Aluminium nanocrystalline conjugate immunopharmaceutical and its preparation method and use

Abstract

The present invention relates to the technical fields of biomedicine and vaccines, in particular to an aluminum nanocrystal composite immuno-drug and its preparation method and use. The aluminum nanocrystal is used as a carrier, the surface of the aluminum nanocrystal is covered with polyethylene glycol, and a functional polypeptide sequence and a cytokine molecule are linked by the polyethylene glycol end group to form a virus-like particle immuno-drug. This drug improves the stability and retention time of cytokine in tumor tissue, and effectively improves the auxiliary anti-tumor effect of cytokine.

Description

[Technical field]

[0001] This application claims priority to Chinese Patent Application No. 202210152866.9, entitled "ALUMINUM NANOCRYSTALLINE COMPOUND IMMUNE DRUG AND PREPARATION METHOD THEREFOR AND USE THEREOF," filed with the China Patent Office on February 18, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the technical fields of biomedicine and vaccines, in particular to an aluminum nanocrystalline conjugate immuno-drug and its preparation method and use. [Background technology]

[0003] So far, theoretical research and related technologies in clinical medicine have made great progress, but tumors (cancer) are still a disease that is difficult to cure and has a high mortality rate. Clinically, there are three traditional treatment regimens for tumors: surgery, chemotherapy, and radiation. Surgical therapy involves directly removing the affected area through surgery. It is more effective in treating early tumors or benign tumors with low metastatic potential. However, in cancer patients with metastasis, the efficacy and prognosis of quality of life are poor. Due to its lack of specificity, chemotherapy also causes significant damage to the patient's own immune system and stem cells while treating tumors. In recent years, continued in-depth research on the growth and development process of tumors has proposed an innovative treatment regimen, immunotherapy, which has greatly promoted the development of cancer treatment. In 2013, it was ranked first among the top ten breakthroughs in natural sciences this year by Science magazine. It is one of the most important research directions in the clinical treatment of cancer in recent years.

[0004] Immunotherapy is a general term for a large class of therapeutic approaches that treat cancer by activating the human immune system. The human immune system has always acted as a "policeman" to ward off foreign invaders and the like from the body. Tumor immunotherapy works in three ways: (1) designing monoclonal antibodies to enhance the immune response to destroy cancer cells, (2) using immune checkpoint inhibitors to help the immune system recognize and attack cancer cells, and (3) synthesizing cancer vaccines to stimulate the immune response to treat and prevent cancer. Unlike other cancer treatment approaches, immunotherapy mainly activates the immune system to give the human body the ability to attack tumor cells. Due to the memory function of the human immune system, this process can be continued for a long time after the initial treatment. In addition, the immune system has the ability to distinguish cancer cells from normal cells, and by activating the immune response, it can selectively attack cancer cells and avoid damaging normal cells.

[0005] Cytokines are one of the major players regulating immune responses, and cytokine-based approaches provide an alternative strategy for cancer immunotherapy. Over the past decades, cytokines and cytokine receptors have been widely studied as targets for cancer therapy. Cytokines are a class of small proteins with a wide range of biological activities that are expressed and secreted by immune cells (monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epithelial cells, fibroblasts, etc.) upon stimulation. They can regulate innate and adaptive immunity, hematopoiesis, cell proliferation, APSC pluripotent cells, as well as the repair of damaged tissues by binding to their respective receptors. Cytokines can be divided into interleukins, interferons, some members of the tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc. Cytokines are the main mediators of intercellular communication in the tumor microenvironment. Unregulated cytokine production by malignant cells, immune cells, and stromal cells is involved in all stages of tumor initiation and development. Certain cytokines are closely related to tumor development, progression, and metastasis, and abnormal production of inflammatory cytokines is a common concomitant result of cancerous changes in non-malignant cells. Therefore, by utilizing the immune stimulating effect of cytokines and neutralizing their effects when cytokines become dysregulated, the treatment of tumor tissue can be effectively achieved. However, systemic administration of cytokines lacks targeting, has certain systemic toxicity, and has potential adverse effects on embryonic tissue. Therefore, local administration of cytokine drugs is a relatively effective and safe method.

