KR20220087880A - Antibody inserted exosome nanoparticle composition and medical uses thereof - Google Patents

Abstract

The present invention relates to an exosome nanoparticle composition that is inserted into a lipid bilayer on the surface of an antibody conjugate comprising a lipid, a biocompatible polymer, and an antibody, and specifically targets T cells, and more particularly, matures by stimulation of ovalbumin Exosome nanoparticles with antibody conjugates inserted into the surface lipid bilayer of exosomes secreted from dendritic cells have the effect of inhibiting tumor growth by targeting T cells and suppressing immune checkpoints and enhancing T cell activity against tumors. As confirmed, the composition containing the exosome into which the antibody conjugate is inserted as an active ingredient can be provided as an immuno-oncology agent and a cancer vaccine.

Description

Antibody inserted exosome nanoparticle composition and medical uses thereof

The present invention relates to an exosome nanoparticle composition in which an antibody conjugate composed of a lipid, a biocompatible polymer and an antibody is inserted into the lipid bilayer on the surface of an exosome and specifically targets T cells, and to a medical use thereof.

Exosomes are naturally secreted nanovesicles with a diameter of 30 to 200 nm, and it is known that they can act as important nanomediators for transporting various substances from derived cells. emerging as a new means of

In particular, exosomes are nanovesicles secreted from living cells and can provide many advantages as cancer vaccines due to their non-immune, non-toxic, long circulation time and unique cell properties that present parental cell identity. A recent report demonstrated that DC-derived exosomes (DEXs) induce an immune response against tumors, which may be a strategic alternative for DC-based cancer vaccines.

However, although the safety of DEX as a therapeutic cancer vaccine was confirmed in clinical investigations, it was confirmed that the actual DEX vaccination showed poor therapeutic effect by inducing specific anti-tumor immunity in only a small number of cancer patients. These causes include insufficient induction of adaptive immune responses and immunosuppressive mechanisms seized by tumors to evade attack of the immune system, such as immune checkpoint components and immunosuppressive cells, are considered major obstacles limiting the efficacy of DEX. .

Cytotoxic T-lymphocyte antigen 4 (CTLA-4), a member of the CD28 receptor family, is specifically expressed on effector CD4+ and CD8+ T cells as well as regulatory T cells (Tregs). CTLA-4 negatively regulates T cell activity during the priming phase of lymph nodes by competing with CD28 for binding to CD80 and CD86 of antigen presenting cells (APCs) with higher affinity.

Accordingly, there is a need for research on anti-CTLA-4 strategies for enhancing the activation of effector T cells, increasing the ratio of effector T cells to regulatory T cells, and transporting activated T cells to tumors.

Republic of Korea Patent Publication No. 10-2018-0005546 (published on January 16, 2018)

The present invention provides exosome nanoparticles into which a costimulatory molecule, a major histocompatibility complex (MHC), an antigenic peptide and an antibody are inserted, thereby inhibiting the immune checkpoint of T cells and enhancing the immune response of T cells to tumor cells. An object of the present invention is to provide an anticancer drug that activates the immune system.

The present invention relates to an antibody conjugate comprising a lipid, a biocompatible polymer and an antibody; and

The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody A labeled exosome nanoparticle composition is provided.

The present invention relates to an antibody conjugate comprising a lipid, a biocompatible polymer and an antibody; and

The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody It provides an immunotherapy composition containing the labeled exosome nanoparticles as an active ingredient.

The present invention relates to an antibody conjugate comprising a lipid, a biocompatible polymer and an antibody; and

The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody It provides a cancer vaccine composition containing the labeled exosome nanoparticles as an active ingredient.

In addition, the present invention comprises the steps of preparing an antibody conjugate by amine coupling reaction between the NHS (N-Hydroxysuccinimide) functional group of DPPE-PEG-NHS and the lysine residue of the antibody (step 1);

Preparing OVA-exosomes by culturing dendritic cells in a medium supplemented with ovalbumin (OVA) and Poly(I:C) (second step); and

It provides a method for producing antibody-labeled exosome nanoparticles comprising the step of culturing the antibody conjugate of the first step and the OVA-exosome of the second step and inserting the antibody conjugate into the exosome surface lipid bilayer (step 3) .

According to the present invention, exosome nanoparticles with antibody conjugates inserted into the surface lipid bilayer of exosomes secreted from dendritic cells matured by ovalbumin stimulation target T cells to suppress immune checkpoints, and T cell activity against tumors As the effect of inhibiting tumor growth is confirmed by improving the , the composition containing the exosome into which the antibody conjugate is inserted as an active ingredient can be provided as an immunotherapy and cancer vaccine.

