Immune microbubble complex and its uses

Abstract

The present invention relates to an immunomicrobubble complex and its uses. The immunomicrobubble complex (IMC) according to the present invention comprises microbubbles conjugated with an antibody, wherein the microbubbles have excellent stability and excellent antibody binding strength, and it has been confirmed that when the immunomicrobubble complex is treated with high-intensity focused ultrasound (HIFU), the anti-tumor effect is significantly enhanced and an immune-enhancing effect is exhibited. Therefore, the immunomicrobubble complex according to the present invention is expected to improve the delivery efficiency of the conjugated antibody and can be used for the diagnosis and treatment of cancer, and exhibits various functions in the field of immunotherapy, including a contrast effect, improvement of half-life, improvement of drug delivery, lymphocyte concentrating effect, cancer immunotherapy, and ultrasound-induced immunotherapy.

Description

Technical Field

[0001] The present invention relates to an immune microbubble complex and uses thereof, the immune microbubble complex comprising microbubbles; and an immune checkpoint inhibitory antibody conjugated to the surface of the microbubbles.

[0002] This application claims the priority and benefit of Korean Patent Application No. 10-2019-0108278, filed on Sep. 2, 2019, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Cancer is one of the incurable diseases that humanity must address, and the whole world is investing heavily in developing a cure for it. In Korea, as the number one disease among the causes of death due to disease, more than 100,000 people are diagnosed with the disease every year and more than 60,000 people die.

[0004] Currently, cancer treatment methods used clinically include chemotherapy, radiotherapy, targeted therapy, and a method of removing the lesion by surgery. Representative examples of chemotherapeutic agents currently used for cancer treatment include doxorubicin, adriamycin, cisplatin, paclitaxel, and 5-fluorouracil, which are widely used in cancer treatment chemotherapy. However, since these methods have limitations and are accompanied by serious side effects and pain for patients rather than completely curing the case, it is very important to develop a cancer treatment technology that can minimize side effects.

[0005] Therefore, cancer immunotherapy has been developed. These methods are methods of killing cancer cells by activating cancer-specific immune cells, and due to the generation of memory immune cells, there is a high possibility of complete cure, it is expected to inhibit recurrence, and surgery is not required, so side effects can be minimized compared to traditional cancer treatment methods. The current cancer immunotherapy market is approximately 4 trillion Korean won (KRW), and it is expected to surge to 10 trillion Korean won (KRW) by 2024. As of now, approximately 15 FDA-approved cancer immunotherapy drugs are being actively applied. Among cancer immunotherapy methods, representative methods include vaccine therapy and immune checkpoint inhibitor therapy, which are currently known as cancer immunotherapy drugs showing good clinical results. However, currently used cancer immunotherapy methods have the phenomenon in which cancer cells produce their own interfering antibodies and reduce the efficacy of cancer immunotherapeutic agents, so the treatment effect decreases due to the loss of a certain amount of the administered cancer immunotherapeutic agent. In addition, cancer immunotherapy has various side effects and drug losses due to the systemic circulation of the drug.

[0006] Meanwhile, a diagnostic agent is a substance capable of diagnosing and treating diseases simultaneously. These agents are generally composed of small-sized substances and usually have such a form that fluorescent dyes, radioactive molecules, etc. are carried in liposomes, polymers or nanoparticles, and drugs or diagnostic markers are introduced to the outside. Recently, research on the synthesis of therapeutic agents composed of lipid structures with excellent biocompatibility has been mainly carried out. In particular, bioimage analysis research using micron-sized microbubbles (MB; microbubble ultrasound agents) composed of lipid structures is actively underway, and various studies on treatment methods of microbubbles, nanoparticle drug delivery systems and combination therapies using them have also been conducted.

[0007] Microbubbles exposed to ultrasound cause cavitation, which leads to the temporary formation of pores in the cell membranes of surrounding cells. Therefore, an increase in lymphocytes near cancer cells is shown, and since dendritic cells are activated due to tumor ablation, an effect of activating the immune system is shown thereby. Therefore, it is expected that the immunotherapy method using ultrasound as an indirect treatment method has a narrower scope or lower treatment effect compared to the treatment method using cancer immunotherapeutic agents alone, but when the unique ultrasound physical mechanism is combined with traditional immunotherapy methods, a synergistic effect can be exhibited.

[0008] Therefore, the present inventors have developed a novel immunotherapy method by combining ultrasound technology with immunotherapy and applying microbubble complexes and ultrasound, which is not yet known in the field of cancer-related research and development such as cancer diagnosis and treatment. Summary of the Invention

[0009] [Technical Problem]

[0010] The present inventors have confirmed that by combining ultrasound technology with a cancer immunotherapy method, when ultrasound treatment is performed on antibody-conjugated microbubbles, excellent anti-tumor effects are exhibited compared to when no ultrasound treatment is performed, and the present invention has been completed based on this.

[0011] Therefore, the present invention aims to provide an immunomicrobubble complex and its use, the immunomicrobubble complex comprising microbubbles; and immune checkpoint inhibitory antibodies conjugated to the surface of the microbubbles.

[0012] However, the technical problems to be solved by the present invention are not limited to the above problems, and those of ordinary skill in the art will fully understand other problems not described herein through the following description.

[0013] [Technical Solution]

[0014] To achieve the object of the present invention, the present invention provides an immune microbubble complex, which comprises microbubbles; and an immune checkpoint inhibitory antibody conjugated to the surface of the microbubbles, and the immune checkpoint inhibitory antibody activates T cells by inhibiting the immune escape of tumor cells in an environment that promotes immune response through ultrasonic sonoporation.

