WO2018124835A1 - 신규 엑소좀 계열 항암제 - Google Patents
신규 엑소좀 계열 항암제 Download PDFInfo
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- WO2018124835A1 WO2018124835A1 PCT/KR2017/015784 KR2017015784W WO2018124835A1 WO 2018124835 A1 WO2018124835 A1 WO 2018124835A1 KR 2017015784 W KR2017015784 W KR 2017015784W WO 2018124835 A1 WO2018124835 A1 WO 2018124835A1
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- exosomes
- sirpα
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Definitions
- the present invention relates to a novel anticancer agent, and more particularly to a novel exosome-based anticancer agent that increases the phagocytosis of cancer cells.
- Tumors avoid immune surveillance by expressing activating and inhibitory ligands that interact with receptors found on the surface of immune cells to survive and proliferate. This interaction between tumors and immune cells prevents tumors from being killed by the immune system (Pardoll DM. Nat Rev Cancer. 12: 252-64. 2012).
- One mechanism of tumor immune evasion is overexpression of CD47, which allows tumors to avoid innate immune surveillance. Dendritic cells "don't eat me” when CD47 binds to the signaling regulatory protein ⁇ (SIRP ⁇ ) of innate immune cells such as macrophages.
- SIRP ⁇ signaling regulatory protein ⁇
- CD47-SIRP ⁇ interactions By activating the signal to induce tumors from phagocytosis, abundant CD47 expression in various malignant cells lowers the survival rate of cancer patients, providing a strong basis for therapeutic targeting of CD47-SIRP ⁇ interactions. Since the N terminus of SIRP ⁇ contains an immunoglobulin superfamily V-like domain that interacts with the N terminus of CD47, several competitive antagonists have been developed to block their interactions with human CD47 blocking monoclonal antibodies (CD47 mAb). Has demonstrated efficacy in a variety of preclinical tumor models and induced T cell mediated destruction of immunogenic tumors (Tseng D, et al. Proc Natl Acad Sci USA. 110: 11103-8. 2013).
- CD47-SIRP ⁇ interaction may have an antagonistic effect with recombinant SIRP ⁇ protein or SIRP ⁇ - FC fusion protein, taking into account that the weak interaction between native CD47 and SIRP ⁇ may limit the usefulness of wild type SIRP ⁇ protein as a therapeutic antagonist.
- high-affinity variants of SIRP ⁇ were shown to antagonize CD47 in cancer cells.
- SIRP ⁇ variants alone acted as adjuvants to tumor-specific antibodies but did not stimulate phagocytosis and tumor growth inhibition of macrophages more than expected (Sockolosky JT et al., Proc Natl Acad Sci USA. 113: E2646-). E54. 2016).
- U.S. Patent Application Publication No. 2015-0376288 discloses a therapeutic method for treating pathogen infection by administering an agent that reduces binding of CD47 to SIRP ⁇ in infected cells on host phagocytic cells.
- the present invention is to solve a variety of problems including the above problems, it is an object of the present invention to provide a novel exosome-based anticancer agent effective in anti-cancer treatment to remove cancer cells by increasing the phagocytosis of macrophages and dendritic cells.
- these problems are exemplary, and the scope of the present invention is not limited thereby.
- a recombinant exosome wherein the phagocytosis promoting protein is presented on an exosome surface.
- a pharmaceutical composition for anticancer comprising a therapeutically effective amount of the recombinant exosomes and a pharmaceutically acceptable carrier.
- the recombinant exosomes presented on the surface of the SiRP according to an embodiment of the present invention is similar to the CD47 protein clustered in the form of lipid rafts on the surface of cancer cells by siRP clustering in the form of lipid rafts (lipid raft) on the surface of the exosomes. Because it binds with high binding avidity, even small amounts of exosomes block SiRP-CD47 interactions, enabling more efficient stimulation of anticancer immune responses. Of course, the scope of the present invention is not limited by these effects.
- SIRP ⁇ variant SIRP ⁇ -exosomes
- FIG. 2 is a Western blot gel photograph (A) confirming the expression of recombinant exosomes (SIRP ⁇ -exosomes) prepared according to one embodiment of the present invention and expressing the expression of SIRP ⁇ on the surface of exosomes through flow cytometry (B) An electron micrograph showing an image of SIRP ⁇ -exosomes (C).
- Figure 3 is a graph analyzing the size of the SIRP ⁇ -exosomes prepared according to an embodiment of the present invention by dynamic light scattering (DLS) method
- A transmission electron micrograph of the SIRP ⁇ -exosomes
- B flow cytometry Graph
- D dynamic light scattering analysis graph
- FIG. 4 is a Western blot gel photograph analyzing the expression of purified recombinant SIRP ⁇ -Myc monomer protein (mSIRP ⁇ ) according to one embodiment of the present invention (A) and a graph quantifying the amount of SIRP ⁇ of recombinant SIRP ⁇ -exosomes ( B).
- mSIRP ⁇ purified recombinant SIRP ⁇ -Myc monomer protein
- Figure 5 is a graph confirming the expression of CD47 on the surface of HT29 (A), Raji (B) and CT26.CL25 cells (C) through a cell binding assay according to an embodiment of the present invention.
- FIG. 6 is a graph analyzing the CD47 binding ability of recombinant SIRP ⁇ according to an embodiment of the present invention (A) and fluorescence intensity of HT29 cells (B) and flow cytometry (B) and Raji and CT26.CL25 cells This is a graph (C) of the fluorescence intensity analysis.
- Figure 7 is a fluorescence micrograph showing the effective binding of recombinant SIRP ⁇ in HT29 cells according to an embodiment of the present invention.
- BMDMs bone marrow-derived macrophages
- A bone marrow-derived macrophages
- BMDMs bone marrow-derived macrophages
- Figure 9 is a graph (A) of the analysis of the increase in phagocytosis by SIRP ⁇ -exosomes according to an embodiment of the present invention and the phagocytosis of Raji and CT26.CL25 cells by bone marrow-derived macrophages (BMDMs)
- One graph (B) is a graph showing phagocytosis index (PI) and a fluorescence micrograph (D) showing the appearance of HT29 cells by bone marrow-derived macrophages (BMDMs).
- 10 is a tumor-bearing immune-deficient mice in accordance with an embodiment of the treatment of SIRP ⁇ -exo to the tumor to observe the anti-tumor effect of tumor growth (A), tumor weight (B) is analyzed It is a graph and a photograph showing an image (C) of an incised tumor.
- Figure 11 is an analysis of the anti-tumor effect of SIRP ⁇ -exosomes according to an embodiment of the present invention (A) and analysis of the biodistribution of Cy5.5-labeled exosomes in HT29 tumor-bearing mice and SIRP ⁇ -exosomes
- the fluorescent image (B) of the tumor 24 hours after injection was a photograph showing the change in the average radiation efficiency of the tumor (C).
- FIG. 13 illustrates the anti-tumor effect of SIRP ⁇ -exosomes using immuno-deficient and immunocompetent mice according to one embodiment of the present invention.