1 is a result of confirming the preparation and characteristics of EXO-OVA-mAb, FIG. 1A is a schematic diagram showing the preparation process of EXO-OVA-mAb targeting lymph nodes to increase tumor-specific T cell response, FIG. 1B is The results of dynamic light scattering analysis confirming the particle sizes of EXO, EXO-OVA, and EXO-OVA-mAb, Figure 1C is a transmission electron microscope image result of EXO-OVA-mAb, Figure 1D is (1) EXO Western blot results confirming CD80, MHC-I, MHC-II, CD63 and Calnexin in dendritic cell lysates to which -OVA and (2) OVA were added, FIG. 1E is anti-CTLA-4 antibody in EXO-OVA-mAb and ELISA quantitative analysis results confirming the surface level of OVA.
2 is a confocal laser scanning microscope image of CD4 ⁺ and CD8 ⁺ T cells after incubation with EXO-OVA-mAb-FITC for 1 hour as a result of confirming the T cell binding ability of EXO-OVA-mAb in vitro.
3 is a result of confirming the effect of exosomes on T cells in vitro, FIG. 3A is a flow cytometric analysis result confirming CD69, a T cell activity marker, after culturing different exosome formulations and CD4+ and CD8+ T cells, respectively, FIG. 3B and 3C are results of confirming CD69 in CD4+ and CD8+ T cells cultured with different exosome formulations, respectively, FIG. 3D is flow cytometry results of CFSE-stained CD4+ and CD8+ T cells treated with other exosome formulations, FIG. 2E And Figure 3F is a result of confirming the proliferation index (proliferation index) after culturing other exosome formulations and CD4+ and CD8+ T cells, the data are mean±SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.
4 is a result of confirming the in vivo transport of cyanine 5.5 (Cy5.5)-labeled EXO-OVA and EXO-OVA-mAb in C57BL/6 mice after administration to the tail, FIG. 4A shows exosomes labeled at different time points Images taken after administration (circle: ILN), FIG. 4B is the in vitro fluorescence image result of ILN and major organs collected 48 hours after injection, and FIG. 4C is the fluorescence of ILN and Cy5.5 per major organ weight (mg). The intensity is quantified, and FIGS. 4D and 4E are flow cytometry results confirming the binding of Cy5.5-labeled exosomes to CD4+ and CD8+ T cells, respectively, and FIG. 4F is Cy5.5-labeled exosome injection 8 As a result of confirming the percentage of Cy5.5+ in CD4+ and CD8+ cells in ILN after time, the result is the mean±SD (n = 3), ***P < 0.001.
5 is a result of confirming the immunostimulatory activity of exosomes, FIGS. 5A and 5B are flow cytometry results confirming TNF-α in CD4+ cells among splenocytes of immunized mice after restimulation with OVA, FIG. 5C and FIG. 5D is a flow cytometric analysis result confirming the expression of IFN-γ in CD8+ cells among splenocytes of the immunized mouse, and FIGS. 5E to 5H are immunochemicals of CD4, CD8, IFN-γ and TNF-α in the spleen and ILN. Results of histological (IHC) analysis (scale bars = 120 μm), FIGS. 5I and 5J are flow cytometry and data analysis results of CD3+ CD8+ CD44+ CD62L- effector memory T cells in the spleen 7 days after the last immunization. After 2 days, immunized mice were euthanized, spleen and ILN were collected, and flow cytometry and IHC analysis were performed. Results were expressed as mean±SD (n = 5), *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6 is the result of confirming the antitumor immune response according to the exosome treatment, Figure 6A is the B16-OVA tumor volume in C57BL/6 mice over time after immunization with EXO, EXO-OVA or EXO-OVA-mAb Results, Fig. 6B is a result of performing immunohistochemical analysis of intratumoral CD4, CD8, IFN-γ and TNF-α, Fig. 6C is a flow cytometric analysis result confirming CD4+ and CD8+ T cells in the tumor, Fig. 6D is It is the result of quantifying the percentage of CD4+ and CD8+ T cells in the tumor, FIG. 6E is a flow cytometry result confirming the ratio of CD8+ T-cytotoxic T lymphocytes (CTL) to regulatory T cells (CTLs) in the tumor, FIG. 6F is the result of the treatment It is the result of confirming the concentration of IFN-γ in the serum of the mouse, and FIG. 6G is the result of confirming the concentration of TNF-α in the serum of the treated mouse. Tumor and serum samples were collected 2 days after the last treatment, and the data are the mean±SD (n = 5), *P < 0.05, **P < 0.01, ***P < 0.001.
7A is the result of confirming the relative change in body weight of the immunized mouse, and FIG. 7B is the histological analysis confirming the heart, liver, spleen, ILN, lung and kidney of exosome-treated mice by performing hematoxylin-eosin staining. It is the result.

Hereinafter, the present invention will be described in more detail.

In the present invention, exosome nanoparticles with DPPE-PEG-CTLA-4-Ab antibody conjugate inserted into the lipid bilayer of exosomes secreted from dendritic cells matured by ovalbumin stimulation target T cells, thereby enabling immune checkpoints of T cells. As it was confirmed to inhibit tumor growth by inhibiting and inducing a tumor-specific immune response to increase the ratio of effector T cells/regulatory T cells in the tumor, the DPPE-PEG-CTLA-4-Ab antibody conjugate was inserted To provide an exosome nanoparticle composition as an immunotherapy agent.

The present invention relates to an antibody conjugate comprising a lipid, a biocompatible polymer and an antibody; and

The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody It is possible to provide a labeled exosome nanoparticle composition.

More specifically, the co-stimulatory molecule may be CD80 or CD86, and the major histocompatibility complex may be a major histocompatibility complex type I and type II.

The antibody conjugate may be fixed to a lipid raft in the lipid bilayer on the surface of the exosome and inserted into the surface of the exosome.

The exosomes may be exosomes secreted from dendritic cells stimulated with ovalbumin and Poly(I:C).

The biocompatible polymer may be one selected from the group consisting of polyethylene glycol, hyaluronic acid, alginate, polyacrylic acid and poloxamer, and more preferably polyethylene glycol, but is not limited thereto.

The antibody may be an anti-immune checkpoint antibody, and more specifically, may be selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody.

The exosome nanoparticles penetrate into lymph nodes and target T cells, thereby suppressing immune checkpoints and activating the immune response of T cells.

The present invention relates to an antibody conjugate comprising a lipid, a biocompatible polymer and an antibody; and

The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody It is possible to provide an immunotherapy composition containing the labeled exosome nanoparticles as an active ingredient.

The exosome nanoparticles may target T cells to suppress immune checkpoints and increase the immune activity of tumor-specific CD4+ and CD8+ T cells.

The immune checkpoint may be selected from the group consisting of CTLA-4 and PD-1, but is not limited thereto.

The present invention relates to an antibody conjugate comprising a lipid, a biocompatible polymer and an antibody; and

The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody It is possible to provide a cancer vaccine composition containing the labeled exosome nanoparticles as an active ingredient.

The present invention comprises the steps of preparing an antibody conjugate by amine coupling reaction between the NHS (N-Hydroxysuccinimide) functional group of DPPE-PEG-NHS and the lysine residue of the antibody (step 1); Preparing OVA-exosomes by culturing dendritic cells in a medium supplemented with ovalbumin (OVA) and Poly(I:C) (second step); and culturing the antibody conjugate of the first step and the OVA-exosome of the second step to insert the antibody conjugate into the exosome surface lipid bilayer (step 3). can do.

The antibody of the first step may be an anti-immune checkpoint antibody.

More specifically, the anti-immune checkpoint antibody may be selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody.

The first step may be to react the antibody and DPPE-PEG-NHS in a molar ratio of 1:2 to 1:8.

