Extracellular vesicle linked to Anti-tfr1 antibody and use thereof

Abstract

The present invention relates to a novel extracellular vesicle linked to an anti-TfR1 antibody and to a use thereof. An extracellular vesicle, according to one aspect, exhibits excellent delivery capability to tumor cells and activates immune cells, thereby being effectively usable as a targeted anticancer therapeutic not limited to specific cancer types.

Description

Extracellular vesicles linked to anti-TFR1 antibodies and uses thereof

[0001] The present invention relates to novel extracellular vesicles linked to anti-TfR1 antibodies and their uses.

[0002] Cancer is a serious disease that requires immediate and effective treatment, and targeted therapy that can accurately deliver anticancer drugs is receiving significant attention.

[0003] Small extracellular vesicles (SEVs), typically 50–200 nm in diameter, can be produced by various cell types. They play a crucial role in intercellular communication, tumorigenesis, and immune response regulation by transporting biologically active substances to recipient cells. Compared to other nanoparticles or small molecules, SEVs exhibit lower toxicity and reduced immunogenicity, leading to active research into their potential as drug delivery vehicles. In particular, immune cell-derived SEVs have been shown to possess various anticancer properties, drawing attention as potential anticancer therapeutics. However, due to their inherent properties, SEVs have a low specific delivery capacity to tumors, necessitating high-dose injection or direct injection into the tumor, and their production presents significant challenges and limitations.

[0004] Meanwhile, transferrin receptor 1 (TfR1), a representative tumor-associated antigen (TAA) encoded by the TFRC gene, is a member of the transferrin receptor family and is also known as CD71. TfR1 is an effective tumor marker that is frequently overexpressed in various cancer types, a result that also coincides with the increased demand for iron ions required for active cell proliferation and tumor growth. Due to its high expression level, TfR1 is a promising target for diagnostic and therapeutic strategies, and its utilization allows for precise targeting of cancer cells. Therefore, antibodies specific for TfR1 or transferrin conjugated with chemicals, toxins, radioisotopes, or nanoparticles are being studied to utilize TfR1 for cancer cell removal. However, existing approaches targeting TfR1 with antibodies or nanoparticles face limitations, such as adverse effects such as immune responses, drug resistance, and iron metabolism disorders at high concentrations, and inefficiency at low concentrations. TfR1-recognizing antibodies may only inhibit the growth of cancer cells in cell models, or when used in animal models, they may bind to certain normal cells, particularly immature reticulocytes and other hematopoietic progenitor cells, and immune cells that express high levels of TfR1, resulting in side effects such as brown urine, decreased red blood cells, and stiffness.

[0005] Our researchers have been working to improve the safety and efficacy of these targeted therapies, and have developed novel anti-TfR1 antibody-conjugated extracellular vesicles with enhanced targeting ability to cancer cells and improved side effects of TfR1-recognizing antibodies.

[0006] One aspect is to provide extracellular vesicles having an antibody or antigen-binding fragment thereof that binds to transferrin receptor 1 (TfR1) linked to their surface.

[0007] Another aspect is to provide a pharmaceutical composition for treating cancer comprising the extracellular vesicles.

[0008] Another aspect provides a method of preventing, ameliorating or treating cancer comprising administering to a subject in need thereof an effective amount of said extracellular vesicles.

[0009] Another aspect provides the use of said extracellular vesicles for the prevention, amelioration or treatment of cancer.

[0010] Another aspect provides the use of said extracellular vesicles for the manufacture of a preparation for the prevention, improvement or treatment of cancer.

[0011] Another aspect is to provide a method for producing the above extracellular vesicles.

[0012] One aspect provides extracellular vesicles having an antibody or antigen-binding fragment thereof that binds to transferrin receptor 1 (TfR1) linked to their surface.

[0013] In this specification, "TfR1 (transferrin receptor 1)" is a member of the transferrin receptor family with two high-affinity binding sites for iron, and is a representative tumor-associated antigen (TAA) encoded by the TFRC gene. TfR1 is an effective cancer marker that is frequently overexpressed in various cancer types.

[0014] As used herein, “antibody or antigen-binding fragment” refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. As used herein, antibody refers to an anti-TfR1 antibody that specifically binds to TfR1.

[0015] As used herein, "extracellular vesicle (EV)" refers to any type of vesicle produced outside the cell, which is a nano-sized membrane-bound structure produced in the endosomal compartment of most eukaryotic cells. "Extracellular vesicle," "extracellular vesicle," "vesicle or vesicle released outside the cell," etc. are all used interchangeably, and extracellular vesicles may include many different types, such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Small extracellular vesicles (sEV) generally refer to extracellular vesicles having a diameter of 50 to 200 nm, and the extracellular vesicles of the present invention may be, but are not limited to, small extracellular vesicles.

[0016] In one specific embodiment, the antibody or antigen-binding fragment thereof may include at least one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody. Preferably, the antibody or antigen-binding fragment thereof may be a scFv-Fc fragment.

[0017] In one specific example, the scFv may comprise an amino acid sequence of SEQ ID NO: 3, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. The Fc may comprise an amino acid sequence of SEQ ID NO: 4, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0018] The extracellular vesicles of the present invention have improved delivery to cancer cells. The extracellular vesicles of the present invention can be effectively delivered to cancer cells by linking the TfR1 antibody to the surface of the extracellular vesicles, thereby expressing the TfR1 antibody on the outside of the extracellular vesicles.

[0019] The extracellular vesicles of the present invention provide anti-TfR1 antibodies in a state linked to the surface of the extracellular vesicles, thereby reducing tumor growth and activity, while reducing side effects such as immunogenicity and systemic toxicity compared to antibody treatment alone.

[0020] In one specific example, the antibody or antigen-binding fragment thereof can be associated with an extracellular vesicle via a transmembrane domain.

[0021] As used herein, the term "transmembrane domain" or "transmembrane domain" refers to a domain located in the cell membrane that connects the extracellular domain and the intracellular domain. The transmembrane domain as used herein refers to a region that functions to connect and fuse an antibody or antigen-binding fragment with an extracellular vesicle and to anchor the antibody or antigen-binding fragment. The transmembrane domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source.

[0022] In one specific example, the transmembrane domain may be a transmembrane domain of TfR1 or a transmembrane domain of another transmembrane protein. For example, the transmembrane protein may be a receptor tyrosine kinase (RTK).

[0023] In one specific example, the transmembrane domain may include at least one transmembrane domain selected from the group consisting of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), tropomyosin receptor kinase (Trk), insulin receptor (IR), Leukocyte receptor tyrosine kinase (LTK), angiopoietin receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF) receptor, receptor tyrosine kinase-like orphan receptors (ROR), discoidin domain receptor (DDR), rearranged during transfection receptor (RETR), tyrosine-protein kinase-like (PTK), related to receptor tyrosine kinase (RYK), and muscle-specific kinase (MuSK). It is not limited. Preferably, the transmembrane domain may be a transmembrane domain of PDGFR.

[0024] In one specific example, the transmembrane domain may comprise an amino acid sequence of SEQ ID NO: 5, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0025] In one specific example, the antibody or antigen-binding fragment thereof may be linked directly to the transmembrane domain or via a linker. The linker may be a short oligopeptide or polypeptide linker, and is not particularly limited in length or type. Any linker known in the art may be applied without limitation.