- Figure 14 is an analysis of the anti-tumor effect of SIRP ⁇ -exosomes according to an embodiment of the present invention is a picture (A) showing the tumor incision from the HT29 mouse model and excised from the CT26.CL25 tumor bearing mouse model It is the photograph (B) which shows the state of a tumor.
- FIG. 15 is a graph analyzing the effect of monomeric SIRP ⁇ protein binding to CD47 according to an embodiment of the present invention (A) and a graph analyzing monomeric SIRP ⁇ mediated phagocytosis in HT29 cells ( B) and graph (C) of monomer SIRP ⁇ mediated phagocytosis in Raji and CT26.CL25 cells.
- exosome is a small body consisting of two layers of membranes secreted by human cells that perform specialized functions such as coagulation, signal transduction between cells, and “waste management” of cells and disease. It forms proteins and specific nucleic acids and is known to be released into body fluids.
- SIRP signal-regulatred protein
- SIRP ⁇ a regulatory membrane glycoprotein among SIRP family proteins that is expressed primarily in bone marrow cells and expressed in stem cells or neurons.
- SIRP ⁇ a regulatory membrane glycoprotein among SIRP family proteins that is expressed primarily in bone marrow cells and expressed in stem cells or neurons.
- SIRP ⁇ a regulatory membrane glycoprotein among SIRP family proteins that is expressed primarily in bone marrow cells and expressed in stem cells or neurons.
- SIRP ⁇ and SIRP ⁇ act as inhibitory receptors and interact with CD47 protein, which is a widely expressed transmembrane protein. It is called a 't eat me)' signal. This interaction negatively regulates the effector effect of innate immune cells, such as host cell phagocytosis. This is similar to the autosignal provided by MHC I family molecules via Ig-like or Ly49 receptors.
- SIRP ⁇ cancer cells that overexpress CD47 activate SIRP ⁇ or SIRP ⁇ to inhibit macrophage-mediated destruction. Recent studies have reported that high affinity variants of SIRP ⁇ increase the phagocytosis of cancer cells by masking CD47 on cancer cells (Weiskopf et al., Science 341 (6141): 88-91, 2013).
- receptor tyrosine kinase is an important group of proteins involved in cell proliferation, differentiation, cancerization, morphogenesis, for example epithelial factor receptor, neuronal factor receptor, insulin. Receptors, hematopoietic stem cell proliferation factor receptors and the like. The receptor activates intracellular tyrosine kinase and transmits signals only when it binds to these proliferation and molecular factors and extracellularly.
- rCD-47 binding domain refers to a domain capable of binding CD47 to N-terminal 112 a.a as an N-terminal domain that binds CD47.
- a recombinant exosome wherein the phagocytosis promoting protein is presented on an exosome surface.
- the phagocytosis protein may be a fusion protein linked to the N-terminus of the transmembrane domain of receptor tyrosine kinase, and the receptor tyrosine kinase may be a platelet-derived growth factor receptor (PDGFR) or epidermal EGFR (EGFR).
- PDGFR platelet-derived growth factor receptor
- EGFR epidermal EGFR
- FGFR growth factor receptor
- FGFR fibroblast growth factor receptor
- VEGFR vascular endothelial growth factor receptor
- HGFR hepatocyte growth factor receptor
- TRK insulin receptor
- LTK leukocyte receptor tyrosine kinase
- Exosomes are a kind of natural substance produced by cells, which can minimize immune responses as biocompatible materials, and can display cell membrane proteins expressed on the surface of cells such as receptors in the same direction as cells and present them on the surface. It is a substance that has a great advantage in presenting and expressing proteins on the surface.
- the phagocytosis protein may be SIRP or a fragment comprising the CD47 binding domain of the SIRP, Surfactant protein A, Surfactant protein D or an anti-CD47 antibody, wherein the SIRP is SIRP ⁇ , SIRP ⁇ or these It may be a high affinity variant of.
- the SIRP may be composed of any one amino acid sequence of SEQ ID NO: 1 to 61 and the exosomes may include an anticancer agent therein and the anticancer agent may be an anticancer protein or an anticancer compound.
- the phagocytic protein may be an anti-CD47 antibody or a SiRP protein, which may block the signaling system due to clustering of the CD47 protein.
- the SIRP presented on the surface of the exosomes clustered in the form of lipid rafts on the surface of the exosomes due to the transmembrane domain of the platelet-derived growth factor receptor (PDGFR) used to express the surface of the exosomes.
- PDGFR platelet-derived growth factor receptor
- binding to CD47 proteins clustering in the form of lipid rafts on the surface of cancer cells has a high binding avidity to block the SIRP-CD47 interaction with only a small amount of exosomes, thereby effectively stimulating the anticancer immune response.
- the synergy of SiRP ⁇ loaded on such exosomes was first identified by the inventors.
- the anticancer protein is an asparaginase, a protein toxin, an antibody specific for a cancer antigen or a fragment of the antibody, a tumor suppressor gene, or an angiogenic factor. Can be.
- the protein toxin is Botulinum toxin, Tetanus toxin, Shiga toxin, Diphtheria toxin (DT), Lysine, Pseudomonas exotoxin, PE ), Cytolysin A (ClyA), ⁇ -Gelonin
- the protein for treating infarct tissue may be an angiogenesis factor
- the angiogenesis factor is VEGF (vscular endothelial growth factor), angiopoie Angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), transforming growth factor (TGF- ⁇ ), integrin, vascular endothelial Cadherin, plasmid Plasminogen activator (PA), ephrin, AC-133, platelet-derived growth factor (PDGF), monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor (FGF) or placenta growth factor (PIGF).
- VEGF vscular
- the tumor suppressor genes are genes that suppress tumor development, and are typically VHL (von Hippel Lindau), APC (Adenomatous polyposis coli), CD95 (cluster of differentiation 95), ST5 (Suppression of tumorigenicity 5), YPEL3 (Yippee like 3) ), And Suppression of tumorigenicity 7 (ST7) and Suppression of tumorigenicity 14 (ST14).
- the anticancer compound In the recombinant exosomes, the anticancer compound, methotrexate, pyrimidine analogs, hydroxy urea, purine analogs, alkylating agents, immunogenic cell death Inducers, mitotic inhibitors, angiogenesis inhibitors, intercalating agents, or radionuclides.
- the anticancer compound may be used as follows.
- VEGF-specific antibodies VEGF-specific antibodies, combretastatin A4, fumagillin, herbimycin
- the immunogenic apoptosis inducing agent is an anthracycline-based anticancer agent, anti-EGFR antibody, BK channel agonist, Bortezomib, cardiac glycoside + non-immunogenic apoptosis inducer, It may be a cyclophosphamide-based anticancer agent, GADD34 / PP1 inhibitor + matomycin, LV-tSMAC, Measles virus, or oxaliplatin and the anthracycline-based anticancer agents are daunorubicin, doxorubicin, epirubicin , Idarubicin, pixantrone, sabarubicin, or valrubicin.