[0015] In one embodiment of the present invention, the conjugation of the microbubbles and the antibody can be an amide bond, a thiol bond or a biotin-avidin bond.

[0016] In another embodiment of the present invention, the microbubbles may comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0017] In yet another embodiment of the present invention, DSPE may be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2000-NHS).

[0018] In addition, the present invention provides a method for preparing an immune microbubble complex, the method comprising: (a) mixing phospholipids with an organic solvent and then hydrating the resulting mixture;

[0019] (b) preparing microbubbles by dispersing and stirring the hydrated liposome precursor obtained in step (a); and

[0020] (c) conjugating an immune checkpoint inhibitory antibody with the microbubbles.

[0021] In one embodiment of the present invention, the phospholipids may comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0022] In another embodiment of the present invention, the molar ratio of DSPC to DSPE may be 6 to 9:1 to 4 (mol).

[0023] In yet another embodiment of the present invention, the immune checkpoint inhibitory antibody may be one or more selected from the group consisting of an anti-programmed death ligand-1 (PD-L1) antibody, an anti-B7-1 antibody and an anti-B7-2 antibody.

[0024] In addition, the present invention provides a drug delivery carrier comprising the immune microbubble complex.

[0025] In addition, the present invention provides a contrast agent composition for cancer cell-specific ultrasonic, magnetic resonance imaging or fluorescence analysis, which comprises the immune microbubble complex.

[0026] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the immunomicrobubble complex.

[0027] In one embodiment of the present invention, the immunomicrobubble complex can be used in combination with ultrasonic treatment.

[0028] In another embodiment of the present invention, the ultrasonic wave can be high-intensity focused ultrasound (HIFU).

[0029] In still another embodiment of the present invention, the immunomicrobubble complex can concentrate lymphocytes near cancer cells through ultrasonic cavitation.

[0030] In addition, the present invention provides a method for providing cancer diagnosis information, which comprises treating a biological sample with the immunomicrobubble complex; and performing ultrasonic treatment.

[0031] In addition, the present invention provides a method for diagnosing cancer, which comprises treating a biological sample with the immunomicrobubble complex; and performing ultrasonic treatment.

[0032] In addition, the present invention provides a method for preventing or treating cancer, which comprises administering a composition comprising the immunomicrobubble complex to a subject in need; and performing ultrasonic treatment.

[0033] In addition, the present invention provides the use of a composition comprising the immunomicrobubble complex for preventing or treating cancer.

[0034] In addition, the present invention provides the use of the immunomicrobubble complex in the preparation of a medicament for cancer treatment.

[0035] [Advantageous Effects]

[0036] The immunomicrobubble complex (IMC) according to the present invention comprises microbubbles conjugated with an antibody, wherein the microbubbles have excellent stability and excellent antibody binding strength, and it has been confirmed that when the immunomicrobubble complex is treated with high-intensity focused ultrasound (HIFU), the anti-tumor effect is significantly increased and an immune-enhancing effect is exhibited. Therefore, the immunomicrobubble complex according to the present invention is expected to improve the delivery efficiency of the conjugated antibody, can be used for the diagnosis and treatment of cancer, and exhibits various functions in the field of immunotherapy, including a contrast effect, improved half-life, improved drug delivery, lymphocyte concentration effect, cancer immunotherapy, and induced immunotherapy using ultrasonic waves. Brief Description of the Drawings

[0037] Figure 1 Shows the size and number of microbubbles (MB) prepared according to the ratio of DSPC:DSPE-PEG2K-NHS according to one embodiment of the present invention.

[0038] Figure 2 Shows the observation results of the stability over time of microbubbles prepared according to the ratio of DSPC:DSPE-PEG2K-NHS according to an embodiment of the present invention.

[0039] Figure 3 Shows the size distribution of microbubbles prepared according to the ratio of DSPC:DSPE-PEG2K-NHS according to an embodiment of the present invention.

[0040] Figure 4 Shows the DSPE-PEG-NHS structure and size distribution of microbubbles prepared at a DSPC:DSPE-PEG2K-NHS ratio of 9:1 according to an embodiment of the present invention.

[0041] Figure 5 Shows the number of microbubbles depending on time and temperature according to an embodiment of the present invention.

[0042] Figure 6 Shows the concentrations of anti-PD-L1 antibody and anti-PD-L1 antibody + micelles in the remaining solution after synthesizing the immunomicrobubble complex (IMC) according to an embodiment of the present invention.

[0043] Figure 7 Shows a set of confocal images of the immunomicrobubble complex according to an embodiment of the present invention.

[0044] Figure 8 Shows the confocal image and DIC image of the immunomicrobubble complex according to an embodiment of the present invention.

[0045] Figure 9 Shows the experimental design for verifying the PD-L1 expression in B16F10 cells according to an embodiment of the present invention.

[0046] Figure 10 Shows the results of verifying the PD-L1 expression in B16F10 cells according to an embodiment of the present invention.

[0047] Figure 11 Shows the anti-tumor effects of microbubbles and immunomicrobubble complexes according to an embodiment of the present invention treated with high-intensity focused ultrasound (HIFU).

[0048] Figure 12 Shows the IFN-γ and cytolytic T cell activities after HIFU treatment of microbubbles and immunomicrobubble complexes according to an embodiment of the present invention. Detailed Description

[0049] The present invention provides an immune microbubble complex, which comprises microbubbles; and an immune checkpoint inhibitory antibody conjugated to the surface of the microbubbles, and the immune checkpoint inhibitory antibody activates T cells by inhibiting the immune escape of tumor cells in an environment that promotes an immune response through sonophoresis.

