WO2024128868A1 - DENDRITIC CELL-MIMETIC FUNCTIONAL NANOSTRUCTURE COMPRISING αPD-1, AND PREPARATION METHOD THEREFOR - Google Patents

Abstract

The present invention relates to a dendritic cell-mimetic nanostructure for inhibiting immune checkpoints, and a preparation method therefor, the nanostructure comprising a nanoparticle core and a shell, which comprises the cell membrane of a lipid molecule derived from dendritic cells, wherein the shell comprises αPD-1 monoclonal antibody, and the strong activation of cytotoxic T cells, caused by immune checkpoint inhibitory activity, increases an immune response, and thus an excellent cancer immunotherapy effect can be provided.

Description

Dendritic cell-mimicking functional nanostructure containing αPD-1 and method for manufacturing the same

The present invention relates to a dendritic cell-mimicking functional nanostructure containing αPD-1 and a method of manufacturing the same.

Cancer ranks first among the causes of death in Korea. Representative cancer treatment methods include surgery to remove the cancer, radiation therapy, and chemical treatment, but these treatment methods have the problem of poor prognosis and serious side effects.

The main cause of cancer development is tumor formation by cancer cells that cannot be eliminated by the immune response due to decreased immunity or immune evasion of cancer cells. Therefore, immunotherapy that can help treat cancer by increasing the patient's own immune response can be a fundamental cancer treatment. Currently developed immunotherapy treatments mostly involve direct injection of drugs that enhance immune function, but drug injection still has limitations in that the delivery efficiency is very low and additional side effects may exist. Accordingly, research is focusing on the development of treatments that can dramatically increase cancer treatment efficiency, reduce recurrence rates and side effects, and increase immune function.

Meanwhile, research on nano-bio convergence technology that combines nanotechnology with the biomedical field continues. However, most nanomaterials are obtained through synthesis, and surface modification using nanomaterials involves the process of adding composites, which can lead to side effects such as toxicity in the body, induction of unwanted immune responses, and induction of cancer. Therefore, by using a surface modification method that directly mimics the living body, side effects can be minimized and various functions can be implemented according to biological characteristics, thereby overcoming the limitations of existing nanotechnology.

In particular, nanoparticle-cellularization technology is a technology that physically cloaks the surface of nanoparticles by using the entire cell membrane of a specific cell as a coating material. It can maintain the complex properties of the cell membrane along with proteins, lipids, and carbohydrates, thereby maintaining the surface of the nanoparticle. The characteristics of specific cells can be implemented as is.

Based on the technology that introduced platelets to the surface of PLGA (poly(lactic-co-glycolic) acid) particles for the first time in 2015 by Liangfang Zhang's research team at the University of California, it has been applied to various cells to this day.

Doxorubicin is loaded onto a PLGA core and then coated with a red blood cell membrane to produce an immunocompatible nanocarrier to remove solid tumors, or the red blood cell membrane and the cell membrane of cancer cells are simultaneously coated with melanin nanoparticles to create nanoparticles with a hybrid cell membrane. A study was reported to increase circulation time in the body and increase tumor targeting ability by manufacturing .

However, most of the conventional cell membrane coating technology was focused on cancer cells and blood cells, and its application was mainly focused on research aimed at stable delivery in the blood. In addition, when cancer cell membranes are directly introduced as antigens and delivered into the body, there are disadvantages such as decreased immune response due to antigen resistance and resistance to cancer cell-derived substances. Therefore, it is very necessary to develop an immunotherapy agent that can have the function of a direct antigen-presenting immune cell and induce differentiation and proliferation of T cells without intermediate processes.

The present disclosure aims to more effectively realize the cancer treatment effect by increasing the immune response by using cells with an antigen presentation function, breaking away from the material limitations of the prior art. Specifically, by modifying the surface of a nanostructure that mimics dendritic cells with the monoclonal antibody αPD-1, which can bind to PD-1, selective killing of cancer cells is achieved by directly activating cytotoxic T cells through inhibition of immune checkpoints. It induces and increases immune response to provide excellent immunotherapy effects.

In order to achieve the above object, the dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to the present disclosure includes a nanoparticle core; and a shell containing a cell membrane of lipid molecules derived from dendritic cells, wherein the shell contains an αPD-1 monoclonal antibody.