[0026] The above linker may be a flexible linker. For example, it may be a peptide linker composed of glycine, serine, alanine, or proline, and more specifically, it may be (GS)n, (GGGGS)n, (GGGGA)n, (GGGGP)n, (GGGA)n, (GGS)n, (GSGGS)n, or (GGGS)n. The copy number "n" is any natural number and can be adjusted taking into account the optimization of the linker.

[0027] In one specific example, the antibody or antigen-binding fragment thereof may comprise the amino acid sequence of SEQ ID NO: 2, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0028] In one specific example, the antibody or antigen-binding fragment thereof may include a fluorescent protein tag. The tag may be included for purposes such as improving the water solubility of the recombinant protein, increasing production, maintaining structural stability, and facilitating separation and purification.

[0029] In one specific example, the fluorescent protein may be at least one selected from the group consisting of mCherry, DsRed2, mScarlet, mStrawberry, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, mKate2, mNeptune, CFP, GFP, Emerald, Superfolder GFP, TagGFP2, mClover2, mClover3, and mEos2, but is not limited thereto.

[0030] In one specific example, the fluorescent protein may comprise an amino acid sequence of SEQ ID NO: 6, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0031] In one specific example, the antibody or antigen-binding fragment thereof may comprise an amino acid sequence of SEQ ID NO: 1, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. The amino acid sequence of SEQ ID NO: 1 refers to a sequence comprising the tag.

[0032] The antibodies and domains of the present disclosure may include each of the antibodies and domains described above, as well as modified forms of each of the antibodies and domains. In this case, the modifications may be performed by substituting, deleting, or adding one or more amino acids from the amino acid sequence of the wild-type antibody and domain without altering the function of the antibodies and domains. Typically, the substitutions may be performed by conservative amino acid substitutions that do not affect the charge, polarity, or hydrophobicity of the entire protein.

[0033] In one specific example, the extracellular vesicles may be derived from immune cells.

[0034] In one specific example, the immune cell may include at least one selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells, γδ T cells, NK cells, NKT cells, and dendritic cells. Preferably, the immune cell is a CD4+ T cell.

[0035] The extracellular vesicles according to the present invention can have enhanced immune sensitivity by being derived from immune cells. The extracellular vesicles, being derived from immune cells, can effectively reduce Rab27a and PD-L1 levels.

[0036] In one specific example, the immune cell may comprise a vector comprising a base sequence encoding an antibody or antigen-binding fragment thereof that binds to TfR1.

[0037] As used herein, the term "vector" refers to any nucleic acid construct capable of delivering or directing the movement of foreign genetic material into a target cell where the polynucleotide can be replicated and / or expressed. The term "vector" as used herein includes the delivered construct. The vector may be a linear molecule or a circular molecule. The vector may be integrating or non-integrating.

[0038] In one specific example, the vector may be a plasmid, an episomal vector, a viral vector, a non-viral vector, a cosmid, or an artificial chromosome. The vector may be used without limitation as long as it can stably produce the extracellular vesicles within a host cell by transducing or transfecting an animal cell, particularly a T cell, through infection.

[0039] In one specific example, the viral vector may be any one selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, and a vaccinia virus, but is not limited thereto.

[0040] In one specific example, the non-viral vector preferably uses a transposon system (Hackett et al., US 6,489,458 B), but is not limited thereto, and it is obvious to those skilled in the art that any non-viral vector that is suitable for the purpose of the present invention among commonly used vectors can be used.

[0041] In one specific example, the viral or non-viral vector may comprise a base sequence encoding an antibody or antigen-binding fragment thereof that binds to TfR1.

[0042] In one specific example, the sequence encoding the antibody or antigen-binding fragment thereof may comprise a base sequence represented by any one of SEQ ID NOs: 7 to 9, a portion thereof, or a base sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. That is, the immune cell may comprise a vector comprising a base sequence represented by any one of SEQ ID NOs: 7 to 9, a portion thereof, or a base sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0043] Even if this application describes "comprising a gene sequence / amino acid sequence of a specific sequence number" or "having a gene sequence / amino acid sequence of a specific sequence number," it is self-evident that gene sequences / amino acid sequences in which some sequences are deleted, modified, substituted, or added may also be used in this application, as long as they have the same or corresponding function as those composed of the gene sequence / amino acid sequence of the corresponding sequence number. Furthermore, gene sequences and base sequences may be used interchangeably in this application.

[0044] For example, if it has the same or corresponding function as the extracellular vesicle, it is obvious that it is within the scope of the present invention even if a meaningless sequence is added to or at the end of the sequence of the sequence number, or a part of the sequence of the sequence number or at the end is deleted.

[0045] Homology and identity refer to the degree to which two given base sequences are related, and can be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences are homologous or identical can be determined using a well-known computer algorithm, such as the "FASTA" program, using default parameters, as in, for example, Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, sequence homology or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information database in the United States.

[0046]

[0047] Another aspect provides a composition for preventing or treating cancer comprising the extracellular vesicles.

[0048] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition of the present invention to a subject. For preventive purposes, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0049] As used herein, the term "treatment" refers to any action that improves the symptoms of a disease or provides benefit by administering the composition of the present invention to a subject. As used herein, the terms "treatment," "palliation," and "improvement" may be used interchangeably. A therapeutic benefit refers to any therapeutically significant improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0050] As used herein, the terms "administering," "introducing," and "transplanting" are used interchangeably and may refer to the placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site. Administration may be by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to a desired location within a viable subject. The survival period of the cells after administration to a subject may be as short as several hours, for example, 24 hours, to several days, or as long as several years.

[0051] The cancer of the present invention may be a cancer that expresses a target antigen recognized by the extracellular vesicle of the present invention. Specifically, the cancer is a cancer that expresses a tumor antigen that can be recognized by the extracellular vesicle of the present invention.

[0052] In one specific example, the cancer may be a cancer that expresses TfR1. That is, the composition may be a composition for preventing or treating cancer, solid tumor, and / or hematological malignancy that expresses TfR1.

[0053] The cancer type is not particularly limited and includes solid cancer and blood cancer. Specifically, the cancer may include at least one selected from the group consisting of breast cancer, lung cancer, skin cancer, kidney cancer, colon cancer, head and neck cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, melanoma, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer, and pancreatic cancer.

[0054] The content of the extracellular vesicles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0055] The composition according to the present invention may further comprise a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. may be used. For injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used. For topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it may be prepared in the form of unit dose ampoules or multiple doses. In addition, the anticancer composition may typically comprise a surfactant that facilitates movement through a membrane. These surfactants include those derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidyl glycerol.

[0056] The composition may be administered in combination with additional anticancer agents. Examples of additional anticancer agents include alkylating agents, antimetabolites, spindle-inhibiting plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of such anticancer agents include compounds used in targeted therapies and conventional chemotherapy. Also, examples of the above antibodies include alemtuzumab, apolizumab, acelizumab, atlizumab, bapineuzumab, bevacizumab, vivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, May include pascolizumab, pekfucituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resaivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadoxizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

[0057] Another aspect provides a method of preventing, ameliorating or treating cancer comprising administering to a subject in need thereof an effective amount of said extracellular vesicles.

[0058] Another aspect provides the use of the extracellular vesicles for the prevention, amelioration or treatment of cancer.

[0059] Another aspect provides the use of said extracellular vesicles for the manufacture of a preparation for the prevention, amelioration or treatment of cancer.

[0060]

[0061] Another aspect is to provide a method for producing the above extracellular vesicles.

