replicates. The different concentrations of L-histidine treatment groups were prepared by mixing L-histidine with phosphate buffer at pH 7.5.
[0030] Cells were cultured at 37°C in a 5% CO2 incubator for 48 h, then digested with trypsin and collected by centrifugation. Cells were resuspended in staining buffer at a density of 1×10⁶ cells / mL. 6 per mL.
[0031] Take 100 μL of cell suspension (1×10 5 Add 100 cells to each flow cytometry tube, along with 5 μL of Annexin V-FITC and 5 μL of PI Solution. Mix gently and incubate at room temperature in the dark for 15 min. Add 400 μL of staining buffer to each tube and mix gently. Analyze the stained cells using flow cytometry. Data were analyzed using FlowJo (version 10.9).
[0032] 1.4 Cell cycle experiment Human colon cancer cell line HT29 cells were seeded in 6-well plates, approximately 1 × 10⁶ cells per well. 6 Cells were divided into a control group (phosphate buffer at pH 7.5) and a 5 mM L-histidine treatment group based on different concentrations of L-histidine. Each group had three replicates, and the 5 mM L-histidine treatment group was prepared by mixing L-histidine with phosphate buffer at pH 7.5.
[0033] Cells were cultured at 37°C in a 5% CO2 incubator for 48 h, then digested with trypsin and collected by centrifugation. 1 mL of pre-chilled 70% ethanol was added to the collected cell pellet, and the mixture was gently agitated. The pellet was fixed at 4°C for 2 h, then centrifuged again to discard the supernatant. Cells were resuspended in 1 mL of pre-chilled PBS and washed, then collected by centrifugation.
[0034] Add 50 μL of PI staining working solution to each cell pellet, resuspend the cell pellet, and incubate at room temperature in the dark for 30 min. Analyze the stained cells using flow cytometry. Data were analyzed using FlowJo (version 10.9).
[0035] 1.5 Experimental Results Table 1 Results of cell proliferation assay
[0036] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0037] Table 2 Results of the Cloning Experiment
[0038] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0039] Table 3 Results of apoptosis experiments
[0040] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0041] Table 4 Results of cell cycle experiments
[0042] Note: Compared to the control group, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001.
[0043] As shown in Tables 1-2 above, the cell proliferation and colony formation assay results indicate that L-histidine has an inhibitory effect on the proliferation of colon cancer cells, and this effect is time- and concentration-dependent. As shown in Tables 3-4 above, L-histidine treatment can promote apoptosis of colon cancer cells, and the effect increases with increasing concentration. Furthermore, L-histidine can inhibit the transition of colon cancer cell lines from G1 to S phase.
[0044] The results showed that L-histidine can inhibit the proliferation of colon cancer cells.
[0045] Example 2: Transcriptomic analysis of the effect of L-histidine on gene expression in colorectal cancer cell lines 2.1 Cell treatment before transcriptomics analysis Human colon cancer cell line HT29 was seeded in 6-well plates, with approximately 1 × 10⁻⁶ cells added to each well. 6 Cells were divided into a control group (phosphate-buffered saline, pH 7.5) and an L-histidine treatment group. Three wells in the control group were treated with normal culture medium, while three wells in the L-histidine treatment group were treated with 5 mM L-histidine (preparation method as in Example 1).
[0046] After culturing the cells at 37°C in a cell culture incubator containing 5% CO2 for 48 h, wash twice with pre-cooled 1×PBS (prepared with DEPC water), add 1 mL of lysis buffer (TRIzol) to each well, and thoroughly lyse the cells using a pipette. Transfer the lysis buffer to 1.5 mL enzyme-free tubes, store at -80°C, and send for transcriptome sequencing analysis.
[0047] 2.2 Transcriptomics Analysis Methods Differential gene expression was analyzed using Deseq2, and differentially expressed genes were screened based on the following criteria: fold change |log2FoldChange| > 1, and statistical significance. P-value <0.05.
[0048] KEGG enrichment analysis was performed using Cluster Profiler. During the analysis, the gene list and gene count for each pathway were calculated using differentially expressed genes annotated with KEGG pathways, and then calculated using the hypergeometric distribution method. P- value (The criteria for significant enrichment are) P-value (<0.05) Identify KEGG pathways with significantly enriched differentially expressed genes compared to the overall genomic background, thereby determining the major biological functions performed by differentially expressed genes.
[0049] 2.3 Transcriptomics Analysis Results Differential gene volcano diagram as shown Figure 1 As shown, the KEGG enrichment pathway is as follows Figure 2 As shown in Table 5, the genes with transcriptomic changes were statistically analyzed.
[0050] Table 5. Genes with Transcriptomic Changes
[0051] The transcriptomic results above indicate that L-histidine can affect oxidative phosphorylation and the metabolism of energy and one-carbon units such as glycine and serine in colorectal cancer cell lines. Furthermore, it can inhibit the proliferation of colorectal cancer cells and promote their death by influencing the classic growth signaling pathway PI3K-Akt / mTOR.
