WO2018201981A1 - 免疫磁性组成物及其制备方法、用途和治疗癌症的试剂盒 - Google Patents

免疫磁性组成物及其制备方法、用途和治疗癌症的试剂盒 Download PDF

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WO2018201981A1
WO2018201981A1 PCT/CN2018/084769 CN2018084769W WO2018201981A1 WO 2018201981 A1 WO2018201981 A1 WO 2018201981A1 CN 2018084769 W CN2018084769 W CN 2018084769W WO 2018201981 A1 WO2018201981 A1 WO 2018201981A1
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Prior art keywords
antibody
immunomagnetic composition
composition according
immunomagnetic
tumor
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PCT/CN2018/084769
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English (en)
French (fr)
Inventor
徐伟成
陈三元
江智圣
谢佳宏
林佑融
蔡长海
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中国医药大学
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Priority claimed from CN201810051881.8A external-priority patent/CN108785668B/zh
Application filed by 中国医药大学 filed Critical 中国医药大学
Priority to EP18793787.5A priority Critical patent/EP3636269A4/en
Priority to CA3062089A priority patent/CA3062089C/en
Priority to AU2018262962A priority patent/AU2018262962B2/en
Publication of WO2018201981A1 publication Critical patent/WO2018201981A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a composition and a preparation method thereof, and more particularly to an immunomagnetic composition characterized by a specific physical form, a preparation method thereof, use thereof, and a kit for treating cancer.
  • Cancer also known as malignant tumor, is a state of abnormal cell proliferation, and these proliferating cells may invade other parts of the body, causing diseases caused by abnormal mechanisms controlling cell division and proliferation.
  • the number of people suffering from cancer in the world is increasing. Cancer is one of the top ten causes of death among Chinese people, and it has been the top ten cause of death for 27 consecutive years.
  • Immunotherapy is another method for treating cancer other than the above treatment method, which is to activate the patient's own immune system and use the tumor cells or tumor antigen substances to induce specific cellular and humoral immune responses of the body, thereby enhancing the body's anticancer ability. Prevent tumor growth, spread and recurrence for the purpose of clearing or controlling the tumor.
  • the immune checkpoint is one of the most valued immunotherapy treatments. Since 2015, more than 50 compound therapies using immunological checkpoint inhibitors have been clinically tested. However, immunosuppressive stimulators will shut down the feedback mechanism of the human immune system, allowing cytotoxic T cells (CD8+ T cells) to attack the cancer cells, as well as autoimmune reactions such as skin ulceration and gastrointestinal ulcers.
  • An object of the present invention is to provide an immunomagnetic composition which can be used for preparing an anticancer drug, a preparation method thereof, use thereof, and a kit for treating cancer.
  • an immunomagnetic composition comprising a core layer, a shell layer and an outer layer.
  • the shell layer is composed of a composite layer coated with a core layer, wherein the composite is composed of fucoidan, dextran and superparamagnetic iron oxide nanoparticles by hydrophobic interaction. And formed.
  • the outer layer comprises at least one antibody, and the antibody is grafted outside the shell to form an outer layer, wherein the antibody is an immunoassay inhibitor and/or a killer T cell proliferation agent.
  • the aforementioned immunomagnetic composition may be a sphere, and the particle diameter of the sphere is between 80 nm and 350 nm.
  • the aforementioned fucoidan extract can be extracted from Undaria pinnatifida, Macrocystis pyrifera or Fucus vesiculosus.
  • the oxidized dextran may have an aldehyde group
  • the oxidized dextran may be prepared from dextran having a molecular mass of between 5 kDa and 270 kDa.
  • the aforementioned immunological test stimulator inhibitor may be selected from the group consisting of a PD-L1 antibody, a PD-1 antibody, a CTLA-4 antibody, and a TIM-3 antibody
  • the aforementioned killer T cell proliferation agent may be A group consisting of a CD3 antibody, a CD28 antibody, and a 4-1BB antibody.
  • the core layer may further contain an active material.
  • the immunomagnetic composition of the present invention utilizes fucoidan, oxidized dextran, and superparamagnetic iron oxide nanoparticles having anticancer activity as a carrier component, thereby forming a nanoparticle having an antibody on the outer layer and capable of coating the active material in the core layer.
  • Grade structure can increase the circulation time in the body, and penetrate into the tumor to enhance the effect of fucoidan on tumor.
  • the outer layer of the antibody may be an immunosuppressive stimulator and/or a killer T cell proliferator, so that the immunomagnetic composition of the present invention can simultaneously be an immune check stimulator and/or in addition to the anticancer function of the material itself.
  • the killer T cell proliferation agent greatly improves the microenvironment of the tumor, and the immunomagnetic composition of the present invention can greatly improve the anticancer effect of immunotherapy with the same antibody alone, and can achieve better tumor suppressing ability with less antibody dosage.
  • the manufactured immunomagnetic composition can be stored in a sterilized form by lyophilization to form a powdery crystal for a long period of time, and can be used as it is, if necessary, it can be used as a solvent, and it exhibits convenience and stability.
  • Another object of the present invention is to provide a process for preparing an immunomagnetic composition
  • a process for preparing an immunomagnetic composition comprising the steps of providing an aqueous phase solution, providing an oil phase solution, performing an emulsification reaction, removing an organic solvent in the emulsion, and performing antibody grafting.
  • the aforementioned aqueous phase solution comprises fucoidan and oxidized dextran.
  • the aforementioned oil phase solution contains an organic solvent and superparamagnetic iron oxide nanoparticles.
  • the aqueous phase solution and the aforementioned oil phase solution are first mixed to form an emulsion.
  • a magnetic fucoidan carrier and at least one antibody are mixed to form an immunomagnetic composition, wherein the antibody may be an immunoassay inhibitor and/or a killer T cell proliferation agent.
  • the immunological check-up inhibitor may be selected from the group consisting of a PD-L1 antibody, a PD-1 antibody, a CTLA-4 antibody, and a TIM-3 antibody
  • the aforementioned killer T cell may be selected from the group consisting of a CD3 antibody, a CD28 antibody, and a 4-1BB antibody.
  • the weight ratio of the fucoidan to the oxidized dextran may be from 1:0.1 to 1:4.
  • the oxidized dextran may have an aldehyde group
  • the oxidized dextran may be prepared from dextran having a molecular mass of between 5 kDa and 270 kDa.
  • the organic solvent may be methane, dichloromethane or chloroform.
  • the preparation method of the immunomagnetic composition of the present invention is different from the complicated manufacturing process of the target carrier, and it is not necessary to use an excess surfactant to stabilize the structure, and the material is obtained and produced quite easily.
  • a further object of the present invention is to provide a use of an immunomagnetic composition as described above, which is a medicament for the preparation of an anticancer.
  • the anticancer drug may be a drug that inhibits proliferation of cancer cells, a drug that inhibits cancer metastasis, or a drug that elicits a tumor immune response.
  • It is still another object of the present invention to provide a kit for treating cancer comprising the immunomagnetic composition as described above and a magnetic field generating device.
  • the kit for treating cancer of the present invention comprises the immunomagnetic composition of the present invention and a magnetic field generating device, and the immunomagnetic composition of the present invention can be collectively accumulated by the magnetic field generating device as an auxiliary tool for magnetic guidance.
  • the affected part achieves the effect of local amplification therapy and avoids a systemic immune response.
  • the kit for treating cancer of the present invention has both physical target and biological target action, so that it can exhibit more excellent use only by using one hundredth of the dose of the known pure antibody. The ability of tumor suppression is increased by more than 2 times the half-life.
  • FIG. 1 is a schematic view showing the structure of an immunomagnetic composition of the present invention
  • FIG. 2 is a flow chart showing the steps of a method for preparing an immunomagnetic composition of the present invention
  • 3A to 3F are structural analysis diagrams of a magnetic brown algae polysaccharide carrier
  • 4A to 4C are structural analysis diagrams of the magnetic carrier IO@Fu;
  • 5A and 5B are structural analysis diagrams of a magnetic carrier IO@Dex
  • 6A to 6C are diagrams showing the results of stability analysis of a magnetic brown algae polysaccharide carrier
  • 7A and 7B are transmission electron micrographs of the magnetic brown algae polysaccharide carrier before and after lyophilization
  • Figure 8 is a structural analysis diagram of an embodiment of the immunomagnetic composition of the present invention.
  • Figure 9 is a graph showing the results of X-ray photoelectron spectroscopy (XPS) analysis of an embodiment of the immunomagnetic composition of the present invention.
  • FIGS. 10A and 10B are structural analysis views of another embodiment of the immunomagnetic composition of the present invention.
  • FIGS. 11A to 11F are diagrams showing analysis results of the target ability and cell binding ability of the immunomagnetic composition of the present invention.
  • 12A to 12D are diagrams showing the results of analysis of the cell binding ability of the immunomagnetic composition of the present invention.
  • FIGS. 13A to 13C are diagrams showing the results of analysis of the accumulation of tumors in a kit for treating cancer in the present invention.
  • Figure 13D is a graph showing the results of hematoxylin-eosin staining and Prussian blue staining of the immunomagnetic composition of the present invention and the therapeutic effect of the kit for treating cancer;
  • 14A to 14E are diagrams showing the results of analysis of inhibition of cancer cell proliferation and cancer metastasis in a mouse model of breast cancer lung metastasis by the immunomagnetic composition of the present invention and a kit for treating cancer;
  • 15A to 15C are diagrams showing the results of analysis of an immunomagnetic composition of the present invention and a kit for treating cancer in inhibiting cancer cell proliferation in a mouse model of colorectal cancer;
  • FIG. 16A to FIG. 16I are diagrams showing analysis results of changes in the number of tumor-infiltrating lymphocytes and changes in cytokine content in a tumor microenvironment after administration of the immunomagnetic composition of the present invention and a kit for treating cancer;
  • 17A and 17B are diagrams showing the results of analysis of reaction sites of the immunomagnetic composition of the present invention and a kit for treating cancer;
  • 17C and 17D are diagrams showing the results of TUNEL detection of the immunomagnetic composition of the present invention and a kit for treating cancer;
  • Fig. 18A to Fig. 18E are graphs showing the degree of infiltration of mouse CD4+ T cells and CD8+ T cells after administration of the immunomagnetic composition of the present invention and a kit for treating cancer;
  • 19A to 19D are diagrams showing results of blood biochemical analysis of mice after administration of the immunomagnetic composition of the present invention and a kit for treating cancer;
  • Figure 20 is a pathological section diagram of a mouse after administration of the immunomagnetic composition of the present invention and a kit for treating cancer.
  • the disclosure of the present specification provides a novel immunomagnetic composition obtained by combining a fucoidan polysaccharide and an oxidized dextran with a hydrophobic force and superparamagnetic iron oxide nanoparticles, and then grafting the antibody to obtain an immunomagnetic property.
  • the composition can greatly improve the anticancer effect of immunotherapy with the same antibody alone, and can achieve better tumor inhibition ability with less antibody dosage.
  • the present specification also discloses a novel kit for treating cancer, which comprises the immunomagnetic composition of the present invention and a magnetic field generating device, which can further enhance the anticancer effect of the immunomagnetic composition of the present invention.
  • the animal model of breast cancer lung metastasis and colorectal cancer is used in the specification to verify the efficacy and mechanism of the immunomagnetic composition of the present invention and the kit for treating cancer in immunotherapy.
  • Fucoidan in the specification is a water-soluble dietary fiber extracted from the unique slip-slip component of the brown seaweed surface. Fucoidan is rich in fucose and is a natural polysaccharide with high biosafety and various biological activities such as anti-oxidation, anti-coagulation, anti-thrombosis, anti-virus and anti-cancer.
  • the aforementioned "dextran” in the specification is a complex and branched dextran (a polysaccharide composed of a plurality of glucose molecules) which may have a molecular mass ranging from 3 Da to 2000 kDa.
  • the linear portion of dextran is composed of glucose molecules linked together by ⁇ -1,6 glycosidic bonds, and the branches are extracted by ⁇ -1,3 glycosidic bonds.
  • oxidized dextran is a surface modification of dextran to oxidize a hydroxyl group on dextran to an aldehyde group, thereby obtaining an oxidized dextran which can further graft an antibody.
  • FIG. 1 shows a schematic diagram of the immunomagnetic composition 100 of the present invention.
  • the immunomagnetic composition 100 includes a core layer 110, a shell layer 120, and an outer layer 130.
  • the core layer 110 may contain an active substance, and the active substance may be a cytokine or an anticancer drug.
  • the shell layer 120 is composed of a composite layer, and the shell layer 120 is coated with the core layer 110, wherein the composite is formed by a combination of hydrophobic interaction of fucoidan, oxidized dextran and superparamagnetic iron oxide nanoparticles.
  • the fucoidan used in the composite constituting the shell layer 120 can be extracted from Undaria pinnatifida, Macrocystis pyrifera or Fucus vesiculosus, and the oxidized dextran used can be used. It has an aldehyde group and it can be prepared from dextran having a molecular mass of between 5 kDa and 270 kDa.