[0062] In one specific example, the manufacturing method may include a step of introducing a gene encoding an antibody or an antigen-binding fragment thereof that binds to TfR1 into an immune cell.

[0063] In one specific example, the manufacturing method may include a step of producing and isolating extracellular vesicles from the immune cells.

[0064]

[0065] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0066] Extracellular vesicles having antibodies or antigen-binding fragments thereof that bind to TfR1 on their surface have excellent delivery to tumor cells and have the potential to be used as targeted cancer therapeutics that are not limited to cancer types by activating immune cells.

[0067] Figure 1 is a schematic diagram showing the process of producing extracellular vesicles (T-EVs) expressing anti-TfR1 scFv-Fc and the process of targeting cancer cells.

[0068] Figure 2 is a graph showing the results of flow cytometry analysis of GFP-positive cell populations in C-EVs and T-EVs.

[0069] Figure 3 is a graph measuring the size distribution of C-EV and T-EV.

[0070] Figure 4 is a TEM image and quantitative graph showing the shape and diameter of C-EV and T-EV.

[0071] Figure 5 is an image showing the results of Western blot analysis of Fc, HSP70, and TSG101 in C-EV and T-EV.

[0072] Figure 6 is an image showing the results of Western blot analysis of Fc and TSG101 after treatment with proteinase K alone or together with TritonX-100 in T-EV.

[0073] Figure 7 is a graph showing the results of flow cytometry analysis of TfR1-positive cell populations in various tumor cells of humans and mice.

[0074] Figure 8 is a graph showing the results of flow cytometry analysis over time in various tumor cells of humans and mice after treatment with PBS, C-EV, or T-EV.

[0075] Figure 9 is a time-lapse image of sEVs bound to the cell membrane in tumor cells taken at 10-minute intervals after C-EV or T-EV treatment.

[0076] Figure 10 is an image measuring the degree of colocalization of sEV and TfR1 in tumor cells after C-EV or T-EV treatment.

[0077] Figures 11 and 12 are images showing changes in sEV and nuclei in tumor cells after treatment with αTfR1 antibody followed by treatment with PBS, C-EV, or T-EV.

[0078] Figure 13 is a graph showing the survival rate of tumor cells after treatment with PBS, C-EV, or T-EV.

[0079] Figure 14 is a graph measuring the mRNA levels of Rab27a and PD-L1 in tumor cells after treatment with PBS, C-EV, or T-EV.

[0080] Figure 15 is a graph measuring the protein expression levels of Rab27a and PD-L1 in tumor cells after treatment with PBS, C-EV, or T-EV.

[0081] Figure 16 is a graph of NTA analysis of the number of sEV particles extracted from tumor cells after treatment with PBS, C-EV, or T-EV.

[0082] Figure 17 is a graph showing the survival rate of tumor cells after treatment with PBS, C-EV, or T-EV in a co-culture analysis with a CD8+ T cell line.

[0083] Figure 18 is a schematic diagram showing the administration schedule of C-EV or T-EV in various mouse cancer models.

[0084] Figure 19 is an image showing the biodistribution of sEVs after administration of C-EV or T-EV in various mouse cancer models.

[0085] Figure 20 is a graph showing the average intensity of fluorescence from tumors after administration of C-EV or T-EV in various mouse cancer models.

[0086] Figure 21 is a fluorescent image of tumor tissue after administration of C-EV or T-EV in various mouse cancer models.

[0087] Figure 22 is a schematic diagram showing the administration schedule of C-EV or T-EV in various mouse cancer models.

[0088] Figure 23 is a graph showing the average tumor volume of mice after administration of C-EV or T-EV in various mouse cancer models.

[0089] Figure 24 is a graph showing tumor weights obtained 10 days after administration of C-EV or T-EV in various mouse cancer models.

[0090] Figure 25 is an image and graph showing tumor bioluminescence after administration of C-EV or T-EV in various mouse cancer models.

[0091] Figure 26 is a graph showing body weight measurements after administration of C-EV or T-EV in various mouse cancer models.

[0092] Figure 27 is an H&E staining image of major organs (heart, lungs, liver, spleen, kidney) after administration of C-EV or T-EV in various mouse cancer models.

[0093] Figure 28 shows the percentage of red blood cell hemolysis and images of red blood cells after administration of C-EV or T-EV in various mouse cancer models.

[0094] Figure 29 is a graph showing the mRNA and protein expression levels of Rab27a and PD-L1 after administration of C-EV or T-EV in a mouse breast cancer model.

[0095] Figure 30 is an image showing the results of IHC analysis of Rab27a and PD-L1 expression after administration of C-EV or T-EV in a mouse breast cancer model.

[0096] Figure 31 is a graph showing the results of flow cytometry analysis on the proliferation (Ki-67) and activity (IFNγ) of CD45+CD3+CD8+ T cells after administration of C-EV or T-EV in a mouse breast cancer model.

[0097] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0098]

[0099] Example 1. Preparation and delivery of lentivirus

[0100] In one embodiment, surface-bound anti-TfR1 antibody-bound extracellular vesicles (T-EVs) were prepared by overexpressing a construct comprising the gene sequences of Table 2, which include an anti-TfR1 scFv targeting TfR1 on the surface of cancer cells, an Fc region for enhancing surface exposure, and a GFP protein for cell selection, in Jurkat T cells via lentiviral infection. The amino acid sequence of the construct is shown in Table 1 below, and the gene sequence is shown in Table 2.

[0101]

[0102] Domain amino acid sequence anti-TfR1 scFv (SEQ ID NO: 3) EVQLQQSGTVLARPGASVKMSCKASGYSFTIYWIHWVKQRPGQGLEWIATIYPGNSDIIYNQKFKGKAKLTAVTSASTAYMELSSLTNEASAVYYCTRQGYDYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVQITQSPSYLAASPGETIIINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPQDFAMYYCQQHNEYPWTFGGGTKLEIKRFc (SEQ ID NO: 4)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPDGFRβ TM domain (SEQ ID NO: 5)AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRTagGFP2 (SEQ ID NO: 6)MSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKLPVPWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVKFEGDT LVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLESFSACCHTHGMDELYR

[0103]

[0104] 영역유전자 서열anti-TfR1 scFv(서열번호 9)GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGTTTTACCATCTACTGGATACACTGGGTAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGCTACTATTTATCCTGGGAATAGTGATATTATTTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCGGTCACATCCGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGCCTCTGCGGTCTATTACTGTACAAGACAGGGGTACGATTATTATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGTGGCGGCTCCGGTGGTGGCGGCTCCGGTGGTGGCGGCTCCGATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATCATTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGgATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTCAAGATTTTGCAATGTATTACTGTCAACAGCATAATGAATACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGFc(서열번호10)GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAPDGFRβ TM domain(서열번호 11)GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGTTagGFP2(서열번호12)ATGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTGATCGAGCTGGACGGCGACGTGCACGGCCACAAGTTCAGCGTGCGCGGCGAGGGCGAGGGCGACGCCGACTACGGCAAGCTGGAGATCAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTCTGCTACGGCATCCAGTGCTTCGCCCGCTACCCCGAGCACATGAAGATGAACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACATCCAGGAGCGCACCATCCAGTTCCAGGACGACGGCAAGTACAAGACCCGCGGCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAAGGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAGCTTCAACAGCCACAACGTGTACATCCGCCCCGACAAGGCCAACAACGGCCTGGAGGCTAACTTCAAGACCCGCCACAACATCGAGGGCGGCGGCGTGCAGCTGGCCGACCACTACCAGACCAACGTGCCCCTGGGCGACGGCCCCGTGCTGATCCCCATCAACCACTACCTGAGCACTCAGACCAAGATCAGCAAGGACCGCAACGAGGCCCGCGACCACATGGTGCTCCTGGAGTCCTTCAGCGCCTGCTGCCACACCCACGGCATGGACGAGCTGTACAGG