In the case of exosomes extracted from cells, the yield is low and the tissue regenerative function is low. When the exosomes are injected in vivo in a disease model, most of them are accumulated in the liver, spleen and kidney, and the targeting ability to the lesion site is very low. There is, in order to solve this problem, it is necessary to modify the surface of the exosomes. The exosome nanoparticle manufacturing method of the present invention has excellent biostability and antibody stability and is economical compared to the conventional method of modifying the exosome surface with an antibody using a crosslinking agent.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Reference example>

The following reference examples are intended to provide reference examples commonly applied to each embodiment according to the present invention.

1. Substance

Poly(I:C) was purchased from InvivoGen (San Diego, CA, USA). InVivoMAb a anti-mouse CTLA-4 antibody was purchased from BioXCell (West Lebanon, NH, USA). RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Hyclone (Logan, UT, USA).

Chicken OVA, 2-mercaptoethanol and exosome-depleted FBS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse granulocyte/macrophage-stimulating factor (GM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA), and 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-PEG-succinimidyl ester (DPPE-PEG-NHS) , 3.4 kDa) were purchased from Biochempeg Scientific (Watertown, MA, USA). All fluorescence-bound antibodies used for flow cytometry were purchased from Biolegend (San Diego, CA, USA).

2. Cells

B16/F10 cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). B16-OVA cells were generated by transfecting the pcDNA3-OVA plasmid (Addgene, Arsenal Way, MA, USA) into B16/F10 cells. The resulting B16/F10 cells were grown in DMEM medium containing 10% FBS and 100 IU/ml penicillin/streptomycin until inoculated into C57BL/6 mice.

CD4 ⁺ and CD8 ⁺ T cells were isolated from the spleen of female C57BL/6 mice using EasySep ^™ mouse CD4 ⁺ and CD8 ⁺ T Cell enrichment kit (Stemcell Technologies, Vancouver, Canada), respectively, according to the manufacturer's instructions.

Briefly, spleens isolated from C57BL/6 mice were digested in PBS solution containing 2% FBS, the cell suspension was passed through a 70-μm cell strainer (Sigma-Aldrich), and centrifuged at 300 × g for 10 min. to obtain cells. The cells were resuspended in PBS containing 2% FBS and 1 mM EDTA at 1.0 × 10 ⁸ cells/ml.

Then, the 1.0 × 10 ⁷ cell aliquot was transferred to a 5 ml polystyrene round-bottom tube. Next, 50 μl of rat serum (Stemcell Technologies) and 50 μl of Enrichment Cocktail were added to the cell suspension and stored on ice for 15 minutes.

Next, 100 μl of CD4+ or CD8+ Selection cocktail was added to the synthesized samples and further incubated for 15 minutes. Finally, magnetic particles were added to the mixture, incubated for 5 minutes, and then a MACS magnet (Miltenyi Biotec, Bergisch Gladbach, Germany) was added. The isolated CD4+ and CD8+ T cells were cultured in RPMI 1640 medium containing 10% FBS, 100 IU/ml penicillin/streptomycin and 30 IU/ml recombinant mouse IL-2 (Peprotech).

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Preparation of exosomes (EXO) and EXO-OVA

Briefly, bone marrow-derived DCs (BMDCs) were prepared from bone marrow cells isolated from femurs of 8-10 week-old C57BL/6 mice with 10% FBS, 100 IU/ml penicillin/stretomycin, 50 μM 2-mercaptoethanol and It was produced by culturing in RPMI-1640 complete medium containing 20 ng/ml murine GM-CSF. After 3 days, an equal volume of fresh complete medium was added to the cells. On day 6, BMDCs were collected and washed with Hank's balanced salt solution (HBSS).

To prepare EXO-OVA, chicken OVA (300 μg/ml) was added to BMDCs, and cells were further cultured for 12 hours. Cells were washed with HBSS buffer to completely remove serum and free OVA, and RPMI-containing 10% exosome-deficient FBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 50 ng/ml poly(I:C) Further incubation in 1640 medium.

After 24 hours, the appropriate OVA and poly(I:C)-treated BMDC medium was collected and centrifuged at 3,000×g, 4°C for 30 minutes. The resulting supernatant was filtered through a 0.22 μm filter (Advantec MFS, Dublin, Ireland), and finally the filtrate was centrifuged at 10,000×g, 4° C. for 120 minutes. The pelleted OVA-supported mature DC-derived exosomes (EXO-OVA) were resuspended in PBS and centrifuged at 10,000×g, 4° C. for 120 minutes.

EXO-OVA was stored at -80°C until further analysis. EXO was prepared in the same way without OVA and poly(I:C) treatment on BMDC.

2. EXO-OVA-mAb Preparation

Surface modification of EXO-OVA using anti-CTLA-4 antibody was performed. First, anti-CTLA-4 antibody (4 mg/ml) and DPPE-PEG-NHS (13333 μM) were reacted in 0.1 M biscarbonate salt buffer (pH 8.5) at room temperature for 1 hour to synthesize. Thereafter, the reaction was stopped by adding Tris buffer (pH 8.0), and the synthesized DPPE-PEG-mAb was purified by dialysis against PBS (MWCO: 10 kDa). Purified DPPE-PEG-mAb was stirred with EXO-OVA corresponding to 100 μg protein in different amounts at 40° C. for 2 hours to insert DPPE-PEG-mAb into the EXO-OVA membrane.

Unbound DPPE-PEG-mAb was removed using 300 kD MWCO Vivaspin tubes (Sartorius, 37079 Goettingen, Germany), and the resulting EXO-OVA-mAb was resuspended and stored in PBS.

3. Confirmation of size and morphological characteristics

The hydrodynamic size and zeta potential of different exosome formulations were confirmed by dynamic light scattering (Zetasizer Nano, Malvern, UK).

The morphology of the exosomes was confirmed using a transmission electron microscope (CM 200 UT; Philips, Andover, MA, USA). Briefly, exosomes were mounted on carbon-coated copper grids, stained with 2% uranyl acetate, and observed using an accelerating voltage of 120 kV.

4. Confirmation of protein properties of exosomes

For western blots, EXO-OVA was centrifuged at 100,000×g, 4°C for 120 min, followed by M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) and an EDTA-free protease inhibitor cocktail (Roche Diagnostics, Risch-Rotkreuz). , Switzerland) was resuspended in the protein extraction solution.

Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were mixed with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) at a 4/1 ratio (v/v), heated at 90°C for 10 minutes, and then 10% sodium dodecyl sulfate polyacry-lamide gel (SDS-PAGE) ) and separated at 80V for 2 hours, and transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, Massachusetts, USA) using Power Supply (PS300-B, Hoefer Inc., MA, USA). Membranes were blocked with 5% skim milk solution for 1 hour and incubated overnight with anti-mouse primary antibodies (BioLegend) against CD80, MHC-I, MHC-II and CD63. The membranes were then washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG (Thermo Fisher Scientific).

Finally, the membrane was exposed to imaging film (Kodak, Rochester, New York, USA). For Coomassie blue staining, free OVA, anti-CTLA-4 mAb, EXO-OVA and EXO-OVA-mAb were loaded onto 6% SDS-PAGE gels and electrophoresed at 60 V for 2 hours. The gel was then stained with 0.25% Coomassie Blue R-250 (Thermo Fisher Scientific) dissolved in a solution containing acetic acid:methanol:H ₂ O (10:50:40, v/v/v) for 30 min at room temperature. did The gel was decolorized with a destaining solution containing acetic acid:methanol:H ₂ O (7.5: 5: 87.5, v/v/v) until the background became transparent and the image was clear.

5. Quantification of Surface-bound OVA

The amount of OVA was confirmed using ELISA. A 96-well ELISA plate (Corning Costar, USA) was incubated overnight with exosomes corresponding to 10 μg of protein. After coating, the plate was blocked with PBS buffer containing 1% bovine serum albumin, and an anti-mouse OVA antibody (BioLegend) and an anti-mouse antibody conjugated with secondary horseradish peroxidase (HRP) (Thermo Fisher Scientific) were added. did Then, HRP-sensitive substrate solution (R&D Systems, Minneapolis, MN, USA) was added, and absorbance was measured at 450 nm in a microplate reader (Thermo Fisher Scientific).

6. Quantification of anti-CTLA-4 antibody on exosome surface

The amount of anti-CTLA-4 contained in the EXO-OVA membrane was indirectly quantified by calculating the concentration of unbound anti-CTLA-4 antibody in the supernatant collected using a 300 kD MWCO Vivaspin ultrafiltration tube.

Briefly, 96-well ELISA plates were coated with goat anti-human IgG Fc antibody (2 μg/ml) (Thermo Fisher Scientific) overnight at room temperature followed by 100 ng/ml recombinant mouse CTLA-4 Fc chimeric protein (R&D Systems) and incubated for 2 hours.

Next, the supernatant containing unbound anti-CTLA-4 antibody was added and further incubated for 2 hours. After adding the secondary HRP-conjugated anti-mouse antibody and HRP-sensitive substrate solution, absorbance was measured at 450 nm in a microplate reader.

To confirm that the anti-CTLA-4 antibody was hybridized to the EXO-OVA membrane, FITC (Thermo Fisher Scientific) was labeled with the antibody according to the manufacturer's instructions before DPPE-PEG-NHS binding. After incubation with EXO-OVA, unbound DPPE-PEG-mAb-FITC was removed in the same manner as in the previous experiment, and FITC-labeled EXO-OVA-mAb (EXO-OVA-mAb-FITC) was applied to U-2800 UV -Vis spectroscopy (Hitachi, Tokyo, Japan) confirmed.

7. Confirmation of T-cell binding ability in vitro of EXO-OVA-mAb

To confirm that EXO, EXO-OVA, and EXO-OVA-mAb bind to T cells, the coverslips were immersed in 0.03% PureCol EZ gel solution (Sigma-Aldrich) overnight at 4°C at 4°C for collagen-coated 12-wells. prepared on a coverslip plate.

After removing the collagen solution, CD4 ⁺ and CD8 ⁺ T cells were added to a collagen-coated 12-well coverslip plate and incubated at 37°C for 24 hours. Prior to incubation with T cells, EXO, EXO-OVA and EXO-OVA-mAbs were labeled with PKH67 green fluorescent cell linker (Sigma-Aldrich) according to the manufacturer's instructions.

Thereafter, T cells and PKH67-labeled EXO, EXO-OVA, and EXO-OVA-mAb were cultured for 1 hour and confirmed with a K1-Fluo Confocal Laser Scanning Microscope (Nanoscope, Daejeon, South Korea).

8. Identification of the effect of exosomes on T cell activation and proliferation in vitro

To determine the effect of exosomes on T cell activation, CD4+ or CD8+ T cells were seeded at 1.0 × 10 ⁶ cells per well in 24-well plates and EXO, EXO-OVA or EXO-OVA- corresponding to 10 μg of protein The amount of mAb was treated for 72 hours.

CD4+ and CD8+ T cells were then collected and PerCP/Cy5.5 anti-mouse CD3 (Clone: 17A2), FITC anti-mouse CD4 (Clone: RM4-5), PE/Cy7 anti-mouse CD8a (Clone: 53-6.7) ) and PE anti-mouse CD69 (Clone: H1.2F3) antibody staining to perform flow cytometry.

Quantification of TNF-α produced by CD4+ and IFN-γ produced by CD8+ according to the manufacturer's instructions using mouse IFN-γ Duoset ELISA kit (R&D Systems) and mouse TNF-α ELISA MAX TM Standard (BioLegend) Conditioned culture medium was collected for this purpose.

T-cell proliferation after exosome treatment was confirmed by flow cytometry using CFSE staining. Before treatment, CD4+ and CD8+ T cells were stained using the CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

The stained cells were seeded in a 24-well plate at 1.0 × 10 ⁶ cells per well and incubated with other exosome formulations for 72 hours. CFSE-stained T cells were then treated with PerCP/Cy5.5 anti-mouse CD3 (Clone: 17A2), APC anti-mouse CD4 (Clone: RM4-4), or PE/Cy7 anti-mouse CD8a antibody (Clone: 53-6.7). ) and subjected to flow cytometry.