  • the hydrophobic interaction between the brown algae polysaccharide, the oxidized dextran, and the superparamagnetic iron oxide nanoparticles can be formed by a method such as emulsification or nanoprecipitation, but the invention is not intended to be limited thereto.
  • the outer layer 130 comprises at least one antibody 131, and the antibody 131 is grafted outside the shell layer 120 to form an outer layer 130.
  • the antibody 131 may be an immunological check stimulator and/or a killer T cell proliferator, and the immunosuppressive stimulator may be selected from the group consisting of a PD-L1 antibody, a PD-1 antibody, a CTLA-4 antibody and a TIM-3 antibody.
  • the cohort T-cell proliferative agent can be selected from the group consisting of a CD3 antibody, a CD28 antibody, and a 4-1BB antibody.
  • the aforementioned immunomagnetic composition 100 may be a sphere, and the sphere has a particle diameter of between 80 nm and 350 nm. Further, the aforementioned immunomagnetic composition has a hollow shape.
  • a flow chart of the steps of the method 300 for preparing an immunomagnetic composition of the present invention is shown.
  • a method 300 for preparing an immunomagnetic composition includes step 310, step 320, step 330, step 340, and step 350.
  • Step 310 is to provide an aqueous phase solution comprising fucoidan and oxidized dextran, wherein the fucoidan extract can be extracted from Undaria pinnatifida, Macrocystis pyrifera or Fucus vesiculosus.
  • the oxidized dextran used may have an aldehyde group which can be prepared from dextran having a molecular mass of between 5 kDa and 270 kDa.
  • the brown algae polysaccharide and the oxidized dextran are mixed in a weight ratio of 1:0.1 to 1:4.
  • Step 320 is to provide an oil phase solution comprising an organic solvent and superparamagnetic iron oxide nanoparticles, wherein the organic solvent may be methane, dichloromethane or chloroform.
  • Step 330 is to carry out an emulsification reaction, and the aqueous phase solution provided in step 310 and the oil phase solution provided in step 320 are mixed to form an emulsion.
  • the organic solvent in the emulsion may be removed by evaporation or the like to form a magnetic fucoidan carrier.
  • step 350 antibody grafting is performed, and the magnetic fucoidan vector and at least one antibody are grafted to form an immunomagnetic composition.
  • the antibody used may be an immunosuppressive stimulator and/or a killer T cell proliferator, wherein the immunosuppressive stimulator may be selected from the group consisting of PD-L1 antibody, PD-1 antibody, CTLA-4 antibody and TIM-3 antibody.
  • the killer T cell proliferation agent can be selected from the group consisting of a CD3 antibody, a CD28 antibody, and a 4-1BB antibody.
  • the immunomagnetic composition prepared by the above method can be used as an anticancer drug in the subsequent use, for example, as a drug for inhibiting cancer cell proliferation, a drug for inhibiting cancer metastasis, and a drug for eliciting a tumor immune response.
  • the immunomagnetic composition prepared by the above method has a hollow shape, so that the active substance can be further coated on the core layer to enhance the anticancer effect of the immunomagnetic composition.
  • the immunomagnetic composition prepared by the foregoing method may be combined with a magnetic field generating device to constitute a kit for treating cancer
  • the magnetic field generating device may be a device capable of generating a magnetic field such as a magnet, a three-dimensional field magnet or a magnetic vibrometer.
  • the magnetic field generated by the magnetic field generating device is used as an auxiliary tool for magnetic guidance, and the immunomagnetic composition of the present invention is concentrated and accumulated in the affected part to achieve the effect of local amplification treatment, so that the kit for treating cancer of the present invention only needs to be used. It is known that one-hundredth of the dose of pure antibody is able to exhibit superior tumor inhibition ability and increase the half-life of more than two times.
  • a magnetic fucoidan carrier of an ungrafted antibody was prepared to test the optimal preparation conditions, and a scanning electron microscopy (SEM) and a transmission electron microscopy (TEM) were used. The morphology of the magnetic brown algae polysaccharide carrier was observed. Analysis of the zeta potential, particle size and magnetic fucoidan carrier in secondary distillation (DDW) and phosphate buffer of magnetic brown algae polysaccharide carrier using a nano-particle size and interface potential analyzer (Delsa Nano C particle analyzer, BECKMAN COULTER) Stability in solution (PBS).
  • DDW secondary distillation
  • phosphate buffer of magnetic brown algae polysaccharide carrier using a nano-particle size and interface potential analyzer (Delsa Nano C particle analyzer, BECKMAN COULTER) Stability in solution (PBS).
  • the superparamagnetic iron oxide nanoparticles and the oxidized dextran were prepared separately, and the oxidized dextran used in the test example has an aldehyde group to facilitate subsequent grafting of the antibody.
  • Design synthesis of superparamagnetic iron oxide nanoparticles (hereafter referred to as "IO").
  • IO superparamagnetic iron oxide nanoparticles
  • NMR Nuclear Magnetic Resonance
  • the magnetic fucoidan carrier was prepared under the following conditions: 0.5 mg/ml of fucoidan (extracted from Fucus sinensis) and 0.5 mg/ml of Dex were mixed as an aqueous phase solution. 2 mg of IO was dissolved in 0.2 ml of dichloromethane as an oil phase solution. The aqueous phase solution and the oil phase solution were mixed, and then emulsified by a homogenizer (Double Eagle Enterprise Co., Ltd.) at a power of 120 W for 50 seconds to obtain an emulsion.
  • a homogenizer Double Eagle Enterprise Co., Ltd.
  • the obtained magnetic brown algae polysaccharide carrier (hereinafter referred to as "IO@FuDex”) was purified by a magnetic separation apparatus (MagniSort, eBioscience), and IO@FuDex was DDW or 0.1.
  • IO@FuDex 0.5 mg/ml of fucoidan or 0.5 mg/ml of Dex was used as the aqueous phase solution, and IO dissolved in dichloromethane was used as the oil phase solution, and the magnetic carrier IO@ was prepared by the same preparation method.
  • IO@FuDex aqueous phase solution is brown algae polysaccharide
  • IO@Dex aqueous phase solution is oxidized dextran having an aldehyde group.
  • the prepared IO@FuDex, IO@Fu and IO@Dex are analyzed by scanning electron microscopy and transmission electron microscopy; the interface potential and particle size are analyzed by nanometer particle size and interface potential analyzer; and superconducting is utilized.
  • a quantum interference magnetometer is used for magnetic analysis.
  • FIG. 3A to 3C, FIG. 3A to FIG. 3F are structural analysis diagrams of a magnetic brown algae polysaccharide carrier, wherein FIG. 3A is a scanning electron microscope photograph of IO@FuDex, and FIG. 3B to FIG. 3F are IO@FuDex penetration. Electron micrograph photo. 4A to 4C are structural analysis views of the magnetic carrier IO@Fu, wherein FIGS. 4A and 4B are scanning electron microscope photographs of IO@Fu, and FIG. 4C is a transmission electron microscope photograph of IO@Fu. 5A and 5B are structural analysis views of the magnetic carrier IO@Dex, wherein FIG. 5A is a scanning electron microscope photograph of IO@Dex, and FIG.
  • FIG. 5B is a transmission electron microscope photograph of IO@Dex.
  • 6A to 6C are diagrams showing the results of stability analysis of the magnetic brown algae polysaccharide carrier, wherein FIG. 6A is a hydrodynamic size distribution diagram of IO@FuDex, IO@Fu, and IO@Dex, and FIG. 6B is IO@FuDex, IO@Fu, and The interface potential analysis diagram of IO@Dex, Figure 6C is the magnetic analysis diagram of IO@FuDex and IO.
  • Fig. 3A show that, by observation by a scanning electron microscope, it can be seen that IO@FuDex has a spherical structure and collapses due to the hollow environment of the electron microscope, which represents a hollow structure. From the results of Fig. 3B, the dark contrast caused by the overlapping layers of IO@FuDex due to collapse can be observed by a transmission electron microscope.
  • Fig. 3C is a transmission electron micrograph showing the enlargement of the shell layer, and many superparamagnetic iron oxide nanoparticles having a size of about 5 nm can also be seen, which proves to be a structure of the shell layer.
  • the apparent hollow structure and the condition in which the iron oxide is distributed in the carrier can be seen again.
  • IO@FuDex exhibits a uniform particle size in the fluid, a particle size between 80 nm and 350 nm, and an average particle size of 141.5 nm, which is less than the average particle size of IO@Fu ( 241nm).
  • IO@FuDex and IO@Fu are composed of brown algae polysaccharides, which contain sulphate which gives IO@FuDex and IO@Fu a strong negative interface potential, but because of the Dex in the body of IO@FuDex, The negative interface potential of IO@FuDex (-32.8mV) is lower than the negative interface potential of IO@Fu (-58.4mV), so the strong repulsive force between IO@FuDex maintains the stability and good dispersion of the colloid. From the results of FIG.
  • the superparamagnetic iron oxide nanoparticles (10) have a relatively high saturation magnetization per unit weight
  • IO@FuDex contains superparamagnetic iron oxide nanoparticles, fucoidan, and oxidized dextran. The value is slightly decreased, but still retains a high value of 57.5 emu g -1 , indicating that the magnetic strength is not lowered by the formation of the composite support structure, which facilitates magnetic purification using a magnetic separation device to obtain a high-yield IO@FuDex.
  • a magnetic fucoidan polysaccharide carrier is prepared by using different molecular weight oxidized dextran, which is prepared by using dextran having a molecular weight of 5 kDa to 270 kDa to prepare oxidized dextran, and then oxidizing dextran and brown algae in the aqueous phase solution provided in step 310 of FIG.
  • the polysaccharides were mixed in a weight ratio of 1:0.2, and the details of the remaining steps 320 to 340 were substantially the same as those described in Test Example 1.1, and are not described herein again.
  • the particle size of the obtained magnetic brown algae polysaccharide carrier was analyzed by nanometer particle size and interface potential analyzer. The results are shown in Table 1 below:
  • a magnetic fucoidan polysaccharide carrier is prepared by mixing different ratios of fucoidan and oxidized dextran, which is provided in the aqueous phase solution as shown in step 310 of FIG. 2, and the fucoidan and dextran are from 1:0.1 to 1:4.
  • the weight ratio is mixed, and the details of the remaining steps 320 to 340 are substantially the same as those described in Test Example 1.1, and are not described herein again.
  • the particle size of the obtained magnetic brown algae polysaccharide carrier was analyzed by nanometer particle size and interface potential analyzer. The results are shown in Table 2 below:
  • Fucoidan dextran (weight ratio) Average particle size (nm) 1:0.1 145 ⁇ 6.9 1:0.2 153 ⁇ 11.6 1:1 130 ⁇ 9.8 1:2 141 ⁇ 16.2 1:3 162 ⁇ 23.6 1:4 176 ⁇ 26.8
  • the prepared IO@FuDex was further freeze-dried to form powdery crystals by using a freeze dryer, and then the crystals after lyophilization were dissolved.
  • the structure of IO@FuDex before and after lyophilization was observed in an aqueous solution using a transmission electron microscope.
  • FIG. 7A is a photomicrograph of a transmission electron microscope before lyophilization of IO@FuDex
  • FIG. 7B is a photomicrograph of a transmission electron microscope after lyophilization of IO@FuDex.
  • the results show that the structure before lyophilization of IO@FuDex is a hollow sphere with a size of between 80 nm and 350 nm.
  • the crystals after lyophilization can be quickly dissolved back into the aqueous solution, and the IO@FuDex structure which is re-dissolved under the transmission electron microscope remains a hollow sphere which is the same size as the carrier before lyophilization.
  • the stability of IO@FuDex is excellent.
  • the immunomagnetic composition of the present invention was further prepared under the conditions described above for optimal preparation of IO@FuDex.
  • the Schiff base is formed with the primary amine on the antibody, and after the reductive amination reaction using sodium cyanoborohydride, the antibody is grafted to a chemically stable state.
  • the obtained immunomagnetic composition was purified using a magnetic separation apparatus.
  • Example 1-3 of the present invention wherein the antibody used in Example 1 is a CD3 antibody and a CD28 antibody, and the antibody used in Example 2 is a PD-L1 antibody, and Examples The antibodies used were the PD-L1 antibody, the CD3 antibody, and the CD28 antibody.
  • the prepared examples were characterized by morphological and elemental analysis by transmission electron microscopy and X-ray photoelectron spectroscopy (XPS) to determine the elemental composition in Example 3.
  • XPS X-ray photoelectron spectroscopy
  • FIG. 8 is a structural analysis diagram of Embodiment 3.
  • the results show that the structure of Example 3 is a spherical hollow structure having a size of about 80 to 350 nm. Elemental analysis by electron microscopy showed that the outer layer of Example 3 showed a clear nitrogen signal (N) uniformly due to the presence of antibodies.
  • the carrier component is only sugar and iron oxide, and there is no nitrogen-bearing material; however, after grafting the antibody, since the antibody has many amine groups and peptide bonds, and has a large amount of nitrogen, it can be proved
  • the immunomagnetic composition of the invention carries an antibody.