[0050] According to the present invention, the microbubbles can promote an immune response through sonophoresis, and the immune checkpoint inhibitory antibody can enable immune cells to recognize target disease cells.

[0051] In the present invention, the microbubbles may comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0052] In the present invention, the antibody can be conjugated to the microbubbles through a linker or an activating functional group on the surface of the microbubbles. The functional group can be a thiol group or an amine group, and the linker can be a compound containing the functional group, and the conjugation of the antibody to the microbubbles can be an amide bond, a bond between thiols, or a biotin-avidin bond.

[0053] In the present invention, a "linker" refers to a substance that connects an antibody or a drug to the microbubbles, and can be, but is not limited to, for example, N-hydroxysuccinimide (NHS) or maleimide. According to an embodiment of the present invention, DSPE can be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2000-NHS) to which NHS as a linker is attached.

[0054] In the present invention, the particle size of the microbubbles can be 0.8 to 1.5 μm, and according to an embodiment of the present invention, the average diameter of the microbubbles can be 1.19 ± 0.245 μm.

[0055] In the present invention, the molar ratio of DSPC to DSPE can be 6 to 9:1 to 4 (mol), 6.5 to 9:1 to 3.5 (mol), or 7 to 9:1 to 3 (mol), and preferably 7:3 (mol) or 9:1 (mol). Most preferably, when the microbubbles are prepared at a molar ratio of 9:1 (mol), the stability of the prepared microbubbles may be the highest, but the present invention is not limited thereto.

[0056] As used herein, the term "ultrasound" refers to sound waves with frequencies above 16 Hz to 20 kHz that are typically audible to the human ear, and high-intensity focused ultrasound (HIFU) can exhibit instantaneous thermal effects (65 to 100 °C), cavitation effects, mechanical effects, and sonochemical effects depending on energy and frequency by introducing focused ultrasound to provide continuous high-intensity ultrasound energy to the focus. Ultrasound is harmless when passing through human tissues, but the high-intensity ultrasound forming the focus generates sufficient energy to cause coagulative necrosis and thermal cauterization regardless of the type of tissue.

[0057] In the present invention, the ultrasound can be HIFU, but the present invention is not limited thereto. In the present invention, the frequency for HIFU can be 0.1 to 10 MHz, 0.1 to 9 MHz, 0.1 to 8 MHz, 0.1 to 7 MHz, 0.1 to 6 MHz, 0.1 to 5 MHz, 0.1 to 4 MHz, 0.1 to 3 MHz, or 0.1 to 2 MHz, and according to one embodiment of the present invention is 1.1 MHz. In the present invention, HIFU can be processed for 10 to 20 seconds under the conditions of 1.1 MHz, 50 to 100 W, 1 to 10 duty cycles, and 40 PRF, but the present invention is not limited thereto.

[0058] As used herein, the term "sonophoresis" utilizes the wave energy of sound waves, typically ultrasound, to improve the transport of substances through a liquid medium, and refers to a drug delivery method that uses ultrasound to increase drug absorption. Here, the compression wave of the sound wave causes "flow" and / or "cavitation" in the liquid medium. When the immune checkpoint inhibitory antibody according to the present invention is treated with ultrasound, an immune response promoting effect is exhibited through sonophoresis, and the antibody can further target target cells such as cancer cells.

[0059] As used herein, the term "cavitation" refers to a phenomenon in which, when there is a low-pressure region in a fluid, the gas contained in the water escapes from the water and accumulates in the low-pressure region, resulting in a cavity space without water, and as ultrasound-induced cavitation occurs in the immune microbubble complex of the present invention, voids are temporarily formed in the cell membranes of surrounding cells, and substances penetrate into the cells through the voids, thereby causing lymphocytes to aggregate near cancer cells.

[0060] In addition, the present invention provides a method for preparing an immune microbubble complex, the method comprising: (a) mixing phospholipids with an organic solvent and then hydrating the resulting mixture;

[0061] (b) preparing microbubbles by dispersing and stirring the hydrated liposome precursor obtained in step (a); and

[0062] (c) conjugating an immune checkpoint inhibitory antibody with the microbubbles.

[0063] In the present invention, the phospholipid may comprise DSPC and DSPE.

[0064] In the present invention, the molar ratio of DSPC and DSPE in the phospholipid may be 6 to 9:1 to 4 (mol), 6.5 to 9:1 to 3.5 (mol), or 7 to 9:1 to 3 (mol), preferably 7:3 (mol) or 9:1, and most preferably 9:1 (mol).

[0065] The term "checkpoint" refers to proteins used in the program of activating or inactivating human immune cells, which include, for example, programmed death ligand-1 (PD-L1), B7-1, and B7-2 located on the surface of cancer cells. In the present invention, "checkpoint inhibition" refers to inhibiting the ability of the immune system to suppress checkpoints, and the blockade of the inhibited immune checkpoint activates the immune system function. In the present invention, an antibody conjugated to a microbubble can be used as a checkpoint inhibitor.

[0066] As used herein, the term "antibody" refers to a polypeptide comprising a framework region derived from an immunoglobulin gene that specifically binds to an antigen or a fragment thereof. The recognized immunoglobulin genes include genes for the kappa (κ), lambda (λ), alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ) constant domains, as well as numerous genes for the immunoglobulin variable domains. The light chain is divided into κ or λ. The heavy chain is divided into γ, μ, α, δ, and ε, indicating the class of immunoglobulin, such as IgG, IgM, IgA, IgD, and IgE. Generally, the antigen-binding region of an antibody is most critical for binding specificity and affinity. In the present invention, the antibody or its fragment may be derived from different subjects, including humans, mice, rats, hamsters, camels, and rabbits, but the present invention is not limited thereto. The antibody of the present invention may include an antibody modified or mutated at one or more amino acid positions to improve or regulate the preferred functions of the antibody (such as glycosylation, expression, antigen recognition, effector function, antigen binding, or specificity).