[0006] Therefore, in order to solve the problems of cytokine stability and local accumulation time in the body, increase the cytokine action time with cells in tumor tissue, and fully play the role of immune regulation, it is of great practical value to develop a composite immune drug based on aluminum nanocrystals. Summary of the Invention

[0007] In view of this, the present invention provides an aluminum nanocrystal composite immunological drug, its preparation method and its use, as well as a functional polypeptide sequence and cytokine linked to the surface of the aluminum nanocrystal in the composite immunological drug based on the aluminum nanocrystal. The aluminum nanocrystal provided by the present invention can be effectively bound to the functional polypeptide sequence and cytokine via polyethylene glycol molecule to construct the composite immunological drug, effectively improving the binding ability of the composite immunological drug to tumor cells at tumor site, and increasing the accumulation time of the composite immunological drug at tumor site by the functional polypeptide sequence, while effectively increasing the stability of cytokines and the time window for them to exert their immunoregulatory ability at tumor site, effectively improving the immune response of the organism to tumor tissue, and inhibiting the proliferation of tumor tissue.

[0008] In order to achieve the above objectives of the present invention, the present invention provides the following technical solutions: The present invention provides an aluminum nanocrystal-conjugated immunopharmaceutical that is constructed by linking a functional polypeptide sequence and an aluminum nanocrystal-based cytokine molecule through a polyethylene glycol molecule.

[0009] Furthermore, in the aluminum nanocrystal-conjugated immuno-drug, the functional polypeptide sequence includes a polypeptide sequence capable of specifically binding to a protein highly expressed on the surface of a tumor cell.

[0010] Furthermore, in the aluminum nanocrystal-conjugated immunodrug, the functional polypeptide sequence is one or both of the cyclic RGD sequence and the TAT sequence, which can assist the immunodrug to enter cells.

[0011] Further, in the aluminum nanocrystalline conjugate immuno-drug, the cytokine molecule is selected from one or more of interleukin factors including IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, gamma interferon IFN-γ, or tumor necrosis factor TNFα.

[0012] Furthermore, the construction is based on the formation of covalent amide bonds between the amino groups of M-PEG-NH2 on the surface of the aluminum nanocrystals and the carboxyl groups on the cytokine molecules.

[0013] Further, the aluminum nanocrystals can be prepared by the following process: The aluminum salt solution and the polyethylene glycol solution are thoroughly mixed by stirring, and the pH value is adjusted by adding NaOH aqueous solution, and then they are allowed to stand to obtain a precipitate, which is then centrifuged, washed and purified to obtain the polyethylene glycol modified aluminum nanocrystals.

[0014] Furthermore, the solute of the aluminum salt solution is one or more of aluminum chloride, aluminum nitrate, aluminum sulfate, and aluminum acetate, and the solvent used for the aluminum salt solution is pure water or a sodium acetate solution having a concentration of 0.01 mol / L.

[0015] Furthermore, the polyethylene glycol is a mixed reagent of polyethylene glycol modified with phosphoserine (PS-PEG) and polyethylene glycol having a maleimide group and a terminal carboxylic acid (M-PEG-NH2).

[0016] Furthermore, an aqueous NaOH solution is added to adjust the pH to 5.0 to 8.0.

[0017] The present invention also provides a method for preparing an aluminum nanocrystalline conjugate immunopharmaceutical, comprising the steps of: (1) adding aluminum nanocrystals and functional polypeptide sequences to a solvent, stirring for reaction, centrifuging, washing and purifying, and then resuspending in ultrapure water to obtain a functional polypeptide sequence modified aluminum nanocrystal solution; (2) adding cytokine molecules to MES buffer, adding EDC / NHS to activate carboxyl groups on cytokine molecules, and then adding aluminum nanocrystalline solution to form covalent amide bonds between the amino groups of M-PEG-NH2 and the carboxyl groups on cytokine molecules, thereby obtaining cytokine molecule-modified aluminum nanocrystalline conjugate immuno-drug.