  • Example 3 corresponds to IO@FuDex with a signal with a bond energy of 399 eV, and this signal is a nitrogen signal, which indicates that in Example 3, the antibody was successfully grafted onto the carrier. .
  • an active substance such as a cytokine or an anticancer drug can be further coated in the core layer.
  • Interleukin-2 IL-2
  • Example 5 coated with IL-2.
  • Example 5 the preparation conditions of Example 5 were as follows: 0.5 mg/ml of fucoidan, 0.5 mg/ml of Dex, and 50 ⁇ g of IL-2 were mixed as an aqueous phase solution. 2 mg of IO was dissolved in 0.2 ml of dichloromethane as an oil phase solution. The aqueous phase solution and the oil phase solution were mixed, and then emulsified by a homogenizer at a power of 120 W for 50 seconds to obtain an emulsion. After removing the dichloromethane using a rotary evaporator, the obtained Example 5 was purified by a magnetic separation apparatus. The prepared examples were analyzed by scanning electron microscopy and analyzed for particle size using a nanoparticle size and interface potential analyzer.
  • FIG. 10A is a scanning electron microscope photograph of the embodiment 5
  • FIG. 10B is a hydrodynamic size distribution diagram of the embodiment 5.
  • the results showed that the IL-2 coating ratio of Example 5 was as high as 99.93%.
  • the average particle size of Example 5 is increased from 161.2 nm to 356.2 nm of the pure carrier, demonstrating that the immunomagnetic composition of the present invention can further coat the active material in the core layer.
  • Active substances such as Doxorubicin, Paclitaxel (Dcetaxel or Paclitaxel) and Astaxanthin (ASTX) can also be coated in the immunomagnetic composition of the present invention in the same manner.
  • the prepared Examples 1-3 were subjected to standard capacity analysis and cell binding ability analysis to verify whether the immunomagnetic composition of the present invention can reverse the decrease in T cell immunity and promote tumor regression in mice.
  • a triple negative breast cancer cell line (4T1) with metastatic ability, invasiveness and expression of PD-L1 was selected as an experimental model.
  • quantum dots were attached to Examples 1-3 of the fluorescence microscope analysis group, respectively.
  • the 1-3 and 4T1 cell lines (4 ⁇ 10 5 ) were incubated in a medium containing bovine serum albumin (BSA) for 30 minutes at 4 ° C, respectively, using flow cytometry. (Novocyte Flow Cytometer, ACEA Biosciences) for analysis.
  • BSA bovine serum albumin
  • IO@FuDex with quantum dots and Example 2 were incubated with 4T1 cell lines for 1, 4, 12, and 24 hours, respectively, using flow cytometry or fluorescence microscopy (Carl Zeiss, Thornwood). Analyze.
  • 11A to 11F are diagrams showing the results of analysis of the target ability and cell binding ability of the immunomagnetic composition of the present invention.
  • 11A is an in vitro target of a PD-L1 antibody having a grafting concentration of 1.4 ⁇ g/ml (low concentration; L), 7 ⁇ g/ml (medium concentration; M), and 35 ⁇ g/ml (high concentration; H). The test results of the ability.
  • Figure 11B is a test result of the in vitro target ability of Example 2 and grafted IgG of IO@FuDex.
  • Figure 11C is a graph showing the results of analysis of the binding ability of IO@FuDex to the 4T1 cell line at 1, 4, 12 and 24 hours.
  • Figure 11D is a graph showing the results of analysis of the ability of Example 2 to bind to a 4T1 cell line at 1, 4, 12 and 24 hours.
  • Figure 11E shows Example 3 of a CD3 antibody/CD28 antibody with a graft concentration of 1.4 ⁇ g/ml (low concentration; L), 7 ⁇ g/ml (medium concentration; M) and 35 ⁇ g/ml (high concentration; H) in vitro. The test results of the ability.
  • Fig. 11F is a graph showing the results of analysis of the binding ability of Example 2 and Example 3 to the 4T1 cell line at the 1st and 24th hours.
  • Figures 11C and 11D show that the amount of IO@FuDex bound to the cells is time-dependent, with significant cellular uptake after the 12th hour after incubation.
  • Example 2 had a phenomenon of cell binding in the first hour after the incubation, and then the MDI showed a slow shift, indicating that the binding amount of Example 2 to the cells increased as the incubation time increased, and the result showed Grafting PD-L1 antibody to IO@FuDex changed the binding ability of the original IO@FuDex and 4T1 cell lines.
  • Example 3 is an immunomagnetic composition obtained by simultaneously grafting PD-L1 antibody, CD3 antibody and CD28 antibody to IO@FuDex. .
  • FIG. 11E the target ability analysis of Example 3 for grafting different concentrations of CD3 antibody/CD28 antibody, the result of FIG. 11E shows that the group of Example 3 (H) has a group after 1 hour of incubation in vitro. The highest MDI, showing that Example 3 (H) group has strong affinity to CD8 + T cells. While the results of Figure 11F show that Example 3 and the 4T1 cell line have similar binding to Example 2, it was shown that grafting multiple antibodies on IO@FuDex did not affect their affinity for the 4T1 cell line.
  • FIGS. 12A to 12D are diagrams showing the results of analysis of the binding ability of Example 3 to CD8 + T cells.
  • Experiment 3 was incubated with CD8 + T cells for 30 minutes with quantum dots, CD8 + T cells were fixedly stained with Alexa Fluor 488 Phalloidin and DAPI, and Example 3 was confirmed to be located in CD8 + T cells using a conjugated focal microscope.
  • Subcellular location. 12A shows the position of the nucleus
  • FIG. 12B shows the position of Example 3
  • FIG. 12C shows the photomicrograph of the bright field
  • FIG. 12D shows the photomicrograph of the combined photo, wherein the scale bar indicates the length of 5 ⁇ m.
  • the results show that a plurality of Example 3 can be seen in CD8 + T cells, demonstrating that Example 3 does bind to CD8 + T cells.
  • This test case further tests whether the immunomagnetic composition of the present invention has an effect of treating cancer in a mouse model of breast cancer lung metastasis and colorectal cancer, and whether it can be accumulated in the affected part via magnetic guidance.
  • Example 4 the kit for treating cancer of the present invention of the present invention was tested, and Example 4 was administered.
  • the formulation of Example 3 of 125 I was labeled and magnetically guided using a magnet having a circular surface (0.5 cm in diameter, 0.5 Tesla). Dynamic accumulation in tumor tissues of Example 3 and Example 4 was subsequently monitored using single photon emission computed tomography (SPECT).
  • SPECT single photon emission computed tomography
  • FIGS. 13A to 13C are diagrams showing the results of analysis of the accumulation of tumors in a kit for treating cancer according to the present invention.
  • Figure 13B is a whole body single photon emission computed tomography scan of the 4T1-Luc tumor mouse at 24 hours after administration of Example 3 or Example 4 labeled 125 I.
  • Example 3 and Example 4 accumulate mainly in tumor, liver, spleen and bladder, while less accumulated in muscle, brain, heart, lung, stomach, kidney, colon and blood (less than 5% ID/g). Comparing Example 3 with Example 4, it can be seen that Example 4 not only increases the accumulation in the tumor, but also significantly reduces its systemic accumulation in the liver and spleen, showing that Example 4 can be effectively concentrated to the site of action.
  • This test example was further administered via IgG (control), IO@FuDex, and 1-4 to 4T1-Luc tumor mice via the right femoral vein, respectively, 8 days after tumor inoculation, and 3 consecutive administrations every 4 days. (q4dx3).
  • Figure 13D the results of hematoxylin-eosin staining with Prussian blue staining for 4 weeks after application of Examples 1-4 for 4T1-Luc tumor mice, wherein the scale bar indicates a length of 50 ⁇ m.
  • the results of Fig. 13D show that the dispersed Prussian blue staining results were observed in the tumor tissues of Example 4 as compared with the other groups.
  • this test example further applies IgG (control group), IO@FuDex via the right femoral vein, respectively, and examples.
  • IgG control group
  • IO@FuDex via the right femoral vein, respectively
  • examples in 1-4 to 4T1-Luc tumor mice, the administration manner was 8 days after tumor inoculation, and administration was continued 3 times every 4 days (q4dx3).
  • the tumor volume was monitored using a digital caliper (mitutoyo) every 2-3 days and the tumor volume was calculated using the following formula I:
  • Biological cold light evaluation was performed using a non-invasive living molecular imaging system (Non Invasion In Vivo Imaging System, IVIS, Xenogen).
  • IVIS Intravection In Vivo Imaging System
  • Xenogen The survival rate of 4T1-Luc tumor mice was analyzed by the Kaplan-Meier method.
  • FIGS. 14A to 14E are diagrams showing the results of analysis of inhibition of cancer cell proliferation and cancer metastasis in a mouse model of breast cancer lung metastasis by the immunomagnetic composition of the present invention and a kit for treating cancer.
  • Figure 14A is a graph showing the results of a non-invasive live molecular imaging system scan of 4T1-Luc tumor mice 24 hours after administration of Examples 1-4.
  • Figure 14B is a graph of tumor volume statistics of 4T1-Luc tumor mice after administration of Examples 1-4.
  • Figure 14C is a survival curve of 4T1-Luc tumor mice after administration of Examples 1-4.
  • Figure 14D is a photographic representation of tumors and lungs of 4T1-Luc tumor mice after administration of Examples 1-4.
  • Figure 14E is a statistical diagram of lung metastasis of 4T1-Luc tumor mice after administration of Examples 1-4.
  • FIGS. 14A and 14B show that IO@FuDex inhibits tumor growth compared to the control group, but does not have statistically significant differences. While Examples 1-4 had not only anti-tumor effects, but also statistically significant differences (* indicates p ⁇ 0.05, ** indicates p ⁇ 0.01), and in particular, Example 4 almost inhibited tumor growth within 30 days.
  • the results of Figure 14C show that the median survival of the 4T1-Luc tumor mice administered the control group, IO@FuDex, Example 1, Example 2, Example 3, and Example 4 were 24 days and 34 days, respectively. 34 days, 43 days and 44 days. The median survival of the 4T1-Luc tumor mice administered Example 4 was significantly extended to 63 days.
  • the PD-L1 antibody dose in Example 4 showed a superior tumor suppressing ability and a half-life of more than 2 times, although it was only one percent of the general dose of the pure antibody. Furthermore, the results of Figure 14D show that Examples 1-4 all have anti-tumor metastatic ability. In Fig. 14D, administration of Example 3 and Example 4 significantly inhibited lung tumor metastasis compared to the other groups, and the results of Fig. 14E showed that the control 4T1-Luc tumor mice had more than 20 lungs. Lung metastases were metastasized, whereas lungs of less than 5 nodules were found on average in the lungs of the 4T1-Luc tumor mice administered Example 4.
  • the above results show that the immunomagnetic composition of the present invention and the kit for treating cancer have remarkable tumor suppressing and antitumor metastasis effects.
  • IO@FuDex did not significantly reduce tumor metastasis, and it is speculated that the immunomagnetic composition of the present invention and the kit for treating cancer have an anti-tumor metastatic effect, in addition to the inhibition of fucoidan, It is also related to the dynamic response of the tumor microenvironment.
  • a CT-26 cell line with a luciferase gene stably expressing a biological luminescent enzyme was used to establish an animal model of colorectal cancer (provided by the Institute of Molecular Medicine of the Hospital of China Medical University), and IgG (control group) IO@FuDex and Example 3 were administered to CT-26 tumor mice via the right femoral vein, respectively, and the experiment was additionally applied to Example 3 and used with a round magnet (0.5 cm in diameter, 0.5 Tesla).
  • Magnetically guided Example 4 (a kit for treating cancer of the present invention); and Comparative Example 1 in which IO@FuDex was applied and magnetically guided using a magnet having a circular surface.
  • the mode of administration was 8 days after tumor inoculation, and 3 consecutive administrations every 4 days (q4dx3).
  • Tumor volume monitoring was performed every 2-3 days using a digital caliper, and the tumor volume was calculated using the aforementioned Formula I, and bioluminescence evaluation was performed using a non-invasive live molecular imaging system. Survival rates of CT-26 tumor mice were analyzed by Kaplan-Meier method.
  • FIGS. 15A to 15C are diagrams showing the results of analysis of an immunomagnetic composition and a kit for treating cancer in a mouse model of colorectal cancer in which a cancer cell proliferation is inhibited.
  • Figure 15A is a graph showing the results of a non-invasive live molecular imaging system scan of CT-26 tumor mice 24 hours after administration of Examples 3-4.
  • Figure 15B is a graph of tumor volume statistics of CT-26 tumor mice after administration of Examples 3-4.
  • Figure 15C is a survival curve of CT-26 tumor mice after administration of Examples 3-4.
  • Figures 15A and 15B show that IO@FuDex can inhibit tumor growth compared to the control group, but there is no statistically significant difference.
  • the comparison of Example 1 with IO@FuDex and magnetic guidance can improve the therapeutic effect of IO@FuDex.
