A pancreatic cancer immunotherapy pharmaceutical composition jointly targeting CD96 and PD-1

Abstract

The application discloses a pancreatic cancer immunotherapy drug composition for jointly targeting CD96 and PD-1, relates to the technical field of biological medicine, and comprises an anti-human CD96 blocking antibody and an anti-human PD-1 blocking antibody. By jointly blocking the double immune checkpoints of CD96 and PD-1, the pancreatic cancer immunotherapy drug composition presents a significant synergistic effect compared with single antibody treatment, can effectively enhance the killing activity of T cells on pancreatic cancer cells, reverses the immune suppression microenvironment of pancreatic cancer, and restores the anti-tumor immune response of the body. The in-vivo experimental results show that the drug composition can significantly inhibit the growth of transplanted tumors, reduce the tumor volume and the tumor weight, and the tumor inhibition effect is obviously better than that of a single drug group. The preparation has stable properties and simple administration, and provides a safe and effective new immunotherapy scheme with a conversion application prospect for clinically refractory pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pancreatic cancer immunotherapy composition that combines CD96 and PD-1. Background Technology

[0002] Pancreatic cancer is a highly malignant digestive system tumor that is difficult to diagnose in its early stages and has an extremely poor prognosis. Most patients are diagnosed at an advanced stage, making radical surgery impossible. Traditional chemotherapy and radiotherapy have limited effectiveness, resulting in a consistently low 5-year survival rate. Immunotherapy has made groundbreaking progress in treating various malignant tumors, including melanoma, lung cancer, and lymphoma. Its core mechanism involves blocking immune checkpoint pathways and restoring the body's own anti-tumor immune response.

[0003] Currently, PD-1 / PD-L1 inhibitors are the most widely used in clinical practice. However, in pancreatic cancer, the response rate of single PD-1 inhibitor treatment is extremely low, and the tumor microenvironment exhibits an immunosuppressive state. Simply blocking the PD-1 pathway is insufficient to achieve effective anti-tumor effects. CD96 is an immunoglobulin transmembrane protein, whose main ligand is CD155, and it is mainly expressed on NK cells and CD8 cells. + T cells participate in the regulation of immune responses. Studies have shown that pancreatic cancer cells highly express CD155, while immune cells highly express CD96. Blocking the CD96-CD155 pathway can restore CD8+. + T-cell killing function.

[0004] However, current technologies have not disclosed drug compositions that combine CD96 blocking antibodies and PD-1 blocking antibodies for the treatment of pancreatic cancer, nor have they demonstrated that the two can synergistically enhance anti-tumor immune effects in pancreatic cancer. Therefore, developing an immunotherapeutic drug composition that can simultaneously target CD96 and PD-1 and is suitable for pancreatic cancer has significant clinical value. Summary of the Invention

[0005] The purpose of this invention is to provide a pancreatic cancer immunotherapy drug composition that combines CD96 and PD-1 to solve the problems of poor efficacy of single PD-1 inhibitors in treating pancreatic cancer and difficulty in effectively reversing pancreatic cancer immune escape in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a pancreatic cancer immunotherapy drug composition that combines CD96 and PD-1 targeting, wherein the drug composition comprises an anti-human CD96 blocking antibody and an anti-human PD-1 blocking antibody;

[0007] The anti-human CD96 blocking antibody specifically binds to CD96 and blocks the interaction between CD96 and CD155;

[0008] The anti-human PD-1 blocking antibody specifically binds to PD-1 and blocks the PD-1 signaling pathway.

[0009] Furthermore, the anti-human CD96 blocking antibody is an anti-human CD96 monoclonal antibody with clone number 5E6C12.

[0010] Furthermore, the anti-human PD-1 blocking antibody is an anti-human PD-1 monoclonal antibody with clone number 1F2.

[0011] Furthermore, the pharmaceutical composition is an injectable formulation prepared by dissolving anti-human CD96 blocking antibody and anti-human PD-1 blocking antibody together in a buffer solution.

