A tumor immunotherapy composition of f1 / f3 polypeptide combined with immune checkpoint inhibitors and application thereof

Abstract

The present application belongs to the technical field of biological medicine and tumor immunotherapy, and relates to a pharmaceutical composition containing F1 / F3 polypeptide and immune checkpoint inhibitor and application thereof in preparation of tumor immunotherapy drugs. The F1 / F3 polypeptide is a composition of Caerin 1.1 and Caerin 1.9, and the immune checkpoint inhibitor is preferably a PD-1 inhibitor. It is found in the present application that the combination of F1 / F3 polypeptide and PD-1 inhibitor can enhance the anti-tumor effect in a colorectal cancer model, inhibit tumor growth and prolong survival; the effect is related to increased neutrophil infiltration, induced chemokine expression, promoted macrophage pro-inflammatory-like transformation and enhanced NK or T cell-related anti-tumor immune response. The present application can be used for preparing a drug for treating tumors or enhancing the efficacy of immune checkpoint inhibitor therapy.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and tumor immunotherapy technology, specifically relating to a pharmaceutical composition comprising an F1 / F3 peptide and an immune checkpoint inhibitor, and the application of the F1 / F3 peptide in the preparation of a drug or drug sensitizer that enhances the anti-tumor efficacy of immune checkpoint inhibitors. Background Technology

[0002] Cancer treatment does not rely on a single method, but rather on a comprehensive plan tailored to the tumor type, stage, and the patient's overall condition. Currently, the mainstream clinical treatment methods include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In recent years, immune checkpoint inhibitor therapy has become an important component of cancer treatment.

[0003] Immune checkpoint inhibitors enhance anti-tumor immune responses by blocking immunosuppressive signals such as CTLA-4, PD-1, or PD-L1, thereby relieving the suppression of T cell function in the tumor microenvironment. Among them, PD-1 / PD-L1 pathway inhibitors have shown clinical value in various solid tumors.

[0004] However, some solid tumors, especially some colorectal cancers, exhibit low response, no response, or treatment resistance to immune checkpoint inhibitor therapy. For these tumors, PD-1 inhibitor monotherapy alone often fails to achieve satisfactory results. Therefore, developing local immunomodulatory drugs that can improve the sensitivity of tumors to immune checkpoint inhibitor therapy has significant clinical and translational value.

[0005] The F1 / F3 peptide is a combination of Caerin 1.1 and Caerin 1.9. Studies have shown that the F1 / F3 peptide has antibacterial activity and certain antitumor activity.

[0006] The existing technology does not disclose any reports on the use of F1 / F3 peptides in combination with immune checkpoint inhibitors to prepare anti-colorectal cancer drugs, nor does it disclose the use of F1 / F3 peptides as sensitizers for immune checkpoint inhibitor therapy. In particular, it does not disclose that F1 / F3 peptides enhance the effect of PD-1 inhibitors in colorectal cancer by inducing neutrophil-related chemokine signal amplification and tumor immune microenvironment remodeling. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, this invention aims to provide an immunotherapy composition for colorectal cancer and its application, addressing the problems of low response, no response, or tolerance to immune checkpoint inhibitor therapy in existing colorectal cancer treatments. This invention enhances the antitumor efficacy of immune checkpoint inhibitors and improves the tumor immune microenvironment by combining F1 / F3 peptides with immune checkpoint inhibitors.

[0008] This invention is achieved through the following technical solution: The present invention provides a tumor immunotherapy composition comprising an F1 / F3 peptide and an immune checkpoint inhibitor.

[0009] The F1 / F3 polypeptide is a composition of Caerin 1.1 and Caerin 1.9. The mass ratio of Caerin 1.1 to Caerin 1.9 is 1:0.2–5, preferably 1:0.5–2, and more preferably 1:1.

[0010] The Caerin 1.1 is as shown in SEQ ID No. 1, and the Caerin 1.9 is as shown in SEQ ID No. 2.

[0011] The immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors; preferably, the immune checkpoint inhibitor is a PD-1 inhibitor; more preferably, the PD-1 inhibitor is an anti-PD-1 monoclonal antibody.

[0012] The antibody is a chimeric antibody, a humanized antibody, or a human antibody. Preferably, it is a monoclonal antibody derived from mammals.

[0013] The anti-PD-1 monoclonal antibody is selected from anti-mouse PD-1 antibody and anti-human PD-1 antibody.

[0014] The mass ratio of the immune checkpoint inhibitor to F1 / F3 is 1:0.5-5.0, preferably 1:0.8-1.5.

[0015] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabilizer, or any combination thereof.

[0016] The F1 / F3 peptide and the immune checkpoint inhibitor are formulated as the same preparation or as separate preparations.

[0017] The F1 / F3 peptide and the immune checkpoint inhibitor can be administered simultaneously, separately, or sequentially.

[0018] The F1 / F3 peptides are used for local administration, intratumoral administration, peritumoral administration, intracavitary administration, or endoscopic local administration, preferably intratumoral administration. The immune checkpoint inhibitors are used for systemic administration, including intravenous administration, subcutaneous administration, or other suitable systemic administration methods; in animal experiments, intraperitoneal administration may be used.

[0019] The tumor is a solid tumor, preferably colorectal cancer. More preferably, the colorectal cancer is colorectal cancer that has a low response, no response, or tolerance to immune checkpoint inhibitor therapy.

[0020] Application of F1 / F3 peptides in the preparation of sensitizers for colorectal cancer immunotherapy.

[0021] Application of F1 / F3 peptides in the preparation of drugs for treating colorectal tumors or enhancing the sensitivity of colorectal tumors to immune checkpoint inhibitor therapy.

[0022] Application of F1 / F3 peptides combined with immune checkpoint inhibitors in the preparation of immunotherapy drugs for colorectal cancer.

[0023] In the experiment, the mass ratio of F1 to F3 administered was 1:1.

[0024] The mass ratio of the immune checkpoint inhibitor to the F1 / F3 peptide is 1:0.5-5.0, preferably 1:0.8-1.5, and more preferably 1:0.8-1.1.

[0025] Experimental results show that intratumoral injection of F1 / F3 peptides induces macrophage repolarization, tumor-stromal remodeling, and NK / T cell activation through neutrophil-mediated chemokine amplification, thereby enhancing the sensitivity of colorectal tumors to PD-1 antibody inhibitors.

[0026] This invention provides the application of F1 / F3 peptide combined with PD-1 antibody inhibitor in the preparation of immunotherapy drugs for colorectal cancer.

[0027] As described above, the combination of F1 / F3 peptides with immune checkpoint inhibitors can increase neutrophil infiltration, induce chemokine expression, promote macrophage transformation to a pro-inflammatory phenotype, and enhance NK cell and / or T cell-related anti-tumor immune responses in syngeneic colorectal cancer models.

[0028] F1 / F3 combined immune checkpoint inhibitors coordinate macrophage repolarization, tumor-stromal remodeling, and NK or T cell-mediated antitumor immune responses by inducing neutrophil-centered chemokine signaling amplification. Chemokines include Ccl2 / Cxcl2, Ccl3 / Cxcl3, Ccl4 / Cxcl4, or any combination thereof.

[0029] When used in combination, at least one dose of the F1 / F3 peptide and / or at least one dose of the PD-1 antibody inhibitor can be administered within a certain time period. This time period can be within a dosing cycle, preferably within 4 weeks, 3 weeks, 2 weeks, 1 week, or within 24 hours.

[0030] Beneficial technical effects of the present invention: This invention provides the application of F1 / F3 peptides combined with immune checkpoint inhibitors in the preparation of immunotherapeutic drugs for colorectal cancer. The composition comprising F1 / F3 peptides and immune checkpoint inhibitors can be used to treat colorectal cancer with low response, no response, or resistance to PD-1 antibody therapy. Compared with using immune checkpoint inhibitors alone, the combination of F1 / F3 peptides and immune checkpoint inhibitors significantly enhances the antitumor effect in colorectal cancer models, manifested as inhibited tumor growth, prolonged survival, and an increased proportion of long-term disease-free survival.

[0031] Furthermore, the pharmaceutical composition of the present invention can increase neutrophil infiltration in tumor tissue, induce chemokine expression, promote the transformation of macrophages to a pro-inflammatory phenotype, and promote the transformation of the tumor immune microenvironment from an immunosuppressive state to an immune-activated state.

[0032] In MC38, CT26, and Colon26 syngeneic colorectal cancer models, the F1 / F3 peptide combined with a PD-1 inhibitor showed stronger antitumor activity than monotherapy. Single-cell RNA sequencing and neutrophil removal experiments further suggested that neutrophil-associated chemokine signaling amplification is one of the important mechanisms by which the F1 / F3 peptide combined with a PD-1 inhibitor remodels the tumor immune microenvironment. Attached Figure Description

[0033] Figure 1 IC50 fitting curves were plotted to show the inhibitory effects of F1, F3, and F1 / F3 on different colorectal cancer cells at different concentrations in Example 1.