- a pharmaceutical composition for anticancer comprising a therapeutically effective amount of the recombinant exosomes and a pharmaceutically acceptable carrier.
- the anticancer pharmaceutical composition may further include one or more anticancer agents.
- the composition comprising a pharmaceutically acceptable carrier may be a variety of oral or parenteral formulations, but is preferably a parenteral formulation.
- diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Mix is prepared.
- Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included.
- Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
- non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
- base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
- the pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.
- the pharmaceutical composition of the present invention may be administered orally or parenterally.
- parenterally it is possible to administer via various routes such as intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, and transdermal absorption. .
- composition of the present invention is administered in a pharmaceutically effective amount.
- the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level of the subject type and severity, age, sex, drug Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical arts.
- the pharmaceutical composition of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg.
- the dosage may be appropriately adjusted according to the age, sex and condition of the patient.
- the pharmaceutical composition of the present invention may be administered as a separate treatment or in combination with other anticancer agents, and may be administered sequentially or simultaneously with other conventional anticancer agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.
- SIRP ⁇ -exosomes plasmid DNA expressing SIRP ⁇ variants that block phagocytosis by blocking CD47 at the surface of tumor cells.
- the SIRP ⁇ variant gene was obtained through a gene synthesis service (Cosmo Genetech Co.) and the DNA sequence (SEQ ID NO: 62) encoding the SIRP ⁇ variant (SEQ ID NO: 1) of the platelet derived growth factor receptor (PDGFR) in the pDisplay vector. Inserted into the frame between the N-terminal signal peptide and the membrane anchor (FIG. 1).
- HEK293T cells (6 x 10 6 ) is a high glucose culture medium (Dulbecco's modified Eagle's medium, DMEM, 4,500 mg / L glucose) added with 10% FBS, 1% antibiotics for exosome separation according to an embodiment of the present invention Insulin-transferrin-selenium (Gibco) when maintained at 37 ° C., 5% CO 2 and exhibited 80-90% cell saturation in a 15-cm dish. Was exchanged with added serum-free DMEM culture. After 2 hours the cells were transfected with plasma DNA (20 ⁇ g) encoding SIRP ⁇ variants using a transfection reagent (lipofectamine 3000, Invitrogen) according to the manufacturer's instructions. Then, 48 hours after transfection to separate the exosomes, the cell culture supernatants were obtained by differential centrifugation, and the detailed method is as follows:
- centrifugation was performed sequentially at 300 g for 10 minutes, 2000 g at 10 minutes, and 10000 g at 30 minutes, and the culture solution was filtered with a 0.22 ⁇ m filter. Subsequently, ultra-centrifugation was performed for 2 hours at 36,900 rpm using a 70 Ti rotor (Beckman Instruments). The obtained recombinant exosomes (SIRP ⁇ -exosomes) were then resuspended in PBS containing a protease inhibitor (Roche), and the protein concentration of the isolated exosomes was measured using a BCA protein assay kit (Bio-Rad). It was.
- SIRP ⁇ -exosomes prepared according to one embodiment of the present invention are as follows: Western blot (WB), flow cytometry, dynamic light scattering (DLS) and transmission electron microscope It was confirmed using (TEM).
- anti-Alix antibody (1: 500, Santa Crus, sc-99010), anti-Tsg101 antibody (1: 500, Santa Crus, sc-22774) and anti-CD63 antibody (1: 500, Santa Crus, sc-15363) was used as an exosome marker.
- HRP-bound secondary antibody (1: 4000, Sigma-Aldrich) was then added to the membrane and visualized by chemiluminescence.
- the expression of SIRP ⁇ on the surface of the exosomes was analyzed using flow cytometry.
- the purified SIRP ⁇ -exosomes had a SIRP ⁇ variant on the surface membrane and contained an exosome marker protein (FIG. 2).
- transmission electron microscopy (TEM) images and dynamic light scattering (DLS) analysis showed that the average size of the recombinant exosomes was round in shape with an average size of 100 nm (FIG. 3).
- the transformed bacterial cells were then incubated in LB medium containing kanamycin at 37 ° C. until OD 600 became 0.5 and 0.5 mM isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) (Bioneer, Korea) was added. After incubation at 20 ° C.
- IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
- the cells were obtained by centrifugation at 6000 g for 10 minutes and after homogenization using an ultrasonic mill, the pellets were dissolved in lysis buffer (1 M Tris-HCl pH 8.0, 150 mM NaCl, 1 mM PMSF) and mSIRP ⁇ were purified by Ni-NTA affinity chromatography and anti-Myc antibodies (1: 5000, Abcam, ab9106) and HRP-linked secondary antibodies (1: 4000, Sigma Aldrich) It was analyzed by Western blot used.
- SIRP ⁇ -exosome 2 ⁇ 10 9 100 ⁇ g of total protein and SIRP ⁇ -exosome 2 ⁇ 10 9 were obtained from HEK293T cells and exosomes using a standard curve from Western blot images of purified recombinant SIRP ⁇ -Myc monomeric protein (mSIRP ⁇ ). The amount of SIRP ⁇ was quantified and the amount of SIRP ⁇ -exosomes measured was found to be less than 5 ng per 1 ⁇ g exosomes (FIG. 4). Information on the primers used for the PCR amplification is shown in Table 1 below.
- Cell binding assay is a HT29 human colon adenocarcinoma (ATCC), Raji human B cell lymphoma (ATCC) and CT26.CL25 mouse colon cancer (ATCC) cells added RPMI with 10% FBS and 1% antibiotic Cultured in -1640 culture medium and maintained at 37 °C and 5% CO 2 conditions.
- HT29 and Raji cells were then cultured with the addition of anti-human CD47 antibody [B6H12.2] (Abcam, ab3283) and CT26.CL25 cells were treated with anti-mouse CD47 antibody (Santa Crus) to detect CD47 expression on the surface of cancer cells. , sc-12731) was added and cultured.
- HT29, Raji and CT26.CL25 cells (1 ⁇ 10 6 ) were incubated at 4 ° C. for 30 minutes with the addition of PBS, exosomes or mSIRP ⁇ .
- the cells were then incubated with the addition of anti-Myc antibodies (1: 400, Abcam, ab9106) and detected by the addition of Alexa fluor 488-bound secondary antibody (1: 800, Jackson ImmunoResearch).
- the cells were then measured using Accuri TM C6 flow cytometer (BD Biosciences) and analyzed using FlowJo_V10 software (FlowJo). Binding specificity of SIRP ⁇ -exosomes to CD47 was analyzed by block experiments by pre-culture of cells to which anti-human CD47 antibodies (1: 100, Abcam, ab3283) were added.
- HT29 cells (2 ⁇ 10 5 ) were seeded in a glass bottom 4 well chamber and anti-Myc antibody (1: 400, Abcam, ab9106) and Alexa fluor 488-bound as described above.
- the secondary antibody (1: 800, Jackson ImmunoResearch) was added and cultured. After removal of residual non-specific signals, the cells were fixed for 7 minutes using 4% paraformaldehyde after nuclear staining at 25 ° C. for 10 minutes using Hoechst 33258.
