Composition of cpg1018 / 1826 / 007 / 2006 in combination with MNR for treating pancreatic cancer

Abstract

The present invention provides a pharmaceutical composition, comprising a CpG oligodeoxynucleotide and the MNR protein. The present invention further provides use of the pharmaceutical composition in the preparation of an anti-tumor drug.

Description

The combination of CpG1018 / 1826 / 007 / 2006 and MNR is used to treat pancreatic cancer.

[0001] Cross-reference to related applications

[0002] This invention claims priority to the earlier application filed on July 8, 2024, with patent application number 202410904731.2 and entitled "Composition of CpG1018 / 1826 / 007 / 2006 in combination with MNR for the treatment of pancreatic cancer". The entire contents of that earlier application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of pharmaceutical formulation technology, and more particularly to a composition of CpG1018 / 1826 / 007 / 2006 combined with MNR for the treatment of pancreatic cancer. Background Technology

[0004] Pancreatic cancer is a type of cancer with a high mortality rate. Because it often presents with no obvious symptoms in its early stages, most patients are diagnosed at an advanced stage, when the cancer may have already metastasized, making surgical intervention difficult. Furthermore, pancreatic cancer responds poorly to current chemotherapy and radiotherapy, which are among the main treatment methods for cancer. Several other challenges exist in the treatment of pancreatic cancer: 1) Lack of targeted therapy: Targeted therapy and immunotherapy for pancreatic cancer are not as effective or widely available as for other types of cancer, primarily due to the tumor microenvironment and genetic heterogeneity of pancreatic cancer. 2) Tumor microenvironment: The tumor microenvironment of pancreatic cancer is complex, with abundant extracellular matrix and fibroblasts, creating obstacles to drug delivery and immune cell penetration. 3) Drug resistance: Even if there is an initial response to treatment, pancreatic cancer cells may rapidly develop resistance to therapeutic drugs. 4) Lack of biomarkers: Currently, there are no effective biomarkers to predict treatment response or monitor the disease. Therefore, current technologies lack effective treatment methods for pancreatic cancer.

[0005] Bacterial DNA has a strong stimulatory effect on the immune system, particularly on unmethylated CpG dideoxynucleotide motifs. This stimulation is achieved through Toll-like receptor 9 (TLR9), a pattern recognition receptor expressed on the endosome surface of immune cells. TLR9 recognizes not only unmethylated CpG motifs in bacterial or viral DNA but also synthetic CpG oligodeoxynucleotides (ODNs). Human B cells and pDC cells express TLR9 and directly respond to CpG stimulation. After CpG binds to TLR9, it ultimately activates various transcription factors, including NF-κB, AP1, CEBP, and CREB, through MYD88, IRAK, and TRAF6. These transcription factors directly upregulate the expression of cytokine and chemokine genes. CpG initiates immune stimulation by activating these cells, ultimately leading to a cascade of indirect maturation, differentiation, and proliferation of natural killer (NK) cells. T cells and monocytes / macrophages secrete cytokines and chemokines, producing pro-inflammatory factors (IL-1, IL-6, IL-18, and TNF) and Th1-biased immunity (IFNγ, IL-12). By upregulating the expression of CD80, CD86, CD40, and MHC molecules on pDC cells, it increases antigen processing / presentation and CD8+ T cell responses, driving Th1-type immunity and CD8+ CTL cytotoxicity. Meanwhile, TLR9-dependent B cell activation leads to increased antigen-specific humoral responses and IgG class switching.