[0018] Further, the solvent is a 0.1 M phosphate buffer or a 0.1 M ammonium acetate buffer.

[0019] Additionally, functional polypeptide sequences include polypeptide sequences capable of specifically binding to proteins highly expressed on the surface of tumor cells.

[0020] Additionally, the functional polypeptide sequence is one or both of the cyclic RGD and TAT sequences, which can assist the immunodrug to enter the cell.

[0021] Additionally, the cytokine molecule is selected from one or more of the interleukin factors including IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, gamma interferon IFN-γ, and tumor necrosis factor TNFα.

[0022] The present invention further provides the use of the aluminum nanocrystal-conjugated immunodrug or the aluminum nanocrystal-conjugated immunodrug prepared by the above-mentioned aluminum nanocrystal-conjugated immunodrug preparation method in immunotherapy in tumor models.

[0023] The use is by intravenous injection, mucosal administration, subcutaneous injection, intradermal injection, peritumoral injection, or intratumoral injection.

[0024] The method for immunotherapy of tumors includes administering an aluminum nanocrystal-conjugated immunodrug or an aluminum nanocrystal-conjugated immunodrug prepared by the preparation method.

[0025] Additionally, modes of administration include intravenous injection, mucosal administration, subcutaneous injection, intradermal injection, peritumoral injection, or intratumoral injection.

[0026] The technical effects of the present invention are: The aluminum nanocrystals prepared via polyethylene glycol provided by the present invention can effectively bind to functional polypeptide sequences and cytokines, effectively increase the stability of cytokines and the time frame for them to exert their immune-modulating ability at tumor sites, improve the immune response of the organism to tumor tissues, and inhibit the proliferation of tumor tissues. [Brief description of the drawings]

[0027] [Figure 1] 4 shows the analysis of the effect of the nano-aluminum crystal composite immuno-drugs constructed in Examples 6 and 7 of the present invention on inhibiting tumor growth. [Diagram 2] 4 shows the analysis of the tumor growth inhibiting effect of the aluminum nanocrystal conjugate immunopharmaceuticals constructed in Examples 8 to 13 of the present invention. [Diagram 3] 4 shows the analysis of cytokine accumulation and stability of the aluminum nanocrystal conjugate immunopharmaceuticals constructed in Examples 6 and 7 of the present invention in a mouse tumor model. [Figure 4] 4 shows the regulation of the aluminum nanocrystal conjugated immunopharmaceuticals constructed in Examples 6 and 7 of the present invention on the proliferation of CD8 T cells in tumor tissue in a mouse tumor model. [Diagram 5] 4 shows the analysis of the effect of the aluminum nanocrystal conjugate immunopharmaceuticals constructed in Examples 6 and 7 of the present invention on inhibiting distant tumor tissue growth. [Figure 6] FIG. 1 shows a schematic diagram of the construction process of the aluminum nanocrystalline conjugate immunopharmaceutical of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present invention discloses an aluminum nanocrystalline composite immuno-drug and its preparation method, as well as a functional polypeptide sequence and cytokine linked to the surface of aluminum nanocrystalline in the composite immuno-drug based on aluminum nanocrystalline. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It is particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and use of the present invention are described through preferred embodiments. Those involved can obviously modify or appropriately modify and combine the method and use described herein to implement and apply the technology of the present invention without departing from the content, spirit and scope of the present invention.

[0029] All the raw materials and reagents used in Examples 1 to 19 provided by the present invention can be purchased from the market.

[0030] The present invention is further described below in conjunction with examples.

[0031] Example 1 Preparation of Aluminum Nanocrystalline (Al-PEG-1) AlCl3·6H2O was dissolved in 0.01 mol / L NaAc to prepare an aluminum salt solution with an aluminum ion content of 0.05 mol / L. To the obtained aluminum salt solution, a mixed solution of PS-PEG and M-PEG-NH2 with a total molecular concentration of 0.05 mol / L in a molar ratio of 1:1 was added, where Al 3+ and phosphoserine in a molar ratio of 1:2. NaOH aqueous solution was added to adjust the pH value to 7.6. After the reaction was completed, the mixture was left to stand for 7 hours, centrifuged, washed, and finally sterilized through high-pressure steam or filtration membrane to obtain aluminum nanocrystalline material called Al-PEG-1.