- the cell binding capacity of exosomes was observed by fluorescence microscope (Nikon Eclipse Ti, Nikon) and analyzed using LAS AF Lite software (Leica).
- SIRP ⁇ -exosomes showed a higher affinity for CD47 in binding of HT29 tumor cell surface compared to exosomes of controls whose effects were confirmed SIRP ⁇ -exosome concentration dependent.
- SIRP ⁇ -exosome binding was reduced by preculture with anti-human CD47 antibodies, indicating that SIRP ⁇ -exosomes specifically bind to CD47 on the surface of tumor cells and that human Raji Burkitt's lymphoma (Raji) and mouse CT26 Similar results of CD47 binding of SIRP ⁇ -exosomes were observed in CL25 colon cancer cells. The results indicate that expression of membrane-associated SIRP ⁇ variants in exosomes has the ability to bind CD47 in cancer cells (FIGS. 6 and 7).
- phagocytosis analysis was performed to observe whether CD47 with antagonism of tumor cells increases macrophage mediated phagocytosis of tumor cells.
- BMDMs bone marrow-derived macrophages
- BALB / c mice were sacrificed and bone marrow cells were isolated from leg bones.
- the isolated bone marrow cells were maintained in RPMI medium added with 10% FBS and 1% antibiotic and differentiated into macrophage colony-stimulating factor (M-CSF) for 7 days.
- Phagocytosis was analyzed by co-culture of BMDM and cancer cells in serum-free RPMI medium for 4 hours.
- differentiated macrophages 2.5 ⁇ 10 5
- differentiated macrophages 2.5 ⁇ 10 5
- BMDM BMDM
- the rate of phagocytosis was assessed as a percentage of double positive signals using Accuri TM C6 flow cytometer (BD biosciences) and FlowJo_V10 software (FlowJo).
- phagocytosis index phagocytosis index
- CellTracker TM the Green BMDM dyed (Thermo fisher scientific) and inoculated on a 35 mm glass-bottom dishes at a density of 2.5 x 10 5
- a mixture of HT29 cells (1 ⁇ 10 6 ) was stained with pHrodo Deep red (Thermo fisher scientific) and exosomes were treated with macrophages.
- the engulfment of HT29 cells by macrophages was analyzed by fluorescence microscopy (Nikon Eclipse Ti, Nikon) as a red positive signal associated with macrophage formation in macrophages.
- phagocytosis of macrophages with double positive signals was shown to increase in a concentration dependent manner in the SIRP ⁇ -exosome treated group compared to the control (PBS-treated or control-exosome-treated).
- FIG. 8 and similarly in Raji and CT26.CL25 colon cancer cells treated with SIRP ⁇ -exosomes.
- the treatment of SIRP ⁇ -exosomes increased the phagocytosis of tumor cells and accordingly PI was significantly increased in the SIRP ⁇ -exosomes treatment group (FIG. 9).
- BMDMs bone marrow-derived macrophages
- the exosomes were labeled with Cy5.5-NHS and the Cy5.5-NHS dye (1 ⁇ g) was treated with 100 ⁇ g of exosomes. Then, the cells were incubated at room temperature for 2 hours, and centrifuged for 45 minutes using an airfuse centrifuge (Beckman coulter). After performing two washes to remove non-bound dye, the labeled exosome pellets were resuspended in PBS and the fluorescence intensity was measured using a fluorescence microplate reader (Infinite M200 Pro, TECAN). And adjusted.
- HT29 tumor bearing BALB / c nude mice were intravenously administered Cy5.5-labeled exosomes (500 ⁇ g), free dye and PBS and the fluorescence intensities of all samples were determined using data obtained with a fluorescent microplate reader. Based on the same value.
- In vivo systemic imaging of mice was performed at various time points (5 minutes, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours) using IVIS spectra (Caliper Life Sciences).
- IVIS spectra Caliper Life Sciences
- total photons per centimeter square per steadian were calculated at the ROI using Analysis Workstation software (Advanced Research Technologies Inc.) and 24 hours post-injection.
- mice were sacrificed and tumors and major organs, including liver, lung, spleen, kidney and heart, were excised and analyzed in the same manner as above.
- immuno-deficient BALB / c nude mice and immunocompetent BALB / c mice were implanted with tumors at 7 weeks of age for in vivo experiments. It was managed in a facility of the Korea Institute of Science and Technology (KIST). Subsequently, HT29 cells (1 ⁇ 10 7 ) were inoculated subcutaneously into the left leg of BALB / c nude mice and tumors were grown for a week, then control-exosomes, SIRP ⁇ -exosomes and control PBS five times every three days. Injected. Thereafter, 100 ⁇ g of exosomes were injected into the tumors of mice for analysis of local anti-tumor effects.
- KIST Korea Institute of Science and Technology
- exosomes 200 ⁇ g
- PBS PBS
- SIRP ⁇ -exo in HT29 tumor bearing BALB / c immune-deficient mice The use of SIRP slightly reduced tumor growth but was not significant, whereas SIRP ⁇ -exosomes in CT26.CL25 tumor bearing immune-sensitive BALB / c mice showed very good anti-tumor effects. .
- administration of recombinant SiRP ⁇ protein (mSIRP ⁇ ) did not properly induce tumor phagocytosis despite being able to efficiently bind to CD47 of cancer cells (FIG. 15).
- recombinant exosomes prepared according to one embodiment of the present invention increase the phagocytosis of macrophages and dendritic cells by blocking CD47-SIRP ⁇ interaction, thereby in vitro conditions as well as animal model experiments. It has been shown to have a significant anti-tumor effect and can be used as a novel anticancer agent for cancer treatment.
Abstract
본 발명은 탐식작용 촉진 단백질이 엑소좀(exosome) 표면에 제시된, 재조합 엑소좀 및 그의 용도를 제공한다.
Description
본 발명은 신규 항암제에 관한 것으로서 보다 구체적으로는 암세포의 식균작용을 증가시키는 신규 엑소좀 계열 항암제에 관한 것이다.