In one embodiment of the present disclosure, the αCTLA-4 monoclonal antibody may be conjugated with a lipid molecule.

In one embodiment of the present disclosure, the nanostructure may be one in which an antigen has been pulsed.

In one embodiment of the present disclosure, the antigen may be a peptide or protein derived from a tumor antigen.

In one embodiment of the present disclosure, the nanostructure may have an average particle diameter of 20 ㎚ to 50 ㎚, and specifically, may have an average particle diameter of 20 ㎚ to 1000 ㎚.

The present disclosure can provide a pharmaceutical composition for cancer treatment containing the dendritic cell-mimicking nanostructure for immune checkpoint inhibition as described above.

The present disclosure provides a method for manufacturing a dendritic cell-mimicking nanostructure for immune checkpoint inhibition, the method comprising: (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing nanoparticles and the liposomes and compressing them through a filter to obtain nanoparticles into which the cell membrane has been introduced; and (S50) introducing the αPD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane has been introduced.

In one embodiment of the present disclosure, after the step (S40), the step of pulsing an antigen to the nanoparticles into which the cell membrane has been introduced may be further included.

The dendritic cell-mimicking nanostructure according to the present disclosure introduces an antigen-presenting function to the surface of the nanoparticle, thereby preserving the antigen-presenting function of dendritic cells and adding the targeting function and photothermal effect function of the nanoparticle without killing them, thereby enhancing the function. It can provide functional immunotherapy effects.

The dendritic cell-mimicking nanostructure according to the present disclosure can stay in the body for a long period of time by mimicking dendritic cells and continuously induce proliferation and differentiation of antigen-specific T cells, thereby providing the effect of increasing the immune response.

The dendritic cell-mimicking nanostructure according to the present disclosure can induce binding to PD-1 present on the surface of cytotoxic T cells by including the monoclonal antibody αPD-1, which can bind to PD-1, on its surface. Through this combination, it inhibits the binding of PD-L1 and PD-1 present in antigen-presenting cells, thereby inducing suppression of the immune checkpoint, significantly activating cytotoxic T cells, inducing selective death of cancer cells, and promoting immune response. It provides excellent immunotherapy effect by increasing the anti-cancer immunotherapy effect.

Figure 1 is a schematic diagram showing a dendritic cell-mimicking nanostructure (αPD-1-conjugated Nano DC) for immune checkpoint inhibition into which αPD-1 monoclonal antibody is introduced. It shows that as PD-1 of cytotoxic T cells binds to αPD-1 of the nanostructure, PD-L1 inhibits the binding of PD-1, thereby activating cytotoxic T cells.

Figure 2 shows a method for producing αPD-1 conjugated phospholipid according to the present disclosure.

Figure 3 is a graph showing the results of surface potential analysis showing that αPD-1 conjugated phospholipid was successfully synthesized.

Figure 4 is a graph showing the results of FT-IR analysis showing that αPD-1 conjugated phospholipid was successfully synthesized.

Figure 5 shows the results of an in vitro experiment on the effect of inducing T cell proliferation and differentiation of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which the αPD-1 monoclonal antibody was introduced according to Experimental Example 1 of the present disclosure.

Figure 6 shows the in vivo test method conditions of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which the αPD-1 monoclonal antibody was introduced according to Experimental Example 2 of the present disclosure.

Figures 7 and 8 show the results of in vivo experiments on the effect of inducing T cell proliferation and differentiation of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which the αPD-1 monoclonal antibody was introduced according to Experimental Example 2 of the present disclosure.

Hereinafter, with reference to the attached drawings and examples, a person skilled in the art can easily perform the dendritic cell-mimicking functional nanostructure containing αPD-1 and its manufacturing method according to the present invention. Please explain in detail so you can. However, the present invention may be implemented in various different forms and is not limited to the implementation examples described herein. Additionally, it is not intended to limit the scope of protection limited by the claims.

Unless otherwise defined, technical terms and scientific terms used in the description of the present invention have meanings commonly understood by those skilled in the art, and the gist of the present invention is summarized in the following description. Descriptions of known functions and configurations that may be unnecessarily obscure are omitted.