[0034] A: MC38 B: CT26 C: Colon26 D: COLO678.

[0035] Figure 2 This study examines the tumor-suppressing effect and survival impact of administering F1 / F3 peptides or PD-1 inhibitors alone to MC38 tumor-bearing mice in Example 2.

[0036] A: Schematic diagram of the experimental protocol for treating MC38 tumor-bearing mouse model with F1 / F3 and PD-1; B: Effects of F1 / F3 and PD-1 on tumor size in MC38 tumor-bearing mice; C: Effects of F1 / F3 and PD-1 on survival curves in MC38 tumor-bearing mice.

[0037] Figure 3 This study examines the antitumor effects and survival impact of PD-1 inhibitors, F1 / F3 peptides, and their combined administration in Example 3 on MC38, CT26, and Colon26 tumor-bearing mice.

[0038] A: Schematic diagram of the experimental protocol for treating MC38 tumor-bearing mouse model with PD-1, F1 / F3, and PD-1+F1 / F3; B: Effects of PD-1, F1 / F3, and PD-1+F1 / F3 on tumor size in MC38 tumor-bearing mice; C: Effects of PD-1, F1 / F3, and PD-1+F1 / F3 on survival curves in MC38 tumor-bearing mice; D: Effects of PD-1, F1 / F3, and PD-1+F1 / F3 on tumor size in CT26 tumor-bearing mice; E: Effects of PD-1, F1 / F3, and PD-1+F1 / F3 on survival curves in CT26 tumor-bearing mice; F: Effects of PD-1, F1 / F3, and PD-1+F1 / F3 on tumor size in Colon26 tumor-bearing mice; G: Effects of PD-1, F1 / F3, and PD-1+F1 / F3 on survival curves in Colon26 tumor-bearing mice.

[0039] Figure 4 In Example 4, the F1 / F3 peptide combined with a PD-1 inhibitor promoted immune cell infiltration, neutrophil aggregation, and macrophage pro-inflammatory remodeling in two colorectal cancer models (MC38 and CT26).

[0040] A: Changes in tumor volume in MC38 tumor-bearing mice after treatment with PD-1 and PD-1+F1 / F3 (Day 28–Day 34). B: Tumor images of MC38 tumor-bearing mice after treatment with PD-1 and PD-1+F1 / F3 (Day 35). C: Tumor weight in each MC38 group (Day 35); D: CD45.2 of tumor cells in each MC38 group. + Cell proportions (right) and representative flow cytometry scatter plot (left); E: The proportion of M1 pro-inflammatory macrophages and M2 resident macrophages in each group of MC38 tumor cells, the upregulation of M1 pro-inflammatory macrophages / M2 resident macrophages (right) and representative flow cytometry scatter plot (left); F: Neutrophil percentage in each group of MC38 tumor cells (right) and representative flow cytometry scatter plot (left); G: Changes in tumor weight in CT26 tumor-bearing mice after treatment with PD-1 and PD-1+F1 / F3; H: CD45.2 of tumor cells in each group of CT26 + The percentage of cells, the percentage of M1 pro-inflammatory macrophages and M2 resident macrophages, the upregulation of M1 pro-inflammatory macrophages / M2 resident macrophages, and the percentage of neutrophils.

[0041] Figure 5 The effect of PD-1 combined with F1 / F3 on the immune system of draining lymph nodes in MC38 tumor-bearing mice in Example 5.

[0042] A: Percentage of CD3+ T cells in the draining lymph nodes of tumors in each MC38 group; B: Percentage of NK cells in the draining lymph nodes of tumors in each MC38 group; C: Percentage of macrophages in the tumor-draining lymph nodes of each MC38 group; D: Percentage of M1 pro-inflammatory macrophages in the draining lymph nodes of each MC38 group; E: Percentage of M2-resident macrophages in the tumor-draining lymph nodes of each MC38 group; F: Upregulation of M1 pro-inflammatory macrophages / M2 resident macrophages in the tumor draining lymph nodes of each MC38 group.

[0043] Figure 6 The results of single-cell RNA sequencing analysis of the MC38 tumor microenvironment by combining F1 / F3 peptides with PD-1 inhibitor in Example 6 are shown.

[0044] A: Effects of PD-1 and PD-1+F1 / F3 on tumor size in MC38 tumor-bearing mice; B: Images of tumors in MC38 tumor-bearing mice after treatment with PD-1 and PD-1+F1 / F3; C: Tumor weight in each MC38 group; D: Quality control screening image for single-cell sequencing; EF: Dimensionality reduction analysis and unsupervised clustering of single-cell RNA sequencing data of MC38 tumors in each group; G: Cellular composition of the tumor microenvironment (TME) in each group; H: The relative abundance of tumor cells in the total captured cells in each group; I: Relative abundance of neutrophils in total captured cells in each group; J: Relative abundance of macrophage subsets in total captured cells in each group; K: Relative abundance of T cell subsets in total captured cells in each group; L: Relative abundance of B cells in total captured cells in each group; M: Relative abundance of dendritic cell subsets in total captured cells in each group; N: The relative abundance of fibroblasts in the total captured cells in each group.

[0045] Figure 7 This refers to the Neu_6 neutrophil state with high expression of chemokines related to the F1 / F3 peptide combined with PD-1 inhibitor-induced therapy in Example 6.

[0046] AB: Reclustering analysis of tumor-infiltrating neutrophils; C: Expression of marker genes for different neutrophil subsets in tumor infiltration; D: Expression profile of Neu_6 chemokine in tumor-infiltrating neutrophil subsets; E: Transcriptomic features of tumor-infiltrating neutrophils transitioning from a suppressor phenotype to an activation / recruitment phenotype; FH: Specific enrichment of NF-κB, TNF, and cytokine interaction pathways in the neutrophil subset Neu_6 effect; I: Intercellular communication analysis.

[0047] Figure 8 In Example 7, neutrophil removal weakened the F1 / F3 peptide combined with a PD-1 inhibitor in MC38 tumor-bearing mice. The effects of anti-tumor efficacy and the tumor immune microenvironment.

[0048] A: Schematic diagram of experimental design and dosing regimen for the effect of neutrophil clearance on the antitumor efficacy of combination therapy; B: Tumor volume curves of tumor-bearing mice in each group of MC38; C: Survival curves of tumor-bearing mice in each group of MC38.

[0049] D: The proportion of neutrophils in each group of MC38 tumors (right) and representative flow cytometry scatter plots (left); E: The proportion of M1 pro-inflammatory macrophages and M2 resident macrophages in each group of MC38 tumors, and the proportion of M1 pro-inflammatory macrophages. Upregulation of cells / M2-resident macrophages (right) and representative flow cytometry scatter plot (left); F: The proportion of CD45.2+ immune cells in each group of MC38 tumors (right) and representative flow cytometry scatter plots (left); G: Expression of chemokine genes such as Ccl2, Cxcl2, Ccl3, Cxcl3, and Ccl4 in tumor tissues of various MC38 tumor groups result.

[0050] Figure 9 The F1 / F3 peptide, in combination with a PD-1 inhibitor, mediates the reprogramming of tumor-associated macrophages, thereby reshaping the tumor immune microenvironment through amplification of related chemokine signals.

[0051] A: Reclustering analysis of tumor-associated macrophages in MC38 tumors; B: Cxcl after combined treatment + Reprogramming of macrophage inflammatory / chemokine networks with high expression; C: Cell type-specific expression matrix of chemokine genes in major cell populations of the tumor microenvironment; D: The upregulation of cell type-specific expression density of chemokine ligands and receptors in the tumor microenvironment by combined therapy; E: Quantitative PCR was used to verify the expression of chemokines in MC38 tumor tissue after treatment.

[0052] Figure 10 yes Figure 7 Enlarged view of A in the middle.

[0053] Figure 11 yes Figure 7 Enlarged view of B in the middle.

[0054] Figure 12 yes Figure 7 Enlarged view of C.

[0055] Figure 13 yes Figure 7 Enlarged view of D.

[0056] Figure 14 yes Figure 7 Enlarged view of the middle E.

[0057] Figure 15 yes Figure 7 Enlarged view of the middle F.

[0058] Figure 16 yes Figure 7 Enlarged view of G in the middle.

[0059] Figure 17 yes Figure 7 Enlarged view of H in the middle.

[0060] Figure 18 yes Figure 7 Enlarged view of the middle section (I).

[0061] Figure 19 yes Figure 9 Enlarged view of A in the middle.

[0062] Figure 20 yes Figure 9Enlarged view of B in the middle.

[0063] Figure 21 yes Figure 9 Enlarged view of C.