[0006] CpG ODNs are short, single-stranded synthetic nucleic acids containing unmethylated cytosine-guanine (CG) dinucleotides in specific base pair sequences, known as CpG motifs. Because CpG ODNs are rapidly degraded by nucleases after entering systemic circulation, their development has focused on nuclease-resistant delivery systems. Synthetic CpG ODNs possess a partially or completely phosphate-thioester backbone, rather than the native phosphodiester backbone. Sometimes, they have one or two poly-G tails at the 3′ and 5′ ends. CpG ODNs have been shown to strongly activate the proliferation of human peripheral blood monocytes and the secretion of pro-inflammatory cytokines. Based on their specific chemical modifications and immunostimulatory effects, CpG ODNs can be classified into three classes: class A (type D), class B (type K), and class C. Those with multiple CpG motifs on the phosphate-thioester backbone are classified as class B and are strong inducers of B cell activation, plasmacytoid dendritic cell maturation, and monocyte maturation. It has relatively low sensitivity to DNases and can remain in the animal body for a longer period of time to exert a lasting effect. Type A CpGs typically contain a single CpG motif linked by natural phosphodiester bonds, with a phosphodiester / thiophosphate diester backbone. The motif is flanked by palindromic sequences and poly-G tails at the 3′ and 5′ ends. This structural feature allows the CpG molecule to form complex polymers in solution. This type of CpG can activate NK cells, promote the maturation of plasmacytoid dendritic cells and the secretion of IFNα, but has no effect on B cells. Type C ODNs are similar to Type B in their CpG units, consisting entirely of thiophosphate nucleotides. Structurally, they contain palindromic CpG motifs similar to those in Type A CpGs, thus forming stem-loop structures or dimers. Type C ODNs can induce the activation of B cells and plasmacytoid dendritic cells and the production of IFNα. The immunostimulatory effects induced by different CpG motifs vary across species, exhibiting species specificity, mainly due to the different structures of the TLR9 receptor protein in different species. Among them, Dynavax used CpG 1018 to enhance the immunization effect of its hepatitis B vaccine.

[0007] CpG1018, a class B ODN composed of 22 unmethylated bases, can increase the immunogenicity of antigens by improving antigen uptake by antigen-presenting cells, activating their functional maturation, and producing cytokines and chemokines. CpG1018 possesses advantages such as stability, low cost, ease of synthesis, high efficiency, and low toxicity. Experiments have demonstrated that CpG1018 can significantly enhance the immunogenicity and accelerate the immune response of aluminum-adjuvanted vaccines against infectious diseases such as hepatitis B, anthrax, and influenza. However, whether it has an effect on improving the efficacy of treatment for pancreatic cancer remains unclear. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a treatment combination of CpG1018 / 1826 / 007 / 2006 and MNR for pancreatic cancer. This combination resolves the problem of poor treatment efficacy for pancreatic cancer in existing technologies.

[0009] In a first aspect, the present invention provides a pharmaceutical composition comprising CpG oligodeoxynucleotides and MNR protein.

[0010] In one embodiment of the present invention, the CpG oligodeoxynucleotide includes one or more of CpG1018, CpG1826, CpG007, and CpG2006. Preferably, the CpG oligodeoxynucleotide is CpG1018.

[0011] In one embodiment of the present invention, the MNR protein comprises a fusion protein of MBP (maltose-binding protein) and MUC1-N (mucin 1).

[0012] In one embodiment of the present invention, the amino acid sequence of MUC1-N is as shown in SEQ ID NO.1, or has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8% identity with the sequence shown in SEQ ID NO:1.

[0013] In one embodiment of the present invention, the amino acid sequence of the MBP is as shown in SEQ ID NO.3, or has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8% identity with the sequence shown in SEQ ID NO:3.

[0014] Preferably, the gene nucleotide sequence of MUC1-N is shown in SEQ ID NO.2, and the gene nucleotide sequence of MBP is shown in SEQ ID NO.4.

[0015] In one specific embodiment of the present invention, the amino acid sequence of the MNR protein is as shown in SEQ ID NO:5, or has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8% identity with the sequence shown in SEQ ID NO:5.

[0016] In one embodiment of the present invention, the mass ratio of the CpG oligodeoxynucleotide to MNR is 3-22.6:5-10.

[0017] In one embodiment of the present invention, the mass ratio of the CpG oligodeoxynucleotide to MNR is 3:10.

[0018] In one embodiment of the present invention, the composition further includes a carrier solution. In a specific embodiment of the present invention, the carrier solution includes one or more of Tris-HCl solution, sodium acetate solution, Arg-HCl solution, and PS80 (polysorbate 80). In a preferred embodiment of the present invention, the carrier solution is a Tris-HCl solution.

[0019] In one embodiment of the present invention, the volume ratio of the composition to the carrier solution is 1:1.