[0067] In the present invention, the antibody may be one or more selected from the group consisting of an anti-PD-L1 antibody, an anti-B7-1 antibody, and an anti-B7-2 antibody, and according to one embodiment of the present invention, the antibody may be an anti-PD-L1 antibody, but the present invention is not limited thereto.

[0068] In the present invention, the anti-PD-L1 antibody can specifically bind to PD-L1, thereby inhibiting PD-L1 binding and preventing the inhibition of the immune response to tumors. Here, the amino acid sequence of PD-L1 can be represented by SEQ ID NO:1 (NCBI GenBank: ADK70950.1), and InVivoPlus anti-mouse PD-L1 (B7-H1) (Catalog #BP0101, Clone 10F.9G2) was purchased as the anti-PD-L1 antibody.

[0069] In addition, the present invention provides a drug delivery carrier comprising the immune microbubble complex.

[0070] As used herein, the term "drug" refers to any compound having the desired biological activity. The desired biological activity includes any activity useful for the diagnosis, cure, alleviation, treatment, or prevention of diseases in humans or other animals.

[0071] In addition, the present invention provides a contrast agent composition for cancer cell-specific ultrasound, magnetic resonance imaging, or fluorescence analysis, which comprises the immune microbubble complex.

[0072] As used herein, the term "contrast agent" refers to a substance administered to an organism to effectively and specifically contrast or image cancer cells in vivo, and is now widely used for image enhancement of tissues and cells in the medical and diagnostic fields. The term "contrast agent" as used herein is not limited to the scope of conventionally known CT, PET, and MRI contrast agents, and includes contrast agents for ultrasound imaging and fluorescence imaging.

[0073] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, which comprises the immune microbubble complex.

[0074] As used herein, the term "cancer" is a general term for diseases caused by cells that have the invasiveness of cell division and growth not restricted by normal growth, the invasiveness of cells penetrating into surrounding tissues, and the metastatic property of cells spreading to other parts of the body.

[0075] In the present invention, the cancer is not particularly limited as long as it is a malignant tumor known in the art, and may be selected from the group consisting of: breast cancer, colorectal cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell cancer, renal pelvic cancer, CNS tumors, primary CNS lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenoma.

[0076] In the present invention, "prevention" refers to all actions of inhibiting cancer or delaying its onset by administering the composition according to the present invention.

[0077] As used herein, "treatment" refers to all actions involved in alleviating or beneficially altering the symptoms of cancer by administering the composition according to the present invention.

[0078] The term "pharmaceutical composition" as used herein refers to a pharmaceutical composition prepared for preventing or treating cancer, and may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating materials, and controlled release additives.

[0079] The pharmaceutical composition according to the present invention can be formulated into the following forms according to conventional methods: powders, granules, sustained release granules, enteric coated granules, solutions and liquids, eye drops, elixirs, emulsions, suspensions, spirits, lozenges, aromatic waters, lemon waters, tablets, sustained release tablets, enteric coated tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusion agents, plasters, lotions, pastes, sprays, inhalants, patches, sterile injections, or topical preparations, such as aerosols, and the topical preparations may have dosage forms such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes, or poultices.

[0080] As the carriers, excipients, and diluents that can be included in the pharmaceutical composition according to the present invention, lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil can be used.

[0081] The composition can be formulated with diluents or excipients such as fillers, thickeners, binders, wetting agents, disintegrants, and surfactants conventionally used.

[0082] As additives for tablets, powders, granules, capsules, pills, and lozenges, excipients can be used, such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica, hydroxypropyl starch; hydroxypropyl methylcellulose 1928, 2208, 2906, 2910; propylene glycol, casein, calcium lactate, and Primojel; binders, such as gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerol, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethyl cellulose, purified shellac, starch powder, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone; disintegrants, such as hydroxypropyl methylcellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicon anhydride, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, starch gel, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, granulated sugar, magnesium aluminum silicate, d-sorbitol solution, and light anhydrous silicic acid; and lubricants, such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, limestone kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000 and 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, polyethylene glycol (Macrogol), synthetic aluminum silicate, silicon anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, di-leucine, and light anhydrous silicic acid.

[0083] Additives for the liquid according to the present invention may be water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitan fatty acid ester (Tween ester), polyoxyethylene monoalkyl ether, lanolin ether, lanolin ester, acetic acid, hydrochloric acid, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, gliadin, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethyl cellulose.

[0084] For the syrup according to the present invention, a sucrose solution, other types of sugars or sweeteners may be used, and, if necessary, flavoring agents, coloring agents, preservatives, stabilizers, suspending agents, emulsifying agents, or thickening agents may be used.

[0085] For the emulsion according to the present invention, emulsifying agents, preservatives, stabilizers, or flavoring agents may be used as needed.

[0086] For the suspension according to the present invention, suspending agents such as gum arabic, tragacanth, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose 1828, 2906, or 2910 may be used, and, if necessary, surfactants, preservatives, stabilizers, coloring agents, and flavoring agents may be used.

[0087] For the injection according to the present invention, the following can be used: solvents such as injectable sterile water, 0.9% sodium chloride for injection, Ringer's solution, glucose for injection, glucose + sodium chloride solution for injection, PEG, lactated Ringer's solution, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, or phenyl benzoate; co-solvents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, phenylbutazone, propylene glycol, Tween, nicotinamide, hexamine, or dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, or gum; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O3), sodium sulfite (Na2SO3), nitrogen gas (N2), or ethylenediaminetetraacetic acid; antioxidants such as 0.1% sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, or sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, or calcium gluconate; or suspending agents such as sodium CMC, sodium alginate, Tween 80, or aluminum monostearate.