[0064] Figure 22 yes Figure 9 Enlarged view of D.

[0065] Figure 23 yes Figure 9 Enlarged view of the middle E. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to the embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. For those skilled in the art, appropriate adjustments can be made to the relevant experimental conditions, dosage, administration frequency, administration method, and detection indicators without departing from the concept of the present invention; such adjustments should still fall within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available.

[0067] The F1 / F3 polypeptide is a composition of F1 (Caerin 1.1) and F3 (Caerin 1.9). In the examples below, unless otherwise specified, the mass ratio of F1 to F3 is 1:1. It was synthesized by Qiangyao Biotechnology Co., Ltd., with a purity far exceeding 90%, and is in powder form, normally stored at -80 degrees Celsius.

[0068] In the attached diagram, M1 is short for M1 pro-inflammatory macrophage; M2 is short for M2 resident macrophage.

[0069] The amino acid sequences of F1 and F3 are shown in SEQ ID No. 1-2.

[0070] The PD-1 inhibitor used in the examples was an anti-mouse PD-1 antibody (CD279), purchased from Selleck.cn, clone number RMP1-14.

[0071] Anti-Ly6G: InVivoMAb anti-mouse Ly6G (purchased from BioXcell, product number: BE0075-1, clone number: 1A8).

[0072] Cell viability was assessed using the MTT assay with an MTT kit (purchased from Beyotime, product number: C0009M).

[0073] Flow cytometry data were analyzed using FlowJo software; statistical analysis was performed using GraphPad Prism software. Unless otherwise stated, data are expressed as mean ± standard error, and P < 0.05 was considered statistically significant.

[0074] C57BL / 6 mice: Female C57BL / 6 (H-2b) mice, 6–8 weeks old, purchased from Guangdong Provincial Animal Resource Center (Guangzhou, China). BALB / c mice: Female BALB / c mice, 6–8 weeks old, purchased from Guangdong Provincial Animal Resource Center (Guangzhou, China).

[0075]

[0076] Example 1: In vitro inhibitory effects of F1, F3, and F1 / F3 peptides on colorectal cancer cells. The MTT assay was used to detect the in vitro inhibitory effects of F1, F3, and F1 / F3 peptides on MC38, CT26, Colon26, and COLO678 colorectal cancer cells. The corresponding cell lines were cultured at a concentration of 1 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates, with 100 μL of cell suspension added to each well. Four replicates were set up for each group. Subsequently, different concentrations of F1, F3, or F1 / F3 peptides were added. After culturing, cell viability was detected using an MTT assay kit, and the IC50 (half-maximal inhibitory concentration) was calculated.

[0077] In MC38 cells, the concentration gradients of F1 were 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg / mL; the concentration gradients of F3 were 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg / mL; and the concentration gradients of F1 / F3 were 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg / mL. Figure 1 Results showed that F1 / F3, F1, and F3 all inhibited MC38 cell proliferation, with F1 / F3 showing the strongest inhibitory effect. The IC50 values ​​for F1 / F3, F1, and F3 were 5.166 μg / mL, 6.703 μg / mL, and 11.14 μg / mL, respectively.

[0078] In CT26 cells, the concentration gradients of F1, F3, and F1 / F3 were 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg / mL. Figure 1Results showed that F1 / F3, F1, and F3 all inhibited CT26 cell proliferation, with F1 / F3 exhibiting the strongest inhibitory effect. The IC50 values ​​for F1 / F3, F1, and F3 were 8 μg / mL, 11.37 μg / mL, and 12.93 μg / mL, respectively.

[0079] In Colon26 cells, the concentration gradients of F1, F3, and F1 / F3 were 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg / mL. Figure 1 The results showed that F1 / F3, F1, and F3 all inhibited the proliferation of Colon26 cells, with F1 / F3 showing the strongest inhibitory effect. The IC50 values ​​for F1 / F3, F1, and F3 were 8.631 μg / mL, 13.80 μg / mL, and 19.91 μg / mL, respectively.

[0080] In human colorectal cancer COLO678 cells, the concentration gradients of F1, F3, and F1 / F3 were 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg / mL. Figure 1 Results showed that F1 / F3, F1, and F3 all inhibited the proliferation of COLO 678 cells, with F1 / F3 showing the strongest inhibitory effect. The IC50 values ​​for F1 / F3, F1, and F3 were 7.786 μg / mL, 10.75 μg / mL, and 10.15 μg / mL, respectively.

[0081] The above results indicate that the F1 / F3 peptide has an in vitro inhibitory effect on various colorectal cancer cells.

[0082] Results are expressed as mean ± standard error and were analyzed using GraphPad Prism 8.4.3 software.

[0083] Example 2: Inhibitory effect of F1 / F3 peptide or PD-1 inhibitor alone on MC38 tumor-bearing mice Model establishment: When MC38 cells reached approximately 70% confluence, they were digested with 0.25% trypsin and washed twice with PBS. 1×10⁶ cells were then... 6 One MC38 cell (dissolved in 0.1 mL PBS) was subcutaneously inoculated into the left flank of C57BL / 6 mice. Tumor volume was measured every 3 days and calculated using the formula: length × width × width / 2.

[0084] When the tumor volume reaches 50mm 3Mice bearing tumors were randomly divided into three groups of 6-7 mice each: a blank control group (intratumoral injection of PBS), an F1 / F3 monotherapy group (intratumoral injection of F1 / F3 solution), and a PD-1 monotherapy group (intraperitoneal injection of PD-1 solution). Mice were sacrificed by CO2 inhalation when the tumor length reached 15 mm to plot survival curves.

[0085] Sample preparation: Preparation of PD-1 solution: Dissolve PD-1 antibody in PBS to prepare a solution containing 200 µg of PD-1 antibody per 100 µL.

[0086] Preparation of F1 / F3 solution: Dissolve 30 μg each of F1 and F3 in PBS to prepare a solution containing 30 μg each of F1 and F3 per 100 µL.

[0087] Dosage regimen: In the MC38 model, each group reached a tumor volume of 50 mm. 3 Start medication on day 3.

[0088] Blank control group: 100µL of PBS was injected into the tumor on day 3.

[0089] PD-1 monotherapy group: 100µL of PD-1 solution was injected intraperitoneally every three days for a total of 5 times (administered on days 3, 6, 9, 12, and 15).

[0090] F1 / F3 monotherapy group (i.e., caerin 1.1 / 1.9): 100 μL of F1 / F3 solution was injected intratumorally daily starting from day 3 for 14 consecutive days.

[0091] Record the size of mouse tumors, plot the mouse tumor volume curve, and simultaneously plot the mouse survival curve.

[0092] Figure 2 Results B showed that in MC38 tumor-bearing mice, there was a significant difference between the blank control group and the F1 / F3 treatment group, indicating that F1 / F3 peptide alone significantly inhibited tumor growth; there was a significant difference between the blank control group and the F1 / F3 treatment group and the PD-1 treatment group; there was no significant difference between the PD-1 treatment group and the F1 / F3 treatment group.

[0093] Figure 2 C-survival curve analysis showed that the median survival times for the PBS, PD-1, and F1 / F3 groups were 35, 76, and 88 days, respectively. Both the PD-1 and F1 / F3 groups showed survival benefits compared to the PBS control group (no significant difference between the two groups); the long-term disease-free survival rates were 42.8% and 50%, respectively, when the MC38 tumor volume reached 50 mm. 3 When administration was initiated, F1 / F3 monotherapy showed antitumor activity comparable to or even superior to PD-1.

[0094] Results are expressed as mean ± standard error. Differences between groups were analyzed using GraphPad Prism 8.4.3 software, and statistical differences between groups were calculated through two-way ANOVA, simple survival analysis (Kaplan–Meier), or multivariate ANOVA.

[0095] ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0096] Example 3: Antitumor effect of F1 / F3 peptide combined with PD-1 inhibitor on MC38, CT26 and Colon26 tumor-bearing mice Modeling: Mouse models of colorectal cancer bearing MC38, CT26, and Colon26 syngeneic cells were constructed. MC38 cells were grown at a rate of 1×10⁻⁶. 6 CT26 and Colon26 cells were inoculated at 5 × 10⁶ cells per mouse on the left flank; 5 One tumor per mouse was injected into the left flank of BALB / c mice. Tumor volume was measured every 3 days and calculated using the formula: length × width × width / 2.

[0097] To more effectively compare drug efficacy, the treatment initiation point was changed from a tumor volume of 50 mm. 3 Delayed up to 100 mm 3 The tumor-bearing mice were then randomly divided into 4 groups, with 5-6 mice in each group.

[0098] When the long diameter of the tumor reached 15 mm, the mice were euthanized by CO2 inhalation to plot tumor volume curves and survival curves.