  • Examples 3-4 of the present invention not only had an antitumor effect, but also had statistically significant differences (* indicates p ⁇ 0.05, ** indicates p ⁇ 0.01), and in particular, Example 4 almost inhibited tumor growth within 30 days.
  • the results of Fig. 15C show that administration of Example 3 and Example 4 significantly prolonged the survival time of the mouse, again demonstrating that the immunomagnetic composition of the present invention has an effect of inhibiting tumor growth and metastasis.
  • This test case further investigates the immunological effects of the immunomagnetic composition of the present invention and a kit for treating cancer in a tumor microenvironment in a 4T1 tumor model. Changes in the number of lymphocytes in tumor, blood, ascites, and spleen of 4T1-Luc tumor mice from early tumor (10 days) to advanced (30 days) were experimentally monitored.
  • 16A to 16I are graphs showing the results of analysis of changes in the number of tumor-infiltrating lymphocytes and changes in cytokine content in the tumor microenvironment after administration of the immunomagnetic composition of the present invention and a kit for treating cancer.
  • the results of Figures 16A and 16B show that administration of the groups of Examples 1-4 can significantly increase the number of anti-tumor lymphocytes, such as CD8 + T cells and CD4 + T cells, particularly the group administered with Example 4 ( p ⁇ 0.01).
  • the content of pro-inflammatory cytokines such as TNF- ⁇ , VEGF and TGF- ⁇ can be significantly reduced, in particular, the group of Example 4 is administered (p ⁇ 0.01).
  • the degree of activation of CD8 + T cells was evaluated by the level of expression of intracellular granzyme B (GrB + ) and Ki67, and it can be seen that the administration of the groups of Examples 1-4 can effectively activate CD8 + .
  • the function of T cell tumor infiltrating lymphocytes, particularly the group administered in Example 4 (p ⁇ 0.01).
  • this test example further explores the immunomagnetic composition of the present invention and a kit for treating cancer for a tumor.
  • Specific immune responses and changes in systemic effects In the experiment, tumors, serum and spleens of different groups of 4T1-Luc tumor mice were collected, and changes of INF- ⁇ + CD44 + T cells and CD8 + CD3 + T cells were monitored, and different groups of 4T1- were observed by TUNEL assay. Apoptosis status of skin tissue of Luc tumor mice.
  • FIGS. 17A and 17B are diagrams showing the results of analysis of reaction sites of the immunomagnetic composition of the present invention and a kit for treating cancer.
  • Figure 17A is a graph showing the results of changes in INF- ⁇ + CD44 + T cells of different groups of 4T1-Luc tumor mice.
  • Figure 17B is a graph showing the results of changes in CD8 + CD3 + T cells of different groups of 4T1-Luc tumor mice.
  • Fig. 17A show that the amount of INF- ⁇ + CD44 + T cells increased after administration of Examples 1-4, particularly the group administered with Example 4 was highly proliferated. While the results of Fig. 17B show that CD8 + CD3 + T cells were amplified in both serum and spleen after administration of Examples 1-3, the group administered with Example 4 was compared with the group to which Example 3 was administered, Magnetic guidance reduces the expansion of CD8 + CD3 + T cells in serum and spleen.
  • the pruritus and leukoplakia caused by dermal toxicity is one of the side effects in the treatment of immunosuppressive snoring inhibitors. Since immune-related adverse events often occur in the long-term, this test case is 4T1-4 weeks after tumor inoculation. TUNEL assays were performed on skin tissue of Luc tumor mice to assess whether infiltrating T cells induced an immune response and caused skin tissue damage.
  • FIGS. 17C and 17D are diagrams showing the results of TUNEL detection of the immunomagnetic composition of the present invention and a kit for treating cancer.
  • Figure 17C is a graphical representation of TUNEL detection of skin tissue apoptotic index in different groups of 4T1-Luc tumor mice, in which the apoptotic index is divided by the proportion of TUNEL + apoptotic nuclei divided by the total number of nuclei (randomly selected microscopic range). Differences between the different groups were assessed by two-factor analysis of variance and Newman-Keuls post hoc comparison test.
  • Figure 17D is a fluorescence micrograph of TUNEL detection of different groups of 4T1-Luc tumor mice, wherein the scale indicates a length of 50 ⁇ m.
  • Fig. 17C and Fig. 17D show that after administration of Examples 1-3, apoptotic cells which can be labeled with TUNEL (green) were increased, wherein the group induced by the administration of Example 3 increased the apoptotic index compared with the control group. 3.3 times. However, the magnetic guidance can effectively reduce the number of apoptotic cells in the skin tissue compared to the group administered with Example 3 and the group of Example 3.
  • This test example was further applied to a 4T1 tumor mouse by administering the immunomagnetic composition of the present invention or a kit for treating cancer, and Example 3 or Example 4 was administered to 4T1 tumor mice experimentally, and 4 weeks after tumor inoculation. 4T1 tumor mice were tested for Immune-related adverse events (irAEs) to analyze the safety of the immunomagnetic composition of the present invention and a kit for treating cancer.
  • irAEs Immune-related adverse events
  • FIG. 18A to FIG. 18E for the analysis of the degree of infiltration of mouse CD4 + T cells and CD8 + T cells after administration of the immunomagnetic composition of the present invention and a kit for treating cancer, wherein FIG. 18A is an analysis result of the liver.
  • Fig. 18B is a graph showing the results of analysis of the lungs
  • Fig. 18C is a graph showing the results of analysis of the spleen
  • Fig. 18D is a graph showing the results of analysis of the kidney
  • Fig. 18A is an analysis result of the liver.
  • Fig. 18B is a graph showing the results of analysis of the lungs
  • Fig. 18C is a graph showing the results of analysis of the spleen
  • Fig. 18D is a graph showing the results of analysis of the kidney