[0088] For the suppositories according to the present invention, matrices can be used, such as cocoa butter, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, mixtures of stearates and oleates, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, Lanette wax, glyceryl monostearate, Tween or Span, Imhausen, monopropylene (propylene glycol monostearate), glycerol, Adeps solidus, Buytyrum Tego-G, CebesPharma 16, hexalide base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Masupol, Masupol-15, neosuppostal-N, paramount-B, supposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Suppostal (N, Es), Wecoby (W, R, S, M, Fs) or Tegester triglyceride base (TG-95, MA, 57).

[0089] Solid preparations for oral administration can be tablets, pills, powders, granules, or capsules, and such solid preparations can be prepared by mixing at least one of excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin with the active ingredient. Moreover, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0090] As liquid preparations for oral administration, suspensions, oral liquids, emulsions, or syrups can be used, and can include simple and commonly used diluents such as water or liquid paraffin, as well as various types of excipients such as wetting agents, sweeteners, flavors, and preservatives. Preparations for parenteral administration can be sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized products, or suppositories. As non-aqueous solvents or suspensions, propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), or injectable esters (such as ethyl oleate) can be used.

[0091] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose can be determined by parameters including: the type of disease of the patient, the severity, the drug activity, the sensitivity to the drug, the administration time, the administration route and the excretion rate, the treatment duration, and the drugs used simultaneously, as well as other parameters well known in the medical field.

[0092] The pharmaceutical composition of the present invention can be administered alone or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, or in a single dose or multiple doses. Considering all the above parameters, it is important to achieve the maximum effect with the minimum dose without side effects, and such a dose can be easily determined by those of ordinary skill in the art.

[0093] The pharmaceutical composition of the present invention can be administered to a subject in need thereof via various routes. All administration routes are contemplated, and the pharmaceutical composition of the present invention can be administered, for example, by oral administration, subcutaneous injection, intraperitoneal administration; intravenous, intramuscular or intrathecal injection; sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, by mouth or nasal spray, cutaneous administration or transdermal administration.

[0094] The pharmaceutical composition of the present invention is determined according to a variety of relevant parameters, which include the disease to be treated, the administration route, the age, sex and weight of the patient, as well as the severity of the disease, and the type of the drug as the active ingredient.

[0095] In the pharmaceutical composition for preventing or treating cancer according to the present invention, the immunomicrobubble complex contained in the composition can be used in combination with ultrasonic treatment, and in addition to the immunomicrobubble complex, the composition can further contain one or more selected from the group consisting of anticancer agents, imaging contrast agents, antibiotics, anti-inflammatory agents, proteins, cytokines, peptides and antibodies.

[0096] In addition, the present invention provides a method for providing cancer diagnosis information, the method comprising treating a biological sample with an immunomicrobubble complex; and performing ultrasonic treatment.

[0097] In addition, the present invention provides a method for diagnosing cancer, the method comprising treating a biological sample with an immunomicrobubble complex; and performing ultrasonic treatment.

[0098] As used herein, the term "diagnosis" refers to confirming the existence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm whether cancer has progressed.

[0099] In addition, the present invention provides a method for treating cancer, the method comprising administering to a subject in need thereof a composition comprising an immunomicrobubble complex; and performing ultrasonic treatment.

[0100] In addition, the present invention provides the use of a composition comprising the immunomicrobubble complex for treating cancer.

[0101] In addition, the present invention provides the use of an immunomicrobubble complex in the preparation of a medicament for cancer treatment.

[0102] In the present invention, "administering" means providing the composition of the present invention to a subject in need thereof by any suitable method.

[0103] In the present invention, the term "subject" as used herein refers to a subject in need of treatment, more specifically a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0104] In one embodiment of the present invention, microbubbles were prepared using different ratios of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2K-NHS), and the physical properties of the microbubbles were compared. It has been confirmed that when microbubbles are prepared using a molar ratio of 9:1 of DSPC and DSPE-PEG2K-NHS, the microbubbles have the highest stability (see Example 2).

[0105] Tumor cells evade the immune response using their physiological methods. On the surface of tumor cells, PD-L1 is usually upregulated, while on the surface of T cells, programmed death 1 (PD-1) induces an immune response to PD-L1 / PD-1 binding and inhibits the participation of the costimulatory molecule CD80. CD80 is a member of the immunoglobulin superfamily and provides an antigen-nonspecific costimulatory signal important for the maximum immune response. When this receptor binds to its ligand, it recruits protein tyrosine phosphatases of the Src homology 2 domain-containing family (SHP), which prevents T cells from releasing granules and perforin regardless of whether they recognize MHC I, inhibits regulatory T cell stimulation, promotes T cell apoptosis and effector T cell activation.

[0106] Inhibition of PD-L1 / PD-1 binding is one of the methods to maintain anti-tumor immune responses by suppressing immunosuppression, and immunotherapy targeting PD-L1 / PD-1 binding can be used.

[0107] Thus, in another embodiment of the present invention, an immunomicrobubble complex (IMC) was prepared by binding an anti-PD-L1 antibody to the prepared microbubbles, and it was confirmed by measuring the conjugation efficiency of the anti-PD-L1 antibody that the antibody was 100% conjugated (see Example 3).

[0108] In yet another embodiment of the present invention, by observing the confocal images of the prepared immunomicrobubble complex (IMC), the antibody conjugated to the surface of the microbubbles was confirmed (see Example 4).