[0099] Blank control group (intratumoral injection of PBS), PD-1 monotherapy group (intraperitoneal injection of PD-1 solution), F1 / F3 monotherapy group (intratumoral injection of F1 / F3 solution), PD-1+F1 / F3 combination therapy group (intraperitoneal injection of PD-1 solution + intratumoral injection of F1 / F3 solution).

[0100] Sample preparation: Preparation of PD-1 solution: Dissolve PD-1 antibody in PBS to prepare a solution containing 200 µg of PD-1 antibody per 100 µL.

[0101] Preparation of F1 / F3 solution: Dissolve 30 μg each of F1 and F3 in PBS to prepare a solution containing 30 μg each of F1 and F3 per 100 µL.

[0102] Dosing regimen: In the MC38, CT26, and Colon26 models, drug administration was initiated when the tumor volume reached 100 mm³. For the MC38 model, the regimen is as follows: Figure 3 A.

[0103] Blank control group: Starting from day 6, 100µL of PBS was injected into the tumor, and this was continued for 14 days.

[0104] PD-1 monotherapy group: PD-1 was administered starting on day 6, with 100µL of PD-1 solution injected intraperitoneally every three days for a total of 5 times (6, 9, 12, 15, 18).

[0105] F1 / F3 monotherapy group (i.e., caerin 1.1 / 1.9): Dosing started on day 6, with 100 μL of F1 / F3 solution injected intratumorally daily for 14 consecutive days.

[0106] Combined treatment group: On day 6, PD-1 solution 100µL was injected intraperitoneally. On days 9, 12, 15 and 18, PD-1 solution 100µL was injected intraperitoneally daily for a total of 5 times. At the same time, F1 / F3 solution 100µL was injected intratumorally when drug administration was started on day 6, and then F1 / F3 solution 100µL was injected intratumorally daily for a total of 14 days.

[0107] The inhibitory effects and survival curves of PD-1, F1 / F3, and PD-1+F1 / F3 on MC38, CT26, and Colon26 tumors are shown in the table below. Figure 3 .

[0108] In the MC38 model ( Figure 3 In cases B and C), tumor volume results showed that, compared with the PBS control group, the PD-1 monotherapy group, the F1 / F3 monotherapy group, and the PD-1+F1 / F3 combination group all significantly inhibited tumor growth (P < 0.0001). Specifically, the antitumor activity of the PD-1+F1 / F3 combination group was significantly better than that of the PD-1 monotherapy group (P < 0.05) and the F1 / F3 monotherapy group (P < 0.0001), while the PD-1 monotherapy group was better than the F1 / F3 monotherapy group (P < 0.001). At day 33 of treatment, the mean tumor volumes in the PBS, PD-1, F1 / F3, and combination groups were 1688, 142.1, 474.0, and 0 mm, respectively. 3 The PD-1 monotherapy group and the F1 / F3 monotherapy group shrank tumors by 91.6% and 71.9%, respectively. Under the experimental conditions, it was observed that the combined use of F1 / F3 and PD-1 could achieve complete tumor regression.

[0109] Survival curve analysis further showed that, compared with the PBS control group, the PD-1 monotherapy group, the F1 / F3 monotherapy group, and the PD-1+F1 / F3 combination group all significantly prolonged tumor survival. Specifically, the antitumor activity of the PD-1+F1 / F3 combination group was significantly superior to that of the PD-1 monotherapy group (P < 0.05) and the F1 / F3 monotherapy group (P < 0.001), while the PD-1 monotherapy group was superior to the F1 / F3 monotherapy group (P < 0.05). The median survival was 26 days in the PBS group (long-term disease-free survival rate of 0%), 63 days in the PD-1 group (long-term disease-free survival rate of 33.3%), and 42 days in the F1 / F3 monotherapy group (long-term disease-free survival rate of 0%). The PD-1+F1 / F3 group achieved 100% long-term disease-free survival, indicating an improved long-term disease-free survival rate in the combination therapy groups.

[0110] In CT26 model ( Figure 3 In cases D and E), tumor volume results showed no significant difference in tumor volume between the PD-1 monotherapy group and the PBS group (this model is insensitive to PD-1 blockade). Compared with the PBS control group, both the F1 / F3 monotherapy group (P < 0.01) and the PD-1 + F1 / F3 combination group (P < 0.0001) significantly inhibited tumor growth. The antitumor activity of the PD-1 + F1 / F3 combination group was significantly better than that of the F1 / F3 monotherapy group (P < 0.0001), indicating that the addition of F1 / F3 significantly enhanced the antitumor effect of the PD-1 inhibitor in the low-response model. On day 28 of treatment, the mean tumor volumes of the PBS control group, PD-1 monotherapy group, F1 / F3 monotherapy group, and combination group were 1772, 1394, 1058, and 330.3 mm, respectively. 3 The combined group showed a tumor volume reduction of 1441.7 mm. 3 (This is) far greater than the sum of the reductions in the two single-drug groups (1092 mm). 3 This result suggests that the combined use of F1 / F3 and PD-1 inhibitors has a synergistic effect.

[0111] Survival curves showed that the median survival in the PBS group and the PD-1 group was 20 days and 22 days, respectively (no significant difference), and the long-term disease-free survival rate in the PD-1 group was 0. The median survival in the F1 / F3 monotherapy group was 28 days, significantly longer than that in the PBS group (P < 0.01), with a long-term disease-free survival rate of 0. The median survival in the PD-1 + F1 / F3 combination group was 47.5 days, significantly longer than that in the PBS group (P < 0.001), with a long-term disease-free survival rate of 33.3%. Compared with the F1 / F3 monotherapy group or the PD-1 group, the survival in the combination group was also significantly improved (P < 0.01), suggesting that the combined use of F1 / F3 and PD-1 inhibitors has a synergistic effect.

[0112] In the Colon26 model ( Figure 3 In F and G), the model was known to be extremely insensitive to PD-1 monotherapy (no significant difference in tumor volume between the PD-1 group and the PBS group). Compared with the PBS control group, both the F1 / F3 monotherapy group (P < 0.05) and the PD-1 + F1 / F3 combination group (P < 0.0001) significantly inhibited tumor growth. Combining F1 / F3 significantly enhanced the antitumor effect of PD-1 inhibitors in the low-response model. On day 34 of treatment, the mean tumor volumes of the PBS control group, PD-1 monotherapy group, F1 / F3 monotherapy group, and combination group were 1688, 1578, 1362, and 281.3 mm, respectively. 3 The tumor volume in the combined group decreased by 1406.7 mm. 3 (This is) far greater than the sum of the reductions in the two single-drug groups (436 mm). 3 This indicates that the combined use of F1 / F3 and PD-1 inhibitors has a significant synergistic effect.

[0113] Survival curve results also showed that the median survival times in the PBS group, PD-1 group, and F1 / F3 group were 19 days, 20 days, and 25.5 days, respectively (no significant difference). The long-term disease-free survival rate was 0 in both the PD-1 and F1 / F3 groups. The long-term disease-free survival rate in the PD-1 + F1 / F3 group was 83.3%, significantly longer than that in the PBS group (P < 0.01). These results suggest that the combined use of F1 / F3 and PD-1 inhibitors has a synergistic effect.

[0114] Results are expressed as mean ± standard error. Differences between groups were analyzed using GraphPad Prism 8.4.3 software, calculated through two-way ANOVA, simple survival analysis (Kaplan–Meier), or multivariate ANOVA. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0115] Example 4: The combination of F1 / F3 peptide and PD-1 inhibitor promoted immune cell infiltration, neutrophil aggregation and macrophage pro-inflammatory remodeling in two colorectal cancer models (MC38 and CT26).

[0116] 1. Test subjects and modeling: MC38 (C57BL / 6 background) and CT26 (BALB / c background) syngeneic colorectal cancer mouse models were constructed according to the method in Example 3. When the MC38 tumor volume reached approximately 380 mm... 3 (Day 28) CT26 The tumor volume reached approximately 300 mm 3On Day 19, the patients were divided into groups, with each group consisting of a PBS control group, a PD-1 inhibitor monotherapy group, and a PD-1 inhibitor + F1 / F3 combination therapy group.

[0117] 2. Dosing regimen: On Day 28, the MC38 tumor was observed to have a size of 380 mm. 3 Dosing should be initiated around 10:00 or 11:00.

[0118] The tumor size of CT26 was observed to reach 300 mm on Day 19. 3 Dosing should be initiated around 10:00 or 11:00.

[0119] PBS group: At the start of drug administration, 100 µL of PBS was injected intratumorally for 7 consecutive days.

[0120] PD-1 group: At the start of dosing, 100 µL of PD-1 solution was injected intraperitoneally once every 3 days for a total of 2 times.

[0121] PD-1+F1 / F3 group: At the start of drug administration, 100 µL of PD-1 solution was injected intraperitoneally (once every 3 days for a total of 2 times), and 100 µL of F1 / F3 solution was injected intratumorally at the same time for 7 consecutive days.