  • 18E is a graph showing the results of analysis of the large intestine. The results showed that the degree of T cell infiltration in the liver, lung, spleen, kidney and large intestine of Example 3 was lower than that of the control group, and Example 4 was more significantly reduced in peripheral accumulation due to the attraction of magnets (liver, lung) , spleen, kidney and large intestine), thereby reducing the infiltration of T cells.
  • FIGS. 19A to 19D are diagrams showing the results of blood biochemical analysis of mice after administration of the immunomagnetic composition of the present invention and a kit for treating cancer, wherein FIG. 19A is an AST value analysis diagram, and FIG. 19B is an ALT value analysis diagram. 19C is a graph of creatinine value analysis, and FIG. 19D is a graph of blood glucose level analysis.
  • FIG. 19A is an AST value analysis diagram
  • FIG. 19B is an ALT value analysis diagram
  • 19C is a graph of creatinine value analysis
  • FIG. 19D is a graph of blood glucose level analysis.
  • the biochemical value of the treatment group administered with Example 3 or Example 4 and the control group remained within the range of normal values, indicating that the immunomagnetic composition of the present invention and the kit for treating cancer have certain The degree of security.
  • the preparation method of the immunomagnetic composition of the present invention is simple in process, and the fucoidan polysaccharide having anticancer activity is used as a carrier component, and the superparamagnetic iron oxide nanoparticles are combined to form an outer layer graftable antibody and can be coated with an activity.
  • the immunomagnetic composition of the substance in the core layer is a nano-scale structure which is sized to penetrate into the tumor and enhance the effect of the fucoidan on the tumor.
  • the outer layer of the antibody may be an immunosuppressive stimulator and/or a killer T cell proliferator, so that the immunomagnetic composition of the present invention can simultaneously be an immune check stimulator and/or in addition to the anticancer function of the material itself.
  • the killer T cell proliferation agent greatly improves the microenvironment of the tumor, and the immunomagnetic composition of the present invention can greatly improve the anticancer effect of immunotherapy with the same antibody alone, and can achieve better tumor suppressing ability with less antibody dosage.
  • the manufactured immunomagnetic composition can be stored in a sterilized form by lyophilization to form a powdery crystal for a long period of time, and can be used as it is, if necessary, it can be used as a solvent, and it exhibits convenience and stability.
  • the kit for treating cancer of the present invention comprises the immunomagnetic composition of the present invention and a magnetic field generating device, which can generate a magnetic field by a magnetic field generating device, and as an auxiliary tool for magnetic guiding, concentrate the immunomagnetic composition of the present invention on The affected part has a strong immune cell proliferation in the tumor, and can reduce the immune response of the systemic circulation, and can further enhance the anticancer effect of the immunomagnetic composition of the present invention. It is confirmed by the aforementioned test data that it has the ability to be treated locally, and the kit for treating cancer of the present invention has both physical target and biological target action, and is helpful for immunotherapy and chemotherapy. Or combination therapy with immunotherapy to avoid serious side effects caused by excessive immune response.

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Abstract

一种免疫磁性组成物及其制备方法、用途和治疗癌症的试剂盒，免疫磁性组成物包含核心层、壳层以及外层。壳层由复合物构成，壳层包覆核心层。复合物由褐藻多糖、氧化右旋糖酐及多个超顺磁性氧化铁纳米粒子经疏水性作用力结合而形成。外层包含至少一种抗体。

Description

免疫磁性组成物及其制备方法、用途和治疗癌症的试剂盒 技术领域
本发明是关于一种组成物及其制备方法，特别是一种以特殊物理形态为特征的免疫磁性组成物及其制备方法、用途和治疗癌症的试剂盒。
背景技术
癌症又名为恶性肿瘤，是一种细胞不正常增生的状态，且这些增生的细胞可能侵犯身体的其他部分，为由控制细胞分裂增殖机制失常而引起的疾病。全世界罹患癌症的人口有不断增加的趋势，癌症乃国人十大死因之一，且已连续二十七年为居十大死因的榜首。
常规的癌症治疗方法包括手术治疗、放射线治疗及化疗等。免疫疗法为上述治疗方法以外的另一种治疗癌症的方法，是通过激活患者自身免疫系统，利用肿瘤细胞或肿瘤抗原物质诱导机体的特异性细胞免疫和体液免疫反应，增强机体的抗癌能力，阻止肿瘤的生长、扩散和复发，以达到清除或控制肿瘤的目的。而免疫检查哨(immune checkpoint)为几种免疫治疗中最受重视的一种，从2015年至今，已经有超过50项利用免疫检查哨抑制剂(immune checkpoint inhibitor)的复方疗法展开了临床试验。但免疫检查哨抑制剂会关闭人类的免疫系统的回馈机制，使细胞毒性T细胞(CD8+T细胞)于攻击癌细胞之余，也会产生皮肤溃烂、肠胃溃疡等自体免疫反应。
增加体内对于肿瘤具有专一性的免疫细胞也被视为癌症治疗中相当具有希望的一环。目前的技术大多从患者身上取得肿瘤中的免疫细胞，并于体外进行培养，利用微米尺寸的结构体(例如微珠microbead)来模仿抗原呈现细胞(antigen presenting cell,APCs)，进行T细胞的增生和训练，最后回输至患者体内用以猎杀癌细胞。但这样的方式耗时且耗材，且患者体内的癌细胞容易产生变异，使回输的免疫细胞失去本来应该发挥的效果。另一方面，用来进行增生的微珠由于尺寸过大只能用于体外培养，无法经由人体血液循环进入目标区域。此外，此类载体除了表面嫁接的抗体或是包覆的有效成分外，形成载体的材料通常为赋形剂，对于治疗本身并没有太大的帮助，且会限制施打时的剂量，成为此类材料天生的缺陷。
发明内容
本发明的目的在于提供一种可用于制备抗癌的药物的免疫磁性组成物及其制备方法、 用途和治疗癌症的试剂盒。
有鉴于此，本发明的一目的是在提供一种免疫磁性组成物，其包含核心层、壳层和外层。前述壳层是由复合物构成，壳层包覆核心层，其中复合物是由褐藻多醣(fucoidan)、氧化右旋糖酐(dextran)及超顺磁性氧化铁纳米粒子经疏水性作用力(hydrophobic interaction)结合而形成。前述外层包含至少一种抗体，且前述抗体嫁接于壳层外构成外层，其中抗体为免疫检查哨抑制剂及/或杀手T细胞增生剂。
依据前述的免疫磁性组成物，其中前述免疫磁性组成物可为球体，且球体的粒径介于80nm至350nm之间。
依据前述的免疫磁性组成物，其中前述褐藻多醣可萃取自裙带菜(Undaria pinnatifida)、梨形囊巨藻(Macrocystis pyrifera)或墨角藻(Fucus vesiculosus)。
依据前述的免疫磁性组成物，其中氧化右旋糖酐可具有醛基，氧化右旋糖酐可由分子质量介于5kDa至270kDa之间的右旋糖酐制备而得。
依据前述的免疫磁性组成物，其中前述免疫检查哨抑制剂可选自PD-L1抗体、PD-1抗体、CTLA-4抗体及TIM-3抗体所组成的群组，前述杀手T细胞增生剂可选自CD3抗体、CD28抗体及4-1BB抗体所组成的群组。
依据前述的免疫磁性组成物，其中前述核心层可另含有活性物质。
借此，本发明的免疫磁性组成物利用具有抗癌活性的褐藻多醣、氧化右旋糖酐以及超顺磁性氧化铁纳米粒子作为载体组成，进而形成外层具有抗体且可在核心层包覆活性物质的纳米等级结构；其大小及表面电荷合适，可增长体内循环时间，并渗透进入肿瘤，增强褐藻多醣对于肿瘤的作用。而外层的抗体可为免疫检查哨抑制剂及/或杀手T细胞增生剂，使本发明的免疫磁性组成物除了本身材料的抗癌功能外，还可以同时为免疫检查哨抑制剂及/或杀手T细胞增生剂，大幅改善肿瘤的微环境，且本发明的免疫磁性组成物可大幅改善单独以相同的抗体进行免疫治疗的抗癌效果，可以更少的抗体用量达到更佳的肿瘤抑制能力。另外，制造完成的免疫磁性组成物可利用冻干形成粉状晶体于无菌下长期保存，在有需要时以溶剂回溶即可使用，显示其方便和稳定性佳的特性。
本发明的另一目的是在提供一种免疫磁性组成物的制备方法，前述方法包含提供水相溶液、提供油相溶液、进行乳化反应、移除乳液中的有机溶剂和进行抗体嫁接等步骤。在提供水相溶液的步骤中，前述水相溶液包含褐藻多醣和氧化右旋糖酐。在提供油相溶液的步骤中，前述油相溶液包含有机溶剂和超顺磁性氧化铁纳米粒子。在进行乳化反应的步骤中，先是混合前述水相溶液和前述油相溶液以形成乳液。再移除乳液中的有机溶剂，以形 成磁性褐藻多醣载体。在进行抗体嫁接的步骤，是混合磁性褐藻多醣载体和至少一种抗体，以形成免疫磁性组成物，其中抗体可为免疫检查哨抑制剂及/或杀手T细胞增生剂。
依据前述的免疫磁性组成物的制备方法，其中前述免疫检查哨抑制剂可选自PD-L1抗体、PD-1抗体、CTLA-4抗体及TIM-3抗体所组成的群组，前述杀手T细胞增生剂可选自CD3抗体、CD28抗体及4-1BB抗体所组成的群组。
依据前述的免疫磁性组成物的制备方法，其中褐藻多醣和氧化右旋糖酐的重量比例可为1:0.1至1:4。
依据前述的免疫磁性组成物的制备方法，其中氧化右旋糖酐可具有醛基，氧化右旋糖酐可由分子质量可介于5kDa至270kDa之间的右旋糖酐制备而得。
依据前述的免疫磁性组成物的制备方法，其中有机溶剂可为甲醇(methane)、二氯甲烷(dichloromethane)或三氯甲烷(chloroform)。
借此，本发明的免疫磁性组成物的制备方法，不同于他种标靶载体的复杂制造过程，也不需要利用多余的界面活性剂来稳固结构，材料的取得与制作也相当容易。
本发明的再一目的是在提供一种如前述免疫磁性组成物的用途，其是用于制备抗癌的药物。
依据前述的免疫磁性组成物的用途，其中前述抗癌的药物可为抑制癌症细胞增生的药物、抑制癌症转移的药物或引发肿瘤免疫反应的药物。
本发明的又一目的是在提供一种用于治疗癌症的试剂盒，其包含如前述免疫磁性组成物和磁场产生装置。