[0105]

[0106] 구체적으로, HEK293FT 세포를 4.6 × 10 5The day after seeding at 10 cells / mL, the cells were cultured with a DNA-lipofectamine solution containing transfer plasmids encoding anti-TfR1 scFv and Fc and packaging plasmids carrying VSVG and pCMV at a 1:1:1 ratio. After 24 h, the cells were switched to fresh medium. The cell supernatant containing lentivirus was collected for 48 h, centrifuged at 300 × g for 5 min to remove debris, and filtered through a 0.45 μm filter. Then, 2 × 10 6 Jurkat T cells were seeded in cells / well to produce T cell-derived sEVs with anti-TfR1 scFv-Fc. Cells were spin-coated with a 1:1 ratio of medium and lentivirus mixture at 300 × g for 90 min, and then replaced with fresh medium. Once the cells were stabilized, sEVs were isolated. A schematic diagram illustrating the process of producing extracellular vesicles and their cancer-specific applications is shown in Figure 1. Jurkat Control The results of flow cytometry analysis of the GFP-positive cell population in JurkatαTfR1 scFv cells are shown in Figure 2.

[0107] As shown in Fig. 2, it was confirmed that a structure according to one specific example was overexpressed in cells infected with lentivirus.

[0108]

[0109] Example 2. Production and purification of T-EV

[0110] In Example 1, when the cells were stabilized, the anti-TfR1 antibody according to one specific example was isolated from the surface-bound extracellular vesicles and produced and purified as follows.

[0111] Jurkat T cells were cultured at 3 × 10 7Cells / dishes were seeded with 500 IU / mL of recombinant IL-2 (Gene Script). After 2 days, the culture medium was replaced with FBS-depleted RPMI-1640 for 24 h according to the MISEV 2023 guidelines. The supernatant was then collected, and sEVs were isolated according to the following method. First, the culture supernatant was sequentially centrifuged at 300 × g for 5 min, 10,000 × g for 30 min, and 25,000 × g for 20 min to remove cell debris and large vesicles. The supernatant was further filtered through a 0.2 μm filter, and sEVs were isolated and characterized using tangential flow filtration (TFF). For sEV staining, DiR (#2533890, Invitrogen) or DiI (#2571429, Invitrogen) dye was incubated with sEV for 30 min at room temperature, after which the buffer was immediately replaced using TFF.

[0112]

[0113] Experimental Example 1. Characteristic Evaluation of T-EV

[0114] The properties of surface-bound extracellular vesicles (T-EVs) containing anti-TfR1 antibodies according to one specific example were evaluated as follows.

[0115] First, the size and shape of T-EVs were observed. SEVs isolated from untransfected Jurkat T cells were used as a control (C-EV). The size distributions of T-EVs and C-EVs were observed using nanoparticle tracking assay (NTA). The shapes and diameters of T-EVs and C-EVs were observed using transmission electron microscopy (TEM). The results are shown in Fig. 3.

[0116] As shown in Fig. 3, there was no difference in sEV size and particle number between C-EV and T-EV.

[0117] As shown in Fig. 4, sEVs were confirmed to be round and intact, and the average diameters were 92.58 ± 20.09 nm for C-EVs and 93.24 ± 34.63 nm for T-EVs, which were indistinguishable and within the normal range. Scale bar = 100 nm.

[0118]

[0119] Additionally, anti-TfR1 scFv-Fc expression was confirmed by Western blot analysis. First, the results of Western blot analysis of Fc, HSP70, and TSG101 in C-EV and T-EV are shown in Fig. 5. Next, to confirm the expression of anti-TfR1 scFv-Fc in the outer membrane of sEV, protease K (PK), a protease that cannot permeate lipid bilayers, was treated alone or together with 10% Triton X-100, and the results of Western blot analysis of Fc and TSG101 are shown in Fig. 6.

[0120] As shown in Figure 5, the anti-TfR1 scFv-Fc construct was expressed with a molecular weight matching the designed structure only in T-EV.

[0121] As shown in Figure 6, PK treatment alone degraded anti-TfR1 scFv-Fc without affecting the internal proteins, confirming that anti-TfR1 scFv-Fc was displayed on the outer membrane of T-EV.

[0122]

[0123] That is, Jurkat T cells transduced with the lentivirus of Example 1 produced sEVs bound to anti-TfR1 scFv-Fc, and it was confirmed that the overexpressed anti-TfR1 scFv-Fc was located on the sEV outer membrane. In addition, this modification did not alter the general characteristics of T-EVs, such as size, production, or morphology, compared to C-EVs.

[0124]

[0125] Experimental Example 2. Confirmation of T-EV's cancer-targeting ability.

[0126] To confirm TfR1-mediated targeting of surface-bound extracellular vesicles (T-EVs) to various cancer types by anti-TfR1 antibodies according to one specific example, the following experiments were performed.

[0127] First, TfR1 expression in various cancer types of humans and mice was evaluated using flow cytometry, and the results are shown in Figure 7.

[0128] Additionally, each cell line was treated with PBS or DiR-labeled sEV, and the proportion of DiR-positive cancer cells was examined by flow cytometry at various time points (0, 2, 4, 6, and 12 hours after treatment) for 12 hours, and the results are shown in Fig. 8.

[0129] As shown in Figure 7, high TfR1 expression was observed in human and mouse cell lines of breast cancer (human: MCF-7, mouse: 4T1), lung cancer (human: A549, mouse: LLC), and melanoma (human: SK-MEL-28, mouse: B16F10).

[0130] As shown in Figure 8, at most time points, the DiR-stained T-EV treatment group showed a higher proportion of DiR-positive cancer cells compared to the DiR-stained C-EV treatment group.

[0131]

[0132] Next, to demonstrate that the high delivery of T-EVs to cancer cells resulted from enhanced binding between sEVs and cells rather than increased cellular uptake of sEVs, time-lapse live cell imaging was performed, and the results are shown in Fig. 9.

[0133] As shown in Figure 9, when both mouse (4T1) and human (MCF-7) breast cancer cell lines were treated with DiI-stained sEVs, T-EVs were observed to bind to the cell surface more rapidly than C-EVs. White arrowheads indicate sEVs bound to the cell membrane. This suggests that the high transduction capacity of T-EVs is due to their superior binding ability. Scale bar = 10 μm.

[0134]

[0135] Additionally, immunocytochemistry (ICC) confirmed that T-EV binds to cancer cells via TfR1-mediated binding. Specifically, the degree of colocalization of sEV and TfR1 in tumor cells after C-EV or T-EV treatment is shown in Fig. 10, and images showing changes in sEV and nuclei in tumor cells after treatment with αTfR1 antibody followed by PBS, C-EV, or T-EV treatment are shown in Figs. 11 and 12.

[0136] As shown in Fig. 10, T-EVs were not only more effectively delivered to cancer cells but also showed significant co-localization with TfR1 in both human and mouse breast cancer cells.