[0020] In one embodiment of the present invention, the composition further includes an adjuvant component, which comprises an aluminum salt adjuvant. In one embodiment of the present invention, the aluminum salt adjuvant includes aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), aluminum hydrochloride, aluminum sulfate, ammonium alum, potassium alum, or aluminum silicate.

[0021] In one embodiment of the present invention, CpG oligodeoxynucleotides and MNR are administered simultaneously or sequentially.

[0022] In one embodiment of the present invention, CpG oligodeoxynucleotides and MNR are administered sequentially or at any time interval, allowing both therapeutic agents to exert their biological activity simultaneously. Preferably, CpG oligodeoxynucleotides and MNR produce a synergistic therapeutic effect.

[0023] In one embodiment of the present invention, the drug can be administered through various routes, including but not limited to oral, intra-arterial, parenteral, intranasal, intravenous, intramuscular, intratracheal, oral, rectal, intraperitoneal, intradermal, subcutaneous, local, transdermal and intrathecal administration, or other methods such as implantation or inhalation.

[0024] In one embodiment of the present invention, the dosage of CpG oligodeoxynucleotides and MNR is the therapeutically effective dosage, and the dosage varies depending on factors such as the type of drug, the target population, and the route of administration.

[0025] In one embodiment of the present invention, the dosage of CpG oligodeoxynucleotides is 1200-10400 μg / kg. In another embodiment, CpG oligodeoxynucleotides are administered using three methods: high dose, medium dose, and low dose. In some embodiments, high doses of CpG oligodeoxynucleotides are administered in the range of 4000-10400 μg / kg, such as 4000-10000 μg / kg, 4000-9000 μg / kg, 4000-8000 μg / kg, 4000-7000 μg / kg, 4000-6000 μg / kg, and 4000-5000 μg / kg. In some embodiments, medium doses of CpG oligodeoxynucleotides are administered in the range of 1500-4000 μg / kg, such as 1500-3500 μg / kg, 1500-3000 μg / kg, 1500-2500 μg / kg, and 1500-2000 μg / kg. In some embodiments, low doses of CpG oligodeoxynucleotides are administered in the range of 1200-1500 μg / kg, such as 1200-1400 μg / kg, 1200-1300 μg / kg, and 1200-1250 μg / kg.

[0026] In one embodiment of the present invention, the dosage of MNR is 1000-5000 μg / kg, for example 4000 μg / kg.

[0027] In one embodiment of the present invention, CpG oligodeoxynucleotides and MNR are administered at the above-mentioned dosage approximately every 2-4 days, for example, twice a week. In another embodiment of the present invention, the administration period is 2-6 weeks, for example, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks.

[0028] In one embodiment of the present invention, the tumor-inhibiting effects of CpG oligodeoxynucleotides and MNRs are enhanced with increasing dosage.

[0029] In one embodiment of the present invention, the MNR protein can combat the growth of pancreatic cancer. In a specific embodiment of the present invention, the MNR protein inhibits the growth of pancreatic cancer cells by mobilizing T cells.

[0030] In one embodiment of the present invention, aluminum adjuvant has adsorption and activation effects on MNR. In another embodiment of the present invention, MNR + aluminum adjuvant has a better tumor-inhibiting effect compared to MNR.

[0031] In one embodiment of the present invention, compared with the aluminum adjuvant + MNR group, the aluminum adjuvant + CpG + MNR group has a better effect on inhibiting pancreatic cancer growth and inhibiting tumor weight growth.

[0032] In one embodiment of the present invention, CpG+MNR has a better tumor-inhibiting effect compared with CpG+MNR+aluminum adjuvant.

[0033] In one embodiment of the present invention, the carrier solution significantly enhances the antitumor effect of MNR+CpG. For example, the tumor inhibition rate of 30 μg CpG-HP007 + 50 μg MNR-Tris is 38.09%, the tumor inhibition rate of 30 μg CpG-HP007 is 29.79%, and the tumor inhibition rate of 50 μg MNR-Tris is 6.81%.

[0034] In one embodiment of the present invention, the composition using Tris-HCl solution as the carrier solution exhibits better antitumor effect than the composition using sodium acetate solution as the carrier solution. For example, the antitumor rate of 30 μg CpG-HP007 + 50 μg MNR-Ac is 27.66%, while the antitumor rate of 30 μg CpG-HP007 + 50 μg MNR-Tris is 38.09%.