Numerical ranges used herein include lower and upper limits and all values within that range, increments logically derived from the shape and width of the range being defined, all doubly defined values, and upper and lower limits of numerical ranges defined in different forms. Includes all possible combinations of Unless otherwise specified in the specification of the present invention, values outside the numerical range that may occur due to experimental error or rounding of values are also included in the defined numerical range.

As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly dictates otherwise.

The term “comprises” in this specification is an open description with the same meaning as expressions such as “comprises,” “contains,” “has,” or “characterized by” elements that are not additionally listed; Does not exclude materials or processes.

Hereinafter, the present invention will be described in detail.

*The dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to the present disclosure includes a nanoparticle core; and a shell containing a cell membrane of lipid molecules derived from dendritic cells, wherein the shell contains an αPD-1 monoclonal antibody.

The dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to the present disclosure contains the monoclonal antibody αPD-1 capable of binding to PD-1 on its surface, thereby inducing binding to PD-1 present on the surface of cytotoxic T cells, By inhibiting the binding of B7-1/2 and PD-1 present in antigen-presenting cells, it can provide the effect of significantly activating cytotoxic T cells by inducing inhibition of immune checkpoints. This further induces the selective death of cancer cells and increases the immune response, demonstrating excellent immunotherapy effects.

To this end, the dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to the present disclosure includes a nanoparticle core; and a shell containing a cell membrane of lipid molecules derived from dendritic cells. In this case, the shell may be characterized as containing an αPD-1 monoclonal antibody.

PD-1 (Programmed cell death protein 1) is a type I transmembrane protein of 55 kDa, which forms part of the Ig superfamily genes, and is well known as a co-inhibitory molecule for T cells belonging to the immunoglobulin molecule group. In normal T cells, PD-1 is not expressed under normal conditions, but its expression increases when activated. PD-1 binds to PD-L1, a ligand present on the surface of cancer cells. The interaction between PD-1 and PD-L1 reduces the number of tumor-invasive lymphocytes, reduces T cell receptor-mediated proliferation, and reduces the number of tumor-invasive lymphocytes. Immune evasion occurs.

αPD-1 antibodies can increase T cell activity by inhibiting the binding to PD-L1 and PD-1 and blocking their signal transduction, which has the effect of strengthening immune function against tumors. can be provided.

αPD-1 antibody is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. It may comprise an antigen-binding moiety or fragment that binds to a receptor and exhibits similar functional properties as a full antibody in inhibiting ligand binding and upregulating the immune system. αPD-1 antibodies known in the art include, but are not limited to, HuMAb, nivolumab, pembrolizumab, and pidilizumab.

The dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to the present disclosure includes an αPD-1 antibody in the shell, thereby blocking signal transduction from binding to the aforementioned PD-L1 and or PD-L2, thereby promoting T cell-mediated immunity. It can provide an effect that strengthens the response.

As an example of the present disclosure, the nanoparticle core may be a one-dimensional or two-dimensional nanostructure. Specific examples of the one-dimensional or two-dimensional nanostructure include carbon nanoribbons, carbon nanotubes, graphene, graphene oxide, reduced graphene oxide, MXene, metal nanorods, metal nanowires, or metal nanoplatelets ( platelet), but is not limited thereto.

In addition, the nanoparticle core includes biocompatible organic polymer nanoparticles, metal organic framework nanoparticles, metal nanoparticles, metal oxide nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, photothermal conversion nanoparticles, and It may be one or two or more selected from the group consisting of nucleic acid-containing nanoparticles and protein-containing nanoparticles. Specifically, it is more desirable if the nanomaterial itself is non-toxic and has light sensitivity so that it can be applied to photothermal therapy.

In one non-limiting example, the nanoparticle core can be a gold nanoparticle.

At this time, the nanoparticle core may have an average particle diameter of 5 to 1000 nm, or 10 to 500 nm. The nanostructure comprising the nanoparticle core and shell may have an average particle diameter of 20 nm to 50 ㎚, specifically 20 ㎚ to 10 ㎚, more specifically 20 ㎚ to 1000 ㎚, more specifically 50 ㎚ to 500 ㎚ It can have an average particle size of .