종양(tumor)은 생존하고 증식하기 위해 면역세포의 표면에서 발견되는 수용체와 상호 작용하는 활성화 및 억제 리간드를 발현함으로써 면역 감시(immune surveillance)를 피한다. 종양과 면역 세포 사이의 이러한 상호 작용은 면역계(immune system)에 의해 종양이 사멸되는 것을 방지한다(Pardoll DM. Nat Rev Cancer. 12:252-64. 2012). 종양 면역 회피의 메커니즘 중 하나는 종양이 선천성 면역 감시를 피할 수 있게 하는 CD47의 과발현으로 CD47이 대식세포와 같은 선천성 면역 세포의 신호 조절 단백질α(SIRPα)과 결합하면 수지상 세포는 "나를 먹지 마시오" 신호를 활성화시켜 종양이 식균작용(phagocytosis)으로부터 피할 수 있게 유도하는데 다양한 악성 세포에서 풍부한 CD47가 발현되어 암 환자의 생존율이 낮아지므로 CD47-SIRPα 상호작용의 치료 표적화를 위한 강력한 근거를 제시한다. SIRPα의 N 말단이 CD47의 N 말단과 상호 작용하는 면역 글로블린 수퍼 패밀리 V-유사 도메인을 포함하기 때문에, 몇몇 경쟁적 길항제가 이들의 상호 작용을 차단하기 위해 개발되었는데 인간 CD47 차단 단일클론 항체(CD47 mAb)는 다양한 전 임상 종양 모델에서 효능을 입증하였고 면역원성 종양의 T 세포 매개의 파괴(destruction)를 유발하였다(Tseng D, et al. Proc Natl Acad Sci USA. 110:11103-8. 2013). 또한, CD47-SIRPα 상호작용은 재조합 SIRPα 단백질이나 SIRPα- FC 융합 단백질과 함께 길항효과를 나타낼 수 있는데 천연 CD47과 SIRPα 사이의 약한 상호작용이 치료 길항제로서 야생형 SIRPα 단백질의 유용성을 제한할 수 있다는 것을 감안할 때, SIRPα의 고친화성(high-affinity) 변이체가 생성되어 암세포에서 CD47을 길항하는 것으로 나타났다. 그러나 SIRPα 변이체만으로 종양 특이적 항체에 대한 보조제(adjuvants)로 작용하였으나 기대이상으로 대식세포의 식균작용과 종양 성장억제를 자극하진 않았다(Sockolosky JT et al., Proc Natl Acad Sci USA. 113:E2646-E54. 2016). 이와 관련하여 미국 공개특허 제2015-0376288호는 숙주 식세포(host phagocytic cell)상의 감염된 세포에서 SIRPα로 CD47의 결합을 감소시키는 약제를 투여하여 병원체 감염을 치료하는 치료방법을 개시하고 있다.
그러나, 상기 선행기술의 경우, 감염성 질환 치료를 위해 항-CD47 제제를 투여하여 SIRPα의 결합을 감소시키므로 항암치료를 위한 치료제로는 부적합하다.
본 발명은 상기 문제점을 포함한 다양한 문제점을 해결하기 위한 것으로서, 대식세포와 수지상세포의 식균작용을 증가시켜 암세포를 제거하는 항암치료에 효과적인 신규 엑소좀 계열 항암제를 제공하는 것을 목적으로 한다. 그러나 이러한 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 한정되는 것은 아니다.
본 발명의 일 관점에 따르면, 탐식작용 촉진 단백질이 엑소좀(exosome) 표면에 제시된, 재조합 엑소좀이 제공된다.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 재조합 엑소좀 및 약학적으로 허용가능한 담체를 포함하는, 항암용 약학적 조성물이 제공된다.
상기한 바와 같이 이루어진 본 발명의 일 실시예에 따르면, 대식세포와 수지상세포의 암세포 탐식능력을 향상시켜 항암 면역 효과가 증가된 효과적인 엑소좀 계열 항암제 생산효과를 구현할 수 있다. 특히 본 발명의 일 실시예에 따른 SiRP 가 표면에 제시된 재조합 엑소좀은 상기 SiRP가 엑소좀 표면에 지질 뗏목(lipid raft) 형태로 클러스터링됨으로써 마찬가지로 암세포 표면에 지질 뗏목 형태로 클러스터링하고 있는 CD47 단백질에 대하여 높은 결합능(high binding avidity)를 가지고 결합하므로 적은 양의 엑소좀만으로도 SiRP-CD47 상호작용을 차단하여 항암면역반응을 보다 효율적으로 자극할 수 있게 한다. 물론 이러한 효과에 의해 본 발명의 범위가 한정되는 것은 아니다.
도 1은 본 발명의 일 실시예에 따라 SIRPα 변이체(SIRPα-엑소좀)를 발현하는 플라스마 DNA의 구조를 개략적으로 나타내는 개요도이다.
도 2는 본 발명의 일 실시예에 따라 제조된 재조합 엑소좀(SIRPα-엑소좀)의 발현을 확인한 웨스턴블랏 겔사진(A)이고 유세포분석(flow cytometry)을 통해 엑소좀 표면의 SIRPα의 발현을 분석한 그래프이며(B) SIRPα-엑소좀의 이미지를 보여주는 전자 현미경사진이다(C).
도 3은 본 발명의 일 실시예에 따라 제조한 SIRPα-엑소좀의 크기를 동적 광 산란(DLS)방법으로 분석한 그래프이고(A) SIRPα-엑소좀의 투과전자현미경 사진이며(B) 유세포분석 그래프이고(C) 동적 광 산란(DLS) 분석 그래프이다(D).
도 4는 본 발명의 일 실시예에 따라 정제된 재조합 SIRPα-Myc 단량체 단백질(mSIRPα)의 발현을 분석한 웨스턴 블랏 겔사진이고(A) 재조합 SIRPα-엑소좀의 SIRPα의 양을 정량한 그래프이다(B).
도 5는 본 발명의 일 실시예에 따라 세포 결합 분석을 통하여 HT29(A), Raji(B) 및 CT26.CL25 세포(C) 표면에서 CD47의 발현을 확인한 그래프이다.
도 6은 본 발명의 일 실시예에 따라 재조합 SIRPα의 CD47 결합 능력을 분석한 것으로 HT29 세포의 형광강도를 분석한 그래프(A)이고 유세포분석을 수행한 그래프(B)이며 Raji 및 CT26.CL25 세포의 형광강도를 분석한 그래프(C)이다.
도 7은 본 발명의 일 실시예에 따라 HT29 세포에서 재조합 SIRPα의 효과적인 결합을 나타내는 형광현미경 사진이다.
도 8은 본 발명의 일 실시예에 따라 골수 유래 대식세포(BMDMs)에 의한 식균작용을 분석한 것으로 골수 유래 대식세포의 분화를 분석한 그래프(A)이고 골수 유래 대식세포(BMDMs)에 의한 HT29 세포의 식균작용을 분석한 FACS 데이터이다(B).
도 9는 본 발명의 일 실시예에 따라 SIRPα-엑소좀에 의해 식균작용이 증가된 것을 분석한 그래프(A)이고 골수 유래 대식세포(BMDMs)에 의해 Raji 및 CT26.CL25 세포의 식균작용을 분석한 그래프(B)이며 식균작용 지수(PI)를 나타내고 있는 그래프(C)이고 골수 유래 대식세포(BMDMs)에 의해 HT29 세포의 모습을 나타내고 있는 형광 현미경 사진(D)이다.
도 10은 본 발명의 일 실시예에 따라 종양 보유 면역-결핍 마우스에서 SIRPα-엑소좀을 종양에 처리하여 항-종양 효과를 관찰한 것으로 종양의 성장(A), 종양의 무게(B)를 분석한 그래프이고 절개된 종양의 이미지(C)를 나타내는 사진이다.