9. In vivo transport of exosomes to lymph nodes

According to the previous experiment (32), EXO-OVA and EXO-OVA-mAb were labeled with cyanine 55 NHS dye (Lumiprobe Corp, Maryland, USA). Briefly, 10 mM Cy55 NHS dye was added to the exosomes in an amount corresponding to 100 μg of total protein and incubated for 2 hours at room temperature under dark conditions.

After removal of unbound dye using Amicon Ultrafiltration Tubes (10 kDa, Merck KGaA, Darmstadt, Germany), Cy55-labeled EXO-OVA and EXO-OVA-mAbs were administered to female 6-8 week-old C57BL/6 mice in the tail region. Injected subcutaneously.

The fluorescence signal of exosomes was confirmed in the inguinal lymph node (ILN) at different time points using a FOBI imaging instrument (NeoScience, Su-won, South Korea). After 48 hours, mice were euthanized, and ILN and polyFMS major organs were collected, washed with saline, and imaged.

To determine whether exosomes were targeted to T-cells in ILN, mice were euthanized 8 hours after exosome injection to collect ILN, cut with small manipulations, and collagen using RPMI-1640 medium containing 10% FBS Degradation enzyme D (Sigma-Aldrich) 1 mg/ml was stirred at 37° C. for 30 minutes at 150 rpm to decompose.

The samples were passed through a 70-μm cell strainer (Sigma-Aldrich) to remove undigested tissue. Single cells were stained with PerCP/Cy55 anti-mouse CD3 (Clone: 17A2), FITC anti-mouse CD4 (Clone: RM4-5) and PE anti-mouse CD8a (Clone: 53-67) antibodies and subjected to flow cytometry. .

10. In vivo anti-tumor identification

To confirm the therapeutic antitumor effect of exosomes, a B16-OVA melanoma tumor model was prepared. Female 6-8 week old C57BL/6 mice were randomly divided into groups and 100,000 B16-OVA cells suspended in DMEM containing 50% Matrigel were injected into the right flank. 7 days after tumor cell inoculation, PBS (control) or other exosome formulations (EXO, EXO-OVA, and EXO-OVA-mAb) were injected subcutaneously into mice 3 times at 5 day intervals in an amount equal to 100 μg exosomal protein. did

The tumor volume (V) was calculated by measuring the major (A) axis and minor (B) axis of the tumor using a digital caliper, and V (mm ³ ) = A×B ² /2.

Two days after the last injection, immunized mice were euthanized and tumors, ILN and major organs were collected for further experiments.

To confirm T-cell activity in vivo, whole splenocytes were prepared from the spleen of euthanized mice, and red blood cells were lysed with RBC lysis buffer (BioLegend) to obtain PerCP/Cy5.5 anti-mouse CD3, FITC anti- Mouse CD4, PE/Cy7 anti-mouse CD8a and PE anti-mouse CD69 antibodies were stained.

To confirm the generation of effector memory T cells, T-cells were transfected with PerCP/Cy5.5 anti-mouse CD3 (Clone: 17A2), PE anti-mouse CD8a (Clone: 53-6.7), PE/Cy7 anti-mouse CD44 (Clone: IM7) and FITC anti-mouse CD62L (Clone: MEL-14) antibodies.

Stained cells were characterized by flow cytometry. To confirm the increase in antigen-activated CD4+ and CD8+ T-cells after immunization, splenocytes isolated from euthanized mice were treated with RPMI-1640 medium containing Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific) and 10 μg/ml OVA for 72 hours. stimulated while

Then, the splenocytes were stained with PerCP/Cy5.5 anti-mouse CD3 (Clone: 17A2), FITC anti-mouse CD4 (Clone: RM4-5) or PE anti-mouse CD8a (Clone: 53-6.7) antibodies. After fixation, the permeability was checked with intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific).

In addition, cells were stained with APC anti-mouse TNF-α (Clone: MP6-XT22) and PE/Cy7 anti-mouse IFN-γ (Clone: XMG1.2) antibodies and characterized by flow cytometry.

To identify intratumoral T cell populations, tumor tissue was collected, cut into small pieces, and composed of 10 U/ml Collagenase I, 400 U/ml Collagenase IV, and 30 U/ml DNAse I (Thermo Fisher Scientific). It was continuously stirred at 150 rpm for 2 hours at 37°C with DMEM tumor degradation medium.

Single cells were stained with PerCP/Cy5.5 anti-mouse CD3, FITC anti-mouse CD4, and PE/Cy7 anti-mouse CD8 antibodies, fixed, permeabilized, and stained with APC anti-mouse Foxp3 antibody for flow cytometry analysis.

To analyze the number of Foxp3+ Treg cells in CD4+ helper T cells in the spleen by flow cytometry, the splenocytes were stained with PerCP/Cy5.5 anti-mouse CD3 and FITC anti-mouse CD4 antibodies, fixed and then permeabilized to APC anti-mouse. After staining with Foxp3 antibody, flow cytometry analysis was performed.

11. Serum Cytokine Quantification

After 48 hours of the last immunization, mouse peripheral blood was collected by retro-orbital puncture, and the mouse IFN-γ Duoset ELISA kit (R&D Systems) and mouse TNF-αELISA MAX TM Standard (BioLegend) were used according to the manufacturer's instructions. Serum IFN-γ and TNF-α levels were analyzed.

12. Histological and Immunohistochemical Analysis

Histological analyzes of tumors, heart, liver, spleen, inguinal lymph nodes (ILN), lungs and kidneys of treated mice were performed in two regions, each in the center of each tumor slice or organ.

Changes in immune activity against CD4, CD8, TNF-α and IFN-γ in tumor slices, spleen, and ILN were evaluated using an avidin-biotin complex-conjugated primary antibody and peroxidase substrate kit (Vector Labs, Burlingame, CA, USA). ) was used to confirm.

Tissues exhibiting 20% or more immune activity for each marker were identified as immunoreactive cells. The average number of immunolabeled cells for CD4, CD8, TNF-α and IFN-γ in tumor slices, spleen and ILN (cells/mm ² ) were determined using an automated image analyzer.

<Example 1> Preparation and characterization of EXO-OVA-mAb

First, an anti-CTLA-4 antibody was bound to DPPE-PEG-NHS through an amine coupling reaction between an N-Hydroxysuccinimide (NHS) functional group and a lysine residue of the antibody.