[0137] As shown in Figures 11 and 12, pretreatment of cells with anti-TfR1 antibodies prior to introduction of T-EVs reduced T-EV delivery to cancer cells. This suggests that the enhanced delivery of T-EVs was mediated by TfR1.

[0138]

[0139] These results indicate that the extracellular vesicles of the present invention, according to one specific example displaying anti-TfR1 scFv on their surface, bind to cancer cells more rapidly in a TfR1-dependent manner and are delivered more efficiently than C-EVs.

[0140]

[0141] Experimental Example 3. Confirmation of the Rab27a and PD-L1 inhibitory effects of T-EV.

[0142] After confirming that surface-bound anti-TfR1 antibodies according to one specific example exhibited enhanced delivery to cancer cells, the following experiments were performed to investigate whether this targeting function enhanced the efficacy of T cell-derived EVs against cancer cells. All experiments were performed 48 hours after treatment with PBS, C-EVs, or T-EVs.

[0143]

[0144] 3.1 Measurement of changes in tumor cell survival rate

[0145] First, changes in the viability of breast cancer cells (4T1-Luc2) treated with C-EV or T-EV were investigated using MTS analysis and luminescence analysis, and the results are shown in Fig. 13 (n = 5-6).

[0146] As shown in Figure 13, the viability of 4T1 cells after 48 hours was unchanged in all treatments with PBS, C-EV, and T-EV.

[0147]

[0148] 3.2 Measurement of changes in Rab27a and PD-L1 expression

[0149] Next, we evaluated changes in Rab27a and PD-L1 expression in 4T1-Luc2 cells. Rab27a and PD-L1 mRNA levels were measured using qRT-PCR, and the results are shown in Figure 14 (n = 5-6). Rab27a and PD-L1 protein expression levels were confirmed using Western blot analysis, and the results are shown in Figure 15 (n = 4). Furthermore, to assess the impact of decreased Rab27a expression, the number of cancer cell-derived sEV particles was measured using NTA, and the results are shown in Figure 16 (n = 3).

[0150] As shown in Figures 14 and 15, it was confirmed that more sEVs were delivered to 4T1 cells, which resulted in a decrease in the mRNA and protein expression of Rab27a and PD-L1.

[0151] As shown in Figure 16, we found that sEV production was significantly reduced in cells treated with C-EV and T-EV compared to cells treated with PBS. In particular, T-EV showed a higher targeting affinity for 4T1 cells, resulting in a further decrease in the number of sEV particles.

[0152]

[0153] 3.3 Co-culture analysis with CD8+ T cell lines

[0154] To investigate whether the reduction in Rab27a expression, resulting inhibition of cancer cell-derived sEV production, and reduction in PD-L1 expression could enhance immune activity, we performed a co-culture assay with the CTLL-2 CD8+ T cell line and 4T1-Luc2 cells. First, 4T1 cells were pre-seeded, and then CTLL-2 cells were seeded with sEV the next day. 4T1-Luc2 cells (target cells; T) and CTLL-2 cells (effector cells; E) were co-cultured at a ratio of T:E = 1:10. Changes in the viability of breast cancer cells (4T1-Luc2) treated with C-EV or T-EV were investigated using MTS assay (n = 6) and luminescence assay (n = 5), and the results are shown in Fig. 17.

[0155] As shown in Figure 17, after 48 hours, the group treated with sEV showed decreased cancer cell viability compared to the group treated with PBS. Furthermore, the group treated with T-EV showed a greater decrease in viability compared to the group treated with C-EV.

[0156] This suggests that enhanced delivery of sEVs enhances their anticancer effects in the presence of CD8+ T cells.

[0157]

[0158] Experimental Example 4. Confirmation of the enhanced transmissibility of T-EVs in a mouse cancer model.

[0159] In a mouse in vivo model, we examined whether surface-bound extracellular vesicles (T-EVs) containing anti-TfR1 antibodies according to one specific example function in complex in vivo systems.

[0160] Specifically, after subcutaneous injection of 4T1 cells into wild-type (WT) BALB / C mice in a breast cancer mouse model, tumor sizes averaged 220 mm 3 When reaching , PBS, DiR-stained C-EVs, or DiR-stained T-EVs were administered intravenously. Fluorescence intensity was analyzed 6 h after injection (n = 6). In a lung metastasis mouse model, A549-Luc2 cells were injected into the tail vein of WT nude mice, and lung bioluminescence was measured to an average of 1.85 × 10 2 p / s / cm 2 When / sr was reached, DiR-labeled C-EVs or DiR-stained T-EVs were intravenously injected. After 2 hours, major organs, including the lungs containing tumors, were dissected to determine fluorescence intensity (n = 5). In a melanoma mouse model, after subcutaneous injection of SK-MEL-28 cells into WT nude mice, tumors grew to an average of 120 mm 3 Upon reaching the target, DiR-labeled sEVs were intravenously injected. Fluorescence intensity was analyzed 7 hours after injection (n=5-7). Figure 18 is a schematic diagram showing the administration schedule of C-EV or T-EV in various mouse cancer models.

[0161] The biodistribution of DiR-labeled sEVs was evaluated and shown in Fig. 19, the average intensity of fluorescence from tumors after sEV administration was shown in Fig. 20, and the fluorescence image of tumor tissue was shown in Fig. 21.

[0162] As shown in Figures 19 to 21, in both tumor tissues and cross-sections of breast cancer models, lung cancer models, and melanoma models, a significant increase in fluorescence intensity was observed in mice injected with T-EVs compared to mice injected with PBS or C-EVs.

[0163]

[0164] Taken together, these results demonstrate that intravenously injected T-EVs are more likely to be delivered to tumors in three mouse tumor models, confirming that the T-EVs of the present invention can be practically delivered in an in vivo system.

[0165]

[0166] Experimental Example 5. Confirmation of the anticancer effect of T-EV.

[0167] 5.1 Confirmation of tumor suppression effect

[0168] To evaluate whether enhanced delivery of surface-bound extracellular vesicles (T-EVs) to mouse cancer tissues led to tumor suppression, the anticancer efficacy of sEVs was determined as follows.

[0169] Specifically, in a breast cancer mouse model, 4T1-Luc2 cells were subcutaneously injected into WT BALB / C mice, and tumor sizes averaged 20 mm 3 When reached, PBS, C-EV, or T-EV were intravenously injected 5 times every 2 days. In the lung cancer mouse model, LLC-Luc2 cells were subcutaneously injected into C57BL / 6 mice, and the tumor size was 30 mm on average. 3 When reached, PBS, C-EV, or T-EV were administered intravenously five times every two days. Figure 22 is a schematic diagram showing the administration schedule of C-EV or T-EV in various mouse cancer models.

[0170] The average tumor volume is shown in Figure 23, the average tumor weight is shown in Figure 24, and the tumor bioluminescence data obtained from each group on day 10 or day 9 are shown in Figure 25 (n = 7).

[0171] As shown in Figures 23 and 24, mice injected with T-EV showed significantly reduced tumor growth and body weight compared to mice treated with PBS or C-EV, whereas the effect of C-EV was no different from that of mice treated with PBS.

[0172] As shown in Figure 25, tumor bioluminescence showed that the bioactivity and viability of cancer cells were reduced in mice injected with T-EV compared to the other groups.

[0173]

[0174] 5.2 Cytotoxicity evaluation

[0175] According to one specific example, the presence of side effects such as in vivo systemic toxicity of surface-bound extracellular vesicles (T-EVs) by anti-TfR1 antibodies was determined.