[0032] Example 2 Preparation of Aluminum Nanocrystalline (Al-PEG-2) Al(NO3)3 was dissolved in pure water to prepare an aluminum salt solution with an aluminum ion content of 0.05 mol / L. A mixed solution of PS-PEG and M-PEG-NH2 with a molar ratio of 1:2 and a total molecular concentration of 0.05 mol / L was added to the obtained aluminum salt solution, where Al 3+ and phosphoserine in a molar ratio of 1:1. NaOH aqueous solution was added to adjust the pH value to 5.0. After the reaction was completed, the mixture was left to stand for 10 hours, centrifuged, washed, and finally sterilized through high-pressure steam or filtration membrane to obtain aluminum nanocrystalline material called Al-PEG-2.

[0033] Example 3 Preparation of Aluminum Nanocrystalline (Al-PEG-3) In this example, aluminum nanocrystalline (Al-PEG-3) was prepared. The preparation method was essentially the same as that in Example 1, except that the aluminum salt used was aluminum acetate, the molar ratio of PS-PEG to M-PEG-NH2 was 2:1, the pH value was adjusted to 8.0 by adding NaOH aqueous solution, and the standing time was 3 hours after the reaction was completed.

[0034] Example 4 Preparation of Aluminum Nanocrystalline (Al-PEG-4) In this example, aluminum nanocrystalline (Al-PEG-4) was prepared. The preparation method was essentially the same as that in Example 1, except that the aluminum salt used was aluminum sulfate, the pH value was adjusted to 6.5 by adding NaOH aqueous solution, and the standing time was 8 hours after the reaction was completed.

[0035] Example 5 The aluminum nanocrystals or aluminum nanocrystalline immunoconjugate drugs obtained in Examples 1 to 4 were tested for particle size.

[0036] At 25° C., the concentration of the aluminum nanocrystals was diluted to 10 μg / mL and the particle size of the aluminum nanocrystals was tested using a nanoparticle size analyzer.

[0037] Here, the physical and chemical properties of the aluminum nanocrystals prepared in Examples 1, 2, 3, and 4 are shown in Table 1 below. [Table 1]

[0038] As can be seen from Table 1, Preparation Examples 1, 2, 3, and 4 of the present invention all obtain nano-sized aluminum nanocrystals with similar particle sizes. Since the main reaction substrates used are similar, the properties of the obtained aluminum nanocrystals are similar.

[0039] Example 6 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-RGD-IL12 (1) The aluminum nanocrystals prepared in Example 1 and a polypeptide sequence c (RGDfC) capable of specifically binding to the functional protein integrin highly expressed on the surface of tumor cells are added to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, in which the ratio of the polypeptide sequence to the maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.2:1; the reaction mixture is stirred for 24 hours, centrifuged, washed and purified to obtain aluminum nanocrystals modified with the RGD peptide sequence; (2) Cytokine IL-12 molecules were added into 50 mM MES buffer, and EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.5:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-RGD-IL12.

[0040] Example 7 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-TAT-TNFα (1) The membrane-permeable peptide sequence TAT, which can effectively deliver the aluminum nanocrystals and nanoparticles prepared in Example 1 into cells, is added to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, where the ratio of the polypeptide sequence to the maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.5:1; the reaction mixture is stirred for 24 hours, centrifuged, washed and purified to obtain aluminum nanocrystals modified with the TAT peptide sequence; (2) Tumor necrosis factor TNFα molecules were added into 50 mM MES buffer, EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.3:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-TAT-TNFα.

[0041] Example 8 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-TAT-IL2 (1) The membrane-permeable peptide sequence TAT, which can effectively deliver the aluminum nanocrystals and nanoparticles prepared in Example 2 into cells, is added to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, in which the ratio of the polypeptide sequence to the maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.1:1; the reaction mixture is stirred for 15 hours, centrifuged, washed and purified to obtain aluminum nanocrystals modified with the TAT peptide sequence; (2) Cytokine IL2 molecules were added into 50 mM MES buffer, EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.2:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-TAT-IL12.