借此，本发明的用于治疗癌症的试剂盒包含本发明的免疫磁性组成物和磁场产生装置，可通过磁场产生装置作为磁导引的辅助工具，将本发明的免疫磁性组成物集中累积于患部，达到局部放大治疗的效果，避免全身性的免疫反应。是以本发明的用于治疗癌症的试剂盒同时具有物理性的标靶和生物性的标靶作用，是以其仅需使用公知施打纯抗体剂量的百分之一，却得以展现更优异的肿瘤抑制能力，并增长2倍以上的半生存期。
上述发明内容旨在提供本发明的简化摘要，以使阅读者对本发明具备基本的理解。此发明内容并非本发明的完整概述，且其用意并非在指出本发明实施例的重要/关键元件或界定本发明的范围。
附图说明
为让本发明的上述和其他目的、特征、优点与实施例能更明显易懂，结合附图说 明如下：
图1绘示本发明的免疫磁性组成物的结构示意图；
图2绘示本发明的免疫磁性组成物的制备方法的步骤流程图；
图3A至图3F为磁性褐藻多醣载体的结构分析图；
图4A至图4C为磁性载体IO@Fu的结构分析图；
图5A和图5B为磁性载体IO@Dex的结构分析图；
图6A至图6C为磁性褐藻多醣载体的稳定性分析结果图；
图7A和图7B为磁性褐藻多醣载体冻干前后的穿透式电子显微镜照片图；
图8为本发明的免疫磁性组成物的一实施方式的结构分析图；
图9为本发明的免疫磁性组成物的一实施方式的X射线光电子能谱学(XPS)分析结果图；
图10A和图10B为本发明的免疫磁性组成物的另一实施方式的结构分析图；
图11A至图11F为本发明的免疫磁性组成物的标靶能力和细胞结合能力的分析结果图；
图12A至图12D为本发明的免疫磁性组成物的细胞结合能力的分析结果图；
图13A至图13C为本发明的用于治疗癌症的试剂盒集中累积至肿瘤的分析结果图；
图13D为本发明的免疫磁性组成物和用于治疗癌症的试剂盒治疗效果的苏木素-伊红染色搭配普鲁士蓝染色结果图；
图14A至图14E为本发明的免疫磁性组成物和用于治疗癌症的试剂盒于乳腺癌肺转移小鼠模型中抑制癌症细胞增生和癌症转移的分析结果图；
图15A至图15C为本发明的免疫磁性组成物和用于治疗癌症的试剂盒于大肠癌小鼠模型中抑制癌症细胞增生的分析结果图；
图16A至图16I为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后，肿瘤微环境中肿瘤浸润淋巴细胞数量变化和细胞因子含量变化的分析结果图；
图17A和图17B为本发明的免疫磁性组成物和用于治疗癌症的试剂盒的反应位点的分析结果图；
图17C和图17D为本发明的免疫磁性组成物和用于治疗癌症的试剂盒的TUNEL检测结果图；
图18A至图18E为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后小 鼠CD4+T细胞和CD8+T细胞浸润程度分析图；
图19A至图19D为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后小鼠血液生化分析结果图；以及
图20为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后小鼠的病理切片图。
具体实施方式
本说明书公开内容提出一种新颖的免疫磁性组成物，其是将褐藻多醣和氧化右旋糖酐以疏水性作用力与超顺磁性氧化铁纳米粒子结合后，再嫁接抗体而得，所制得的免疫磁性组成物可大幅改善单独以相同的抗体进行免疫治疗的抗癌效果，可以更少的抗体用量达到更佳的肿瘤抑制能力。此外，本说明书也公开一种新颖用于治疗癌症的试剂盒，其是包含本案的免疫磁性组成物和磁场产生装置，可进一步提升本发明的免疫磁性组成物的抗癌效果。说明书中以乳腺癌肺转移和大肠癌的小鼠动物模型，来验证本案的免疫磁性组成物和用于治疗癌症的试剂盒在免疫治疗上的功效和机制。
以下为本说明书中所用特定名词的说明：
说明书中前述的“褐藻多醣(fucoidan)”是一种水溶性食物纤维，萃取自褐色海藻表面独有的黏滑成分。褐藻多醣富含岩藻糖，为一种生物安全性高，且抗氧化、抗凝血、抗血栓、抗病毒及抗癌等多种生物活性的天然多醣。
说明书中前述的“右旋糖酐(dextran)”是一种复合且支链的葡聚糖(由许多葡萄醣分子构成的多醣)，其构成的分子质量可从3Da至2000kDa不等。右旋糖酐中的直链部分由经α-1,6糖苷键相连在一起的葡萄糖分子组成，而支链由α-1,3糖苷键引出。而说明书中前述的“氧化右旋糖酐(oxidized dextran)”是将右旋糖酐进行表面修饰，使右旋糖酐上的羟基氧化成醛基，得到后续可进一步嫁接抗体的氧化右旋糖酐。
请参照图1，其绘示本发明的免疫磁性组成物100的示意图。免疫磁性组成物100包含核心层110、壳层120和外层130。
核心层110可含有活性物质，而活性物质可为细胞因子或抗癌药物。
壳层120是由复合物构成，壳层120包复核心层110，其中复合物是由褐藻多醣、氧化右旋糖酐及超顺磁性氧化铁纳米粒子经疏水性作用力结合而形成。进一步地说，构成壳层120的复合物所使用的褐藻多醣可萃取自裙带菜(Undaria pinnatifida)、梨形囊巨藻(Macrocystis pyrifera)或墨角藻(Fucus vesiculosus)，所使用的氧化右旋糖酐可具有醛基，且其可由分子质量介于5kDa至270kDa之间的右旋糖酐制备而得。而褐藻多醣、氧化右旋糖酐及超顺磁性氧化铁纳米粒子之间的疏水性作用力可经由乳化作用或纳米沉淀法等方法形成，但本发明并不欲以此为限。
外层130包含至少一种抗体131，且抗体131嫁接于壳层120外构成外层130。其中抗体131可为免疫检查哨抑制剂及/或杀手T细胞增生剂，而免疫检查哨抑制剂可选自PD-L1抗体、PD-1抗体、CTLA-4抗体及TIM-3抗体所组成的群组，杀手T细胞增生剂可选自CD3抗体、CD28抗体及4-1BB抗体所组成的群组。
进一步地说，前述免疫磁性组成物100可为球体，且球体的粒径介于80nm至350nm之间。此外，前述免疫磁性组成物呈空心状。
请参照图2，其绘示本发明的免疫磁性组成物的制备方法300的步骤流程图。在图2中，免疫磁性组成物的制备方法300包含步骤310、步骤320、步骤330、步骤340和步骤350。
步骤310是提供水相溶液，水相溶液包含褐藻多醣和氧化右旋糖酐，其中所使用的褐藻多醣可萃取自裙带菜(Undaria pinnatifida)、梨形囊巨藻(Macrocystis pyrifera)或墨角藻(Fucus vesiculosus)，所使用的氧化右旋糖酐可具有醛基，其可由分子质量介于5kDa至270kDa之间的右旋糖酐制备而得。褐藻多醣和氧化右旋糖酐以1:0.1至1:4的重量比例混合。
步骤320是提供油相溶液，油相溶液包含有机溶剂和超顺磁性氧化铁纳米粒子，其中有机溶剂可为甲醇(methane)、二氯甲烷(dichloromethane)或三氯甲烷(chloroform)。
步骤330是进行乳化反应，将步骤310所提供的水相溶液和步骤320所提供的油相溶液混合形成乳液。
在步骤340中，可通过减压蒸发等方法移除乳液中的有机溶剂，以形成磁性褐藻多醣载体。
步骤350是进行抗体嫁接，将磁性褐藻多醣载体和至少一种抗体嫁接，以形成免疫磁性组成物。所使用的抗体可为免疫检查哨抑制剂及/或杀手T细胞增生剂，其中免疫检查哨抑制剂可选自PD-L1抗体、PD-1抗体、CTLA-4抗体及TIM-3抗体所组成的群组，杀手T细胞增生剂可选自CD3抗体、CD28抗体及4-1BB抗体所组成的群组。
据此，经由前述方法制备而得的免疫磁性组成物可于后续用作为抗癌的药物，例如可作为抑制癌症细胞增生的药物、抑制癌症转移的药物和引发肿瘤免疫反应的药物。且经由前述方法制备而得的免疫磁性组成物呈空心状，因此可进一步包覆活性物质于核心层，以增强免疫磁性组成物的抗癌效果。
此外，经由前述方法制备而得的免疫磁性组成物可搭配磁场产生装置构成用于治疗癌症的试剂盒，所述磁场产生装置可为磁铁、三维场磁铁或磁振造影仪等可以产生磁场的装置，通过磁场产生装置产生的磁场作为磁导引的辅助工具，将本发明的免疫磁性组成物集中累积于患部，达到局部放大治疗的效果，使本发明的用于治疗癌症的试剂盒仅需使用公知施打纯抗体剂量的百分之一，却得以展现更优异的肿瘤抑制能力，并增长2倍以上的半生存期。
兹以下列具体试验例进一步示范说明本发明，用以有利于本发明所属技术领域的技术人员，可在不需过度解读的情形下完整利用并实践本发明，而不应将这些试验例视为对本发明范围的限制，但用于说明如何实施本发明的材料及方法。
<试验例>
一、本发明的免疫磁性组成物及其制备方法
1.1 磁性褐藻多醣载体的结构和稳定性分析
在本试验例中，先制备未嫁接抗体的磁性褐藻多醣载体，以测试最佳制备条件，并利用扫描式电子显微镜(scanning electron microscopy；SEM)和穿透式电子显微镜(transmission electron microscopy；TEM)观察磁性褐藻多醣载体的形态。利用纳米粒径及介面电位分析仪(Delsa Nano C particle analyzer,BECKMAN COULTER)分析磁性褐藻多醣载体的介面电位(zeta potential)、粒径和磁性褐藻多醣载体在二次蒸馏(DDW)和磷酸盐缓冲溶液(PBS)中的稳定性。
在制备磁性褐藻多醣载体前，先分别制备超顺磁性氧化铁纳米粒子和氧化右旋糖酐，在本试验例中所使用的氧化右旋糖酐具有醛基，以利后续嫁接抗体。超顺磁性氧化铁纳米粒子(下文中将以“IO”表示)的设计合成参考自2004年由Shouheng Sun团队所发表的文献(Shouheng Sun et al.,Monodisperse MFe 2O 4(M＝Fe,Co,Mn)Nanoparticles.Journal of the American Chemical Society 2004,126(1):273-279)，制备条件为将2mmol的Fe(acac) 3、10mmol的1,2-十六烷二醇、6mmol的油酸和6mmol的乙酰胺在20ml的芐基醚20ml中混合后，在氮气环境下于100℃回流30分钟。再将前述反应物加热至200℃1小时后，再加热至285℃30分钟以完成IO的成核和生长。将反应物冷却至室温后，以6000rpm离心10分钟收集IO，并以乙醇纯化3次，以完成IO的制备。
具有醛基的氧化右旋糖酐(下文中将以“Dex”表示)的制备方法如下：将右旋糖酐(分子量为5kDa至270kDa)在室温下避光溶解于含有过碘酸钠溶液(10mM)的含水氧化缓冲液(0.5-10mg ml-1，pH＝5.5)中氧化30分钟。利用Amicon(分子量为3kDa)透析修饰后Dex以除去过碘酸钠。并利用冷冻干燥机(FreeZone 1L Benchtop Freeze Dry Systems,Labconco,Kansas)再分散和冻干前述Dex。所制得Dex利用核磁共振(Nuclear Magnetic Resonance；NMR)分析结构，并利用比色法醛基测定试剂盒(MAK140,sigma)分析其修饰的程度。
在本试验例中，磁性褐藻多醣载体的制备条件如下：将0.5mg/ml的褐藻多醣(萃取自墨角藻)和0.5mg/ml的Dex混合作为水相溶液。将2mg的IO溶于0.2ml的二氯甲烷中作为油相溶液。将前述水相溶液和油相溶液混合后，以均质机(Double Eagle Enterprise Co，Ltd)以120W的功率乳化50秒，以得到乳液。使用旋转蒸发器除去二氯甲烷后，利用磁选设备(MagniSort，eBioscience)纯化所制得的磁性褐藻多醣载体(下文中将以“IO@FuDex”表示)，并将IO@FuDex以DDW或0.1M的PBS(pH＝6)重新悬浮以进一步进行抗体嫁接。此外，本试验例中另分别以0.5mg/ml的褐藻多醣或0.5mg/ml的Dex作为水相溶液，以溶于二氯甲烷的IO作为油相溶液，利用相同制备方法制备磁性载体IO@Fu(水相溶液为褐藻多醣)或IO@Dex(水相溶液为具有醛基的氧化右旋糖酐)。所制备好的IO@FuDex、IO@Fu和IO@Dex利用扫描式电子显微镜和穿透式电子显微镜分析的形态；利用纳米粒径及介面电位分析仪分析介面电位和粒径；以及利用超导量子干涉磁量仪来进行磁性分析。
请参照图3A至图6C，图3A至图3F为磁性褐藻多醣载体的结构分析图，其中图3A为IO@FuDex的扫描式电子显微镜照片图，图3B至图3F为IO@FuDex的穿透式电子显微镜照片图。图4A至图4C为磁性载体IO@Fu的结构分析图，其中图4A和图4B为IO@Fu的扫描式电子显微镜照片图，图4C为IO@Fu的穿透式电子显微镜照片图。图5A和图5B为磁性载体IO@Dex的结构分析图，其中图5A为IO@Dex的扫描式电子显微镜照片图，图5B为IO@Dex的穿透式电子显微镜照片图。图6A至图6C为磁性褐藻多醣载体的稳定性分析结果图，其中图6A为IO@FuDex、IO@Fu和IO@Dex的流体力学尺寸分布图，图6B为IO@FuDex、IO@Fu和IO@Dex的介面电位分析图，图6C为IO@FuDex和IO的磁性分析图。
图3A的结果显示，通过扫描式电子显微镜的观察，可以看出IO@FuDex为球状结构，并因为电子显微镜的中空环境而有塌陷的现象，代表其为中空结构。由图3B的结果显示，通过穿透式电子显微镜可观察到IO@FuDex因塌陷而重迭的壳层所造成的暗对比。图3C为将壳层处放大的穿透式电子显微镜照片图，也可以见到许多大小约为5nm的超顺磁性氧化铁纳米粒子，证明其为壳层的组成的结构。而在图3D的暗场观测以及图3E和图3F的元素分布探测下，则可再次看出明显的中空结构以及氧化铁分布于载体中的状况。
图4A至图5B的结果显示，IO@Fu和IO@FuDex一样具有均匀的结构，而IO@Dex则不稳定，在蒸发的过程趋于聚集在一起，导致可观察到广泛的粒径分布和随机形态外观。
而图6A和图6B的结果显示，IO@FuDex在流体中呈现均一粒径，粒径大小介于80nm至350nm之间，且平均粒径为141.5nm，其小于IO@Fu的平均粒径(241nm)。而IO@FuDex和IO@Fu因主体由褐藻多醣所组成，其中所含的硫酸盐使IO@FuDex和IO@Fu具有强的负介面电位，但因IO@FuDex的主体中另具有Dex，使得IO@FuDex的负介面电位(-32.8mV)低于IO@Fu的负介面电位(-58.4mV)，因此IO@FuDex之间强力的排斥力可保持胶体的稳定性和良好的分散性。而由图6C的结果显示，超顺磁性氧化铁纳米粒子(IO)具有相当高的单位重量的饱和磁化强度，而IO@FuDex因包含超顺磁性氧化铁纳米粒子、褐藻多醣以及氧化右旋糖酐，其数值略为下降，但仍保有57.5emu g -1的高数值，显示其磁性的强度并不因为形成复合载体结构而下降，有利于使用磁选设备进行磁性纯化，以获得高产量的IO@FuDex。
本试验例中另以不同分子量的氧化右旋糖酐制备磁性褐藻多醣载体，其是如图2中步骤310提供水相溶液中，使用分子量为5kDa至270kDa的右旋糖酐来制备氧化右旋糖酐，再将氧化右旋糖酐与褐藻多醣以1:0.2的重量比例混合，其余步骤320至步骤340的细节与试验例1.1所述大致上相同，在此不再赘述。再以纳米粒径及介面电位分析仪分析所制得的磁性褐藻多醣载体的粒径大小，其结果如下表一所示：
表一、以不同分子量的右旋糖酐所制成的磁性褐藻多醣载体的平均粒径