With respect to PEGylated antibodies, it has been reported that a high ratio of bound PEG per antibody may have a negative effect on the binding activity of the antibody, and when the molar ratio of IgG and DOPE-PEG-NHS is 1:5 , as it was confirmed that the binding of DOPE-PEG-NHS and IgG shows a high Fc-positive ratio, DPPE-PEG-mAb was synthesized by selecting an anti-CTLA-4 antibody and DPPE-PEG-NHS in a molar ratio of 1:5 .

As it was confirmed that the band of DPPE-PEG-mAb exhibited high molecular weight compared to the free mAb, successful preparation of DPPE-PEG-mAb was confirmed.

EXO-OVA-mAb was fabricated through three main steps as shown in Fig. 1A.

First, immature BMDCs were cultured with polyinosinic acid:polycytidylic acid [poly(I:C)], a TLR-3 agonist and OVA to generate mature OVA-mounted BMDCs (mBMDC-OVA). .

Next, BMDC-derived exosomes containing OVA peptide (EXO-OVA) were collected from the mBMDC-OVA culture medium by performing continuous centrifugation.

Finally, the obtained EXO-OVA was combined with anti-CTLA-4 mAb 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine)-poly(ethylene glycol) [1,2-dipalmitoyl-sn -glycero-3-phosphoethanolamine)-poly(ethylene glycol), DPPE-PEG-mAb] and the anti-CTLA-4 mAb was further modified. DPPE-PEG-mAb containing an alkyl chain was automatically immobilized on the lipid raft of the exosome membrane to form EXO-OVA-mAb.

To confirm the characteristics of the prepared EXO-OVA-mAb, dynamic light scattering analysis was performed. As a result, it was confirmed that the average hydrodynamic size of EXO-OVA (81.3 nm) and EXO-OVA-mAb (95.6 nm) was smaller than 100 nm as shown in FIG. 1B . In addition, as a result of transmission electron microscopy, it was confirmed that EXO-OVA-mAb had an oval shape and a diameter in the range of 30-100 nm as shown in FIG. 1C.

Next, the cell surface marker profile of EXO-OVA-mAb was evaluated through Western blot and flow cytometry.

As a result, as shown in FIG. 1D , the costimulatory molecules CD80 and MHC-I/II were found at similar levels in mBMDC-OVA and EXO-OVA-mAb. On the other hand, the exosome positive marker CD63 and the negative marker Calnexin were identified only in EXO-OVA-mAb and mBMDC-OVA, respectively, and the purity of the isolated exosome was confirmed using the marker.

In addition, high frequencies of CD80 and OVA peptide (SIINFEKL) were confirmed on the surface of EXO-OVA-mAb, but not in EXO.

The successful removal of free OVA from EXO-OVA during the separation step and hybridization of DPPE-PEG-mAb on the exosome surface performed free OVA, free mAb, EXO-OVA and EXO-OVA-mAb electrophoresis using polyacrylamide gel. After that, it was confirmed by performing Coomassie blue staining under non-reducing and reducing conditions.

As a result, it was confirmed that the OVA band was present in the EXO-OVA sample, and it was confirmed that free OVA was completely removed from the result. In addition, the presence of cleaved Fc and Fab fragments was confirmed under reducing conditions, whereas no mAb band was identified due to stable covalent bonding of DPPE-PEG-mAb under non-reducing conditions. was confirmed to exist.

In addition, to confirm that DPPE-PEG-mAb was successfully immobilized on the EXO-OVA membrane, an anti-CTLA-4 antibody was conjugated with FITC (fluorescein) prior to conjugation with DPPE-PEG-NHS (DPPE-PEG-mAb-FITC). After labeling with isothiocyanate) fluorescence reagent, it was mixed with EXO-OVA.

Finally, UV-VIS spectra of mAb-FITC, DPPE-PEG-mAb-FITC and FITC-labeled EXO-OVA-mAb (EXO-OVA-mAb-FITC) were checked.

As a result, the FITC absorption peak was confirmed at 490 nm for all FITC-labeled mAb samples in the UV-VIS spectrum.

From the above results, it was confirmed that EXO-OVA-mAb was successfully prepared.

On the other hand, additional enzyme-linked immunosorbent assay (ELISA) analysis was performed to confirm OVA peptide hybridization and the presence of anti-CTLA-4 antibody on the surface of EXO-OVA-mAb.

As a result, the presence of anti-CTLA-4 antibody in EXO-OVA-mAb was confirmed as shown in FIG. 1E.

The successful binding of the immunostimulatory component and anti-CTLA-4 antibody identified from the above results may suggest the potential of exosomes to block immunosuppressive CTLA-4 checkpoints in lymph nodes as well as direct T cell stimulation.

<Example 2> Confirmation of in vitro T cell stimulation by EXO-OVA-mAb

The specific binding ability of EXO-OVA-mAb to T cells in vitro was confirmed using confocal laser scanning microscopy.

Referring to FIG. 2 , it was confirmed that the FITC-labeled EXO-OVA-mAb had a very high binding capacity to CD4+ and CD8+ T cells compared to EXO and EXO-OVA.

In addition, EXO-OVA-mAb mostly bound to the surface of T cells, and the amount internalized by the cells was very small.

The ability of exosomes to activate T cells and induce proliferation in vitro was confirmed by performing flow cytometry analysis for CD69, surface T-cell activation markers, and carboxyfluorescein succinimidyl ester (CFSE) staining.

As a result, as shown in FIGS. 3A to 3C , exosomes isolated from dendritic cells (DC) not treated with OVA and poly(I:C) (EXO) did not exhibit a significant T cell activation effect. On the other hand, exosomes loaded with OVA (EXO-OVA and EXO-OVA-mAb) significantly induced the activity of both CD4+ and CD8+ T cells. In particular, it was confirmed that EXO-OVA-mAb stimulated T cells more effectively than EXO-OVA.

In addition, the levels of the surface marker CD69 as well as the cytokines TNF-α and IFN-γ accompanying CD4+ and CD8+ T cell activity were confirmed by ELISA in the conditioned medium of exosome-treated CD4+ and CD8+ T cells, respectively.