[0012] Furthermore, the buffer solution is a sterile, sodium azide-free, low-endotoxin phosphate buffer solution with a pH of 7.0-7.4.

[0013] Furthermore, the anti-human CD96 blocking antibody and the anti-human PD-1 blocking antibody are either pre-mixed single formulations or separately prepared and packaged, and then mixed at a mass ratio of 1:1 before use.

[0014] Furthermore, the pharmaceutical composition is a preparation aseptically dispensed into vials and stored at 2-8°C in the dark.

[0015] Furthermore, the route of administration of the pharmaceutical composition is intravenous injection or intraperitoneal injection.

[0016] Compared with existing technologies, the present invention provides a pancreatic cancer immunotherapy composition that combines CD96 and PD-1 targeting. By jointly blocking the CD96 and PD-1 dual immune checkpoints, it exhibits a significant synergistic effect compared with single antibody therapy. It can effectively enhance the killing activity of T cells against pancreatic cancer cells, reverse the immunosuppressive microenvironment of pancreatic cancer, and restore the body's anti-tumor immune response. In vivo experiments have demonstrated that the drug composition can significantly inhibit the growth of transplanted tumors, reduce tumor volume and weight, and the formulation is stable and easy to administer. It provides a safe, effective, and promising new immunotherapy option for clinically refractory pancreatic cancer. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0018] Figure 1A representative graph of T cell killing of CFSE-labeled PANC-1 cells by flow cytometry provided in an embodiment of the present invention;

[0019] Figure 2 A statistical result graph showing the specific kill rate of different treatment groups provided in the embodiments of the present invention;

[0020] Figure 3 Animal experiment flowchart provided for embodiments of the present invention;

[0021] Figure 4 An image showing the tumor condition in an animal experiment provided for an embodiment of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Antibodies: anti-human CD96 blocking antibody (clone number 5E6C12), anti-human PD-1 blocking antibody (clone number 1F2), anti-mouse CD96 blocking antibody (clone number 3.3), anti-mouse PD-1 blocking antibody (clone number RMP1-14) and corresponding isotype control antibodies (the first two were purchased from Huaan Biotechnology, and the rest were purchased from Bio X Cell).

[0024] Kit and main reagents:

[0025] Lymphocyte separation solution (purchased from Baidi Biotechnology);

[0026] CFSE (purchased from ambeed);

[0027] PI (purchased from Ambeed);

[0028] Leukocyte Activation Cocktail (containing Brefeldin A, purchased from BD Biosciences).

[0029] Example 1:

[0030] Buffer solution: Sterile, sodium azide-free, low endotoxin PBS (phosphate buffer), pH 7.0-7.4.

[0031] Antibody preparation: The purified anti-CD96 antibody and / or anti-PD-1 antibody (monoclonal antibody) were diluted to the target concentrations using the buffer solution described above. This buffer solution was used to dilute the antibodies in subsequent cell and animal experiments.

[0032] Mixing preparation: Mix the two antibodies in a certain proportion so that both antibodies are within their working concentration range.

[0033] Formulation: It can be prepared as a single mixed formulation, or the two antibodies can be aliquoted separately and mixed before use.

[0034] Preparation aliquoting and storage: Under aseptic conditions, aliquot the prepared antibody solution or antibody mixture into vials, then cap and seal. Store the finished product at 2-8°C, protected from light.

[0035] When used to treat pancreatic cancer, this drug composition can be administered via intravenous or intraperitoneal injection. For combination drug compositions, the dosage can be calculated based on the patient's weight, for example, at a dose of 10 mg / kg for each antibody. The anti-CD96 antibody and the anti-PD-1 antibody can also be formulated and dispensed separately, and mixed immediately before use or injected separately in the same dosing regimen.

[0036] Example 2:

[0037] Please see Figure 1-2 This embodiment, based on Embodiment 1, performs in vitro T cell killing function detection (CFSE / PI method).

[0038] 1. Preparation of effector cells

[0039] Fresh peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Paque PLUS density gradient centrifugation. All donors signed informed consent forms, and blood collection was approved by the ethics committee.