도 11은 본 발명의 일 실시예에 따라 SIRPα-엑소좀의 항종양 효과를 분석한 것으로 HT29 종양 보유 마우스에서 Cy5.5 표지된 엑소좀의 생체분포를 분석한 사진(A)이고 SIRPα-엑소좀을 주입 후 24시간 경과시점에서 종양의 형광 이미지(B)이며 종양의 평균 방사 효율의 변화를 나타내는 사진이다(C).
도 12는 본 발명의 일 실시예에 따라 마우스에 Cy5.5 표지된 엑소좀을 주사한 후 장기별로 분석한 IVIS 이미지다.
도 13은 본 발명의 일 실시예에 따라 면역-결핍(immuno-deficient) 및 면역 감응(immunocompetent) 마우스를 이용하여 SIRPα-엑소좀의 항-종양효과를 관찰한 것으로 HT29 종양 보유 BALB/c 면역-결핍 마우스에 SIRPα-엑소좀을 주입하여 종양 성장 억제를 분석한 그래프(A) 및 상기 면역-결핍 마우스 모델로부터 절개된 종양의 중량을 분석한 그래프(B), CT26.CL25 종양 보유 면역 감응 BALB/c 마우스의 종양 성장에 있어 SIRPα-엑소좀의 주입에 따른 항-종양효과를 분석한 그래프(C) 및 상기 CT26.CL25 종양 보유 면역 감응 마우스 모델로부터 절개된 종양의 중량을 분석한 그래프(D)이다.
도 14는 본 발명의 일 실시예에 따라 SIRPα-엑소좀의 항종양 효과를 분석한 것으로 HT29 마우스 모델로부터 절개된 종양을 모습을 나타내고 있는 사진(A)이고 CT26.CL25 종양 보유 마우스 모델로부터 절개된 종양의 모습을 나타내고 있는 사진(B)이다.
도 15는 본 발명의 일 실시예에 따라 단량체 SIRPα 단백질의 효과를 분석한 것으로 단량체 SIRPα 단백질이 CD47에 결합하는 정도를 분석한 그래프(A)이고 HT29 세포에서 단량체 SIRPα 매개 식균작용을 분석한 그래프(B)이며 Raji 및 CT26.CL25 세포에서 단량체 SIRPα 매개 식균작용을 분석한 그래프(C)이다.
용어의 정의
본 문서에서 사용되는 용어 "엑소좀(exosome)"은 인간 세포에서 분비되는 두 층의 막으로 이루어진 작은 소체로 이들은 응고, 세포 간의 신호 전달 및 세포의 "폐기물 관리"와 같은 전문 기능을 수행하고 질병 특이적 핵산과 단백질을 형성하며 체액으로 방출되는 것으로 알려져 있다.
본 문서에서 사용되는 용어 "SIRP(signal-regulatred protein)"는 골수세포에서 주로 발현되고 그리고 줄기세포 또는 신경세포에서 발현되는 SIRP족 단백질 중 조절성 막 당단백질이다. 상기 SIRP에는 SIRPα, SIRPβ, SIRPγ 및 SIRPδ의 네가지가 알려져 있는데 이들중 SIRPα 및 SIRPγ는 억제성 수용체로 작용하며, 광범위하게 발현되는 막통과 단백질인 CD47 단백질과 상호작용하는데 이는 이른 바 "날 먹지마(don't eat me)" 신호로 불린다. 이러한 상호작용은 숙주세포 탐식작용과 같은 선천적 면역세포의 효과기 작용을 음성적으로 조절한다. 이는 Ig-유사 또는 Ly49 수용체를 경유한 MHC I 계열 분자에 의해 제공되는 자가신호와 유사하다. CD47를 과발현하는 암세포는 SIRPα 또는 SIRPγ를 활성화시켜 대식세포-매개 파괴를 억제한다. 최근 연구에 의하면 SIRPα의 고친화성 변이체가 암세포 상에서 CD47을 마스킹하여 암세포에 대한 탐식작용을 증가시킨다는 보고가 있다(Weiskopf et al., Science 341(6141): 88-91, 2013).
본 문서에서 사용되는 용어 "수용체 티로신 카이네이즈(receptor tyrosine kinase, RTK)"는 세포의 증식, 분화, 암화, 형태형성 등에 관여하는 중요한 단백질군으로 예를 들면 상피증식인자수용체, 신경생장인자수용체, 인슐린수용체, 조혈간세포증식인자수용체 등 다수가 있다. 수용체는 이들 증식 및 분자인자와 세포 외에서 결합할 때에만 세포내 티로신인산화효소를 활성화하여 신호를 전달한다.
본 문서에서 사용되는 용어 "CD-47 결합 도메인(rCD-47 binding domain)"은 CD47과 결합하는 N-말단 도메인으로 N-말단의 112 a.a까지로 CD47과 결합할 수 있는 도메인을 의미한다.
발명의 상세한 설명
본 발명의 일 관점에 따르면, 탐식작용 촉진 단백질이 엑소좀(exosome) 표면에 제시된, 재조합 엑소좀이 제공된다.
상기 재조합 엑소좀에 있어서, 상기 탐식작용 촉진 단백질은 수용체 티로신 카이네이즈의 막통과 도메인의 N-말단에 연결된 융합단백질일 수 있고, 상기 수용체 티로신 카이네이즈는 PDGFR(platelent-derived growth factor receptor), EGFR(epidermal growth factor receptor), FGFR(fibroblast growth factor receptor), VEGFR(vascular endothelial growth factor receptor), HGFR(hepatocyte growth factor receptor), Trk(tropomyosin receptor kinase), IR(insulin receptor), LTK(Leukocyte receptor tyrosine kinase), 안지오포이에틴 수용체(angiopoietin receptor), ROR(receptor tyrosine kinase-like orphan receptors), DDR(discoidin domain receptor), RETR(rearranged during transfection receptor), PTK(tyrosine-protein kinase-like), RYK(related to receptor tyrosine kinase), 또는 MuSK(muscle-specific kinase)일 수 있다.
엑소좀은 세포에 의해 생성되는 일종의 천연물질로서 생체친화적인 물질로서 면역반응을 최소화할 수 있으며, 수용체와 같은 세포 표면에 발현되는 세포막 단백질을 세포와 동일한 방향으로 정향시켜 표면에 제시할 수 있기 때문에, 세포 표면제시 단백질을 표면에 제시하여 표출시키는데 큰 장점을 가지고 있는 물질이다.
상기 재조합 엑소좀에 있어서, 상기 탐식작용 촉진 단백질은 SIRP 또는 상기 SIRP의 CD47 결합도메인을 포함하는 단편, Surfactant protein A, Surfactant protein D 또는 항-CD47 항체일 수 있고, 상기 SIRP는 SIRPα, SIRPγ 또는 이들의 고친화성 변이체일 수 있다.