[0176] In the two mouse transgenic models of Experimental Example 5.1, body weights were measured on the 10th day after intravenous injection of PBS, C-EV, or T-EV, and the results are shown in Fig. 26. On the same day, major organs such as the heart, lungs, liver, spleen, and kidneys were collected from the mice, and immunohistological damage was confirmed through H&E staining, and the results are shown in Fig. 27. In addition, the percentage (%) of red blood cell hemolysis and images of red blood cells in mouse blood were observed, and the results are shown in Fig. 28.

[0177] As shown in Figures 26 to 28, there was no difference in body weight between the groups, and no histological damage was observed. Furthermore, no anemia due to decreased erythropoiesis was observed, and the erythrocyte morphology remained normal, maintaining a disc-like shape.

[0178]

[0179] 5.3 Investigation of the anticancer mechanism of T-EV

[0180] To further investigate the mechanisms underlying the anticancer effects of surface-bound extracellular vesicles (T-EVs) of anti-TfR1 antibodies according to one specific example, tumor samples from the 4T1-Luc2 transgenic mouse model were further analyzed.

[0181] Specifically, the results of Rab27a and PD-L1 analysis using qRT-PCR and Western blotting in tumors of the 4T1-Luc2 homologous mouse model are shown in Figure 29, and the results of IHC analysis of Rab27a and PD-L1 expression in tumor sections are shown in Figure 30 (n = 7).

[0182] As shown in Figures 29 and 30, consistent with the in vitro data, T-EV treatment significantly reduced the expression of Rab27a and PD-L1 in tumor tissues compared to PBS and C-EV treatments.

[0183]

[0184] Additionally, because previous studies have shown that T-cell-derived sEVs affect T-cell activation by downregulating both Rab27a, which reduces tumor-derived sEV production, and PD-L1, a major immune checkpoint inhibitor, CD8+ T-cell activation was assessed. Specifically, flow cytometric analysis results for the proliferation (Ki-67) and activation (IFNγ) of CD45+CD3+CD8+ T cells in tumor-infiltrating lymphocytes (TILs), draining lymph nodes (DLN), and peripheral blood mononuclear cells (PBMCs) are shown in Figure 31 (n = 7).

[0185] As shown in Figure 31, flow cytometry analysis of CD8+ T cells in TIL, DLN, and PBMC showed that positivity for IFNγ and Ki-67 increased, confirming enhanced cytotoxicity and cell proliferation, respectively.

[0186]

[0187] In summary, T-EVs specifically delivered to tumor cells by anti-TfR1 antibodies exhibit anticancer effects without systemic toxicity in various mouse cancer models. These results suggest that surface-bound extracellular vesicles containing anti-TfR1 antibodies, according to one specific example, have the potential to activate CD8+ T cells and serve as a targeted cancer treatment beyond cancer.

[0188]

[0189] Reference example

[0190]

[0191] Reference Example 1. Cell Culture

[0192] Breast cancer cell lines (4T1, 4T1-Luc2, and MCF7), lung cancer cell lines (LLC, LLC-Luc2, A549, and A549-Luc2), melanoma cell lines (B16F10 and SK-MEL-28), T cell lines (Jurkat and CTLL-2), and HEK293FT cell lines were maintained at 37°C with 5% CO2. 4T1, 4T1-Luc2, LLC, LLC-Luc2, B16F10, and HEK293FT cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic-antimycotic (AA; #CA002-010, GenDEPOT). MCF7, A549, A549-Luc2, CTLL-2, and Jurkat T cells were cultured in Roswell Park Memorial Institute medium (RPMI-1640; Hyclone) containing 10% FBS and 1% AA. SK-MEL-28 cells were cultured in Minimum Essential Medium (MEM; Hyclone) containing Earle's Balanced Salts supplemented with 10% FBS and 1% AA. CTLL-2 cells were additionally treated with 20 IU / mL of recombinant IL-2 (R&D Systems).

[0193]

[0194] Reference Example 2. Nanoparticle tracking analysis (NTA)

[0195] Samples containing sEVs were introduced into the NanoSight LM10 analysis chamber using a sterile syringe. Measurements were performed at 30 frames per second using a 405 nm laser beam to determine vesicle size based on Brownian motion. The captured vesicle motion was analyzed using NTA software version 2.3. Post-acquisition settings for NTA were optimized for each sample but maintained consistently across all measurements.

[0196]

[0197] Reference Example 3. Transmission electron microscopy (TEM) analysis

[0198] Before mounting sEVs on TEM grids for detailed observation, they were stabilized using a cell fixative. The fixative used was a mixture of 1.25% glutaraldehyde (GA) and 2.65% formaldehyde (FA) in 0.2 M sodium cacodylate buffer (SC; pH 7.2). This fixative was cooled to below 4°C and then mixed with the sEV emulsion in a 1:1 ratio. The mixture was kept at below 4°C for 1 h and stirred every 10 min to ensure uniform fixation. Then, 10 μL of the fixed sEV solution was applied to a plasma-cleaned Ni TEM grid (200 mesh, TED Pella) for 10 min. Excess solution was removed using filter paper, and then washed with 10 μL of SC. This washing and blotting procedure was repeated 15 times every 10 min to effectively remove the fixative. To remove residual salts from the SC, the grid was washed with deionized water using the same blotting method, repeated at 10-minute intervals for at least 15 times. The Ni TEM grid with immobilized sEVs was then slowly dried at atmospheric pressure. All handling and washing steps were performed at temperatures below 4°C. Images obtained from the TEM were analyzed using ImageJ software (National Institutes of Health, USA).

[0199]

[0200] Reference Example 4. Antibodies and Reagents

[0201] The antibodies used in the experiment are shown in Table 3 below.

[0202]

[0203] Target antigenIsotypeDilutionSourceApplicationβ-actinRabbit1:2000Cell Signaling (#4970)WBCD3-APCRat1:200Cell Signaling Technology (#24265S)FCD8-PERat1:200Cell Signaling Technology (#56984S)FCD45-PE-Cy7Rat1:200Cell Signaling Technology (#60943S)FFc-HRPGoat1:2000Abcam (#ab97225)WBFc-HRPGoat1:2000Abcam (#ab97224)FHSP70Mouse1:2000Abcam (#ab2787)WBIFN-γRabbit1:500Abcam (#ab9657)FKi-67-FITCRabbit1:200Cell Signaling Technology (#11882S)FPD-L1Rabbit1:100Cell signaling (13684)IHCPD-L1Mouse1:1000Abcam (#ab210934)WBRab27aMouse1:100Abcam (#ab55667)IHCRab27aMouse1:1000Abnova (#H00005873-M02)WBTfR1Mouse1:2000 (F), 1:500 (ICC)Invitrogen (#13-6800)F, ICCTSG101Rabbit1:1000Abcam (#ab30871)WBRabbit-HRPGoat1:200 (IHC), 1:3000 (WB)Cell Signaling Technology (#7074)IHC, WBMouse IgG-HRPHorse1:200 (IHC), 1:3000 (WB)Cell Signaling Technology (#7076)IHC, WBMouse IgG-Alexa Fluor 488GoatICC (1:500),F (1:2000)Invitrogen (#A-11001)ICC, FRabbit IgG-Alexa Fluor 488Goat1:2000Invitrogen (#A-11008)F

[0204] * F: Flow cytometry, ICC: Immunocytochemistry, IHC: Immunohistochemistry, WB: Western blot

[0205]

[0206] Reference Example 5. Cell Viability Test

[0207] Cell viability was assessed by MTS assay using CellTiter 96 Aqueous One Solution Cell Proliferation Assay System (#G3582, Promega) or bioluminescence using an in vivo imaging system (IVIS; Perkin Elmer). 4T1 and 4T1-Luc2 cells (5 × 10 3 cells / well) were seeded in clear or dark 96-well plates in DMEM medium supplemented with 10% FBS and 1% AA and cultured overnight. The cells were then incubated with sEVs (4.3 × 10 10 (particles / mL) for 48 h. MTS reagent was added, incubated at 37°C for 1 h, and absorbance was measured at 490 nm using a VersaMax Microplate Reader (Molecular Devices). D-luciferin (#122799, PerkinElmer) was added for bioluminescence, and the signal was analyzed using IVIS and IVIS software.