[0122] The preparation methods for PD-1 solution and F1 / F3 solution are the same as in Example 3.

[0123] The MC38 model was initiated on Day 28 (PD-1 was administered on Day 28 and Day 31; F1 / F3 was administered on Day 28–Day 34), and tumor samples were collected on Day 35 for flow cytometry analysis.

[0124] The CT26 model was initiated with drug administration on Day 19 (PD-1 was administered on Day 19 and Day 22; F1 / F3 was administered on Day 19–Day 25), and tumor samples were harvested on Day 26 for flow cytometry analysis.

[0125] 3. Flow cytometry analysis of the microenvironment: Test method: Tumor tissue was used to prepare a single-cell suspension, and the concentration was adjusted to 1×10⁻⁶. 6 Cells / mL, add 1 μL of each of the following fluorescently labeled antibodies: FVS510, FITC-CD45.2 (clone 104), PE-F4 / 80 (clone BM8), Percp-cy5.5-CD11b (clone M1 / 70), APC-Ly6G (clone 1A8), and BV421-Ly6C (clone AL-21), and incubate at 4°C in the dark for 30 min. Data were acquired using a BDFACSAria II flow cytometer.

[0126] Flow cytometry data were analyzed using Flowjo 10 software. Results are expressed as mean ± standard deviation. Differences between groups were analyzed using GraphPad Prism 8.4.3 software, and statistical differences between groups were calculated using one-way ANOVA or multivariate ANOVA. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0127] 4. Experimental Results: Figure 4 A: During Day 28–Day 34, the changes in MC38 tumor volume after treatment were monitored. The results showed that the combination therapy of PD-1 and F1 / F3 was significantly better than PD-1 monotherapy in inhibiting tumor growth (P=0.0166).

[0128] Figure 4 BC: On Day 35, mice were sacrificed and the tumor weight of MC38 was collected. The results showed that intratumoral injection of F1 / F3 combined with intraperitoneal injection of PD-1 was significantly more effective than PD-1 alone (P=0.0314). These results indicate that intratumoral injection of PD-1 combined with F1 / F3 can significantly enhance the antitumor effect of PD-1 and inhibit the progression of colorectal cancer.

[0129] Figure 4 D: Showing the percentage (%) of CD45.2+ cells in each group of MC38 tumors and representative flow cytometry scatter plots: PBS group: 9.52%, PD-1 group: 15.0%, PD-1+F1 / F3 group: 29.3%.

[0130] Figure 4 E: Presenting the proportions of M1 pro-inflammatory macrophages and M2 resident macrophages in MC38 tumor cells across different groups, as well as the upregulation of the M1 pro-inflammatory macrophage / M2 resident macrophage ratio: The proportion of M1 pro-inflammatory macrophages in the PBS, PD-1, and PD-1+F1 / F3 groups was 8.368%, 11.97%, and 19.18%, respectively; the proportion of M2 resident macrophages in the PBS, PD-1, and PD-1+F1 / F3 groups was 61.87%, 60.60%, and 32.73%, respectively. The M1 pro-inflammatory macrophage / M2 resident macrophage ratio in the PBS, PD-1, and PD-1+F1 / F3 groups was 0.1341, 0.2010, and 0.5761, respectively. These results suggest that the combination of F1 / F3 peptides and PD-1 inhibitors can promote the transformation of tumor-associated macrophages to a pro-inflammatory phenotype.

[0131] Figure 4F: Showing the proportion of neutrophils in each group of MC38 tumors in the PBS, PD-1, and PD-1+F1 / F3 groups. The PD-1+F1 / F3 group (23.13%) was significantly higher than that in the PBS group (5.19%) and the PD-1 group (2.37%). This suggests that the combination of F1 / F3 peptide and PD-1 inhibitor can increase neutrophil infiltration in tumor tissue.

[0132] Figure 4 G: On Day 26, the tumors of CT26 tumor-bearing mice were sacrificed and weighed. The combined treatment group was significantly lower than that of the PBS group (P = 0.0486), while there was no significant difference between the PBS group and the PD-1 monotherapy group, which is consistent with the low response of the CT26 model to PD-1 monotherapy.

[0133] Figure 4 H: CT26 tumor flow cytometry analysis showed that the proportions of CD45.2+ immune cells in the PBS group, PD-1 inhibitor group, and PD-1+F1 / F3 group were 39.18%, 48.98%, and 69.70%, respectively. The upregulation of the proportions of M1 pro-inflammatory macrophages and M2 resident macrophages, as well as the M1 pro-inflammatory macrophage / M2 resident macrophage ratio, was as follows: the proportion of M1 pro-inflammatory macrophages in the PBS, PD-1, and PD-1+F groups were 8.368%, 11.97%, and 19.18%, respectively; the proportion of M2 resident macrophages was 61.87%, 60.60%, and 32.73%, respectively. The M1 pro-inflammatory macrophage / M2 resident macrophage ratio in the PBS, PD-1, and PD-1+F groups were 0.3845, 0.3185, and 1.010, respectively. The proportions of neutrophils were 11.60%, 17.18%, and 30.20%, respectively. In the CT26 model, the combination of F1 / F3 peptides and PD-1 inhibitors also promoted immune cell infiltration, neutrophil aggregation, and macrophage pro-inflammatory remodeling in tumor tissue.

[0134] Conclusion: In both MC38 and CT26 syngeneic colorectal cancer models, the combination of F1 / F3 peptides and PD-1 inhibitors significantly enhanced antitumor efficacy. The mechanism involves remodeling the tumor immune microenvironment, driving the recruitment of CD45.2⁺ immune cells and neutrophils, and promoting pro-inflammatory remodeling of macrophages, thus achieving a shift from "immunosuppression" to "immunoactivation." Particularly noteworthy is that in the CT26 model, which was unresponsive to PD-1 inhibitor monotherapy, the combination therapy effectively reversed the immunosuppressive state, suggesting that the F1 / F3 peptides enhance the antitumor response to PD-1 inhibitors by remodeling the tumor immune microenvironment.

[0135] Example 5: Effect of F1 / F3 peptide combined with PD-1 inhibitor on the composition of immune cells in draining lymph nodes of MC38 tumor-bearing mice 1. Test subjects and modeling: After constructing the MC38 tumor-bearing model according to Example 3, the tumor volume reached 100 mm. 3 On Day 9, the experimental groups were divided as follows: PBS group, PD-1 group, and PD-1+F1 / F3 group.

[0136] 2. Dosage regimen: On Day 9, the tumor size reached 100 mm. 3 Dosing should be initiated around 10:00 or 11:00.

[0137] PBS group: 100µL of PBS was injected intratumorally at the start of drug administration, and the intratumoral injection continued for 14 days.

[0138] PD-1 group: 100 μL of PD-1 solution was injected intraperitoneally at the start of dosing, once every three days, for a total of 5 doses (i.e., on days 9, 12, 15, 18, and 21).

[0139] PD-1+F1 / F3 group: PD-1 solution 100 μL was injected intraperitoneally at the start of drug administration, once every three days for a total of 5 times. At the same time, F1 / F3 solution 100 μL was injected intratumorally at the start of drug administration, and the intratumoral injection was continued for 14 days.

[0140] Tumor size was observed, and flow cytometry analysis was performed on Day 23.

[0141] 3. Test methods: The administration was completed on Day 22. The day after the administration ended, single-cell suspensions were prepared from the tumor drainage lymph nodes and the concentration was adjusted to 1×10⁻⁶. 6 Cells / mL, add 1 μL of each of the following fluorescently labeled antibodies: FVS510, FITC CD45.2 (clone:104), APC-cy7-CD3e (clone:145-2C11), PE-F4 / 80 (clone:BM8), PE-NK (clone:PK136), Percp-cy5.5-CD11b (clone:m1 / 70), BV421-Ly6C (clone:AI 21), mix thoroughly by pipetting, and incubate at 4°C for 30 min. Perform flow cytometry analysis.

[0142] Flow cytometry data were analyzed using Flowjo 10 software. Results are expressed as mean ± standard error. Differences between groups were analyzed using GraphPad Prism 8.4.3 software, and statistical differences between groups were calculated using one-way ANOVA or multivariate ANOVA. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0143] Experimental results: Figure 5 A: The percentage of CD3+ T cells in each group: PBS group: 15.85%, PD-1 group: 19.40%, PD-1+F group: 24.48%.

[0144] Figure 5 B: Percentage of NK cells in each group: PBS group: 0.79%, PD-1 group: 1.51%, PD-1+F1 / F3 group: 1.87%.

[0145] Figure 5 C: The proportion of macrophages in each group: The proportion of macrophages in the PD-1+F1 / F3 group (0.925%) was higher than that in the PBS group (0.3425%) and the PD-1 group (0.89%).