[0109] In yet another embodiment of the present invention, as a result of confirming the anti-tumor effect and the activities of IFN-γ and cytotoxic T cells [shown by treating the prepared immunomicrobubble complex (IMC) with HIFU], it was confirmed that when the IMC was treated with HIFU, an immune-enhancing effect was shown (see Example 5).

[0110] Hereinafter, in order to assist in understanding the present invention, exemplary embodiments will be presented. However, the following embodiments are provided only for easier understanding of the present invention and do not limit the present invention.

[0111] Example 1. Preparation of Materials

[0112] 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was obtained from Coatsome, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2000-NHS) was purchased from Nanocs. In addition, rat IgG2b isotype control (7.62 mg / ml) and anti-mouse PD-L1 (B7-H4) (6.76 mg / ml) were obtained from BioXcell.

[0113] Example 2. Preparation of Microbubbles According to DSPE-PEG2K Ratio and Verification of Microbubble Properties

[0114] 2-1. Preparation of Microbubbles

[0115] According to the method of hydrating the phospholipid membrane using 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2K-NHS), microbubbles (hereinafter referred to as MB) were synthesized, and at this time, the properties of the microbubbles prepared according to the ratio of DSPC and DSPE-PEG2K-NHS were compared.

[0116] Therefore, DSPC and DSPE-PEG2K-NHS were dissolved in chloroform at molar ratios of 9:1, 7:3, 6:4, 5:5, or 3:7. Subsequently, the chloroform was evaporated, thereby forming a phospholipid film. The 1.0 mg / ml phospholipid film was disrupted using a bath-type sonicator and hydrated with 0.01 M PBS at the phase transition temperature of DSPC. Then, the hydrated product was placed in a vial and filled with sulfur hexafluoride gas (SF6). Subsequently, in order to convert the hydrated liposome precursor into microbubbles, the hydrated liposome precursor was agitated for 45 seconds using Vialmix TM (Definity, USA) to prepare microbubbles.

[0117] The size distribution and zeta potential of the microbubbles prepared according to the DSPC:DSPE-PEG2K-NHS ratio by the above method were measured using a Malvern Zetasizer Nano (Malvern Instrument Ltd., Worcesterchire, U.K.). The microbubbles were dissolved in PBS (pH 7.4) at 100 μg / ml, and five measurements were performed at 25°C.

[0118] As a result, the sizes and numbers of the microbubbles prepared at five different ratios were as Figure 1 shown. And it was confirmed that when the molar ratio of DSPC:DSPE-PEG2K-NHS was 9:1, the generated microbubbles were smaller and more numerous.

[0119] 2-2. Comparison of Microbubble Stability

[0120] The stability of the microbubbles prepared according to the DSPC:DSPE-PEG2K-NHS ratio in Example 2-1 was observed over time.

[0121] Specifically, microbubbles were prepared at five molar ratios as shown in Example 2-1, and the degree of disappearance of the microbubbles at 0, 3, 6, and 24 hours was visually confirmed at room temperature.

[0122] As a result, as Figure 2 shown, over time, as the solution became transparent in the order of DSPC:DSPE-PEG2K-NHS ratios of 3:7, 5:5, and 6:4, the disappearance of the microbubbles was observed, thereby confirming that the microbubbles were stable when prepared at ratios of 7:3 and 9:1.

[0123] In addition, as Figure 3As shown, as a confirmation result of the size distribution of microbubbles prepared at five different ratios, in the cases of 3:7 and 5:5 ratios, except for the peak at 1 h, no size distribution was shown in the ζ size due to low stability, and in the case of 6:4 ratio, it was confirmed that the microbubbles were unstable at 24 h. On the other hand, it was confirmed that in the cases of 7:3 and 9:1 ratios, the size distribution of the microbubbles was stable, and especially in the case of 9:1 ratio, the microbubbles had the highest stability.

[0124] Therefore, in the present invention, microbubbles were prepared at a DSPC:DSPE-PEG2K-NHS ratio of 9:1 and used for experiments. Here, the microbubbles were prepared by the method described in Example 2-1, and the concentration of the phospholipid membrane was 0.5 mg / ml.

[0125] The structure of DSPE-PEG-NHS used for preparing microbubbles and the size distribution of microbubbles prepared at a DSPC:DSPE-PEG2K-NHS ratio of 9:1 are shown in Figure 4 where the size of the microbubbles was 1.19 ± 0.245 μm and the number average of the microbubbles was 1.99x10 9 .

[0126] 2-3. Confirm the stability of microbubbles according to time and temperature

[0127] To test the stability of microbubbles (MB) according to time and temperature, the number of microbubbles filled with air (MB with air) and microbubbles filled with SF6 gas (MB with SF6) prepared after filling with air was monitored over time at 4 °C and 25 °C under an optical microscope.

[0128] As a result, as shown in Figure 5 , it was confirmed that in the case of MB with SF6 (4 °C), the largest number of microbubbles was observed and the microbubbles were highly stable.

[0129] Example 3. Preparation of Immune Microbubble Complexes

[0130] 3-1. Conditions of microbubble antibody particles

[0131] An immunomicrobubble complex (IMC) was prepared by conjugating anti-PD-L1 antibody with N-hydroxysuccinimide (NHS) of the microbubbles prepared in Example 2-1. Here, assuming a 1:1 reaction of anti-PD-L1 antibody with NHS, the conditions of anti-PD-L1 antibody used for preparing IMC were confirmed.

[0132] In this case, as shown in Table 1, the number of NHS molecules was approximately 100 times the number of PD-L1 antibody molecules. Therefore, it was expected that when the single dose of anti-PD-L1 antibody was 100 to 200 μg, the antibody bound to MB would be sufficient.