In one example of the present disclosure, the αPD-1 monoclonal antibody may be conjugated with a lipid molecule. The lipid molecule may be used without limitation as long as it is a biocompatible lipid molecule, and may specifically be a phospholipid, and more specifically, may be a phosphatidylethanolamine-based phospholipid.

According to one example, phosphatidylethanolamine-based phospholipids and αPD-1 monoclonal antibody may be covalently linked through a coupling agent. αPD-1 monoclonal antibody covalently bound to phospholipids can be easily inserted into the shell containing the cell membrane of lipid molecules using the spontaneous cell membrane insertion phenomenon depending on the interaction with the phospholipid and the lipid bilayer present in the cell membrane, and thus It can be stably introduced to the surface of the nanostructure.

Dendritic cells are powerful antigen-presenting cells, with major histocompatibility complex I/II (MHC I/II) and co-stimulatory molecules such as CD80 and CD86 and ICAM-1 on their cell membrane surface. Cell adhesion molecules are highly expressed, and by secreting large amounts of various cytokines such as interferon, IL-12, and IL-18 related to T cell activation, they can perform a powerful antigen presentation function. Accordingly, the nanostructure according to the present disclosure may preferably be one in which the antigen has been pulsed.

The antigen may be a peptide or protein derived from a tumor antigen for use in cancer treatment. Specifically, for example, in a mouse cancer model, it may be a protein or peptide derived from ovalbumin (OVA), LCMV (Lymphocytic choriomeningitis mammarenavirus) glycoprotein, or retrovirus protein. More specifically, it may be a cancer antigen peptide or protein of the OVA _257-264 , GP _33-41 , or p15E model.

Also, in human cancer antigens, (1) HER2/Neu, tyrosinase, gp100, MART, HPV E6/E7, EBV EBNA-1, carcinoembryonic antigen, a-fetoprotein, GM2, GD2, testis antigen, prostate antigen, and CD20 It may be a peptide or protein derived from (2) a tumor-specific antigen, including a tumor-related antigen and (2) a neoantigen that can be produced by various mutations.

As a non-limiting example, the tumor antigen-derived peptide may be GP _33-41 .

The shell of the nanostructure according to the present disclosure may be labeled with a bioactive polymer, bioactive component, or protein in addition to the αPD-1 monoclonal antibody.

Specifically, the bioactive ingredient may refer to a cytokine for cell signaling. Non-limiting examples may include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, monokines, and colony stimulating factors. More specifically, cytokines include the BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (C-X-C motif ligand ligand) family, and GDF (Growth/differentiation factor) family. Family, growth hormone, IFN (Interferon) family, IL (Interleukin) family, TNF (Tumor necrosis factors) family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6/uPAR-Related Protein 1), SLUPR-2 ( It may be any one selected from the group consisting of Secreted Ly-6/uPAR-Related Protein 2) and combinations thereof.

The cytokine induces or suppresses transcription factors or growth factors essential for T cell differentiation, and mediates the growth and differentiation of other immune cells, thereby enhancing the effect of the dendritic cell mimicking structure according to the present disclosure for immunotherapy. there is.

Specifically, the bioactive ingredient is not limited as long as it is an immune checkpoint inhibitor such as αCTLA-4 or αPD-L1 other than the αPD-1 monoclonal antibody, or an anticancer drug such as a chemotherapy drug using the same.

The present disclosure can provide a pharmaceutical composition for cancer treatment containing the dendritic cell-mimicking nanostructure for immune checkpoint inhibition as described above.

The dendritic cell-mimicking nanostructure for immune checkpoint inhibition introduced with the αPD-1 monoclonal antibody is very effective in inducing the proliferation and differentiation of cytotoxic T cells and activates T cells, making the pharmaceutical composition for cancer treatment a powerful immuno-anticancer treatment. It can provide an effect.

Cancer is a general term for various malignant solid tumors that can expand locally and through metastasis by invasion. Specific examples include B-cell lymphoma, non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, skin squamous cell carcinoma, colorectal cancer, and melanoma. It may be, but is not limited to, tumor, head and neck squamous carcinoma, hepatocellular carcinoma, gastric cancer, sarcoma, gastroesophageal cancer, renal cell carcinoma, glioblastoma, pancreatic cancer, bladder cancer, prostate cancer, breast cancer, cutaneous T-cell lymphoma, Merkel cell carcinoma, or multiple myeloma. No.