[0035] In one embodiment of the present invention, the composition (CpG+MNR-Tris) using Tris-HCl solution as the carrier solution exhibits better tumor-suppressing effect compared to CpG or MNR-Tris. In another embodiment of the present invention, CpG and MNR-Tris have a synergistic effect.

[0036] In a second aspect, the present invention provides a formulation comprising the above-described composition.

[0037] In one embodiment of the present invention, the formulation further includes a pharmaceutically acceptable carrier.

[0038] The pharmaceutically acceptable carrier may be selected from any carrier commonly used in pharmaceutical formulations. For example, a pharmaceutically acceptable carrier may be selected from one or more of the following: lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. The pharmaceutical composition may also contain one or more of the following commonly used in the manufacture of pharmaceutical compositions: diluents, excipients, lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, preservatives, etc. Additionally, the composition may be administered using an optional device capable of delivering the active ingredient to target cells.

[0039] In a third aspect, the present invention provides the use of the above-described compositions and formulations in the preparation of antitumor drugs.

[0040] In one embodiment of the present invention, the tumor is a MUC1-positive tumor.

[0041] In one embodiment of the present invention, the tumor includes breast cancer, endometrial cancer, gastric cancer, and pancreatic cancer. Preferably, the tumor is pancreatic cancer.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1) This invention is the first to discover that the aluminum adjuvant + CpG + MNR composition has an enhanced anti-pancreatic cancer effect, and that CpG and MNR have a synergistic effect. CpG not only improves the anti-tumor effect of MNR, but also improves the anti-tumor effect of MNR + aluminum adjuvant.

[0044] 2) This invention is the first to discover that the addition of Tris-HCl buffer to the CpG+MNR composition significantly enhances its anti-pancreatic cancer effect, which is better than the effects of CpG and MNR alone.

[0045] 3) This invention unexpectedly discovered that the CpG+MNR composition has a better anti-pancreatic cancer effect than the aluminum adjuvant+CpG+MNR composition. Attached Figure Description

[0046] Figure 1 shows the anti-Pan-02 pancreatic cancer effect of MNR and the changes in white blood cells;

[0047] Figure 2 shows the changes in immune organs and tumor cells induced by MNR immunization.

[0048] Figure 3 shows the tumor weight changes in pancreatic cancer treated with MNR combined with CpG / Alum. Detailed Implementation

[0049] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0050] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0051] Technical terms

[0052] "CpG ODN (CpG motif oligonucleotide)," or simply CpG, is an oligodeoxynucleotide with a core of nonmethylated cytosine and guanine dinucleotides. It is a synthetically produced 18-30 bp repeating DNA sequence of nonmethylated cytosine-guanine dinucleotides with immunostimulatory activity. To improve its stability and prolong its in vivo half-life, some or all phosphodiester bonds are replaced by phosphothiodiester bonds. Based on its structural characteristics, CpG can induce cellular and humoral immunity to varying degrees, enhancing the body's immune response. The CpGs used in this invention include CpG1018, CpG1826, CpG007, and CpG2006.

[0053] Mucin 1 (MUC-1), also known as DF3 antigen, is a type I transmembrane protein. Its synthesis and secretion are characteristic of glandular epithelial tissue, playing a crucial role in epithelial renewal and differentiation, maintaining epithelial integrity, and the occurrence and metastasis of cancer. Under normal circumstances, MUC-1 is mainly expressed on the apical surface of epithelial cells near the lumen or glandular lumen in various tissues and organs, exhibiting polar distribution. MUC-1 is also expressed in various cells of the hematopoietic system (T, B, dendritic cells, etc.). It is highly expressed in breast cancer, endometrioid adenocarcinoma, gastric cancer, and pancreatic ductal adenocarcinoma.

[0054] The "MNR protein" is MUC1-N (the extracellular N-terminal domain of mucin 1) containing an MBP (maltose-binding protein) tag protein, expressed via recombinant E. coli. In one embodiment of the present invention, the MNR protein is a fusion protein composed of maltose-binding protein MBP and mucin MUC1-N linked together. In one embodiment of the present invention, the amino acid sequence of the MNR protein is shown in SEQ ID NO:1.