[0040] PBMCs at 2×10 6 Cells / mL were resuspended in complete RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), and Leukocyte Activation Cocktail (containing Brefeldin A) was added and activated in a 37°C, 5% CO2 incubator for 24 h to serve as effector cells.

[0041] 2. Target cell labeling

[0042] Human pancreatic cancer cell line: PANC-1 cells (purchased from Bio-Tech) were routinely cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated in a constant temperature incubator at 37°C and 5% CO2.

[0043] Human pancreatic cancer cell line PANC-1 in logarithmic growth phase was taken, washed with phosphate-buffered saline (PBS), and resuspended in PBS containing 5 μM carboxyfluorescein succinimide (CFSE). The cells were incubated at 37°C in the dark for 20 min. After incubation, the cells were washed three times with pre-cooled PBS to remove excess dye and were then used as target cells.

[0044] 3. Co-cultivation and treatment

[0045] Activated effector cells (PBMCs) and CFSE-labeled target cells (PANC-1) were seeded in 96-well U-plates at an effector cell:target cell ratio of 10:1 (1 × 10⁶ effector cells). 5 cells / well, target cells 1×102 4 Cells / well). The following treatments were performed (3 replicates per group), as shown in Table 1.

[0046] Table 1 Dosing Regimen

[0047] Group Anti-CD96 Anti-PD-1 isotype control Total protein concentration isotype control 0 0 10 μg / mL 10 μg / mL Anti-CD96 monotherapy group 5 μg / mL 0 5 μg / mL 10 μg / mL Anti-PD-1 monotherapy group 0 5 μg / mL 5 μg / mL 10 μg / mL Joint Group 5 μg / mL 5 μg / mL 0 10 μg / mL

[0048] A separate well containing only target cells was set up as a spontaneous death control. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 5 min.

[0049] 4. Flow cytometry detection and data analysis

[0050] Fifteen minutes before the end of co-culture, propidium iodide (PI) was added to each well to a final concentration of 1 μg / mL. Cells were collected, washed once with FACS buffer (PBS containing 2% fetal bovine serum), and immediately analyzed by flow cytometry (BD FACSCanto II). At least 10,000 CFSE-positive cells were collected per sample. Data were analyzed using FlowJo software (v10.8), with the proportion of PI⁺ cells within the CFSE⁺ phylum as the target cell, as the overall mortality rate. The specific killing rate was calculated using the following formula:

[0051]

[0052] 5. Results

[0053] Figure 1 This is a representative image for flow cytometry analysis of T cell killing of CFSE-labeled PANC-1 cells, showing the target cells (CFSE) in different treatment groups. + ) mortality (PI) + The results showed that the target cell death rate was lower in the isotype control group, while the PI in the anti-CD96 antibody monotherapy group and the anti-PD-1 antibody monotherapy group was higher. + The proportion of cells increased significantly, indicating that blocking any immune checkpoint with a single drug can partially restore the killing function of T cells. In particular, the PI in the anti-CD96 and anti-PD-1 antibody combination therapy group was significantly higher. + The highest proportion of cells suggests that combined drug therapy can synergistically enhance T cell-mediated tumor cell killing.

[0054] Figure 2 The statistical results of the specific killing rate of different treatment groups (effective cell:target cell ratio = 10:1) show that the specific killing rate of the anti-CD96 antibody monotherapy group was significantly higher than that of the isotype control group; the anti-PD-1 antibody monotherapy group also showed a significant killing enhancement effect; the specific killing rate of the combination therapy group was significantly better than that of any single drug group, showing a clear synergistic effect.

[0055] Example 3:

[0056] Please see Figure 3-4 This embodiment provides the establishment of a mouse subcutaneous pancreatic cancer xenograft model and its in vivo treatment, based on Embodiment 1.