[0208]

[0209] Reference Example 6. T cell-mediated cytotoxicity assay (co-culture assay)

[0210] 4T1 and 4T1-Luc2 cells (5 × 10 3 cells / well) were seeded into clear or black 96-well plates. The next day, CTLL-2 cells (5 × 10 4 sEVs (4.3 × 10 cells / well) were seeded onto plates containing 4T1 or 4T1-Luc2 cells. 10particles / mL) or PBS. The viability of 4T1 and 4T1-Luc2 cells was measured using MTS and bioluminescence assays.

[0211]

[0212] Reference Example 7. Polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR)

[0213] RNA extraction from cells and tumor tissues was performed using the MiniBEST Universal RNA Extraction Kit (#9767, TaKaRa) according to the manufacturer's protocol. Total RNA was reverse transcribed using the PrimeScript™ 1st Strand cDNA Synthesis Kit (#6110A, TaKaRa). Gene expression analysis was performed using PCR or qRT-PCR using the gene-specific primers (SEQ ID NOs: 13 to 22) listed in Table 4. qRT-PCR was performed on an ABI StepOne Plus instrument (Applied Biosystems) using TB Green® Premix Ex Taq™ II (#RR820B, Takara).

[0214]

[0215] Gene nameSpeciesForward (5'-3')Reverse (5'-3')GAPDHHumanGAATTTGGCTACAGCAACAG(SEQ ID NO: 13)TGAGGGTCTCTCTCTTCCTC(SEQ ID NO: 14)GapdhMouseTCACCACCATGGAGAAGGC(SEQ ID NO: 15)GCTAAGCAGTTGGTGGTGCA(SEQ ID NO: 16)Rab27aMouseTTCCTGCTTCTGTTCGACC(SEQ ID NO: 17)TTTCACTGCCCTCTGGTCTT(SEQ ID NO: 18)Pd-l1MouseTGCGGACTACAAGCGAATCACG(SEQ ID NO: 19)CTCAGCTTCTGGATAACCCTCG(SEQ ID NO: 20)Anti-TfR1 scFv-GAGGTTCAGCTCCAGCAGTC(SEQ ID NO: 21)TTGCCCTTGAACTTCTGGTT(SEQ ID NO: 22)

[0216]

[0217] Reference Example 8. Western Blotting

[0218] Samples were lysed using RIPA buffer (#9806, Cell Signaling Technology) supplemented with protease and phosphatase inhibitors (#5872S, Cell Signaling Technology). Proteins were separated on Bolt™ 4-12% Bis-Tris Plus gels (#NW04120BOX, Invitrogen) and transferred to iBlot™ 2 Transfer Stacks (#IB230023, Invitrogen). The membrane was blocked with 5% nonfat milk (#9809, Cell Signaling Technology) in PBST for 1 hour, probed with specific primary antibodies, and then probed with HRP-conjugated secondary antibodies. A chemiluminescent substrate (Thermo Fisher Scientific) was applied to visualize protein bands. Protein expression levels were quantified using ImageJ (version 1.54g, National Institutes of Health, Bethesda, MD, USA).

[0219]

[0220] Reference Example 9. Flow cytometry analysis

[0221] Cells suspended in PBS were fixed with 4% paraformaldehyde (PFA; #P2031, Biosesang) for 10 minutes at 4°C. If permeabilization was required, cells were incubated with 0.1% Triton X-100 in PBS for 10 minutes at 4°C. After permeabilization, cells were washed and blocked with PBST containing 5% goat serum and 1% BSA. Cells were then incubated with primary antibodies diluted in blocking buffer for 1 hour at 4°C. After washing to remove unbound primary antibodies, cells were incubated with the appropriate fluorescently labeled secondary antibodies for 1 hour at 4°C in the dark. Finally, stained samples were analyzed using a Beckman Coulter CytoFLEX flow cytometer, and data were processed using FlowJo software.

[0222]

[0223] Reference Example 10. Immunocytochemistry (ICC)

[0224] MCF7 or 4T1 cells (1×10 4 cells / well) were cultured with DiR-labeled sEVs (5 × 10 9 sEV-treated cells were cultured for 6 h in the dark with 100 μg / mL of sEV-treated cells. After incubation, sEV-treated cells were washed with PBS and fixed with 4% PFA for 10 min at room temperature (RT). After fixation, cells were washed twice and blocked with PBST containing 5% goat serum and 5% BSA for 1 h at RT. Cells were then incubated with primary antibodies, followed by corresponding secondary antibodies for 1 h each at RT. After staining, cells were washed, stained with DAPI, and mounted using mounting solution. Fluorescent images were captured using a ZEISS fluorescence microscope.

[0225]

[0226] Reference Example 11. Live cell imaging

[0227] MCF7 or 4T1 cells were seeded in confocal dishes (1 × 10 4 cells / plate). After plating, cells were cultured in complete medium at 37°C and 5% CO2. The next day, DiI-labeled sEVs (1 × 10 10 (particles / mL) was added, and live-cell imaging of DiI fluorescence was performed immediately using a ZEISS LSM 900 equipped with a 37°C chamber and 5% CO2. Images were taken every 10 minutes for 60 minutes.

[0228]

[0229] Reference Example 12. In vivo mouse study for sEV targeting analysis.

[0230] To establish an allogeneic tumor model, 5 × 10 5 4T1 cells were injected subcutaneously into 5-week-old female wild-type BALB / c mice. Mice with similar tumor volumes were randomly grouped 10 days after inoculation. DiR-labeled sEVs (8 × 10 9 particles / mouse) or PBS were intravenously injected. Fluorescence analysis was performed using the IVIS imaging system 6 hours after administration (n = 6).

[0231] 3 × 10 in 100 μL of cold PBS 6 A syngeneic tumor model was established by subcutaneously injecting A549-Luc2 cells into 5-week-old male wild-type nude mice. Mice with similar bioluminescence were randomly grouped 25 days after inoculation. DiR-labeled sEVs (1.2 × 10 11 particles / mouse) or PBS were injected intravenously. Fluorescence analysis using the IVIS imaging system was performed 2 hours after administration (n = 5).

[0232] To investigate sEV targeting in cancer-free mice, 13-week-old male wild-type nude mice were randomly grouped. DiR-labeled sEVs (1.2 × 10 11 particles / mouse) or PBS were injected intravenously, and mice were analyzed using the same method as the A549-Luc2 syngeneic tumor model (n = 3).