[0055] "Administration" means the physical introduction of the product of this disclosure into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. In one embodiment of the invention, it refers to intramuscular injection of MNR protein and / or CpG oligonucleotides into C57BL / 6 mice.

[0056] A "fusion protein" is a chimeric protein that comprises the amino acid sequences of two or more different proteins. Typically, fusion proteins are produced using in vitro recombinant techniques well known in the art.

[0057] A "therapeutic effective dose" or "therapeutic dose" is a quantity sufficient to achieve the desired clinical outcome (i.e., to achieve therapeutic efficacy). A therapeutic effective dose may be administered once or multiple times.

[0058] Example 1: MNR protein immunization can combat the growth of murine Pan02 pancreatic cancer.

[0059] First, murine Pan02 pancreatic cancer cells were subcutaneously inoculated into C57BL / 6 mice. The experiment consisted of four groups: 0.2, 2, and 8 mg / kg MNR experimental groups (LD, MD, HD) and an aluminum adjuvant control group. Each group received intramuscular immunization twice weekly for four consecutive weeks. Results showed no animal deaths or significant drug toxicity during the treatment period, and the treatment was well-tolerated. Ten days after vaccination, the MD group showed better anti-cancer activity, with tumors measuring 359±79 mm. 3 From shrinking to 286±62mm 3 (p<0.05); 14 days after inoculation, the LD group showed better anti-cancer effects, with tumor size 408±91mm. 3 From shrinking to 332±68mm 3 (p<0.05) (Figure 1A). Analysis of leukocytes (CD45+), B cells (CD45+ / CD19+), total T cells (CD45+ / CD19- / CD3+), helper T cells (CD45+ / CD19- / CD3+ / CD4+ / CD8-), cytotoxic T cells (CD45+ / CD19- / CD3+ / CD4- / CD8+), and macrophages (CD45+ / CD19- / CD3- / CD11b+ / F4 / 80+) in the blood of patients with LD on day D19. The number of NK cells (CD45+ / CD19- / CD3- / CD335+) and DC cells (CD45+ / CD19- / CD3- / CD11c+) was analyzed. A significant increase in white blood cell count was observed, from 97.2±1.4% to 98.6±0.6% (p<0.05) (Figure 1B), while the proportion of DC cells in white blood cells significantly decreased, from 1.65±0.49% to 1.15±0.29% (p<0.05) (Figure 1C). There was a trend of increasing B cells in the blood (p>0.05) (Figure 1D), a slight increase in T lymphocytes (p>0.05) (Figure 1E), and a trend of increasing toxic T lymphocytes (p>0.05) (Figure 1F).

[0060] MNR protein-mediated immunity against the growth of murine Pan02 pancreatic cancer is achieved by mobilizing T cells. Three days after the completion of the above experiment (D31), flow cytometry analysis of the number of immune cells in mouse tumors, spleen, lymph nodes, and blood revealed an increase in leukocytes in the tumor (p<0.05) (Figure 2A), particularly an increase in toxic T lymphocytes (Figure 2B), with an increased proportion of toxic T lymphocytes among total T lymphocytes (Figure 2C). In contrast, leukocytes in lymph nodes also increased (Figure 2D), with the most significant increase in helper T cells (Figure 2E), and an increased proportion of helper T cells among total lymphocytes (Figure 2F). The number of T lymphocytes in the spleen also increased significantly (Figure 2G), with the most significant increase in helper T cells (Figure 2H), but the proportion of helper T cells among total T cells did not increase (Figure 2I). A slight increase in peripheral blood leukocytes was observed, rising from 90.1±10.5% to 96.9±2.8% (p>0.05), while the percentage of dendritic cells (DCs) in leukocytes showed a slight increase, rising from 2.50±1.07% to 2.82±1.21% (p>0.05). A slight increase in B cells was observed (p>0.05), a slight increase in T lymphocytes was observed (p>0.05), and a slight decrease in toxic T lymphocytes was observed (p>0.05).