[0146] Figure 5 D: The percentage of M1 pro-inflammatory macrophages, which was 2.045%, 6.295%, and 27.45% in the PBS, PD-1, and PD-1+F1 / F3 groups, respectively.

[0147] Figure 5 E: The percentage of M2-resident macrophages, which was 22.95%, 37.38%, and 27.25% in the PBS, PD-1, and PD-1+F1 / F3 groups, respectively.

[0148] Figure 5 F: The ratio of M1 pro-inflammatory macrophages to M2 resident macrophages was 0.09185, 0.1705, and 1.013 in the PBS, PD-1, and PD-1+F1 / F3 groups, respectively.

[0149] In tumor-draining lymph nodes, PD-1+F1 / F3 combination therapy was accompanied by changes in the proportions of CD3+ T cells, NK cells, and pro-inflammatory macrophages, suggesting that it may affect the local and draining lymph node immune status. These changes correspond to the increased neutrophil infiltration and the transformation of M2 resident macrophages into M1 pro-inflammatory macrophages observed in tumor tissue, indicating that F1 / F3 not only acts locally on the tumor but may also remotely regulate the immune system through lymph nodes, thereby enhancing the therapeutic effect of PD-1 blockade.

[0150] Example 6: Single-cell RNA sequencing revealed that F1 / F3 peptides combined with PD-1 inhibitors remodeled the MC38 tumor microenvironment. 1. Test subjects and modeling: The MC38 tumor-bearing model was constructed according to Example 3, and the tumor volume reached 300 mm on Day 13. 3 The experiment was divided into two groups, left and right. The groups are as follows: PBS, PD-1, PD-1+F.

[0151] 2. Dosing regimen: The tumor size was observed to reach 300 mm. 3 Dosing should be initiated around 10:00 or 11:00.

[0152] PBS group: 100µL of PBS was injected intratumorally at the start of drug administration, and the intratumoral injection continued for 7 days.

[0153] PD-1 group: 100 μL of PD-1 solution was injected intraperitoneally at the start of dosing, once every three days, for a total of 2 doses (i.e., on days 13 and 16).

[0154] PD-1+F1 / F3 group: PD-1 solution 100 μL was injected intraperitoneally at the start of drug administration, once every three days for a total of 2 times. At the same time, F1 / F3 solution 100 μL was injected intratumorally at the start of drug administration for 7 consecutive days.

[0155] Observe the size of the tumor in the mouse.

[0156] 3. Single-cell sequencing assay method: On Day 21, the tumor tissue was weighed. After a significant difference was found, the tumor tissue sample was collected and sent to Guangzhou Gediao Biotechnology Co., Ltd. The company prepared single-cell suspension, detected cell viability, captured the cells, synthesized and amplified cDNA, constructed a library, performed quality control, sequenced the cells, and finally analyzed the data.

[0157] 4. Experimental Results: Figure 6 A: Changes in tumor volume of MC38 in different treatment groups as shown by single-cell sequencing.

[0158] Figure 6 BC: The MC38 tumor weight of different treatment groups submitted for single-cell sequencing. There were significant differences among the groups, meeting the submission requirements.

[0159] Figure 6 D: This is the quality control screening chart for single-cell sequencing, indicating that the UMI of all 19,842 collected cells (7,430 in PBS, 6,625 in PD-1, and 5,687 in the combined group) is >11,000, indicating that the data quality is qualified and meets the requirements for downstream analysis.

[0160] Figure 6 EF: Dimensionality reduction and unsupervised clustering analysis were performed on single-cell RNA sequencing data of MC38 tumors from different treatment groups. Combined treatments altered the cellular composition of the MC38 tumor microenvironment.

[0161] Figure 6G: Based on the annotated single-cell RNA sequencing clustering results, the cellular composition of the tumor microenvironment (TME) in each treatment group is presented. The results show that the proportion of Neutrophil cells increased in the three groups, reaching the maximum in PD-1+F1 / F3 (31.74%); while the proportion of cancer cells decreased in the three groups, reaching the minimum in PD-1+F1 / F3 (47.49%), indicating that the combination therapy of PD-1+F1 / F3 has a more significant tumor-suppressive effect compared with PD-1 monotherapy.

[0162] Figure 6 H: The relative abundance of tumor cells in the total captured cells in each treatment group.

[0163] Figure 6 I: Relative abundance of neutrophils in total captured cells in each treatment group.

[0164] Figure 6 J: Relative abundance of macrophage subsets in total captured cells in each treatment group.

[0165] Figure 6 K: The relative abundance of T cell subsets in total captured cells in each treatment group.

[0166] Figure 6 L: The relative abundance of B cells in the total captured cells in each treatment group.

[0167] Figure 6 M: The relative abundance of dendritic cell subsets in total captured cells in each treatment group.

[0168] Figure 6 N: The relative abundance of fibroblasts in total captured cells in each treatment group.

[0169] The columns represent the unsupervised clustering results labeled as different cell types, and the top color bar indicates the cell type identity; Single-cell sequencing results showed that the combination therapy significantly altered the proportion and distribution of various immune cells in the MC38 tumor microenvironment, suggesting that the combination of F1 / F3 peptides and PD-1 inhibitors can effectively reshape the cellular composition of the tumor microenvironment. Specifically, the proportion of neutrophils increased, while the proportion of tumor cells decreased; this result was consistent with the in vivo tumor volume and weight results.

[0170] Example 7: Neutrophil state with high expression of the chemokine Neutralization associated with F1 / F3 peptide combined with PD-1 inhibitor-induced therapy. Experimental methods: Based on the single-cell RNA sequencing data from Example 6, a re-clustering analysis was performed on tumor-infiltrating neutrophils.

[0171] Experimental results: Figure 7 A–B: Reclustering analysis of tumor-infiltrating neutrophils. t-SNE visualization showed a significant remodeling of neutrophil distribution after combination therapy, with the Neu_6 subset mainly appearing in the PD-1 combined with F1 / F3 treatment group. Quantitative analysis showed a significant increase in the total number of neutrophils after combination therapy, with significant expansion of the Neu_4 and Neu_6 subsets.

[0172] Figure 7 C: Showing the expression of marker genes in different neutrophil subsets. The results showed that the Neu_6 and Neu_4 subsets exhibited obvious inflammatory features and chemokine enrichment phenotypes, characterized by high expression of S100a8, S100a9, Il1b, Cxcl2, and Cxcl3.

[0173] Figure 7 D: Indicates the comparison of expression of key chemokine genes among different neutrophil subsets and treatment groups. Compared with the corresponding subsets in the control group and the PD-1 monotherapy group, the expression of Cxcl2, Cxcl3, Ccl3 and Ccl4 genes was significantly enriched in Neu_6 cells of the PD-1 combined with F1 / F3 treatment group.

[0174] Figure 7 E: Represents a heatmap of differentially expressed genes in neutrophils across treatment groups. The combination therapy induced a significant transcriptional program characterized by chemokines, chemokine receptors, and inflammatory mediators.

[0175] Figure 7 FH: Indicates enrichment analysis of treatment-related neutrophil signaling pathways. KEGG and GSEA analyses showed significant enrichment of NF-κB signaling pathway, chemokine signaling pathway, TNF signaling pathway, and cytokine-cytokine receptor interaction pathway in neutrophils (especially the Neu_6 subset) in the combination therapy group.

[0176] Figure 7 I: indicates ligand-receptor interaction, and the results reveal neutrophil-related signaling pairs that may be involved in immune cell recruitment and tumor microenvironment remodeling.

[0177] The distribution of neutrophils changed significantly after combination therapy, with high expression of the Neu_6 subset in the PD-1 inhibitor + F1 / F3 combination therapy group. Marker gene analysis showed that the Neu_6 and Neu_4 subsets exhibited enrichment in inflammation and chemokines, manifested by increased expression of genes such as S100a8, S100a9, Il1b, Cxcl2, and Cxcl3. Pathway enrichment analysis revealed that neutrophils in the combination therapy group, particularly the Neu_6 subset, were enriched in the NF-κB signaling pathway, chemokine signaling pathway, TNF signaling pathway, and cytokine-cytokine receptor interaction-related pathways. Further analysis showed increased expression of chemokine genes such as Cxcl2, Cxcl3, Ccl3, and Ccl4 in Neu_6 cells in the combination therapy group, suggesting that the combination of F1 / F3 peptides and PD-1 inhibitors can induce a treatment-related chemokine-rich neutrophil state.

[0178] Example 8: Neutrophil removal weakens the antitumor efficacy of F1 / F3 peptide combined with PD-1 inhibitor and its effect on the tumor immune microenvironment in MC38 tumor-bearing mice.