[0042] Example 9 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-RGD-IL15 (1) Add the aluminum nanocrystals prepared in Example 4 and c(RGDfC) to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, where the ratio of polypeptide sequence to maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.5:1, and stir the reaction mixture for 24 hours, centrifuging, washing and purifying to obtain aluminum nanocrystals modified with RGD peptide sequence; (2) Cytokine IL15 molecules were added into 50 mM MES buffer, EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.3:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-RGD-IL15.

[0043] Example 10 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-TAT-IL18 (1) The membrane-permeable peptide sequence TAT, which can effectively deliver the aluminum nanocrystals and nanoparticles prepared in Example 3 into cells, is added to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, in which the ratio of the polypeptide sequence to the maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.3:1; the reaction mixture is stirred for 20 hours, centrifuged, washed and purified to obtain aluminum nanocrystals modified with the TAT peptide sequence; (2) Cytokine IL18 molecules were added into 50 mM MES buffer, EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.4:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-TAT-IL18.

[0044] Example 11 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-RGD-IL21 (1) The aluminum nanocrystals prepared in Example 2 and c(RGDfC) are added to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, in which the ratio of polypeptide sequence to maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.5:1, and the reaction is stirred for 24 hours, centrifuged, washed and purified to obtain aluminum nanocrystals modified with RGD peptide sequence; (2) Cytokine IL21 molecules were added into 50 mM MES buffer, and EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.3:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-RGD-IL21.

[0045] Example 12 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-RT-IFN-γ (1) Add the aluminum nanocrystals prepared in Example 4 and c(RGDfC) with TAT peptide (1:1 molar ratio) to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, where the ratio of polypeptide sequence to maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals is 1.3:1, and stir the reaction mixture for 24 hours, centrifuging, washing and purifying to obtain aluminum nanocrystals modified with RGD peptide sequence; (2) Interferon gamma IFN-γ molecules were added into 50 mM MES buffer, EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystalline solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystalline to cytokine molecules was 1.5:1, thereby obtaining an aluminum nanocrystalline conjugated immuno-drug further modified with cytokine molecules, which was named Al-RT-IFN-γ.

[0046] Example 13 Preparation of aluminum nanocrystalline conjugate immunopharmaceutical Al-RT-IL7 (1) The aluminum nanocrystals prepared in Example 1 (the product obtained in Example 1 was only used as a representative for the subsequent experiments, and did not mean that only the aluminum nanocrystals obtained in Example 1 were used in the subsequent experiments) and c(RGDfC) having TAT peptide (molar ratio 1.5:1) were added to 0.1M phosphate buffer or 0.1M ammonium acetate buffer, where the ratio of the polypeptide sequence to the maleimide contained in the PEG molecule on the surface of the aluminum nanocrystals was 1.5:1, and the reaction was stirred for 24 hours, centrifuged, washed and purified to obtain aluminum nanocrystals modified with RGD peptide sequence; (2) Cytokine IL7 molecules were added into 50 mM MES buffer, EDC / NHS was added to activate the carboxyl groups on the molecules, and then the aluminum nanocrystal solution obtained in step (1) was added, in which the ratio of M-PEG-NH2 on the surface of the aluminum nanocrystal to cytokine molecules was 1.3:1, thereby obtaining an aluminum nanocrystal conjugated immuno-drug further modified with cytokine molecules, which was named Al-RT-IL7.

[0047] Example 14 After detecting the hydrated particle size similar to that in Example 5, the physical and chemical properties of the products obtained in Examples 6 to 13 are shown in Table 2 below. The data in the table show that the particle sizes of the aluminum nanocrystalline conjugated immunopharmaceuticals prepared with different cytokines or interferons are all significantly increased. [Table 2]

[0048] Example 15 The aluminum nanocrystal conjugate immunodrugs constructed above (represented by Al-RGD-IL12 conjugate immunodrug constructed in Example 6 and Al-TAT-TNFα conjugate immunodrug constructed in Example 7) were used in immunotherapy in tumor models to evaluate tumor growth inhibition.