Pharmaceutical compositions for the treatment of cancer include formulation substances to modify, maintain or preserve pH, osmolarity, viscosity, sterility, clarity, color, isotonicity, odor, stability, rate of dissolution or release, adsorption or penetration. can do. The pharmaceutical composition may be administered orally or parenterally. Examples of parenteral include: intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intravascular, intradermal, skin patch, or transdermal. It can also be administered through the skin using iontophoresis.

The pharmaceutical composition may be administered as an individual treatment or in combination with other anticancer agents, and may be administered sequentially or simultaneously with conventional anticancer agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

In addition, according to the present disclosure, a method of treating cancer in an individual can be provided, which includes the step of administering the dendritic cell-mimicking nanostructure for immune checkpoint inhibition or a pharmaceutical composition containing the same to an individual suffering from cancer. By administering the pharmaceutical composition to a subject, a tumor-specific immune response can be induced in the subject, or symptoms of cancer can be treated and/or alleviated in the subject. At this time, the subject may be a human or non-human mammal.

In addition, the present disclosure presents the antigen on the surface by pulsing the antigen to a dendritic cell-mimicking nanostructure (anDC), inserts phospholipid conjugated with αPD-1 into the cell membrane, and finally creates an immune checkpoint into which the αPD-1 monoclonal antibody is introduced. A method of manufacturing a dendritic cell-mimicking nanostructure for inhibition can be provided.

Specifically, the manufacturing method includes (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing nanoparticles and the liposomes and compressing them through a filter to obtain nanoparticles into which the cell membrane has been introduced; and (S50) introducing the αPD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane has been introduced.

(S10) The step of purifying cell membranes from dendritic cells can be performed without limitation using methods known in the art, and, as an example, can be performed through rapid freezing-thawing and centrifugation. (S20) Energy can be applied to the purified cell membrane to form a cell membrane suspension. At this time, as an example, a cell membrane suspension of hundreds of nanometers or several micrometers can be obtained by treating it with ultrasound. Afterwards (S30), the cell membrane suspension is filtered through a membrane filter to obtain cell membrane liposomes of the desired size. Although not limited thereto, liposomes having an average particle diameter of specifically 10 to 200 nm, more specifically 20 to 150 nm, can be obtained. Thereafter, the cell membrane liposome and nanoparticles are mixed and co-extruded to obtain a form in which the cell membrane of dendritic cells is coated on the surface of the nanoparticles.

After the step (S40), the method may further include pulsing an antigen to the nanoparticles into which the cell membrane has been introduced. The antigen may be a peptide or protein derived from a tumor antigen for use in cancer treatment, and various tumor-specific antigens may be utilized as described above. In the present disclosure, GP _33-41 can be used as a non-limiting example.

In step (S50), the lipid molecule may be used without limitation as long as it is a biocompatible lipid molecule, and may specifically be a phospholipid, and more specifically, may be a phosphatidylethanolamine-based phospholipid. A specific example of the phosphatidylethanolamine-based phospholipid may be 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Figure 2 shows the process of obtaining αPD-1 conjugated to a lipid molecule. One end of PEG may be conjugated to the hydrophilic group of DSPE, and αPD-1 may be conjugated to the other end of PEG. The weight average molecular weight of PEG may be 200 to 10,000 g/mol or 500 to 5,000 g/mol.

αPD-1 conjugated with phospholipids can be easily inserted according to lipid-lipid interactions in the cell membrane of dendritic cell nanostructures, effectively inducing the activation of T cells.

As described above, the present invention has been described with specific details and limited embodiments, but these are provided only to facilitate a more general understanding of the present invention, and the present invention is not limited to the above embodiments, and the field to which the present invention pertains is not limited to the above embodiments. Those skilled in the art can make various modifications and variations from this description.

Accordingly, the spirit of the present invention should not be limited to the described embodiments, and the scope of the patent claims described later as well as all things that are equivalent or equivalent to the scope of this patent claim shall fall within the scope of the spirit of the present invention. .