상기 재조합 엑소좀에 있어서, 상기 SIRP는 서열번호 1 내지 61 중 어느 하나의 아미노산 서열로 구성될 수 있으며 상기 엑소좀은 내부에 항암제를 포함할 수 있고 상기 항암제는 항암 단백질 또는 항암 화합물일 수 있다. 특히 상기 탐식작용 촉진 단백질은 CD47 단백질의 클러스터링(clustering)에 따른 신호전달체계를 차단할 수 있는 것으로서 항-CD47 항체나 SiRP 단백질인 것이 바람직하다. 특히 엑소좀 표면에 제시된 SIRP는 SIRP의 엑소좀 표면제시에 사용된 PDGFR(platelet-derived growth factor receptor)의 막통과 도메인(transmembrane domain)에 기인하여 엑소좀 표면에 지질 뗏목(lipid raft) 형태로 클러스터링됨으로써 마찬가지로 암세포 표면에 지질 뗏목 형태로 클러스터링하고 있는 CD47 단백질에 대하여 높은 결합능(high binding avidity)를 가지고 결합함으로써 적은 양의 엑소좀만으로도 SIRP-CD47 상호작용을 차단함으로써 항암면역반응을 보다 효율적으로 자극할 수 있게 한다. 상기와 같은 엑소좀에 적재된 SiRPα의 상승작용은 본 발명자들에 의해 처음으로 규명된 것이다.
상기 재조합 엑소좀에 있어서, 상기 항암 단백질은 아스파라기네이즈(asparaginase), 단백질 독소, 암항원에 특이적인 항체 또는 상기 항체의 단편, 종양억제 유전자(tumor suppressor gene) 또는 항혈관생성인자(antiangiogenic factor)일 수 있다. 이 때, 상기 단백질 독소는 보툴리눔 독소(Botulinum toxin), 테타누스 독소(Tetanus toxin), 시가 독소(Shiga toxin), 디프테리아 독소(Diphtheria toxin, DT), 리신(ricin), 슈도모나스 외독소(Pseudomonas exotoxin, PE), 사이토라이신 A(cytolysin A, ClyA), γ-Gelonin일 수 있고, 상기 경색조직 조직 치료용 단백질은 혈관생성인자일 수 있으며, 상기 혈관생성인자는 VEGF(vscular endothelial growth factor), 안지오포이에틴1(angiopoietin 1, Ang1), 안지오포이에틴2(Ang2), 형질전환 성장인자-(transforming growth factor-, TGF-β), 인테그린(integrin), 혈관 내피 캐드헤드린(VE-cadherin), 플라스미노겐 활성제(plasminogen activator, PA), 에프린(ephrin), AC-133, 혈소판 유래 성장인자(PDGF), 단핵구 주화성 단백질-1(MCP-1, monocyte chemotactic protein-1), 섬유아세포 성장인자(FGF) 또는 태반성장인자(placenta growth factor, PIGF)일 수 있다. 상기 종양 억제 유전자는 종양의 발생을 억제하는 유전자로서, 대표적으로 VHL(von HippelLindau), APC(Adenomatous polyposis coli), CD95(cluster of differentiation 95), ST5(Suppression of tumorigenicity 5), YPEL3(Yippee like 3), ST7(Suppression of tumorigenicity 7) 및 ST14(Suppression of tumorigenicity 14)일 수 있다.
상기 재조합 엑소좀에 있어서, 상기 항암 화합물은, 메토트렉세이트(methotrexate), 피리미딘 유사체(pyrimidine analogs), 히드록시우레아(hydroxy urea), 퓨린 유사체(purine analogs), 알킬화제(alkylating agents), 면역원성 세포사멸 유도제, 유사분열 억제제(mitotic inhibitors), 신생혈관억제제, 삽입성 물질(ntercalating agents) 또는 방사성 핵종(radionuclides)일 수 있다.
상기 항암 화합물은 하기의 것들이 사용될 수 있다.
(i) 메토트렉세이트(methotrexate);
(ii) 피리미딘 유사체(pyrimidine analogs)
5-플루오로우라실(5-fluorouracil), 젬시타빈(gemcitabine) 및 아라비노실시토신(arabinosylcytosine);
(iii) 히드록시우레아(hydroxy urea);
(iv) 퓨린 유사체(purine analogs)
머캅토퓨린(mercaptopurine) 및 티오구아닌(thioguanine);
(v) 알킬화제(alkylating agents)
니트로겐 머스타드(nitrogen mustad) 및 사이클로스포라미드(cyclosporamide);
(vi) 항생제(antibiotics)
안트라사이클린(anthracycline), 독소루비신(doxorubicin), 다우노루비신(daunorubicin), 이다루비신
(idarubicin) 및 악티노마이신 D(actinomycin D);
(vii) 유사분열 억제제(mitotic inhibitors)
빈크리스틴(vincristine) 및 탁솔(taxol);
(viii) 항혈관생성제
VEGF에 특이적인 항체, 콤브레타스타틴 A4(combretastatin A4), 푸마길린(Fumagillin), 허비마이신
A(herbimycin A), 2-메톡시에스트라디올(2-methoxyestradiol), OGT 2115, TNP 470, 트라닐라스트(tranilast),
XRP44X, 탈리도마이드(thalidomide), 엔도스타틴(endostatin), 살모신(salmosin), 안지오스타틴(angiostatin)
또는 플라스미노겐(plasminogen) 또는 아포리포단백질(apolipoprotein)의 크링글 도메인(kringle domain);
(ix) 삽입성 물질(intercalating agents)
카르보틀라틴(carboplatin) 및 시스플라틴(cisplatin); 및
(x) 방사성 핵종(radionuclides)
18F, 90Y, 188Re, 32P, 89Sr, 165Dy, 186Re, 198Au, 153Sm, 131I, 169Er, 125I, 99Tc 및 166Ho, 등.
상기 재조합 엑소좀에 있어서, 상기 면역원성 세포사멸 유도제는 안트라사이클린 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside) + 비-면역원성 세포사멸 유도제, 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제 + 마토마이신, LV-tSMAC, Measles 바이러스, 또는 옥살리플라틴일 수 있고 상기 안트라사이클린 계열 항암제는 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin), 또는 발루비신(valrubicin)일 수 있다.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 상기 재조합 엑소좀 및 약학적으로 허용가능한 담체를 포함하는, 항암용 약학적 조성물이 제공된다.
상기 항암용 약학적 조성물에 있어서, 하나 이상의 항암제를 추가로 포함할 수 있다. 약학적으로 허용 가능한 담체를 포함하는 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있으나, 비경구를 위한 제형인 것이 바람직하다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있다.
본 발명의 약학적 조성물은 경구 또는 비경구로 투여될 수 있는데, 비경구로 투여되는 경우, 정맥내 주사, 비강내 흡입, 근육내 투여, 복강내 투여, 경피흡수 등 다양한 경로를 통해 투여하는 것이 가능하다.
상기 본 발명의 조성물은 약학적으로 유효한 양으로 투여된다.
본 문서에서 사용되는 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 0.1 mg/kg 내지 1 g/kg의 용량으로 투여될 수 있으며, 더 바람직하게는 1 mg/kg 내지 500 mg/kg의 투여량으로 투여된다. 한편, 상기 투여량은 환자의 나이, 성별 및 상태에 따라 적절히 조절될 수 있다.