右旋糖酐分子量(kDa) 平均粒径(nm)
5 132±14.5
12 130±9.8
25 141±16.2
50 162±23.6
80 176±26.8
150 203±48.6
270 264±32.6
此外，本试验例中以不同的褐藻多醣和氧化右旋糖酐混合比例制备磁性褐藻多醣载体，其是如图2中步骤310提供水相溶液中，将褐藻多醣和右旋糖酐以1:0.1至1:4的重量比例混合，其余步骤320至步骤340的细节与试验例1.1所述大致上相同，在此不再赘述。再以纳米粒径及介面电位分析仪分析所制得的磁性褐藻多醣载体的粒径大小，其结果如下表二所示：
表二、以不同重量比的褐藻多醣和氧化右旋糖酐所制成的磁性褐藻多醣载体的平均粒径
褐藻多醣：右旋糖酐(重量比) 平均粒径(nm)
1:0.1 145±6.9
1:0.2 153±11.6
1:1 130±9.8
1:2 141±16.2
1:3 162±23.6
1:4 176±26.8
为测试冻干后再回溶的IO@FuDex是否仍具有相同的结构，本试验例进一步利用冷冻干燥机将制备完成的IO@FuDex冻干形成粉状晶体，再将冻干后的晶体回溶于水溶液中，并利用穿透式电子显微镜观察IO@FuDex冻干前和冻干后的结构。
请参照图7A和图7B，图7A为IO@FuDex冻干前的穿透式电子显微镜照片图，图7B为IO@FuDex冻干后再回溶的穿透式电子显微镜照片图。结果显示，IO@FuDex冻干前的结构为中空的球型，大小约为80nm至350nm之间。而冻干后的晶体，可以快速的回溶于水溶液中，且再回溶的IO@FuDex结构于穿透式电子显微镜下，仍保持为中空、且大小与冻干前载体相符的球体。显示IO@FuDex的稳定性极佳。
1.2实施例1-3的制备
以前述最佳制备IO@FuDex的条件进一步制备本发明的免疫磁性组成物。将制备好的IO@FuDex分别与含有不同抗体且含有氰基硼氢化钠(5μM)的缓冲溶液(0.1M,pH＝6)在4℃下进行抗体嫁接4-6小时，Dex中的醛基与抗体上的一级胺会形成席夫碱，在使用氰基硼氢化钠进行还原胺化反应后，会使抗体嫁接达到化学稳定状态。最后使用磁选设备纯化所制得的免疫磁性组成物。请参照下表三，为本发明的实施例1-3所使用的抗体，其中实施例1所使用的抗体为CD3抗体和CD28抗体，实施例2所使用的抗体为PD-L1抗体，实施例3所使用的抗体为PD-L1抗体、CD3抗体和CD28抗体。
表三
PD-L1抗体 CD3抗体 CD28抗体
IO@FuDex - - -
实施例1 - + +
实施例2 + - -
实施例3 + + +
所制备好的实施例利用穿透式电子显微镜分析的形态和元素分析，以及X射线光电子能谱学(XPS)测定实施例3中的元素构成。
请参照图8，为实施例3的结构分析图。结果显示，实施例3的结构为尺寸约为80-350nm的球形中空结构。并可通过电子显微镜的元素分析，看到实施例3外层因为有抗体的存在，而均匀的出现清晰的氮讯号(N)。在未嫁接抗体前，载体成分只有醣类以及氧化铁，并无带有氮的材料；然而，嫁接抗体后，因为抗体具有许多的胺基以及肽键，而带有大量的氮，可证明本发明的免疫磁性组成物带有抗体。
请再参照图9，为本发明的实施例3的X射线光电子能谱学分析结果图。结果显示，可以看出实施例3相对应于IO@FuDex多出了键结能位于399eV的一根信号，而此信号即为氮信号，代表在实施例3中，抗体成功的嫁接于载体上。
1.3实施例5的制备和结构确认
本发明的免疫磁性组成物因呈空心状，因此可进一步于核心层中包覆如细胞因子或抗癌药物等活性物质。在本试验例中的活性物质以介白素-2(Interleukin-2,IL-2)为例，进一步制备包覆IL-2的实施例5。
在本试验例中，实施例5的制备条件如下：将0.5mg/ml的褐藻多醣、0.5mg/ml的Dex和50μg的IL-2混合作为水相溶液。将2mg的IO溶于0.2ml的二氯甲烷中作为油相溶液。将前述水相溶液和油相溶液混合后，以均质机以120W的功率乳化50秒，以得到乳液。使用旋转蒸发器除去二氯甲烷后，利用磁选设备纯化所制得的实施例5。所制备好的实施例利用扫描式电子显微镜分析的形态，并利用纳米粒径及介面电位分析仪分析其粒径。
请参照图10A和图10B，为实施例5的结构分析图，其中图10A为实施例5的扫描式电子显微镜照片图，图10B为实施例5的流体力学尺寸分布图。结果显示，实施例5的IL-2包覆率高达99.93％。而因为IL-2填满核心层，使实施例5的平均粒径大小由纯载体的161.2nm增加至356.2nm，证明本发明的免疫磁性组成物可进一步地于核心层中包覆活性物质，例如小红莓药物(Doxorubicin)、紫杉醇药物(Docetaxel或Paclitaxel)以及虾红素(Astaxanthin；ASTX)等活性物质，也可利用相同的方式包覆在本发明的免疫磁性组成物中。
1.4本发明的免疫磁性组成物的标把能力分析
本试验例将制备完成的实施例1-3进行标把能力分析和细胞结合能力分析，以验证本发明的免疫磁性组成物是否能逆转T细胞免疫力下降的现象，并促进小鼠的肿瘤衰退。试验上选择具有转移能力、侵袭性和表达PD-L1的三阴性乳腺癌细胞株(4T1)作为实验模型。为了方便于荧光显微镜下观察，在荧光显微镜分析组别的实施例1-3分别接上量子点。
为评估标靶效率，试验上分别将实施例1-3和4T1细胞株(4×10 5)在含牛血清白蛋白(BSA)的培养基于4℃下培育30分钟，再利用流式细胞仪(Novocyte Flow Cytometer,ACEA Biosciences)进行分析。而为评估细胞结合能力，将接有量子点的IO@FuDex和实施例2分 别与4T1细胞株培育1、4、12和24小时，再利用流式细胞仪或荧光显微镜(Carl Zeiss,Thornwood)进行分析。
请参照图11A至图11F，为本发明的免疫磁性组成物的标靶能力和细胞结合能力的分析结果图。其中图11A为嫁接浓度为1.4μg/ml(低浓度；L)、7μg/ml(中浓度；M)和35μg/ml(高浓度；H)的PD-L1抗体的实施例2于体外标靶能力的测试结果。图11B为实施例2和嫁接IgG的IO@FuDex于体外标靶能力的测试结果。图11C为IO@FuDex在第1、4、12和24小时与4T1细胞株结合能力的分析结果图。图11D为实施例2在第1、4、12和24小时与4T1细胞株结合能力的分析结果图。图11E为嫁接浓度为1.4μg/ml(低浓度；L)、7μg/ml(中浓度；M)和35μg/ml(高浓度；H)的CD3抗体/CD28抗体的实施例3于体外标靶能力的测试结果。图11F为实施例2和实施例3在第1和24小时与4T1细胞株结合能力的分析结果图。
图11A的结果显示，经过1小时的培育后，实施例2(H)的组别具有最高的平均荧光强度(median fluorescence index,MDI)显示实施例2与4T1细胞株(其表达PD-L1)结合的量与其表面抗体密度密切相关，因此后续试验实施例2所嫁接的PD-L1浓度为高浓度(35μg/ml)。而在图11B的结果显示，接有IgG的IO@FuDex没有观察到MDI移位，显示实施例2对4T1细胞株的亲和力来自PD-L1抗体，而非与IO@FuDex的任何非特异性相互作用。
而图11C和图11D的结果显示，IO@FuDex与细胞结合的量具有时间依赖性，其在培育后第12小时后有明显的细胞吸收。相比之下，实施例2在培育后第1小时中即具有细胞结合的现象，随后MDI呈现缓慢移位，显示随着培育时间的增加，实施例2与细胞的结合量增加，此结果显示将PD-L1抗体嫁接于IO@FuDex后，会改变原本IO@FuDex和4T1细胞株的结合能力。
为了使本发明的免疫磁性组成物可同时为免疫检查哨抑制剂和杀手T细胞增生剂，实施例3为同时嫁接PD-L1抗体、CD3抗体和CD28抗体于IO@FuDex所得的免疫磁性组成物。请参照图11E，为嫁接不同浓度的CD3抗体/CD28抗体的实施例3的标靶能力分析，图11E的结果显示，于体外经过1小时的培育后，实施例3(H)的组别具有最高的MDI，显示实施例3(H)组别与CD8 +T细胞具有强亲和力。而图11F的结果显示，实施例3与4T1细胞株之间具有和实施例2相似的结合力，显示于IO@FuDex上嫁接多个抗体不会影响其对于4T1细胞株的亲和力。
请参照图12A至图12D，为实施例3与CD8 +T细胞结合能力的分析结果图。试验上将接有量子点的实施例3与CD8 +T细胞培育30分钟，使用Alexa Fluor 488Phalloidin和DAPI对CD8 +T细胞进行固定染色，并使用共轭焦显微镜确认实施例3位于CD8 +T细胞的亚细胞位置。其中图12A表明细胞核的位置，图12B表明实施例3的位置，图12C为明视野下的显微照片图，图12D为合并后的显微照片图，其中比例尺表示5μm的长度。结果显示，在CD8 +T细胞中可见多个实施例3，证明实施例3确实能与CD8 +T细胞结合。
二、免疫磁性组成物的用途
2.1本发明的免疫磁性组成物的治疗癌症的疗效
本试验例以乳腺癌肺转移和大肠癌的小鼠模型进一步测试本发明的免疫磁性组成物是否具有治疗癌症的功效，以及其是否可经由磁导引累积于患部。
2.1.1本发明的免疫磁性组成物治疗乳腺癌肺转移的疗效
本试验例使用带有稳定表达生物性冷光酵素的luciferase基因的4T1-Luc肿瘤小鼠为乳腺癌肺转移的动物模型(其是由中国医药大学附设医院分子医学中心所提供)，将含有标记 125I的实施例1-3的制剂经由右股静脉施用于4T1-Luc肿瘤小鼠中，而试验上另有一实施例4(本发明的用于治疗癌症的试剂盒)，实施例4为施用含有标记 125I的实施例3制剂，并搭配利用表面圆形的磁体(直径为0.5cm,0.5Tesla)进行磁导引。后续使用单一光子发射电脑断层扫描(SPECT)监测实施例3和实施例4的在肿瘤组织中的动态累积。
请参照图13A至图13C，为本发明的用于治疗癌症的试剂盒集中累积至肿瘤的分析结果图。图13A为标记有 125I的实施例3或实施例4在4T1-Luc肿瘤小鼠中第0、4、6、12和24小时的时间活性曲线，其中n＝4。图13B为4T1-Luc肿瘤小鼠在施用标记有 125I的实施例3或实施例4后，第24小时的全身单一光子发射电脑断层扫描图。图13C为4T1-Luc肿瘤小鼠在施用标记有 125I的实施例3或实施例4后，第24小时的生物分布定量图，其中n＝4，*表示具有统计上的显著差异(p<0.05)。
图13A的结果显示，实施例3的在肿瘤的累积在施用后第6小时达到每克组织注射剂量的最大浓度(5.08％ID/g)。相较之下，实施例4通过磁导引可以促进其在肿瘤的剂量区域性，可见实施例4在24小时内于肿瘤中持续累积，在第24小时，实施例4在肿瘤中的累积量为实 施例3的3.6倍。而由图13B的结果显示，在施用实施例3或实施例4后24小时，实施例3和实施例4之间的放射性同位素具有不同的生物分布模式。图13C的生物分布定量图显示，实施例3和实施例4主要在肿瘤、肝脏、脾脏和膀胱中累积，而肌肉、脑、心脏、肺脏、胃、肾脏、结肠和血液中积累较少(小于5％ID/g)。其中实施例3和实施例4相比，可见实施例4不仅增加了在肿瘤的累积，更显著降低其在肝脏和脾脏的系统累积，显示实施例4可以有效地集中到作用部位。
本试验例进一步分别经由右股静脉施用IgG(对照组)、IO@FuDex、实施例1-4至4T1-Luc肿瘤小鼠中，施用方式为肿瘤接种后8天，每4天连续施用3次(q4dx3)。请参照图13D，为4T1-Luc肿瘤小鼠施用实施例1-4后4周的苏木素-伊红染色搭配普鲁士蓝染色结果图，其中比例尺表示50μm的长度。图13D的结果显示，其他组别相较，实施例4在肿瘤组织中可观察到分散的普鲁士蓝染色结果。
为了确认本发明的免疫磁性组成物和用于治疗癌症的试剂盒对于4T1-Luc肿瘤小鼠具有治疗效果，本试验例进一步分别经由右股静脉施用IgG(对照组)、IO@FuDex、实施例1-4至4T1-Luc肿瘤小鼠中，施用方式为肿瘤接种后8天，每4天连续施用3次(q4dx3)。而肿瘤体积为每2-3天使用数字卡尺(mitutoyo)进行监测，并以下述公式I计算肿瘤体积：
Figure PCTCN2018084769-appb-000001
并以非侵入式活体分子影像系统(Non Invasion In Vivo Imaging System,IVIS,Xenogen)进行生物冷光评估。4T1-Luc肿瘤小鼠的存活率以Kaplan-Meier法进行分析。
请参照图14A至图14E，为本发明的免疫磁性组成物和用于治疗癌症的试剂盒于乳腺癌肺转移小鼠模型中抑制癌症细胞增生和癌症转移的分析结果图。图14A为4T1-Luc肿瘤小鼠在施用实施例1-4后24小时的非侵入式活体分子影像系统扫描结果图。图14B为4T1-Luc肿瘤小鼠在施用实施例1-4后的肿瘤体积统计图。图14C为4T1-Luc肿瘤小鼠在施用实施例1-4后的生存曲线。图14D为4T1-Luc肿瘤小鼠在施用实施例1-4后的肿瘤和肺脏的照片图。图14E为4T1-Luc肿瘤小鼠在施用实施例1-4后肺转移的统计图。
图14A和图14B的结果显示，和对照组相比，IO@FuDex可以抑制肿瘤生长，但不具统计上的显著差异。而实施例1-4不但具抗肿瘤效果，且具有统计上显著的差异(*表示p<0.05， **表示p<0.01)，特别是实施例4在30天内几乎抑制肿瘤生长。图14C的结果显示，施用对照组、IO@FuDex、实施例1、实施例2、实施例3和实施例4的4T1-Luc肿瘤小鼠的中位数存活期分别为24天、34天、34天、43天和44天。而施用实施例4的4T1-Luc肿瘤小鼠的中位数存活期则可显著延长到63天。实施例4中的PD-L1抗体剂量虽然只有一般施打纯抗体剂量的百分之一，却得以展现更优异的肿瘤抑制能力，以及增长2倍以上的半生存期。此外，图14D的结果显示，实施例1-4皆具有抗肿瘤转移能力。在图14D中，与其他组别相比，施用实施例3和实施例4显著地抑制肺部肿瘤转移，另由图14E的结果显示，对照组4T1-Luc肿瘤小鼠的肺部有超过20个结节的肺转移，而施用实施例4的4T1-Luc肿瘤小鼠的肺部平均发现少于5个结节的肺转移。因此，上述的结果显示，本发明的免疫磁性组成物和用于治疗癌症的试剂盒具有显著的抑制肿瘤和抗肿瘤转移作用。而与对照组相比，IO@FuDex未显著的降低肿瘤的转移，推测本发明的免疫磁性组成物和用于治疗癌症的试剂盒所具有抗肿瘤转移作用，除了褐藻多醣的抑制作用外，可能也与肿瘤微环境的动态反应有关。
2.1.2本发明的免疫磁性组成物治疗大肠癌的疗效
本试验例使用带有稳定表达生物性冷光酵素的luciferase基因的CT-26细胞株来建立大肠癌的动物模型(其是由中国医药大学附设医院分子医学中心所提供)，将IgG(对照组)、IO@FuDex和实施例3分别经由右股静脉施用于CT-26肿瘤小鼠中，而试验上另有施用实施例3并搭配利用表面圆形的磁体(直径为0.5cm,0.5Tesla)进行磁导引的实施例4(本发明的用于治疗癌症的试剂盒)；以及施用IO@FuDex并搭配利用表面圆形的磁体进行磁导引的比较例1。施用方式为肿瘤接种后8天，每4天连续施用3次(q4dx3)。每2-3天使用数字卡尺进行肿瘤体积监测，并以前述的公式I计算肿瘤体积，并以非侵入式活体分子影像系统进行生物冷光评估。CT-26肿瘤小鼠的存活率以Kaplan-Meier法进行分析。