[0179] Object and model: The MC38 tumor-bearing mouse model (C57BL / 6 background) was constructed according to the method in Example 2. Tumor volume was measured every 3 days and calculated using the formula length × width × width / 2.

[0180] Experiment 1: The effect of neutrophil removal on the antitumor efficacy of combination therapy When the tumor volume reaches 300 mm 3 At approximately 10:00 AM, tumor-bearing mice were randomly divided into 5 groups: ( Figure 8 A) The control group consisted of five to six mice: blank control group (intratumoral injection of PBS), negative control group (intratumoral injection of PBS + intraperitoneal injection of lgG2a), PD-1+F1 / F3 group (intratumoral injection of F1 / F3 + intraperitoneal injection of PD-1), PD-1+F1 / F3+lgG2a group (intratumoral injection of F1 / F3 + intraperitoneal injection of lgG2a + intraperitoneal injection of PD-1), and PD-1+F1 / F3+anti-ly6G group (intratumoral injection of F1 / F3 + intraperitoneal injection of anti-ly6G + intraperitoneal injection of PD-1).

[0181] Sample preparation: Preparation of PD-1 solution: Dissolve PD-1 antibody in PBS to prepare a solution containing 200 µg of PD-1 antibody per 100 µL.

[0182] Preparation of F1 / F3 solution: Dissolve 30 μg each of F1 and F3 in PBS to prepare a solution containing 30 μg each of F1 and F3 per 100 µL.

[0183] Preparation of anti-ly6G antibody (neutrophil removal) solution: Dissolve anti-ly6G antibody in PBS to prepare a solution containing 400 µg of anti-ly6G antibody per 100 µL.

[0184] Preparation of anti-lgG2a antibody (rat type 2a immunoglobulin G isotype control antibody): Anti-lgG2a antibody was dissolved in PBS to prepare a solution containing 400 µg of anti-lgG2a antibody per 100 µL.

[0185] The dosing regimen is as follows: Blank control group: Day 17: 100µL of PBS was injected into the tumor, and this was repeated for 14 consecutive days.

[0186] Negative control group: Day 13: 100µL of anti-IgG2a antibody solution was injected intraperitoneally, once every five days for a total of four times; Day 17: 100µL of PBS was injected intratumorally, for 14 consecutive days.

[0187] PD-1+F1 / F3 group: On Day 17, 100 µL of F1 / F3 solution was injected intratumorally, and 100 µL of PD-1 antibody solution was injected intraperitoneally. Subsequently, F1 / F3 solution was injected intratumorally once a day for 14 consecutive days; PD-1 antibody solution was injected intraperitoneally once every 3 days (100 µL) for a total of 5 times.

[0188] PD-1+F1 / F3+lgG2a group: Day 13: 100 µL of anti-lgG2a antibody solution was injected intraperitoneally, once every five days for a total of four times; Day 17: 100 µL of F1 / F3 solution was injected intratumorally, once daily thereafter for 14 consecutive days; Day 17: 100 µL of PD-1 antibody solution was injected intraperitoneally, once every three days thereafter for a total of five times.

[0189] PD-1+F1 / F3+anti-ly6G group: Day 13: Intraperitoneal injection of 100 µL of anti-ly6G solution, once every five days for a total of four injections; Day 17: Intratumoral injection of 100 µL of F1 / F3 solution, once daily thereafter for 14 consecutive days; Day 17: Intraperitoneal injection of 100 µL of PD-1 antibody solution, once every three days thereafter for a total of five injections.

[0190] Experimental methods: Changes in tumor volume and survival time were observed and recorded. When the tumor diameter reached 15 mm, mice were sacrificed using CO2 inhalation. The results were used to plot tumor volume change curves and survival time curves. Figure 8 BC) Experiment 2: Effects of neutrophil removal on immune microenvironment remodeling and chemokine expression induced by combination therapy When the tumor size reaches 300mm 3 Experimental groups were formed at approximately 10:00 AM and 10:00 PM, as follows: PBS group, PD-1 group, PD-1+F1 / F3 group, and PD-1+F1 / F3+Ly6G (anti-ly6G) group.

[0191] The dosing regimen is as follows: PBS group: Day 34: 100 μL of PBS was injected intraperitoneally.

[0192] PD-1 group: 100 μL of PD-1 solution was injected intraperitoneally on Day 34 and Day 37 respectively.

[0193] PD-1+F1 / F3 group: PD-1 solution 100μL was injected intraperitoneally on Day 34 and Day 37 respectively. F1 / F3 peptide solution 100μL was injected starting on Day 34, and the injections continued for 7 days.

[0194] PD-1+F1 / F3+Ly6G group: 100 μL of anti-ly6G solution was injected intraperitoneally on Day 30, Day 35 and Day 40 respectively. 100 μL of PD-1 solution was injected intraperitoneally on Day 34 and Day 37 respectively. 100 μL of F1 / F3 peptide solution was injected starting on Day 34, and the injections continued for 7 days.

[0195] Experimental Methods: Mice were euthanized on Day 41 after drug administration. Tumor tissue was collected to prepare single-cell suspensions for flow cytometry analysis. Total RNA was extracted from tumor tissue for qPCR detection. The flow cytometry antibody staining protocol was as follows: FVS510, FITC-CD45.2 (clone: ​​104), PE-F4 / 80 (clone: ​​BM8), PerCP-Cy5.5-CD11b (clone: ​​M1 / 70), APC-Ly6G (clone: ​​1A8), and APC-Ly6C (clone: ​​AL-21). 1 µL of each antibody was added, and the cells were incubated at 4°C in the dark for 30 min. Data were acquired using a BD FACSAria II flow cytometer and analyzed using Flowjo 10 software. Tumor RNA was extracted using an RNA extraction kit (Goonie, Guangzhou). cDNA was synthesized using HiScript® IV All-in-One Ultra reverse transcription supermixer (Vazyme). qPCR was performed on a LightCycler 480 system (Roche). GAPDH was used as an internal reference gene, and relative gene expression levels were calculated using the 2⁻ΔΔCt method. The PBS control group was used as a calibrator for each target gene (value set to 1).

[0196] Primer sequences are shown in Table 1:

[0197] Experimental results: Figure 8 A: Schematic diagram of the experimental design and dosing regimen for the effect of neutrophil clearance on the antitumor efficacy of combination therapy.

[0198] Figure 8 B: Tumor volume curves of MC38 tumor-bearing mice in each group. The results showed that the tumors in the PBS group and the PBS+IgG2a isotype control group progressed rapidly; the PD-1+F1 / F3 combination group significantly inhibited tumor growth, with a highly significant difference compared with the PBS and PBS+IgG2a control groups; however, the use of the neutrophil neutralizing antibody anti-Ly6G (to clear neutrophils) on the basis of the combination regimen significantly reduced the tumor-suppressing effect and significantly increased the tumor volume; there was no statistically significant difference between the PBS and PBS+IgG2a groups.

[0199] Figure 8 C: Survival curves of tumor-bearing mice in each group of MC38. The results showed that the mice in the PBS group and the PBS+IgG2a group had the shortest survival time, with median survival of 30 days and 28.5 days, respectively. PD-1+F1 / F3 combination therapy significantly prolonged the survival time of tumor-bearing mice, greatly increasing the median survival time, and the long-term tumor-free survival rate was 60%. However, after neutrophil clearance (anti-Ly6G), the tumor-suppressive effect of the combination therapy was significantly reversed, the tumor burden rebounded, and the median survival time of mice was greatly shortened to 40 days. The survival benefit of mice was significantly weakened, and there was no significant difference in efficacy between the blank control group and the isotype IgG2a control group.

[0200] Figure 8 D: Showing the percentage of neutrophils in each group: PBS group: 2.633%, PD-1 group: 7.498%, PD-1+F1 / F3 group: 58.85%, PD-1+F1 / F3+Ly6G group: 0.3225%.

[0201] Figure 8E: The percentage of M1 pro-inflammatory macrophages and M2 resident macrophages, and the upregulation of the M1 pro-inflammatory macrophage / M2 resident macrophage ratio. The percentages of M1 pro-inflammatory macrophages in the PBS, PD-1, PD-1+F1 / F3, and PD-1+F1 / F3+Ly6G groups were 19.68%, 17.93%, 23.3%, and 23.75%, respectively; the percentages of M2 resident macrophages in the PBS, PD-1, PD-1+F1 / F3, and PD-1+F1 / F3+Ly6G groups were 38.45%, 23.03%, 3.098%, and 23.3%, respectively; and the ratio of M1 pro-inflammatory macrophages to M2 resident macrophages in the PBS, PD-1, PD-1+F1 / F3, and PD-1+F1 / F3+Ly6G groups were 0.5120, 0.7799, 6.816, and 1.020, respectively.