As a result, TNF-α and IFN-γ secretion was confirmed at significantly higher levels in EXO-OVA and EXO-OVA-mAb-treated T cells than in EXO-treated T cells, similar to the CD69 results. Importantly, CTLA-4 antibody functionalization of the exosome membrane markedly increased cytokine production.

3D to 3F, the proliferation of CD4+ and CD8+ T cells confirmed using CFSE fluorescence was significantly improved after incubation with EXO-OVA and EXO-OVA-mAb compared to EXO treatment, and similarly , EXO-OVA-mAb enhanced the proliferation index of both CD4+ and CD8+ T cells more strongly than EXO-OVA (proliferation index of each CD4+ T cell: ~1.30 vs. 1.25 and the proliferation index of CD8+ T cells: ~1.36) vs. 1.25).

From the above results, it was confirmed that exosomes that deliver a sufficient set of stimulatory signals, such as MHC-OVA peptide complex and costimulatory molecules, can effectively prepare T cell activation and proliferation. In particular, hybridization of CTLA-4 antibodies on the surface of EXO-OVA for blockade of the CTLA-4 immune checkpoint could enable distinct stimulation and proliferation of both CD4+ and CD8+ T cells.

<Example 3> Confirmation of exchange into lymph nodes in vivo

As it was confirmed that the size of the exosome was included within the optimal range for lymphatic delivery, it was labeled with a cyanine 5.5 (Cy5.5) fluorescence signal to confirm its ability to exchange to lymph nodes in vivo.

Cy5.5-labeled EXO-OVA and EXO-OVA-mAbs were injected subcutaneously into the tail region of C57BL/6 mice, and images were viewed at various time points to assess penetration into inguinal lymph nodes (ILNs).

As a result, as shown in FIG. 4A, Cy5.5 signal was confirmed in ILN of mice injected with EXO-OVA-mAb 1 hour after administration, whereas fluorescence was not observed in mice injected with EXO-OVA. Cy5.5 fluorescence increased with time in both treatment groups, but stronger fluorescence was confirmed in mice injected with EXO-OVA-mAb.

From the above results, it was confirmed that EXO-OVA-mAb showed a higher affinity for ILN than EXO-OVA, and rapidly reached ILN through lymphatic vessels.

After 48 hours, mice were euthanized, and ILN and major organs were extracted to further confirm the distribution of exosomes in vivo.

As a result, as shown in Fig. 4B, it was confirmed that the EXO-OVA-mAb treatment exhibited significantly higher accumulation in ILN than the EXO-OVA treatment. In particular, after administration of EXO-OVA-mAb, Cy5.5 fluorescence was confirmed only in the liver and ILN, whereas fluorescence was also confirmed in the kidneys in mice injected with EXO-OVA.

From the above results, it was confirmed that the anti-CTLA-4 antibody functionalization effectively reduced the non-specific effect.

In addition, referring to Figure 4C, it was confirmed that both exosome formulations preferentially accumulate in ILN in the distribution comparison based on organ weight (mg).

To determine whether CTLA-4 functionalization in lymph nodes can more specifically guide exosomes to T cells, flow cytometry analysis was performed 8 hours after exosome administration to CD4+ Cy5.5+ and CD8+ Cy5.5+ The frequency of cells was quantified.

As a result, as shown in FIGS. 4D to 4F, it was confirmed that a significantly higher amount of EXO-OVA-mAb than that of EXO-OVA targeted CD4+ and CD8+ T cells.

From the above results, it was confirmed that EXO-OVA-mAb easily permeated into lymphatic vessels and migrated to lymph nodes to effectively target T cells.

<Example 4> Confirmation of immunostimulatory activity of EXO-OVA-mAb

As previous experiments confirmed the potential of EXO-OVA-mAb to induce efficient targeting of T cells in lymph nodes as well as T cell activation and proliferation in vitro, after administration of exosomes to B16-OVA tumor-bearing mice Stimulation of CD4+ and CD8+ T cells was confirmed.

As the CD69 expression increase was confirmed, it was confirmed that the OVA-loaded exosomes induced significant CD4+ and CD8+ T cell activity in vivo. In addition, as a result of flow cytometry, it was confirmed that EXO-OVA-mAb activated T cells more than any other formulations, consistent with the in vitro results.

The strong induction of T cell activity in vivo upon treatment with EXO-OVA-mAb may be due to T cell targeting and blockade of immunosuppressive receptors by CTLA-4 antibody functionalization of EXO-OVA.

Next, after restimulation of splenocytes collected from mice immunized with OVA, intracellular staining of TNF-α in CD4 + T cells and IFN-γ in CD8 + T cells followed by flow cytometry analysis was performed to perform OVA-specific Th1 after immunization. and the start of the CTL reaction.

As a result, as compared to other formulations including EXO-OVA as shown in FIGS. 5A to 5D, significantly improved CD4+ TNF-α+ and CD8+ IFN-γ+ fractions were confirmed, so immunization with EXO-OVA-mAb was OVA It was confirmed to exhibit strong Th1 and CTL responses to antigens.

In contrast to EXO-OVA and EXO-OVA-mAb, immunization with EXO barely stimulated Th1 and CTL responses to OVA compared to controls, suggesting that antigenic OVA peptides and costimulatory molecules on the exosome surface It can be suggested that this is due to the lack of

Meanwhile, immunohistochemical analysis of CD4+, CD8+, TNF-α+ and IFN-γ+ cells in ILN and spleen of treated mice was performed to further confirm the acquisition of intracellular immunity to OVA peptide.

As a result, in mice treated with exosome formulations containing two OVAs in the order of EXO-OVA-mAb > EXO-OVA, CD4-, CD8-, TNF-α in the spleen compared to the control group as shown in FIGS. 5E to 5H . - and IFN-γ-immunoactive cell number and tdLN significantly increased (P < 0.01).

Furthermore, in addition to the antigen specificity of cytotoxicity, another important feature of vaccination is the memory feature that allows for a long-term tumor response based on the activity of memory T cells.

Accordingly, splenocytes in immunized mice at the end of treatment for the presence of effector memory T cells (T _EM ) (CD3 + CD8 + CD44 + CD62L−), a subset of T cells that provide an immediate response to previously met antigens were isolated. confirmed

As a result, the fraction of T _EM cells was significantly increased in the mice treated with the exosome formulation containing OVA as shown in _FIGS . confirmed (~25% vs. ~19%). In contrast, no change in T _EM cells was observed in the EXO-treated group compared to the control mice.