[0133] [Table 1]

[0134]

[0135] 3-2. Preparation of Immunomicrobubble Complex

[0136] After purifying the microbubbles prepared in Example 2-1, 200 μg of anti-PD-L1 antibody (hereinafter referred to as PD-L1 Ab; BioXcell, USA) was conjugated to 1.5x10 9 microbubbles. To remove the unconjugated PD-L1 Ab, purification of the microbubble antibody conjugate was performed by gradient centrifugation at 300 rpm for 5 minutes, and the size and surface charge were measured by dynamic light scanning (Malvern Zetasizer Nano series, England).

[0137] 3-3. Measurement of Binding Efficiency of Anti-PD-L1 Antibody

[0138] After IMC synthesis, considering the measurement error of binding strength that may be caused by the residual liposomes + antibody (Ab) in the filtrate, the concentrations of (1) lipid (micelle) + Ab and (2) Ab were measured first. As a result, as Figure 6 shown, it was confirmed that there was almost no difference between the two groups, and the antibody conjugation efficiency was confirmed by measuring the unbound antibody present in the filtrate after IMC synthesis by the Bradford assay method.

[0139] Specifically, after waiting sufficiently to avoid mixing the antibody into the filtrate as much as possible, the filtrate was separated by syringe, mixed with Bradford solution and left for 5 minutes, and then the antibody was measured.

[0140] As a result, in the STD curve, there was no difference from the control group (without Ab), and as shown in Table 2 below, by the Bradford assay method, the antibody conjugation efficiency was 99.9%, and there was no difference between the filtrate and the control, indicating that 200 μg of the antibody was 100% conjugated.

[0141] [Table 2]

[0142]

[0143] In addition, as a result of detecting the conjugation with thiol using the Bradford assay method, the antibody was measured at 0.581 nm and 0.576 nm as shown in Table 3 below.

[0144] [Table 3]

[0145]

[0146] Example 4. Confirmation of Confocal Images of Immune Microbubble Complexes (IMCs)

[0147] 4-1. Confirmation of Confocal Images According to Antibody Concentration

[0148] To confirm the conjugation of the antibody to the microbubble, confocal images of the IMC were confirmed.

[0149] Specifically, the IMC prepared by conjugating 10 μL (0.01 mg / ml) of MB with 20 to 160 μg of the primary antibody (mouse PD-L1 antibody) was treated with a FITC-labeled secondary antibody (binding to the Fc region of the mouse PD-L1 antibody) in an amount twice that of the primary antibody at 4°C for 60 minutes. The IMC solution conjugated with the secondary antibody was purified by centrifugation at 3,000 rpm for 5 minutes, and then the purified product was dropped onto a glass slide. Then, the IMC was observed using a confocal laser scanning microscope (CLSM).

[0150] As a result, as Figure 7 shown, as the antibody concentration increased, the fluorescence became more obvious, and a high binding strength of the anti-PD-L1 antibody to the MB was observed.

[0151] 4-2. Comparison of GFP and DIC Images

[0152] The IMC prepared by conjugating 1 mg of MB with 1 mg of the primary antibody (anti-PD-L1 antibody) was treated with 1 mg of the GFP-labeled secondary antibody, and then its confocal image was observed by the method described in Example 4-1. The images observed using a differential interference contrast (DIC) microscope and the images in which GFP and DIC were combined are shown in Figure 8 .

[0153] As Figure 8 shown, by observing the fluorescence on the surface of the microbubble, it was confirmed that in the IMC according to the present invention, the antibody was conjugated to the surface of the microbubble.

[0154] Example 5. Effects Caused by IMC and HIFU

[0155] 5-1. Confirmation of the Expression of PD-L1 in B16F10 Cells

[0156] As Figure 9 shown, an experiment was designed to confirm the expression of PD-L1 in B16F10 cells.

[0157] Specifically, B16F10 cells were lysed in the wells, and to detect the expressed PD-L1, 50 μg of anti-PD-L1 antibody was added. Subsequently, after washing twice with PBS or medium and confirming with a confocal microscope, 50 to 100 μg of a fluorescently labeled PD-L1 secondary antibody was added and left for approximately 2 hours. After that, it was washed 2 or 3 times again and confocal imaging was performed.

[0158] As a result, as Figure 10 shown, as a result of confirming the expression level of PD-L1, through fluorescent PD-L1, by observing PD-L1 stained red, it was confirmed that PD-L1 was highly expressed on the cell surface.

[0159] 5-2. Antitumor activities of IMC and HIFU

[0160] To confirm the antitumor activity of the IMC prepared in Example 3-2 or when the IMC was treated with HIFU, a BALB / c mouse model bearing CT26wt tumors was used, which was established by subcutaneously injecting a CT26wt cell suspension (1x10 6 cells per mouse) into the right flank of 4-week-old female BALB / c mice. After the tumor volume reached 50 mm 3 , the mice were randomly grouped for treatment. The experimental groups were defined as follows:

[0161] G1: Isotype Ab only (0.3 mg)

[0162] G2: PD-L1 Ab only (0.3 mg)

[0163] G3: PBS

[0164] G4: HIFU only

[0165] G5: Isotype Ab + MB (0.3 mg)

[0166] G6: Isotype Ab + MB + HIFU (0.3 mg)

[0167] G7: PD-L1 + MB (0.3 mg)

[0168] G8: IMC + HIFU (0.3 mg)

[0169] As the isotype Ab, a homologous IgG2 Ab without PD-L1 Ab function was used as a control.

[0170] The tumor size was measured with a digital caliper, and the tumor tissues were excised at the end of the experiment for histological analysis.