[0202] Figure 8 F: The percentage of CD45.2+ immune cells was higher in the PD-1+F1 / F3 group (83.48%) than in the PBS group (23.15%), the PD-1 group (43.93%), and the PD-1+F1 / F3+Ly6G group (28.48%).

[0203] Figure 8 G: PD-1+F1 / F3 combination therapy significantly upregulated the expression of chemokine genes such as Ccl2, Cxcl2, Ccl3, Cxcl3, and Ccl4 in MC38 tumor tissue; while after neutrophil removal, the expression levels of the above chemokines significantly decreased, suggesting that neutrophils are the key factor in the enhanced chemokine expression induced by the combination therapy.

[0204] Neutrophils are crucial cells involved in the anti-tumor and survival-prolonging effects of PD-1 + F1 / F3 combination therapy. This combination therapy recruits neutrophils, driving macrophage M1 / M2 polarization balance and immune cell infiltration, thus reshaping the tumor immune microenvironment. Upon neutrophil depletion, the microenvironment reverses to an immunosuppressive state, and the efficacy and survival benefits of the combination therapy are significantly diminished. This indicates that neutrophils are key cells involved in the activation of the chemokine network and the remodeling of the tumor immune microenvironment induced by F1 / F3 peptides combined with PD-1 inhibitors.

[0205] Example 9: F1 / F3 peptide combined with PD-1 inhibitor mediates tumor-associated macrophage reprogramming, reshaping the tumor immune microenvironment through amplification of related chemokine signals. Experimental Methods: Based on the single-cell RNA sequencing data from Example 6, the regulatory relationships between tumor-infiltrating macrophages, microenvironment cell populations, and chemokine networks were investigated. Gene expression detection of tumor-associated chemokines was based on Example 8.

[0206] Experimental results: Figure 9 A: Based on t-SNE visualization analysis and unsupervised clustering methods, tumor-associated macrophages in MC38 tumors from different treatment groups were re-clustered, and four macrophage subpopulations with unique transcriptional programs were identified.

[0207] Figure 9 B: The combined drug therapy induced the synchronous upregulation of pro-inflammatory factors (such as Stat1, Cxcl9, Cxcl10, Il1b, Cxcl2, and Cxcl3) in the Cxcl subset. This suggests that the combined action of F1 / F3 and PD-1 inhibitors enhances the anti-tumor immune response by globally driving macrophages from an immunosuppressive state to an immune-activated state (pro-inflammatory phenotype).

[0208] Figure 9 C: Cell type-specific expression matrix of key chemokine genes in the tumor microenvironment cell population. Increased expression patterns of chemokines in neutrophils, NK cells, macrophages, monocytes, T cells, and mDC cells indicate that these genes play an important role in tumor immunity.

[0209] Figure 9 D: t-SNE density plots showed that the expression density of chemokine ligands and receptors was significantly higher in the combination therapy group compared with the monotherapy group and the control group.

[0210] Figure 9 E: Real-time quantitative PCR was used to verify the expression of chemokines in MC38 tumor tissue after treatment.

[0211] The above results indicate that the combination of F1 / F3 peptides and PD-1 inhibitors drives macrophages to transform into a pro-inflammatory phenotype and activates a multicellular population chemokine network. The amplification of chemokine signals reshapes the tumor immune microenvironment, thereby achieving immune activation and enhanced anti-tumor efficacy.

[0212] Application overview of the present invention: This invention provides a pharmaceutical composition comprising an F1 / F3 peptide and an immune checkpoint inhibitor. This composition can be used to prepare a medicament for treating colorectal cancer. The F1 / F3 peptide can act as a sensitizer for immune checkpoint inhibitor therapy, particularly as a sensitizer for PD-1 inhibitor therapy in colorectal cancer.

[0213] The technical solution of this invention is particularly applicable to colorectal cancer with low response, no response, or tolerance to PD-1 inhibitor treatment. The F1 / F3 peptide is preferably administered intratumorally or locally, and the PD-1 inhibitor is preferably administered systemically. Both can be administered simultaneously, separately, or sequentially. When the F1 / F3 peptide is combined with a PD-1 antibody for antitumor treatment, the dose ratio of the PD-1 antibody to the F1 / F3 peptide is 1:0.5-5.0, preferably 1:0.8-1.5. In mice, good antitumor activity was observed at doses of 350-1100 μg for the PD-1 antibody and 400-900 μg for the F1 / F3 peptide. Significant antitumor activity was particularly observed at doses of 350-450 μg for the PD-1 antibody and 400-450 μg for the F1 / F3 peptide, or at doses of 900-1100 μg for the PD-1 antibody and 800-900 μg for the F1 / F3 peptide.

[0214] The above embodiments are preferred embodiments of the present invention and are not intended to limit the present invention. All equivalent substitutions, conventional adjustments, or non-substantial changes made based on the content of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for tumor immunotherapy, characterized in that, The pharmaceutical composition comprises F1 / F3 and an immune checkpoint inhibitor; wherein the tumor is preferably colorectal cancer.

2. The pharmaceutical composition according to claim 1, characterized in that, The F1 / F3 is a composition of Caerin 1.1 and Caerin 1.9; wherein the mass ratio of Caerin 1.1 to Caerin 1.9 is 1:0.2–5, preferably 1:0.5–2, and more preferably 1:

1.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The amino acid sequence of Caerin 1.1 is shown in SEQ ID No. 1, and the amino acid sequence of Caerin 1.9 is shown in SEQ ID No.

2.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor, preferably a PD-1 inhibitor, and more preferably an anti-PD-1 monoclonal antibody.

5. The pharmaceutical composition according to any one of claims 1-4, characterized in that, The mass ratio of the immune checkpoint inhibitor to F1 / F3 is 1:0.5-5.0, preferably 1:0.8-1.

5.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, buffer, stabilizer, or any combination thereof.

7. The pharmaceutical composition according to any one of claims 1-6, characterized in that, The F1 / F3 and the immune checkpoint inhibitor are either formulated as the same formulation or formulated as separate formulations.

8. The pharmaceutical composition according to any one of claims 1-7, characterized in that, The F1 / F3 and the immune checkpoint inhibitors are used for simultaneous, separate, or sequential administration.

9. The pharmaceutical composition according to any one of claims 1-8, characterized in that, The F1 / F3 is used for local administration, intratumoral administration, peritumoral administration, intracavitary administration, or endoscopic local administration, preferably intratumoral administration; the immune checkpoint inhibitor is used for systemic administration, preferably intravenous or subcutaneous administration.

10. The pharmaceutical composition according to any one of claims 1-9, characterized in that, The colorectal cancer referred to is colorectal cancer that has a low response, no response, or resistance to immune checkpoint inhibitor therapy.

11. Use of the pharmaceutical composition according to any one of claims 1-10 in the preparation of a medicament for treating colorectal cancer.

12. Application of F1 / F3 peptides in the preparation of drugs for the treatment of colorectal cancer or for the preparation of drugs that enhance the sensitivity of colorectal cancer to immune checkpoint inhibitor therapy.

13. Application of F1 / F3 peptide combined with immune checkpoint inhibitors in the preparation of immunotherapy drugs for colorectal cancer.

14. The application according to claim 13, characterized in that, The mass ratio of F1 to F3 is 1:0.2-5, preferably 1:0.5-2, and more preferably 1:

1.

15. The application according to claim 13 or 14, characterized in that, The F1 / F3 is used for local administration, intratumoral administration, peritumoral administration, intracavitary administration, or endoscopic local administration, preferably intratumoral administration. The PD-1 inhibitor is used for systemic administration, preferably intravenous or subcutaneous administration.

16. The application according to any one of claims 13-15, characterized in that, The immune checkpoint inhibitor is a PD-1 inhibitor, preferably an anti-PD-1 monoclonal antibody.

17. The application according to any one of claims 13-16, characterized in that, The colorectal cancer referred to is colorectal cancer that has a low response, no response, or resistance to PD-1 inhibitor treatment.

18. The application according to any one of claims 13-17, characterized in that, The F1 / F3 peptides exert their immunotherapeutic effect on colorectal cancer by increasing neutrophil infiltration in tumor tissue, inducing chemokine expression, promoting macrophage transformation to a pro-inflammatory phenotype, and enhancing NK cell or T cell-related anti-tumor immune responses, thereby improving the sensitivity of colorectal cancer to immune checkpoint inhibitor therapy.

19. The application according to claim 18, characterized in that, The chemokines include Ccl2 / Cxcl2, Ccl3 / Cxcl3, Ccl4 / Cxcl4, or any combination thereof.

20. The application according to any one of claims 13-19, characterized in that, The F1 / F3 combined immune checkpoint inhibitor exerts its immunotherapeutic effect on colorectal cancer by inducing chemokine signal amplification centered on neutrophils, coordinating macrophage repolarization, tumor-stromal remodeling, and NK cell or T cell-mediated anti-tumor immune responses.

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