Preparation Example 1. Preparation of dendritic cell-mimicking nanostructure

Bone marrow cells were collected from the hind limbs of 6- to 8-week-old Naive 6 mice (Orient Bio), treated with ACK lysis buffer for 3 minutes at room temperature to remove red blood cells (RBCs), and only bone marrow cells from which RBCs were removed were obtained. Count the total number of bone marrow cells and seed ^them in a 6 well non-tissue _plate at 7.5 did. On the 3rd day, 2 ml of GM-CSF (20 ng/ml) media was added and maintained until the 6th day to induce differentiation into dendritic cells. On day 6, dendritic cells with completed differentiation were obtained and seeded at 4×10 ⁶ cells/well in media supplemented with LPS (Lipopolysaccaride) (50 ng/ml). At this time, 4 ml of LPS-added media is used in each 6 well non-tissue plate. On day 7, dendritic cells that had finally completed maturation were obtained.

Dendritic cells that had completed maturation were centrifuged at 2,000 rpm for 5 minutes to isolate only pure dendritic cells, then treated with Protease Inhibitor Tablet-PBS buffer and dispersed at a concentration of 1 to 2Х10 ⁶ cells/ml. Then, only the cell membrane was purified through rapid freezing at -70°C, thawing at room temperature, and centrifugation. Afterwards, ultrasonic waves (VC505, Sonics & Materials) were processed at an amplitude of 20%, performing a total of 60 rounds of 3 seconds with 3 seconds on/off and a 2 second cooling period between cycles to obtain a micro-scale cell membrane suspension. , this was filtered through a polycarbonate membrane with a nano-sized filter (pore size 1 ㎛, 400 ㎚, 100 ㎚) to generate nano liposomes with the diameter of each pore size. Considering the surface area of the cell membrane coated on the nanoparticle surface, it was assumed that X _DM = 1 for the case where nanoparticles and liposomes have an optimized mixing ratio.

[Equation 1]

Mole fraction (X _DM )=(surface area of cell membrane)/(surface area of nanoparticle)

After mixing 5 ㎕ of 60 nm gold nanoparticles and 1 ㎖ of liposomes _so that By treating the prepared dendritic cell-mimicking nanostructure with 0.2 μg/ml of GP _33-41 antigen at 37°C for about 30 minutes, the antigen was induced to be presented on the surface of MHC class I and II, and the antigen was pulsed to the dendritic cells. Mimetic nanostructures were prepared.

Example 1. Preparation of dendritic cell-mimicking nanostructure for immune checkpoint inhibition introduced with αPD-1 monoclonal antibody

According to the method shown in Figure 2, the αPD-1 monoclonal antibody was prepared by conjugating the other end of PEG to a molecule in which the PEG end was conjugated to the hydrophilic group of 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). And, by introducing the αPD-1 monoclonal antibody bound to phospholipids into the cell membrane of the dendritic cell-mimicking nanostructure prepared according to Preparation Example 1 above using the spontaneous cell membrane insertion phenomenon, a dendritic cell-mimicking nanostructure for immune checkpoint inhibition was prepared. did.

Experimental Example 1. Evaluation of T cell proliferation and differentiation inducing effects of dendritic cell-mimicking nanostructures for immune checkpoint inhibition

In a 96 well U bottom plate, 1 × 10 ⁴ each of dendritic cell-mimicking nanostructure (αPD-1-anDC), dendritic cell-mimicking nanostructure (anDC), and dendritic cell (BMDC) for immune checkpoint inhibition prepared according to Example 1. Regarding this, 5 × 10 ⁴ CD8 T cells isolated from P14 mouse were added to the same well with 200 ㎕ of 10% RPMI culture medium, and co-cultured at 37°C for 3 days. Six hours before the end of co-culture, restimulation was performed using GP, GS, DNase1, and GP _33-41 peptides. Six hours later, the plate was recovered from the incubator, stained with a fluorescent antibody, and the level of surface protein and CD8 T cell activation was analyzed using flow cytometry. The results are shown in Figure 5.

Unlike the group in which only T cells were cultured, when T cells were cultured with anDC, it was confirmed that proliferation of T cells was induced, similar to BMDC, and expression of CD44 and the expression of cytokines IFNγ, TNFα, and IL-2 were induced. . These results show that anDC functions as an antigen-presenting cell.