[0233] Melanoma xenograft tumor models were prepared by mixing 100 μL of Matrigel in 100 μL of cold PBS and adding 2 × 10 6 SK-MEL-28 cells were established by subcutaneous injection into 5-week-old male wild-type nude mice. Mice with similar tumor volumes were randomly grouped 27 days after inoculation. DiR-labeled sEVs (1.2 × 10 11 particles / mouse) or PBS were injected intravenously. Fluorescence analysis was performed using an IVIS imaging system 7 hours after administration (n = 5-7).

[0234] To analyze fluorescence signals from mouse tissues, mice were euthanized and specific tissues were collected. The tissues were washed three times with PBS, fixed overnight in 4% PFA, and then washed again with PBS. They were then immersed in a 30% sucrose solution in PBS for 24 hours and then washed again with PBS. The tissues were embedded in tissue freezing medium (#14020108926, Leica) and frozen. The blocks were sectioned at 20 μm thickness, stained with DAPI, and mounted using mounting solution. The frozen sections were analyzed using a confocal microscope (#FV1200, Olympus).

[0235] Mice were purchased from Orient Bio (Republic of Korea) and treated according to a protocol approved by the DGIST Institutional Animal Care and Use Committee (IACUC, Approval Number: DGIST-IACUC-24022807-0001).

[0236]

[0237] Reference Example 13. In vivo mouse study to investigate the anticancer effect of sEV.

[0238] To evaluate the anticancer effect of sEV, 5 × 10 5 4T1-Luc2 cells were injected subcutaneously into 5-week-old female wild-type BALB / c mice. After 10 days, mice with similar tumor volumes were randomly divided into groups. sEV (3 × 10 10 particles / mouse) or PBS were injected intravenously every 2 days (n = 4). Tumor size was measured every 2 days with a digital caliper and calculated using the ellipsoid formula: V = 1 / 2 (length × width 2 ) On the last day of the experiment, the mice were weighed and euthanized, and tumor tissue, specific tissue, and blood samples were collected for further analysis.

[0239] In a separate experiment, 1 × 10 6 LLC-Luc2 cells were injected subcutaneously into 5-week-old male wild-type C57BL / 6 mice. After 2 weeks, the mice were randomly divided into groups. sEV (3 × 10 10 (particles / mouse) or PBS were injected intravenously every 2 days (n = 4). Tumor size was measured using the same method. On the final day, mice were weighed, euthanized, and tumor tissues were analyzed.

[0240] Mice were purchased from Orient Bio (Korea), and all procedures were performed according to protocols approved by the DGIST Institutional Animal Care and Use Committee (IACUC; approval number: DGIST-IACUC-24022807-0001).

[0241]

[0242] Reference Example 14. Histopathological Method

[0243] Histological analysis of mouse tissues was performed using hematoxylin (#CS700, Dako) and eosin (#CS701, Dako) according to the manufacturer's protocol. Tissues were initially fixed in 4% PFA and then embedded in paraffin blocks according to standard procedures. The blocks were sectioned into 5-μm-thick slices using a rotary microtome (#RM2255, Leica Biosystems). For H&E staining, sections were processed according to the manufacturer's instructions. For Rab27a and PD-L1 staining, deparaffinized sections were subjected to antigen retrieval with antigen retrieval buffer (#ab93678, Abcam) and then blocked with blocking solution for 1 hour at room temperature. Sections were then incubated with the corresponding primary antibodies and then with HRP-conjugated secondary antibodies. DAB staining (#SK-4100, Vector Laboratories) was performed according to the manufacturer's instructions, and the sections were mounted. Images were captured using an Eclipse Ni-E microscope (Nikon) and analyzed with NIS-Elements software. Further image analysis was performed using ImageJ.

[0244]

[0245] Reference Example 15. Blood Compatibility Test

[0246] Blood samples were collected in anticoagulant-coated tubes and centrifuged at 500 × g for 5 min. Plasma was removed, and RBCs were washed twice with PBS. After resuspension, 500 μL of RBCs were incubated at 37°C for 30 min. After incubation, the samples were centrifuged at 500 × g for 5 min, and the supernatant was carefully transferred to a microplate. The absorbance of the supernatant was measured at 540 nm. A mixture of PBS and RBC suspension served as a negative control, and a mixture of 1% Triton X-100 and RBC suspension served as a positive control. Hemolysis (%) was calculated using the following formula: Hemolysis (%) = (A s -A0) / (A100 -A0) × 100(%). A0, A 100 , A s represents the absorbance of the negative control, positive control, and test sample, respectively. To examine RBC morphology, the samples were diluted with PBS and analyzed using an optical microscope.

[0247]

[0248] Reference Example 16. Statistical Analysis

[0249] Statistical analyses were performed using PRISM 8 software (GraphPad Software, Inc.). Data are expressed as the mean ± standard error of the mean, as indicated in the figure legends. Significance levels were determined as follows: ns = not significant, p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed p values ​​are provided in the figure legends.

[0250]

[0251] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Extracellular vesicles with an antibody or antigen-binding fragment thereof that binds to TfR1 (transferrin receptor 1) attached to the surface.

2. An extracellular vesicle according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises at least one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

3. An extracellular vesicle according to claim 1, wherein the antibody or antigen-binding fragment thereof is linked to the extracellular vesicle via a transmembrane domain.

4. In claim 3, the transmembrane domain comprises at least one transmembrane domain selected from the group consisting of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), tropomyosin receptor kinase (Trk), insulin receptor (IR), Leukocyte receptor tyrosine kinase (LTK), angiopoietin receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF) receptor, receptor tyrosine kinase-like orphan receptors (ROR), discoidin domain receptor (DDR), rearranged during transfection receptor (RETR), tyrosine-protein kinase-like (PTK), related to receptor tyrosine kinase (RYK), and muscle-specific kinase (MuSK). Extracellular vesicles.

5. An extracellular vesicle according to claim 3, wherein the antibody or antigen-binding fragment thereof is linked directly or through a linker to the transmembrane domain.

6. An extracellular vesicle according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3.

7. In claim 1, the extracellular vesicle is derived from an immune cell.

8. An extracellular vesicle according to claim 7, wherein the immune cell comprises at least one selected from the group consisting of CD4+ T cells, CD8+ T cells, regulatory T cells, γδ T cells, NK cells, NKT cells, and dendritic cells.

9. An extracellular vesicle according to claim 7, wherein the immune cell comprises a vector containing a base sequence encoding an antibody or an antigen-binding fragment thereof that binds to TfR1.

10. An extracellular vesicle according to claim 9, wherein the vector comprises at least one selected from the group consisting of a plasmid, a cosmid, a virus, a phage, a recombinant expression cassette, and a transposon.

11. An extracellular vesicle according to claim 7, wherein the immune cell comprises a vector comprising a base sequence represented by any one of SEQ ID NOs: 7 to 9.

12. A pharmaceutical composition for treating cancer comprising the extracellular vesicle of any one of claims 1 to 11.

13. A pharmaceutical composition according to claim 12, wherein the cancer comprises at least one selected from the group consisting of breast cancer, lung cancer, skin cancer, kidney cancer, colon cancer, head and neck cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, melanoma, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer, and pancreatic cancer.

14. A step of introducing a gene encoding an antibody or an antigen-binding fragment thereof that binds to TfR1 (transferrin receptor 1) into an immune cell; and A method for producing extracellular vesicles, comprising the step of producing and isolating extracellular vesicles from the above-mentioned immune cells.

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