[0057] 1. Laboratory animals

[0058] Female C57BL / 6J mice, aged 6-8 weeks and weighing 18-22 g, were housed in a specific pathogen-free (SPF) grade animal facility with free access to food and water. Experiments were conducted after one week of acclimatization. All animal experimental procedures were approved by the Laboratory Animal Ethics Committee. (Ethics Approval Number: SLBH-202512160002)

[0059] 2. Tumor cell culture

[0060] The mouse pancreatic ductal adenocarcinoma cell line Pan02 was routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for subsequent experiments.

[0061] 3. Establishment of subcutaneous xenograft model

[0062] Pan02 cells in the logarithmic growth phase were washed twice with sterile phosphate-buffered saline (PBS), resuspended in PBS, and mixed with an equal volume of Matrigel (a growth factor-reducing formulation) to adjust the cell density to 1 × 10⁻⁶ cells / year. 7 cells / mL. 100 μL of cell suspension (i.e., 1 × 10⁻⁶ cells / mL) was subcutaneously injected into the right back of each C57BL / 6J mouse. 6 (cells / mouse). Observe the condition of mice and tumor growth daily after inoculation.

[0063] 4. Animal grouping and administration

[0064] When the subcutaneous tumor grows to approximately 50-100 mm 3 At approximately 7-10 days after inoculation, tumor-bearing mice were randomly divided into 4 groups of 6-8 mice each according to tumor volume, and each group received the following intraperitoneal injection treatment (see Table 2), twice a week for three consecutive weeks.

[0065] Table 2 Dosing Regimen

[0066] Group Anti-CD96 Anti-PD-1 isotype control Total antibody dose isotype control 0 0 20 mg / kg 20 mg / kg Anti-CD96 monotherapy group 10 mg / kg 0 10 mg / kg 20 mg / kg Anti-PD-1 monotherapy group 0 10 mg / kg 10 mg / kg 20 mg / kg Joint Group 10 mg / kg 10 mg / kg 0 20 mg / kg

[0067] All antibodies were dissolved in sterile phosphate-buffered saline (PBS), and the intraperitoneal injection volume was 200 μL per mouse.

[0068] 5. Tumor volume and body weight monitoring

[0069] During treatment, the tumor's long diameter (L, in mm) and short diameter (W, in mm) were measured every 2-3 days using electronic calipers, and the tumor volume was calculated using the following formula:

[0070]

[0071] Simultaneously, record changes in mouse weight and closely observe the animals' mental state, activity level, feeding behavior, and injection site reactions. Mice were considered at the experimental endpoint and euthanized if any of the following conditions were observed:

[0072] Tumor volume exceeding 1500 mm 3 ;

[0073] The tumor develops ulcers, breaks down, or becomes infected;

[0074] Weight loss exceeding 20%;

[0075] The animals exhibited obvious signs of distress, such as severe weight loss, arched back, and reduced activity. A complete animal experimental procedure is as follows: Figure 3 As shown.

[0076] 6. Final sample collection and tumor weighing

[0077] At the end of the experiment, the mice were euthanized, the tumor tissue was completely dissected, surface blood was blotted off with filter paper, and the tumor weight was recorded and photographed. A portion of the tumor tissue was used for subsequent flow cytometry, immunohistochemistry, or molecular biological analysis.

[0078] 7. Data Statistical Analysis

[0079] All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA or Kruskal-Wallis test was used for comparisons among multiple groups, and Mann-Whitney test was used for comparisons between two groups. Two-way ANOVA was used for comparisons of tumor growth curves. Kaplan-Meier method and Log-rank test were used for survival analysis. A p-value < 0.05 was considered statistically significant.