[0061] Example 2: Efficacy of CpG1826 combined with MNR / aluminum adjuvant in the treatment of Pan02 pancreatic cancer

[0062] The MNR buffer consisted of 19 mM Tris-HCl, pH 7.0, 133 mM Arg-HCl, 0.02% PS80, with added aluminum adjuvant at 1.0 mg / mL and added CpG1826 at 0.3 mg / mL. First, C57BL / 6 mice were subcutaneously inoculated with murine Pan02 pancreatic cancer cells. The experiment consisted of eight groups: a control group, an aluminum adjuvant (Alum or A) group, a low-dose aluminum adjuvant + MNR group, a high-dose aluminum adjuvant + MNR group, an aluminum adjuvant + CpG group, a low-dose aluminum adjuvant + CpG + MNR group, a high-dose aluminum adjuvant + CpG + MNR group, and a PD-1 antibody group (n=8 per group). The mice were administered buffer, aluminum adjuvant, aluminum adjuvant + 50 μg MNR, aluminum adjuvant + 100 μg MNR, aluminum adjuvant + CpG, aluminum adjuvant + CpG + 50 μg MNR, aluminum adjuvant + CpG + 100 μg MNR, and PD-1 antibody, respectively. The first seven groups were administered intramuscularly, while the PD-1 antibody was administered intraperitoneally. The treatment was administered twice weekly for three consecutive weeks. Tumor volumes in some mice reached 2000 mm². 3The endpoint of the experiment was determined by the following data. On day 18 post-vaccination, the tumor weights of the control group, aluminum adjuvant group, low-dose aluminum adjuvant + MNR group, high-dose aluminum adjuvant + MNR group, aluminum adjuvant + CpG group, low-dose aluminum adjuvant + CpG + MNR group, high-dose aluminum adjuvant + CpG + MNR group, and PD-1 antibody group were 1.234±0.064g, 1.058±0.129g, 1.129±0.136g, 1.015±0.112g, 0.901±0.130g, 0.947±0.070g, 0.703±0.122g, and 0.664±0.099g, respectively. In the low-dose group of aluminum adjuvant CpG + MNR and the high-dose group of aluminum adjuvant + CpG + MNR, the size of pancreatic cancer cells decreased significantly (p<0.01); in the PD-1 antibody group, the size decreased significantly (p<0.001) (Figure 3). Therefore, CpG1826 combined with MNR / aluminum adjuvant is more effective in treating Pan02 pancreatic cancer than MNR / aluminum adjuvant alone.

[0063] Example 3: Anticancer effect of MNR / CpG1826 on Pan02 pancreatic cancer

[0064] Mouse-derived Pan02 pancreatic cancer cells were subcutaneously inoculated into C57BL / 6 mice. The experiment consisted of eight groups: a control group, a CpG1826 group, a high-dose CpG1826+MNR group, a high-dose CpG1826+MNR+aluminum adjuvant group, a CpG-007 group, a high-dose CpG-007+MNR+aluminum adjuvant group, and a PD-1 antibody group (n=8 per group). The respective treatments included vector solution, 30 μg CpG1826, 30 μg CpG1826+100 μg MNR, aluminum adjuvant+30 μg CpG1826+100 μg MNR, 30 μg CpG-007, 30 μg CpG-007+100 μg MNR, and aluminum adjuvant+30 μg CpG1826+100 μg MNR. MNR and PD-1 antibody were administered intramuscularly to the first seven groups, followed by intraperitoneal injection of PD-1 antibody. This was repeated twice weekly for three consecutive weeks. In some mice, tumor volume reached 2000 mm². 3The endpoint of the experiment was determined 19 days after drug injection. At this point, no therapeutic effect was observed in the high-dose aluminum adjuvant + CpG + MNR group, possibly due to the large standard deviation of tumors in the animals. Both the CpG1826 and CpG1826 + MNR high-dose groups significantly inhibited pancreatic cancer growth, reducing tumor size from 1.230±0.131g to 0.639±0.123g and 0.647±0.097g, respectively (p<0.01, p<0.01), with tumor growth inhibition rates (TGI) of 48% and 47%, respectively. The CpG-HP007 + MNR high-dose group significantly inhibited pancreatic cancer growth, reducing tumor size from 1.230±0.131g to 0.829±0.091g (p<0.05), with a TGI of 33%. In addition, PD-1 showed efficacy in treating pancreatic cancer, but not as much as the high-dose CpG1826 and CpG1826+MNR groups. The results are shown in Table 1. Therefore, the anti-cancer effect of MNR / CpG1826 on Pan02 pancreatic cancer is better than that of MNR / CpG1826 combined with aluminum adjuvant.