In addition, in the case of anDC, compared to BMDC, the MFI value of CD44 expressed by T cells is low and the rate of cytokine expression is low, so it can be seen that the ability to activate T cells is relatively low. On the other hand, when αPD-1 was conjugated to anDC, the MFI value of CD44 increased and the rate of cytokine expression increased compared to anDC. The rate of cytokine expression was similar to that of BMDC, and in the case of IL-2, appeared higher. Through CTV+ frequency, it can be seen that more than 90% of T cells in the three groups of BMDC, anDC, and anDC conjugated with aPD-1 proliferated well.

Experimental Example 2. Evaluation of T cell proliferation and differentiation inducing effects of dendritic cell-mimicking nanostructures for immune checkpoint inhibition (in vivo)

After isolating CD8 T cells from a P14 mouse with a congenic marker (Ly5.1), adoptive immune cell transfer was performed into a naive recipient mouse, and after 24 hours, dendritic cells (BMDCs) and dendritic cell-mimicking nanostructures (anDCs) were performed. ) and dendritic cell-mimicking nanostructures for immune checkpoint inhibition (αCTLA4 conjugated anDC, αPD-1 conjugated anDC) were subjected to adoptive immune cell transfection under different conditions for each group. Specific conditions are shown in Figure 6. After 48 hours, immune cells were isolated from the spleen of the mouse, stained with a fluorescent antibody, and the degree of CD8 T cell activation was analyzed using a flow cytometer. The results are shown in Figures 7 and 8.

Unlike the control group, BMDC and anDC, which were not loaded with T cell-specific antigens, when BMDC/GP33 and anDC/GP33 pulsed with antigen were added, proliferation of T cells was induced, and the expression of CD44 and the cytokines IFNγ, TNFα, It can be confirmed that the expression of IL-2 is induced. These results show that anDC functions as an antigen-presenting cell.

In the case of anDC, the proportion of proliferated T cells was low compared to BMDC, and in the case of anDC, compared to BMDC, the MFI value of CD44 expressed by T cells was low and the rate of cytokine expression was low, so the proportion of proliferated T cells was relatively low. It can be seen that the ability to activate cells is reduced. On the other hand, when αPD-1 was conjugated to anDC, the MFI value of CD44 increased and the rate of cytokine expression increased compared to anDC. In other words, it was confirmed that the T cell activation ability was significantly improved when the antibody was combined, and these results suggest that the anticancer activity through the dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to the present disclosure will be very excellent.

Claims (10)

Nanoparticle core; A nanostructure comprising a shell containing a cell membrane of lipid molecules derived from dendritic cells, The shell is a dendritic cell-mimicking nanostructure for immune checkpoint inhibition, characterized in that it contains an αPD-1 monoclonal antibody. According to clause 1, The αPD-1 monoclonal antibody is a dendritic cell-mimetic nanostructure conjugated with a lipid molecule. According to clause 1, The nanostructure is a dendritic cell-mimicking nanostructure in which an antigen is pulsed. According to clause 1, The antigen is a dendritic cell-mimicking nanostructure, wherein the antigen is a peptide or protein derived from a tumor antigen. According to clause 1, The nanostructure is a dendritic cell-mimicking nanostructure having an average particle diameter of 20 ㎚ to 50 ㎛. According to clause 1, The nanostructure is a dendritic cell-mimicking nanostructure having an average particle diameter of 20 ㎚ to 1000 ㎚. According to clause 1, A dendritic cell-mimicking nanostructure wherein the surface of the shell is labeled with a bioactive polymer, bioactive ingredient, or protein. A pharmaceutical composition for treating cancer comprising the dendritic cell-mimicking nanostructure for immune checkpoint inhibition according to claim 1. (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing nanoparticles and the liposomes and compressing them through a filter to obtain nanoparticles into which the cell membrane has been introduced; and (S50) introducing αPD-1 monoclonal antibody conjugated with a lipid molecule into the cell membrane-introduced nanoparticle; A method of manufacturing a dendritic cell-mimicking nanostructure for immune checkpoint inhibition, comprising: According to clause 9, After the step (S40), the method of producing a dendritic cell-mimicking nanostructure for immune checkpoint inhibition further includes the step of pulsing an antigen to the nanoparticles into which the cell membrane has been introduced.

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