[0080] 8. Results

[0081] To validate our in vitro findings in a physiologically relevant environment, we inoculated C57BL / 6J mice with Pan02 pancreatic cancer cells and randomly assigned the mice to receive treatment for three weeks with either an isotype control, an anti-CD96 monoclonal antibody, an anti-PD-1 monoclonal antibody, or a combination of both antibodies. Figure 4 (A) shows the terminal tumor images of mice in each group. Figure 4 (B) shows the statistical results of tumor weight in each group of mice. The figure shows that the tumors in the isotype control group exhibited rapid progressive growth, reaching a weight of 0.51±0.04 g at the treatment endpoint. Tumor growth in the anti-CD96 antibody monotherapy group was inhibited to some extent, with a final tumor weight of 0.34±0.02 g, which was statistically significant compared to the control group. The anti-PD-1 antibody monotherapy group showed a similar tumor-suppressing effect, with a final tumor weight of 0.21±0.04 g. The combination therapy group showed the most significant inhibition of tumor growth, with a final tumor weight of 0.08±0.01 g, and most tumors remained stable or regressed during treatment. Two-way ANOVA showed a significant interaction between the two treatments, indicating a synergistic anti-tumor effect from the combination therapy.

[0082] Figure 4 (C) Statistical results of tumor volume changes in mice of each group. The results showed that the tumors in the isotype control group mice grew rapidly and progressively, reaching a volume of nearly 1500 mm² by the end of treatment. 3 Tumor growth was inhibited to some extent in the anti-CD96 antibody monotherapy group, with a terminal tumor volume of 1000 mm. 3 The difference was statistically significant compared with the control group; the anti-PD-1 antibody monotherapy group showed a similar tumor-suppressing effect, with a final tumor volume of 750 mm. 3 Tumor growth was most significantly inhibited in the anti-CD96 and anti-PD-1 treatment groups, with a final tumor volume of 500 mm. 3 Most tumors stabilized or regressed during treatment. Two-way ANOVA revealed a significant interaction between the two treatments, indicating a synergistic anti-tumor effect from the combined therapy.

[0083] In summary, compared with the isotype control group, anti-CD96 monotherapy produced a moderate but significant inhibition of tumor growth. The combination therapy, however, achieved a significant and synergistic tumor control effect, with the final tumor weight in the combination therapy group being significantly lower than that in either monotherapy group (p < 0.01 for both). This indicates that dual blocking of CD96 and PD-1 synergistically inhibits pancreatic tumor growth and prolongs survival in vivo.

[0084] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A combination immunotherapy drug composition for pancreatic cancer targeting CD96 and PD-1, characterized in that, The pharmaceutical composition comprises an anti-human CD96 blocking antibody and an anti-human PD-1 blocking antibody; The anti-human CD96 blocking antibody specifically binds to CD96 and blocks the interaction between CD96 and CD155; The anti-human PD-1 blocking antibody specifically binds to PD-1 and blocks the PD-1 signaling pathway.

2. The pancreatic cancer immunotherapy composition combining CD96 and PD-1 according to claim 1, characterized in that, The anti-human CD96 blocking antibody is an anti-human CD96 monoclonal antibody with clone number 5E6C12.

3. The pancreatic cancer immunotherapy composition combining CD96 and PD-1 according to claim 1, characterized in that, The anti-human PD-1 blocking antibody is an anti-human PD-1 monoclonal antibody with clone number 1F2.

4. The pancreatic cancer immunotherapy composition combining CD96 and PD-1 according to claim 1, characterized in that, The pharmaceutical composition is an injectable formulation prepared by dissolving anti-human CD96 blocking antibody and anti-human PD-1 blocking antibody together in a buffer solution.

5. The pancreatic cancer immunotherapy composition targeting CD96 and PD-1 according to claim 4, characterized in that, The buffer solution used is a sterile, sodium azide-free, low-endotoxin phosphate buffer solution with a pH of 7.0-7.

4.

6. The pancreatic cancer immunotherapy composition targeting CD96 and PD-1 according to claim 1, characterized in that, The anti-human CD96 blocking antibody and the anti-human PD-1 blocking antibody are either pre-mixed single formulations or separately prepared and packaged, and then mixed at a mass ratio of 1:1 before use.

7. The pancreatic cancer immunotherapy composition combining CD96 and PD-1 according to claim 1, characterized in that, The pharmaceutical composition is a preparation aseptically dispensed into vials and stored at 2-8°C in the dark.

8. The pancreatic cancer immunotherapy composition combining CD96 and PD-1 according to claim 1, characterized in that, The drug composition is administered via intravenous injection or intraperitoneal injection.

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