[0049] In compliance with the National Animal Health Protocol, 6-8 week-old BALB / c mice were selected for dorsal subcutaneous tumor implantation. The tumor model was selected from B16F10 mouse melanoma highly metastatic cells. The implanted tumor tissue was approximately 100 mm 3 When the mice reached maturity, they were randomly divided into 5 groups and injected intratumorally with 50 μL of the following drugs:

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[0050] The results are shown in FIG. 1, which is an analysis of the effect of the constructed aluminum nanocrystal-conjugated immuno-drug on inhibiting tumor growth.

[0051] As shown in Figure 1, on day 20, the average tumor size in the control group (Ctrl group) was 1942 mm 3 The mean tumor volume size in the Alum-IL12 group was 1,364 mm 3 , whereas the mean tumor volume size in the Al-RGD-IL12 group was 183 mm 3 The mean tumor volume size in the Alum-TNFα group was 1540 mm 3 , whereas the mean tumor volume size in the Al-TAT-TNFα group was 204 mm 3 It can be seen that the constructed cytokine-loaded aluminum nanocrystal conjugate immuno-drug can effectively inhibit tumor growth.

[0052] Example 16 In compliance with the National Animal Health Protocol, 6-8 week-old BALB / c mice were selected for dorsal subcutaneous tumor implantation. The tumor model was selected from B16F10 mouse melanoma highly metastatic cells. The implanted tumor tissue was approximately 100 mm 3 When the tumors were fully grown, the mice were randomly divided into 5 groups and intratumorally injected with 50 μL of aluminum nanocrystal composite immunopharmaceutical drug containing 0.25 μg of cytokine or interferon gamma constructed in Examples 8 to 13. The changes in tumor volume of the mice were recorded.

[0053] The results are shown in FIG. 2, which is an analysis of the tumor growth inhibiting effect of the aluminum nanocrystal conjugate immunopharmaceuticals constructed in Examples 8 to 13.

[0054] As shown in FIG. 2, on the 20th day, the average tumor volume size of the mice treated with the aluminum nanocrystal-conjugated immunopharmaceuticals constructed in Examples 8 to 13 was 181 mm 3 ~254mm 3 This is significantly lower than the average tumor volume of the control group shown in Example 15, indicating that the aluminum nanocrystalline conjugate immuno-drugs constructed in Examples 8 to 13 can also effectively inhibit tumor growth.

[0055] Tumor inhibition ability is the most direct index to verify delivery efficiency. All of the aluminum nanocrystalline conjugate immunodrugs obtained in Examples 6 to 13 of the present invention can significantly inhibit the growth of melanoma. Since their components are similar and their action mechanisms are substantially similar, only the Al-RGD-IL12 conjugate immunodrug constructed in Example 5 and the Al-TAT-TNFα conjugate immunodrug constructed in Example 6 are selected for further mechanism verification thereafter.

[0056] Example 17 For each mouse group of Example 15, the accumulation and accumulation of cytokines in the tumor tissue was analyzed 24 hours and 48 hours after the intratumoral injection of the drug.

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[0057] The results are shown in Figure 3, which is an analysis of cytokine accumulation in the mouse tumor model of Example 15. As shown in Figure 3, at 24 hours, the stock level of interleukin 12 in tumor tissue is 24.1 pg / mL in the Alum-IL12 experimental group and 60.9 pg / mL in the Al-RGD-IL12 experimental group. At 48 hours, the stock level of interleukin 12 in tumor tissue is 11.4 pg / mL in the Alum-IL12 experimental group and 52.3 pg / mL in the Al-RGD-IL12 experimental group. At 24 hours, the stock level of tumor necrosis factor in tumor tissue is 18.2 pg / mL in the Alum-TNFα experimental group and 46.7 pg / mL in the Al-TAT-TNFα experimental group. At 48 hours, the stock level of tumor necrosis factor in tumor tissue was 8.4 pg / mL in the Alum-TNFα experimental group and 41.5 pg / mL in the Al-TAT-TNFα experimental group. The data show that the constructed aluminum nanocrystalline conjugate immuno-drug can significantly improve the stability and accumulation time of cytokines in the tumor environment.