[0052] Example 3: L-histidine Inhibition of Colorectal Cancer Growth Experiment 3.1 Experimental Procedure Six-week-old C57BL / 6J mice were purchased and divided into a control group and an L-histidine treatment group (n=7 / group). After one week of normal feeding, the experiment began. On day 1 of the experiment, both groups of mice were injected with mouse-derived microsatellite unstable colorectal cancer cells MC381×102 into the right axilla. 6 Each animal was given L-histidine (L-histidine-phosphate buffer solution, pH 7.5) via gavage every other day, 1.55 mg / animal each time, until the end of the experiment. The control group received an equal volume of phosphate buffer solution. Tumor size was measured and recorded every 3 days. The experiment was terminated on day 18, and subcutaneous tumors were harvested, flash-frozen in liquid nitrogen, and stored at -80°C. Tumor volume and weight were used as evaluation criteria.
[0053] Note: The concentration of L-histidine in the L-histidine treatment group was 15.5 mg / mL.
[0054] 3.2 Experimental Results Table 6 Tumor Growth Volume
[0055] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0056] Table 7 Tumor Weight
[0057] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0058] As shown in Tables 6 and 7 above, using tumor volume and weight as evaluation criteria, L-histidine treatment can significantly inhibit the growth of subcutaneous tumors of colon cancer cells MC38, and reduce the final tumor volume (by about 51.5%) and weight (by about 47.1%).
[0059] The results showed that L-histidine can inhibit the growth of subcutaneous tumors in colon cancer.
[0060] Example 4: Application of L-histidine as a PD-1 antibody sensitizer for microsatellite-stabilized colon cancer 4.1 Experimental Procedure Six-week-old BALB / c mice were purchased and randomly divided into a control group, an L-histidine group, a PD-1 antibody group, and an L-histidine + PD-1 antibody group (n=7 / group). After one week of normal feeding, experiments began. On day 1 of the experiment, mice in each group were injected with 1×10 microsatellite-derived stable murine colorectal cancer cell line CT26 in the right axilla. 6 Animals were divided into two groups: the L-histidine + PD-1 antibody group and the L-histidine group. L-histidine (15.5 mg / mL, prepared by mixing L-histidine with a phosphate buffer solution at pH 7.5) was administered by gavage every other day at a dose of 1.55 mg / animal until the experimental endpoint. The PD-1 antibody group and the control group received an equal volume of phosphate buffer solution at pH 7.5. On the third day after gavage, the L-histidine + PD-1 antibody group and the PD-1 antibody group received an intraperitoneal injection of PD-1 antibody (1 mg / mL), 100 μg / animal, every three days for a total of four injections. Tumor size was measured and recorded every three days. The experiment was terminated on the 15th day. The control group and the L-histidine group received an equal volume of phosphate buffer solution at pH 7.5. After the experiment, subcutaneous tumors were harvested, flash-frozen in liquid nitrogen, and stored at -80°C. Tumor volume and weight were used as evaluation criteria.
[0061] Note: In the L-histidine + PD-1 antibody group, the mass ratio of PD-1 antibody to L-histidine in each individual treatment of mice was 1:15.5. The total mass ratio of PD-1 antibody to L-histidine in the entire experiment was 1:31. The buffer solution in the PD-1 antibody group contained trace amounts of histidine, and the mass ratio of PD-1 antibody to L-histidine was 16.49:1.
[0062] 4.2 Experimental Results Table 8 Tumor Growth Volume
[0063] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0064] Table 9 Tumor Weight
[0065] Note: * represents the difference between the control group and the control group. P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.
[0066] As shown in Tables 8 and 9 above, using tumor volume and weight as evaluation criteria, although L-histidine cannot inhibit the progression of microsatellite stable colorectal cancer, the combined treatment of L-histidine and PD-1 antibody can significantly inhibit the growth of microsatellite stable colorectal cancer and reduce tumor volume and weight.
[0067] The results showed that L-histidine could sensitize the inhibitory effect of PD-1 antibody on microsatellite stable colorectal cancer, and the therapeutic effect was excellent.
[0068] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. Application of L-histidine as a sensitizer for PD-1 antibody therapy in microsatellite stable colorectal cancer.
2. The application as described in claim 1, characterized in that, The mass ratio of the PD-1 antibody to L-histidine is 1:
31.
3. The application as described in claim 1, characterized in that, The L-histidine is prepared as an L-histidine-phosphate buffer solution for application.
4. The application as described in claim 1, characterized in that, The PD-1 antibody is formulated into a PD-1 antibody solution for application.
5. The application as described in claim 1, characterized in that, The concentration of L-histidine in the L-histidine-phosphate buffer solution is 15.5 mg / mL.
6. The application as described in claim 1, characterized in that, The concentration of PD-1 antibody in the PD-1 antibody solution is 1 mg / mL.
7. The application according to claim 3, characterized in that, The L-histidine-phosphate buffer solution is obtained by mixing L-histidine and phosphate buffer solution.
8. The application according to claim 4, characterized in that, The PD-1 antibody solution comprises PD-1 antibody, phosphate buffer solution, and L-histidine.
9. The application according to claim 8, characterized in that, The mass ratio of PD-1 antibody to L-histidine in the PD-1 antibody solution is 16~16.5:
1.
10. A composition comprising L-histidine and PD-1 antibody as used in any one of claims 1 to 9.