[0171] As a result, as Figure 11As shown, compared with G2, the tumor size of G1 was slightly smaller, but there was no significant difference ( Figure 11 upper figure), compared with G3, the tumor size of G4 was slightly smaller ( Figure 11 middle figure), and when both the isotype Ab+MB group (G5) and the PD-L1 Ab+MB group (G7) were treated with HIFU, the tumor size decreased significantly. Especially in the G8 group treated with HIFU for PD-L1 Ab+MB (IMC), the largest decrease in tumor size was shown ( Figure 11 lower figure).

[0172] Therefore, from the above results, it was found that HIFU treatment of the immunomicrobubble complex (IMC) prepared in the present invention led to excellent anti-tumor effects.

[0173] 5-3. Activity of IFN-γ and cytotoxic T cells

[0174] IFN-γ assays were performed by extracting plasma or ascites from mice and evaluating them by ELISA using a Quantikine mouse IFN-γ assay kit (R&D Systems, Minneapolis, MN).

[0175] In addition, for cytotoxic T cells, after resuspending cancer cells in PBS, 10 μM carboxyfluorescein succinimidyl ester (CFSE) was added to the resulting suspension, reacted with the cells at 37 °C for 10 minutes, and treated with an equal volume of FCS at room temperature for 2 minutes to stop the reaction. Subsequently, after washing twice with AIM-V (+5% human A / B serum), the resulting cells were resuspended in AIM-V + IL2, IL-7, and IL-15 (+5% human A / B serum) and incubated with a 1:10 mixture of dendritic cells (DC) and cytotoxic T cells (CTL) at 37 °C for 6 hours. After that, 1 μg / ml propidium iodide was added, and 10 μl of calibration beads were added before measurement.

[0176] As a result, as Figure 12 shown, similar to the results of Example 5-2, it was confirmed that the G8 group treated with HIFU for PD-L1 Ab+MB (IMC) showed the highest IFN-γ specific points and the highest cytotoxic T cell activity.

[0177] Therefore, it can be confirmed that HIFU treatment of the immunomicrobubble complex according to the present invention results in an immune-enhancing effect.

[0178] Those of ordinary skill in the art should understand that the above description of the present invention is exemplary, and without departing from the technical spirit or essential characteristics of the present invention, the exemplary embodiments disclosed herein can be easily modified into other specific forms. Therefore, the above exemplary embodiments should be construed as illustrative rather than limiting in any way.

[0179] Industrial applicability

[0180] The immunomicrobubble complex according to the present invention can improve the delivery efficiency of conjugated antibodies and can be used for the diagnosis and treatment of cancer, and is expected to be used in the field of immunotherapy, including contrast effect, half-life improvement, improved drug delivery, lymphocyte concentrating effect, cancer immunotherapy, and ultrasonic wave-induced immunotherapy.

Claims

1. An immune microbubble complex, comprising: Microbubbles; and An immune checkpoint inhibitory antibody conjugated to the surface of the microbubbles, wherein the immune microbubble complex activates T cells by inhibiting the immune escape of tumor cells in an environment that promotes an immune response through sonophoresis, Among them, The conjugation is an amide bond between the immune checkpoint inhibitory antibody and the microbubbles, Wherein the immune checkpoint inhibitory antibody has the ability to inhibit immune checkpoints, thereby activating the immune system function, Wherein the immune checkpoint inhibitory antibody is one or more selected from the group consisting of anti-programmed death ligand 1 (PD-L1) antibody, anti-B7-1 antibody, and anti-B7-2 antibody, and Wherein the microbubbles comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

2. The complex according to claim 1, wherein the DSPE is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG2000-NHS).

3. The complex according to claim 1, wherein the molar ratio of DSPC to DSPE is 6 to 9:1 to 4 (mol).

4. The complex according to claim 1, wherein the ultrasound is high-intensity focused ultrasound (HIFU).

5. A method for preparing an immune microbubble complex, the method comprising: (a) Mixing phospholipids with an organic solvent and then hydrating the resulting mixture; (b) Preparing microbubbles by dispersing and stirring the hydrated liposome precursor obtained in step (a); and (c) Conjugating an immune checkpoint inhibitory antibody to the microbubbles, Wherein the conjugation is an amide bond between the immune checkpoint inhibitory antibody and the microbubbles, Wherein the immune checkpoint inhibitory antibody has the ability to inhibit immune checkpoints, thereby activating the immune system function, Wherein the immune checkpoint inhibitory antibody is one or more selected from the group consisting of anti-programmed death ligand 1 (PD-L1) antibody, anti-B7-1 antibody, and anti-B7-2 antibody, and Wherein the microbubbles comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

6. The method according to claim 5, wherein the molar ratio of DSPC to DSPE is 6 to 9:1 to 4 (mol).

7. A drug delivery carrier comprising the immune microbubble complex according to any one of claims 1 to 4.

8. A contrast agent composition for cancer cell-specific ultrasound, magnetic resonance imaging, or fluorescence analysis, the contrast agent composition comprising the immune microbubble complex according to any one of claims 1 to 4.

9. A pharmaceutical composition for preventing or treating cancer, comprising the immune microbubble complex according to any one of claims 1 to 4.

10. The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the immunomicrobubble complex is used in combination with ultrasonic treatment.

11. The pharmaceutical composition for preventing or treating cancer according to claim 10, wherein the ultrasonic wave is high-intensity focused ultrasound (HIFU).

12. The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the immunomicrobubble complex focuses lymphocytes near cancer cells through ultrasonic cavitation.

13. Use of the immunomicrobubble complex according to any one of claims 1 to 4 in the preparation of a medicament for cancer treatment.

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