请参照图15A至图15C，为本发明的免疫磁性组成物和用于治疗癌症的试剂盒于大肠癌小鼠模型中抑制癌症细胞增生的分析结果图。图15A为CT-26肿瘤小鼠在施用实施例3-4后24小时的非侵入式活体分子影像系统扫描结果图。图15B为CT-26肿瘤小鼠在施用实施例3-4后的肿瘤体积统计图。图15C为CT-26肿瘤小鼠在施用实施例3-4后的生存曲线。
图15A和图15B的结果显示，和对照组相比，IO@FuDex可以抑制肿瘤生长，但不具统计上的显著差异。施用IO@FuDex并搭配磁引导的比较例1可以提高IO@FuDex的治疗效 果。而本发明的实施例3-4不但具抗肿瘤效果，且具有统计上显著的差异(*表示p<0.05，**表示p<0.01)，特别是实施例4在30天内几乎抑制肿瘤生长。图15C的结果显示，施用实施例3和实施例4显著地延长了小鼠的生存时间，再次证明本发明的免疫磁性组成物具有抑制肿瘤生长和转移的效果。
2.2施用本发明的免疫磁性组成物后的免疫作用分析
本试验例进一步以4T1肿瘤模型，探讨本发明的免疫磁性组成物和用于治疗癌症的试剂盒在肿瘤内微环境的免疫作用。试验上监测早期肿瘤(10天)到晚期(30天)4T1-Luc肿瘤小鼠的肿瘤、血液、腹水和脾脏中淋巴球数量的变化。
请参照图16A至图16I，为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后，肿瘤微环境中肿瘤浸润淋巴细胞数量变化和细胞因子含量变化的分析结果图。图16A和图16B的结果显示，施用实施例1-4的组别可以显著地增加抗肿瘤淋巴细胞，如CD8 +T细胞和CD4 +T细胞的数量，特别是施用实施例4的组别(p<0.01)。而图16C和图16D的结果显示，施用实施例1-4的组别可以显著地降低前肿瘤抗肿瘤淋巴细胞，如CD4 +CD225 +Foxp3 +Treg(调节性T细胞)和CD11b +CD206 +F4/80 +TAM(肿瘤相关巨噬细胞)的数量，特别是施用实施例4的组别可大幅降低调节性T细胞(Treg)数量(p<0.01)。而在图16E至图16G中，收集不同组别的4T1-Luc肿瘤小鼠血清，分析由TAM分泌的TNF-α、VEGF和TGF-β的含量，可发现施用实施例1-4的组别可以显著地降低TNF-α、VEGF和TGF-β等促炎细胞因子的含量，特别是施用实施例4的组别(p<0.01)。而在图16H和图16I中，通过细胞内颗粒酶B(GrB +)和Ki67的表现的水平评估CD8 +T细胞的活化程度，可见施用实施例1-4的组别可以有效地激活CD8 +T细胞肿瘤浸润淋巴细胞的功能，特别是施用实施例4的组别(p<0.01)。
在前述试验例证实施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒可以改变肿瘤微环境后，本试验例进一步探讨本发明的免疫磁性组成物和用于治疗癌症的试剂盒对于肿瘤特异性免疫反应和全身效应的变化。试验上，收集不同组别的4T1-Luc肿瘤小鼠的肿瘤、血清、脾脏，监测INF-γ +CD44 +T细胞和CD8 +CD3 +T细胞的变化，以及利用TUNEL检测观察不同组别4T1-Luc肿瘤小鼠的皮肤组织的细胞凋亡状况。
请参照图17A和图17B，为本发明的免疫磁性组成物和用于治疗癌症的试剂盒的反应位点的分析结果图。图17A为不同组别的4T1-Luc肿瘤小鼠的INF-γ +CD44 +T细胞的变化结果图。图17B为不同组别的4T1-Luc肿瘤小鼠的CD8 +CD3 +T细胞的变化结果图。
图17A的结果显示，INF-γ +CD44 +T细胞在施用实施例1-4后增值的数量提高，特别是施用实施例4的组别高度增殖。而图17B的结果显示，施用实施例1-3后，CD8 +CD3 +T细胞在血清和脾脏中均有扩增，但施用实施例4的组别和施用实施例3的组别相比，磁导引会降低CD8 +CD3 +T细胞在血清和脾脏中的扩增。
皮肤毒性所引起的搔痒症和白斑症是免疫检查哨抑制剂治疗中的副作用之一，由于免疫相关的不良事件往往出现在长期的情况下，本试验例在肿瘤接种后4周，对4T1-Luc肿瘤小鼠的皮肤组织进行TUNEL检测，以评估浸润的T细胞是否诱导免疫应答并引起皮肤组织损伤。
请参照图17C和图17D，为本发明的免疫磁性组成物和用于治疗癌症的试剂盒的TUNEL检测结果图。图17C为TUNEL检测不同组别4T1-Luc肿瘤小鼠的皮肤组织凋亡指数统计结果图，其中凋亡指数是以TUNEL +凋亡细胞核的比例除以细胞核总数(随机选择的镜检范围)，并通过二因子的变异数分析和Newman-Keuls事后比较检验评估不同组之间的差异。图17D为TUNEL检测不同组别的4T1-Luc肿瘤小鼠的荧光显微照片图，其中比例尺表示50μm的长度。
图17C和图17D的结果显示，施用实施例1-3后，会增加可被TUNEL标记(绿色)的凋亡细胞，其中施用实施例3的组别诱导凋亡指数与对照组相比增加了3.3倍。但施用实施例4的组别和施用实施例3的组别相比，磁导引可以有效降低皮肤组织中凋亡细胞的数量。
2.3本发明的免疫磁性组成物的免疫相关不良事件测试
本试验例进一步以施用本发明的免疫磁性组成物或用于治疗癌症的试剂盒的4T1肿瘤模型，试验上将实施例3或实施例4施用于4T1肿瘤小鼠，并在肿瘤接种后4周，对4T1肿瘤小鼠进行免疫相关不良事件(Immune-related adverse events,irAEs)测试，分析本发明的免疫磁性组成物和用于治疗癌症的试剂盒的安全性。
本试验例通过观察经由实施例3以及实施例4治疗后四周的小鼠，其CD4 +T细胞以及CD8 +T细胞于主要器官的浸润程度来判断副作用的影响。请参照图18A至图18E，为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后小鼠CD4 +T细胞和CD8 +T细胞浸润程度分析图，其中图18A为肝脏的分析结果图，图18B为肺脏的分析结果图，图18C为脾脏的分析结果图，图18D为肾脏的分析结果图，图18E为大肠的分析结果图。结果显示，实施例3于肝脏、肺脏、脾脏、肾脏和大肠的T细胞浸润程度低于对照组，而实施例4因为具有磁铁的吸引作用，更能显著地减少于周边的累积(肝脏、肺脏、脾脏、肾脏和大肠)，进而减低T细胞的浸润。
本试验例进一步通过血液生化分析，分析肝功能指数(AST和ALT)、肾功能指数(肌酸酐)以及血糖来判断副作用的影响。请参照图19A至图19D，为施用本发明的免疫磁性组成物和用于治疗癌症的试剂盒后小鼠血液生化分析结果图，其中图19A为AST值分析图，图19B为ALT值分析图，图19C为肌酸酐值分析图，图19D为血糖值分析图。由图可知，施用实施例3或实施例4的治疗组别与对照组的生化值仍保持于正常值的范围之中，显示本发明的免疫磁性组成物和用于治疗癌症的试剂盒具有一定程度的安全性。
此外，小鼠肿瘤接种后四周进行病理切片分析，请参照图20，为施用实施例3和实施例4后小鼠的病理切片图。结果显示，施用实施例3或实施例4的治疗组别在肝脏、肺脏、脾脏、肾脏、和大肠中相对于对照组没有明显的组织损伤，显示本发明的免疫磁性组成物和用于治疗癌症的试剂盒具有一定程度的安全性。
综合上述，本发明的免疫磁性组成物的制备方法的制程简单，利用具有抗癌活性的褐藻多醣作为载体组成，并结合超顺磁性氧化铁纳米粒子，形成外层可嫁接抗体并可包覆活性物质于核心层的免疫磁性组成物，所制备出的免疫磁性组成物为纳米等级结构，其大小合适可渗透进入肿瘤，增强褐藻多醣对于肿瘤的作用。而外层的抗体可为免疫检查哨抑制剂及/或杀手T细胞增生剂，使本发明的免疫磁性组成物除了本身材料的抗癌功能外，还可以同时为免疫检查哨抑制剂及/或杀手T细胞增生剂，大幅改善肿瘤的微环境，且本发明的免疫磁性组成物可大幅改善单独以相同的抗体进行免疫治疗的抗癌效果，可以更少的抗体用量达到更佳的肿瘤抑制能力。另外，制造完成的免疫磁性组成物可利用冻干形成粉状晶体于无菌下长期保存，在有需要时以溶剂回溶即可使用，显示其方便和稳定性佳的特性。
本发明的用于治疗癌症的试剂盒包含本发明的免疫磁性组成物和磁场产生装置，可通过磁场产生装置产生磁场，作为磁导引的辅助工具，将本发明的免疫磁性组成物集中累积于患部，在肿瘤中具有旺盛的免疫细胞增生，且可降低体循环的免疫反应，可进一步提升本发明的免疫磁性组成物的抗癌效果。由前述试验数据确认其具有针对局部进行治疗的能力，是以本发明的用于治疗癌症的试剂盒同时具有物理性的标靶和生物性的标靶作用，有助于免疫治疗以及搭配化疗，或免疫治疗的复方治疗，避免过强的免疫反应造成严重副作用。
然本发明已以实施方式公开如上，然其并非用以限定本发明，任何所属技术领域的技术人员，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视权利要求所界定的为准。

Claims (20)

  1. 一种免疫磁性组成物，其特征在于，包含：
    核心层；
    壳层，是由复合物构成，所述壳层包覆所述核心层，其中所述复合物是由褐藻多醣、氧化右旋糖酐及多个超顺磁性氧化铁纳米粒子经疏水性作用力结合而形成；以及
    外层，包含至少一种抗体，且所述至少一种抗体嫁接于所述壳层外构成所述外层，其中所述至少一种抗体为免疫检查哨抑制剂及/或杀手T细胞增生剂。
  2. 如权利要求1所述的免疫磁性组成物，其特征在于，所述免疫磁性组成物为球体，且所述球体的粒径介于80nm至350nm之间。
  3. 如权利要求1所述的免疫磁性组成物，其特征在于，所述褐藻多醣是萃取自裙带菜、梨形囊巨藻或墨角藻。
  4. 如权利要求1所述的免疫磁性组成物，其特征在于，所述氧化右旋糖酐具有醛基。
  5. 如权利要求4所述的免疫磁性组成物，其特征在于，所述氧化右旋糖酐是由分子质量介于5kDa至270kDa之间的右旋糖酐制备而得。
  6. 如权利要求1所述的免疫磁性组成物，其特征在于，所述免疫检查哨抑制剂是选自PD-L1抗体、PD-1抗体、CTLA-4抗体及TIM-3抗体所组成的群组。
  7. 如权利要求1所述的免疫磁性组成物，其特征在于，所述杀手T细胞增生剂是选自CD3抗体、CD28抗体及4-1BB抗体所组成的群组。
  8. 如权利要求1所述的免疫磁性组成物，其特征在于，所述核心层中另含有活性物质。
  9. 一种免疫磁性组成物的制备方法，其特征在于，包含：
    提供水相溶液，所述水相溶液包含褐藻多醣和氧化右旋糖酐；
    提供油相溶液，所述油相溶液包含有机溶剂和超顺磁性氧化铁纳米粒子；
    进行乳化反应，是混合所述水相溶液和所述油相溶液以形成乳液；
    移除所述乳液中的所述有机溶剂，以形成磁性褐藻多醣载体；以及
    进行抗体嫁接，是混合所述磁性褐藻多醣载体和至少一种抗体，以形成所述免疫磁性组成物，其中所述至少一种抗体为免疫检查哨抑制剂及/或杀手T细胞增生剂。
  10. 如权利要求9所述的免疫磁性组成物的制备方法，其特征在于，所述免疫检查哨抑制剂是选自PD-L1抗体、PD-1抗体、CTLA-4抗体及TIM-3抗体所组成的群组。
  11. 如权利要求9所述的免疫磁性组成物的制备方法，其特征在于，所述杀手T细胞增生剂是选自CD3抗体、CD28抗体及4-1BB抗体所组成的群组。
  12. 如权利要求9所述的免疫磁性组成物的制备方法，其特征在于，所述褐藻多醣和所述氧化右旋糖酐的重量比例是1:0.1至1:4。
  13. 如权利要求9所述的免疫磁性组成物的制备方法，其特征在于，所述氧化右旋糖酐具有醛基。
  14. 如权利要求9所述的免疫磁性组成物的制备方法，其特征在于，所述氧化右旋糖酐是由分子质量介于5kDa至270kDa之间的右旋糖酐制备而得。
  15. 如权利要求9所述的免疫磁性组成物的制备方法，其特征在于，所述有机溶剂为甲醇、二氯甲烷或三氯甲烷。
  16. 一种如权利要求1至8任一项所述的免疫磁性组成物的用途，其特征在于，其是用于制备抗癌的药物。
  17. 如权利要求16所述的免疫磁性组成物的用途，其特征在于，所述抗癌的药物为抑制癌症细胞增生的药物。
  18. 如权利要求16所述的免疫磁性组成物的用途，其特征在于，所述抗癌的药物为抑制癌症转移的药物。
  19. 如权利要求16所述的免疫磁性组成物的用途，其特征在于，所述抗癌的药物为引发肿瘤免疫反应的药物。
  20. 一种用于治疗癌症的试剂盒，其特征在于，包含：
    如权利要求1至8任一项所述的免疫磁性组成物；以及
    磁场产生装置。
PCT/CN2018/084769 2017-05-01 2018-04-27 免疫磁性组成物及其制备方法、用途和治疗癌症的试剂盒 WO2018201981A1 (zh)

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AU2018262962A AU2018262962B2 (en) 2017-05-01 2018-04-27 Immunomagnetic nanocapsule, fabrication method and use thereof, and kit for treating cancer

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WO2006091180A2 (en) * 2005-02-25 2006-08-31 Ali Demir Sezer Fucoidan multiparticulate drug carrier systems
US20120065158A1 (en) * 2009-03-23 2012-03-15 Yoshiharu Okamoto Fucoidan having antitumor activity
CN102716414A (zh) * 2012-05-28 2012-10-10 大连圣弘医药有限公司 一种含褐藻多糖硫酸酯的抗肿瘤药物组合物及其制备方法
CN104013640A (zh) * 2013-03-01 2014-09-03 赖媛淑 褐藻糖胶在制备治疗恶病质及癌症的药物中的应用

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WO2006091180A2 (en) * 2005-02-25 2006-08-31 Ali Demir Sezer Fucoidan multiparticulate drug carrier systems
US20120065158A1 (en) * 2009-03-23 2012-03-15 Yoshiharu Okamoto Fucoidan having antitumor activity
CN102716414A (zh) * 2012-05-28 2012-10-10 大连圣弘医药有限公司 一种含褐藻多糖硫酸酯的抗肿瘤药物组合物及其制备方法
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