[0058] Example 18 The proliferation of CD8+ T cells was studied in tumor tissues of mice after drug injection extracted in Example 15. A portion of the tumor tissue was weighed, crushed, and then filtered through a 100 μm cell filter. The cell suspension was stained for surface markers with anti-CD8, and then analyzed by flow cytometer. The results are shown in Figure 4, which shows the proliferation of CD8 T cells in tumor tissues in the mouse tumor model of Example 15. As shown in Figure 4, at 24 hours, the percentage levels of CD8 T cells in tumor tissues are 1.5% in the Alum-IL12 experimental group, 7.1% in the Al-RGD-IL12 experimental group, 1.7% in the Alum-TNFα experimental group, and 6.6% in the Al-TAT-TNFα experimental group. At 48 hours, the percentage levels of CD8 T cells in tumor tissues are 2.4% in the Alum-IL12 experimental group, 9.0% in the Al-RGD-IL12 experimental group, 2.3% in the Alum-TNFα experimental group, and 8.4% in the Al-TAT-TNFα experimental group. The data show that the constructed aluminum nanocrystalline composite immune drug can significantly improve the proliferation ability of CD8 T cells and effectively regulate the immune response of tumor tissues.

[0059] Example 19 Aluminum nanocrystalline conjugated immunopharmaceuticals inhibit distant tumor growth.

[0060] B16F10 mouse melanoma cells were inoculated subcutaneously into the right hind leg of Balb / c mice, which were recorded as in-situ tumors. Half of the B16F10 mouse melanoma cells were inoculated subcutaneously into the left hind leg of Balb / c mice, which were recorded as distal tumors. The mean volume of the in-situ tumors was 100 mm 3 When the tumors grew to 10 mm, the mean volume size of the distal tumors was approximately 50 mm. 3 The mice were then randomly divided into 5 groups and injected into the in situ tumors with 50 μL of the following agents:

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[0061] The results are shown in Figure 5, which is an analysis of the effect of the constructed aluminum nanocrystalline conjugate immuno-drug in inhibiting distant tumor tissue growth. As shown in Figure 5, on the 20th day, the average size of the distant tumors in the control group mice was 1286 mm 3 and the mean volume size of distal tumors in the Alum-IL12 group was 984 mm 3, whereas the mean volumetric size of distal tumors in the Al-RGD-IL12 group was 253 mm 3 The mean volume size of distal tumors in the Alum-TAT-TNFα group was 880 mm 3 , whereas the mean volumetric size of distal tumors in the Al-TAT-TNFα group was 382 mm 3 It can be seen that the constructed cytokine-loaded aluminum nanocrystal composite immuno-drug can effectively inhibit the growth of distant tumors, which indicates that the constructed aluminum nanocrystal composite immuno-drug not only has a significant inhibitory effect on the growth of existing tumors, but also has a significant inhibitory effect on the growth of neoplasms.

[0062] The above is a detailed overview of the aluminum nanocrystalline composite immuno-drug provided by the present invention, its preparation method and its use.This document uses specific examples to illustrate the principle and embodiment of the present invention.The above description of examples is only used to help understand the method of the present invention and its central concept.It should be noted that those skilled in the art can make some modifications and improvements without departing from the concept of the present invention, and all of them fall within the protection scope of the present invention.

Claims

1. An aluminum nanocrystal-conjugated immunopharmaceutical is constructed by linking a functional polypeptide sequence with an aluminum nanocrystal-based cytokine molecule via a polyethylene glycol molecule.

2. The aluminum nanocrystal conjugate immunodrug of claim 1, wherein the functional polypeptide sequence comprises a polypeptide sequence capable of specifically binding to a protein highly expressed on the surface of a tumor cell.