These results are consistent with the prior opinion that the combination of vaccine and anti-CTLA-4 treatment significantly promotes the expansion and maintenance of the T _EM fraction without negatively affecting total endogenous T cells.

From the above results, the ability of EXO-OVA to induce an immune response against OVA-positive tumors is synergistically increased through the addition of anti-CTLA-4 antibody in an exosome platform for suppressing immunosuppressive components that inhibit T cell activity. It has been confirmed that it can be

<Example 5> Confirmation of antitumor effect of EXO-OVA-mAb

The therapeutic effect of EXO-OVA-mAb was confirmed in the B16-OVA tumor model. On day 7 after B16-OVA cell inoculation, EXO, EXO-OVA, or EXO-OVA-mAb was injected three times at 5-day intervals subcutaneously in the tail region of mice with tumors.

As a result, as shown in FIG. 6A, EXO did not delay tumor growth due to the lack of antigenic OVA peptides and costimulatory molecules. However, EXO-OVA showed significant antitumor ability compared to EXO. In particular, a synergistic increase (P < 0.01) was confirmed in the EXO-OVA-mAb modified with the anti-CTLA-4 antibody in the immune response to OVA-positive tumors.

To confirm the tumor suppressive effect of exosome treatment, flow cytometry and immunohistochemical analysis were performed to identify the profile of tumor-infiltrating lymphocytes, which are considered to be major factors contributing to successful immunotherapy.

As a result, as shown in FIGS. 6B to 6D , the OVA-carrying exosomes significantly promoted the infiltration of both CD4+ and CD8+ cells into the tumor compared to the control treatment group, and very high in the EXO-OVA-mAb vaccinated group. Levels of infiltrating cells were identified. In contrast, there was no significant T cell migration to the tumor site in the EXO-inoculated group.

From the above results, an important role of antigenic OVA for increasing tumor-specific T cell response was confirmed.

In addition to the evaluation of the priming T cell response to the tumor, the ability of EXO-OVA-mAb on the increased cytotoxic T lymphocyte (CTLs)/regulatory T cell (Treg) ratio in the tumor was confirmed.

As a result, it was confirmed that only EXO-OVA-mAb exhibited a significant increase in the CTLs/Treg ratio in the tumor site compared to all other exosome formulations as shown in FIG. 6E.

In addition, in order to confirm the increase in T cell activity against tumors, the levels of IFN-γ and TNF-α, which are major markers of cellular immune responses in serum and tumor sites, were confirmed by ELISA and immunohistochemical analysis, respectively.

As a result, referring to FIGS. 5F to 6G, consistent with the previously confirmed experiments, the T cell activity and tumor infiltration increased by EXO-OVA-mAb treatment of IFN-γ and TNF-α in both serum and tumor High production was confirmed.

Finally, the safety of exosome treatment was confirmed by checking the weight of the treated mice and by checking the organ damage caused by hematoxylin-eosin staining.

As a result, as shown in FIG. 7, a significant difference in body weight was not confirmed in all four treatment groups, and no significant histopathological findings were found in the heart, liver, spleen, ILN, lung and kidney of mice.

From the above results, it was confirmed that the exosome formulation did not induce any serious toxicity in mice.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

an antibody conjugate comprising a lipid, a biocompatible polymer, and an antibody; and
The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody A labeled exosome nanoparticle composition. The method according to claim 1, wherein the antibody conjugate is an exosome nanoparticle composition, characterized in that it is fixed to the lipid raft (lipid raft) in the exosome surface lipid bilayer is inserted into the exosome surface. The exosome nanoparticle composition according to claim 1, wherein the exosomes are exosomes secreted from dendritic cells stimulated with ovalbumin and Poly(I:C). The method according to claim 1, wherein the biocompatible polymer is exosome nanoparticle composition, characterized in that selected from the group consisting of polyethylene glycol, hyaluronic acid, alginate, polyacrylic acid and poloxamer. The exosome nanoparticle composition according to claim 1, wherein the antibody is an anti-immune checkpoint antibody. The exosome nanoparticle composition according to claim 5, wherein the immune checkpoint is selected from the group consisting of CTLA-4 and PD-1. The exosome nanoparticle composition according to claim 1, wherein the exosome nanoparticles penetrate into lymph nodes and target T cells, thereby suppressing immune checkpoints and activating the immune response of T cells. an antibody conjugate comprising a lipid, a biocompatible polymer, and an antibody; and
The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody An immunotherapy composition comprising labeled exosome nanoparticles as an active ingredient. The immunotherapy composition according to claim 8, wherein the exosome nanoparticles target T cells to suppress immune checkpoints and increase the immune activity of tumor-specific CD4+ and CD8+ T cells. The immuno-oncology composition according to claim 9, wherein the immune checkpoint is selected from the group consisting of CTLA-4 and PD-1. an antibody conjugate comprising a lipid, a biocompatible polymer, and an antibody; and
The costimulatory molecule and major histocompatibility complex (MHC)-ovalbumin (OVA) complex are composed of exosomes inserted into the exosome surface lipid bilayer, respectively, and the lipid of the antibody conjugate is inserted into the exosome surface lipid bilayer and the antibody A cancer vaccine composition comprising labeled exosome nanoparticles as an active ingredient. preparing an antibody conjugate by amine coupling reaction between the NHS (N-Hydroxysuccinimide) functional group of DPPE-PEG-NHS and the lysine residue of the antibody (step 1);
Preparing OVA-exosomes by culturing dendritic cells in a medium supplemented with ovalbumin (OVA) and Poly(I:C) (second step); and
A method for producing antibody-labeled exosome nanoparticles comprising the step of culturing the antibody conjugate of the first step and the OVA-exosome of the second step and inserting the antibody conjugate into the exosome surface lipid bilayer (step 3). The method according to claim 12, wherein the antibody of the first step is an anti-immune checkpoint antibody. The method according to claim 12, wherein in the first step, the antibody and DPPE-PEG-NHS are reacted in a molar ratio of 1:2 to 1:8.

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