[0065] Table 1. Treatment of Pan02 pancreatic cancer with different CpG combinations using MNR antigen. *: p < 0.05; **: p < 0.01.

[0066] Example 4: Anticancer effect of MNR / CpG on Pan02 pancreatic cancer

[0067] C57BL / 6 mice were subcutaneously inoculated with murine Pan02 pancreatic cancer cells. The experiment consisted of nine groups: Control (1), MNR- group (2), LD007+MT group (3), HD007+MT group (4), LD007 group (5), HD007 group (6), LD007+MA group (7), LD2006+MA group (8), and LD1018+MA group (9), with eight mice in each group. The mice were administered the following solutions: Tris vector solution, 50 μg MNR-Tris, 30 μg CpG-HP007+50 μg MNR-Tris, 90 μg CpG-HP007+50 μg MNR-Tris, 30 μg CpG-HP007, 90 μg CpG-HP007, 30 μg CpG-HP007+50 μg MNR-Ac, and 30 μg CpG2006+50 μg MNR-Ac. MNR-A, 30 μg CpG1018 + 50 μg MNR-Ac. Intramuscular injection, twice a week for 3 consecutive weeks. Tumor volume in some animals exceeded 2000 mm3, reaching the experimental endpoint. Results are shown in Table 2. The experiment ended 20 days after administration. The results showed that HD007+MT (4) and HD007 group (6) significantly inhibited the growth and proliferation of mouse pancreatic cancer PANC0 allogeneic xenografts in C57BL / 6 mice (P<0.001, P<0.001). The LD007+MT group (3), LD007 group (5), LD007+MA group (7), LD2006+MA group (8), and LD1018+MA group (9) significantly inhibited the growth and proliferation of pancreatic cancer PANC 02 (also known as Pan02) allografts in C57BL / 6 mice (P<0.05, P<0.05, P<0.05, P<0.05, P<0.05).

[0068] Table 2. Effects of different doses of CpG, different types of CpG, and different buffer solutions on the anticancer effect of MNR combined with CpG *: p < 0.05; ***: p < 0.001.

[0069] Example 5: Anticancer effect of MNR+226μg CpG1018 on Pan02 pancreatic cancer

[0070] C57BL / 6 mice were subcutaneously inoculated with murine Pan02 pancreatic cancer cells. The experiment consisted of 9 groups: a control group, and mice with the following cell lines: YC1018K-90, YC1018K-226, YC1018Ap-90, YC1018Ap-226, YC2006K-90, YC2006K-226, YT007-226, and YT1018K-90 (8 mice per group). The respective cell lines used were: sodium acetate carrier solution, 90 μg CpG 1018K + 50 μg MNR-Ac, 226 μg CpG 1018K + 50 μg MNR-Ac, 90 μg CpG 1018Ap + 50 μg MNR-Ac, 226 μg CpG 1018Ap + 50 μg MNR-Ac, and 90 μg CpG... 2006K+50μg MNR-Ac, 226μg CpG; 2006K+50μg MNR-Ac, 226μg CpG; HP007+50μg MNR-T, 90μg CpG; 1018K+50μg MNR-T. Intramuscular injection was administered twice weekly for 3 consecutive weeks. The experimental endpoint was reached when the tumor volume in some animals reached 2000 mm³. Results are shown in Table 3. During the experiment, one animal died in the YC1018Ap-226 monotherapy group. At the end of the experiment, gross autopsies revealed abnormal enlargement of the liver and spleen in the YC1018K-226, YC1018Ap-226, YC2006K-226, and YT007-226 monotherapy groups, presumably related to drug toxicity. %TGITW were 75.18, 84.95, 73.52, and 48.63, respectively. YT1018K-90, YC1018K-90, YC1018Ap-90, and YC2006K-90 also effectively inhibited the growth and proliferation of pancreatic cancer PANC 02 allograft tumors in C57BL / 6 mice (%TGITW were 64.33, 56.01, 54.34, and 58.76, respectively).