3. The aluminum nanocrystal conjugated immuno-drug of claim 2, wherein the functional polypeptide sequence is one or both of a cyclic RGD sequence and a TAT sequence, which can assist the immuno-drug to enter cells.

4. 2. The aluminum nanocrystalline conjugate immuno-drug of claim 1, wherein the cytokine molecule is selected from one or more of interleukin factors including IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, gamma interferon IFN-γ, or tumor necrosis factor TNFα.

5. The aluminum nanocrystals are prepared by the following process: The aluminum nanocrystal composite immuno-drug of claim 1, which is prepared by the steps of mixing aluminum salt solution and polyethylene glycol solution by stirring, adjusting the pH value by adding NaOH aqueous solution, and then allowing them to stand to obtain precipitates, which are then centrifuged, washed and purified to obtain polyethylene glycol modified aluminum nanocrystals.

6. The aluminum nanocrystalline conjugated immuno-drug of claim 5, wherein the solute of the aluminum salt solution is one or more of aluminum chloride, aluminum nitrate, aluminum sulfate, and aluminum acetate, and the solvent used for the aluminum salt solution is pure water or a sodium acetate solution having a concentration of 0.01 mol / L.

7. The aluminum nanocrystal conjugated immuno-drug of claim 5, wherein the polyethylene glycol solution is a mixed reagent of polyethylene glycol modified with phosphoserine and polyethylene glycol having a maleimide group and a terminal carboxylic acid.

8. The nano-aluminum conjugated immuno-drug of claim 5, wherein the pH is adjusted to 5.0-8.0 by adding NaOH aqueous solution.

9. A method for preparing the aluminum nanocrystalline conjugated immunopharmaceutical of any one of claims 1 to 8, comprising the following steps: (1) adding aluminum nanocrystals and functional polypeptide sequences to a solvent, stirring for reaction, centrifuging, washing and purifying, and then resuspending in ultrapure water to obtain a functional polypeptide sequence modified aluminum nanocrystal solution; (2) Add cytokine molecules to MES buffer, add EDC / NHS to activate the carboxyl groups on the cytokine molecules, then add the aluminum nanocrystal solution to activate M-PEG-NH ２ and forming a covalent amide bond between the amino group of and the carboxyl group on the cytokine molecule, thereby obtaining a cytokine molecule-modified aluminum nanocrystalline conjugate immuno-drug.

10. The method for preparing an aluminum nanocrystalline conjugated immuno-drug according to claim 9, wherein the solvent is 0.1M phosphate buffer or 0.1M ammonium acetate buffer.

11. The method for preparing an aluminum nanocrystalline conjugate immuno-drug according to claim 9, wherein the functional polypeptide sequence comprises a polypeptide sequence capable of specifically binding to a protein highly expressed on the surface of the tumor cell.

12. The method for preparing an aluminum nanocrystalline conjugate immuno-drug according to claim 9, wherein the cytokine molecule is selected from one or more of interleukin factors including IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, gamma interferon IFN-γ, or tumor necrosis factor TNFα.

13. The method for preparing an aluminum nanocrystalline conjugated immuno-drug according to claim 9, wherein the functional polypeptide sequence is one or both of a cyclic RGD sequence and a TAT sequence, which can assist the immuno-drug to enter cells.

14. Use of the aluminum nanocrystal-conjugated immunopharmaceutical of any one of claims 1 to 8 or the aluminum nanocrystal-conjugated immunopharmaceutical prepared by the preparation method of any one of claims 9 to 13 in immunotherapy in tumor models.

15. 15. The use according to claim 14, wherein the use is intravenous injection, mucosal administration, subcutaneous injection, intradermal injection, peritumoral injection, or intratumoral injection.

16. A method for tumor immunotherapy, comprising administering an aluminum nanocrystalline conjugated immunodrug according to any one of claims 1 to 8 or an aluminum nanocrystalline conjugated immunodrug prepared by the preparation method according to any one of claims 9 to 13.

17. 16. The method of claim 15, wherein the mode of administration comprises intravenous injection, mucosal administration, subcutaneous injection, intradermal injection, peritumoral injection, or intratumoral injection.

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