[0071] Table 3. Effects of different doses of CpG, different manufacturers, and different buffer solutions on the anticancer effect of MNR combined with CpG *: vs control, p<0.05; **: vs control, p<0.01; ***: vs control, p<0.001.****: vs control, p<0.0001; *****: vs control, p<0.00001

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0073] sequence list

Claims

1. A pharmaceutical composition, characterized in that: This includes CpG oligodeoxynucleotides and MNR proteins.

2. The pharmaceutical composition according to claim 1, characterized in that: The CpG oligodeoxynucleotide includes one or more of CpG1018, CpG1826, CpG007, and CpG2006; preferably, the CpG oligodeoxynucleotide is CpG1018.

3. The pharmaceutical composition according to claim 1, characterized in that: The MNR protein includes a fusion protein of MBP and MUC1-N; Preferably, the amino acid sequence of MUC1-N is as shown in SEQ ID NO.1, or has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8% identity with the sequence shown in SEQ ID NO:1; Preferably, the amino acid sequence of the MBP is as shown in SEQ ID NO.3, or has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8% identity with the sequence shown in SEQ ID NO:3; Preferably, the amino acid sequence of the MNR protein is as shown in SEQ ID NO:5, or has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8% identity with the sequence shown in SEQ ID NO:

5.

4. The pharmaceutical composition according to claim 1, characterized in that: The mass ratio of the CpG oligodeoxynucleotide to MNR is 3-22.6:5-10; Preferably, the mass ratio of the CpG oligodeoxynucleotide to MNR is 3:

10.

5. The pharmaceutical composition according to claim 1, characterized in that: The composition further includes a carrier solution; Preferably, the carrier solution includes one or more of Tris-HCl solution, sodium acetate solution, Arg-HCl solution, and PS80 (polysorbate 80); more preferably, the carrier solution is Tris-HCl solution.

6. The pharmaceutical composition according to claim 1, characterized in that: The composition further includes an adjuvant component, which includes an aluminum salt adjuvant; preferably, the aluminum salt adjuvant includes aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), aluminum hydrochloride, aluminum sulfate, ammonium alum, potassium alum, or aluminum silicate.

7. The pharmaceutical composition according to claim 1, characterized in that: The CpG oligodeoxynucleotide and MNR are administered simultaneously or sequentially.

8. The pharmaceutical composition according to claim 1, characterized in that: The dosage of CpG oligodeoxynucleotides is 1200-10400 μg / kg; Preferably, the dosage of MNR is 1000-5000 μg / kg; Preferably, the MNR protein can combat the growth of pancreatic cancer; more preferably, the MNR protein inhibits the growth of pancreatic cancer cells by mobilizing T cells. Preferably, aluminum adjuvant has an adsorption and activation effect on MNR; more preferably, MNR + aluminum adjuvant has a better tumor-inhibiting effect compared with MNR. Preferably, compared with the aluminum adjuvant + MNR group, the aluminum adjuvant + CpG + MNR group has a better effect on inhibiting pancreatic cancer growth and tumor weight increase; Preferably, CpG+MNR has a better tumor-inhibiting effect compared with CpG+MNR+aluminum adjuvant. Preferably, the carrier solution significantly enhances the antitumor effect of MNR+CpG; more preferably, the composition using Tris-HCl solution as the carrier solution has a better antitumor effect than the composition using sodium acetate solution as the carrier solution. Preferably, the composition using Tris-HCl solution as a carrier solution has a better antitumor effect compared with CpG or MNR-Tris; more preferably, CpG and MNR-Tris have a synergistic effect.

9. A formulation comprising the pharmaceutical composition according to any one of claims 1-9, Preferably, it also includes a pharmaceutically acceptable carrier.

10. The use of the pharmaceutical composition according to any one of claims 1-8, or the formulation according to claim 9, in the preparation of an antitumor drug. Preferably, the tumor is a MUC1-positive tumor; Preferably, the tumor includes breast cancer, endometrial cancer, gastric cancer, and pancreatic cancer; more preferably, the tumor is pancreatic cancer.

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