본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 항암제와 병용하여 투여될 수 있고, 종래의 다른 항암제와 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.
이하, 실시예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예 및 실험예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예 및 실험예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다.
실시예
1: 플라스마 DNA 구축
본 발명의 일 실시예에 따라 종양 세포 표면에서 CD47을 차단하여 식균작용을 증가시키는 SIRPα 변이체(SIRPα-엑소좀s)를 발현하는 플라스미드 DNA를 구축하였다. 구체적으로 SIRPα 변이체 유전자는 유전자 합성 서비스(Cosmo Genetech Co.)를 통하여 수득하였고 상기 SIRPα 변이체(서열번호 1)를 인코딩하는 DNA 서열(서열번호 62)을 pDisplay 벡터에서 혈소판 유래 성장 인자 수용체(PDGFR)의 N- 말단의 신호 펩타이드 및 멤브레인 앵커 사이의 프레임에 삽입하였다(도 1).
실시예
2:
엑소좀
(
exosome
) 분리
본 발명의 일 실시예에 따라 엑소좀 분리를 위하여 HEK293T 세포(6 x 106)는 10% FBS, 1% 항생제가 첨가된 고포도당 배양배지(Dulbecco's modified Eagle's medium, DMEM, 4,500 mg/L glucose)에서 배양하였고, 37℃, 5 % CO2 조건에서 유지되었으며 15-cm 배양접시에서 80~90%의 세포포화도(confluency)을 나타낼 때, 인슐린-트랜스페린-셀레닌(insulin-transferrin-selenium, Gibco)을 첨가한 무-혈청 DMEM 배양액으로 교환해주었다. 2시간 경과 후 상기 세포를 제조사의 지침에 따라 형질감염시약(lipofectamine 3000, Invitrogen)을 사용하여 SIRPα 변이체를 인코딩하는 플라스마 DNA(20 μg)로 형질감염(transfection)시켰다. 그 후, 엑소좀을 분리하기 위하여 형질주입 48시간 후 세포배양 상층액을 분별원심분리(differential centrifugation) 방법으로 수득하였고 상세한 방법은 하기와 같다:
먼저, 엑소좀을 포함하는 배양액에서 세포 찌꺼기와 다른 세포 성분을 제거하기 위하여 300 g으로 10분, 2000 g으로 10분 및 10000 g으로 30분간 순차적으로 원심분리를 하였고 상기 배양액을 0.22 μm 필터로 여과 후 70 Ti rotor(Beckman Instruments)를 이용하여 36,900 rpm에서 2시간 동안 초원심분리(ultra-centrifugation)를 수행하였다. 이후 수득한 재조합 엑소좀(SIRPα-엑소좀)은 단백질 분해효소 억제제(Roche)를 포함하는 PBS에 재현탁하였고 BCA 단백질 분석키트(Bio-Rad)를 이용하여 상기 분리된 엑소좀의 단백질 농도를 측정하였다.
실시예
3: 재조합
엑소좀의
특성 분석
본 발명의 일 실시예에 따라 제조된 재조합 엑소좀(SIRPα-엑소좀)의 품질과 특징은 하기와 같이 웨스턴블랏(WB), 유세포분석(flow cytometry), 동적 광 산란(DLS) 및 투과전자현미경(TEM)을 이용하여 확인하였다.
우선, 웨스턴 블랏 분석을 위해 상기 초-원심분리된 재조합 엑소좀 펠렛은 프로테이즈 저해제 칵테일(Protease Inhibitor Cocktail, Calbiochem)을 포함하는 RIPA 완충액(Cell Signaling Technology)를 이용하여 용해하였고 엑소좀 단백질 동량(10 μg)을 SDS-PAGE로 분석하였으며, 나이트로셀룰로스 막(membranes)으로 전사시켰다. 그 후, SIRPα 발현을 탐지하기 위하여 블랏에 항-Myc 항체(1:3000, Abcam, ab9106) 및 항-HA 항체(1:500, Santa Crus, sc-805)를 첨가한 후 4℃의 조건으로 하룻밤 동안 방치하였고 항-Alix 항체(1:500, Santa Crus, sc-99010), 항-Tsg101 항체(1:500, Santa Crus, sc-22774) 및 항-CD63 항체(1:500, Santa Crus, sc-15363)를 엑소좀 마커(exosome marker)로 사용하였다. 그 후 상기 막에 HRP-결합된 2차 항체(1:4000, Sigma-Aldrich)를 첨가하였고 화학발광(chemiluminescence)에 의해 시각화되었다. 또한, 엑소좀 표면의 SIRPα의 발현을 유세포분석(flow cytometry)을 이용하여 분석하였다. 먼저, 엑소좀 10 μg을 상온에서 2시간동안 최종 볼륨 1 ml PBS에서 4 μm 알데하이드/설페이트 라텍스 비드(Invitrogen)로 삽입하였고 그 후, 0.5 % BSA를 첨가한 PBS로 2번 세척하였으며 Alexa fluor 488-표지된 2차 항체(1:800, Jackson ImmunoResearch)와 배양한 후 4℃에서 1시간동안 항-Myc 항체(1:400, Abcam, ab9106)와 함께 배양하여 SIRPα를 염색하였다. 형광신호(Fluorescence signals)는 AccuriTM C6 유세포 분석기(BD biosciences) 및 FlowJo_V10 소프트웨어(FlowJo)를 사용하여 분석하였다.
아울러, 재조합 엑소좀의 형태는 먼저, 샘플을 탄소 필름(Electron microscopy science)이 구비된 구리격자(copper grids)에 위치하였고 아세트산 우라닐 용액을 이용하여 음성으로 염색하였다. SIRPα의 내부 발현은 면역 전자 현미경 이미지를 통하여 확인하였고 표면 SIRPα는 항-Myc 항체(1:100, Abcam, ab9106) 및 금 결합 항체(1:50, Aurion)를 이용하여 캡처하였으며 투과전자현미경(Tecnai)을 이용하여 분석하였다. 마지막으로, 재조합 엑소좀의 크기 분포는 Zetasizer Nano ZS(Malvern Instruments, Ltd., UK)이용한 동적 광산란 분석법(dynamic light scattering, DLS)을 통하여 분석하였고 엑소좀 크기는 장비에서 제공된 소프트웨어를 이용하여 25℃조건으로 173°의 고정된 각도에서 수 비율(z-average)을 통해 분석하였다.
그 결과, 상기 정제한 SIRPα-엑소좀은 표면 막 상에 SIRPα 변이체(variant)를 보유하고 있고 엑소좀 마커 단백질을 함유하고 있는 것을 관찰하였다(도 2). 또한, 투과 전자 현미경(TEM) 이미지와 동적 광 산란(DLS) 분석 결과 재조합 엑소좀의 평균 크기가 100 nm의 평균 크기를 가진 둥근 모양인 것을 확인하였다(도 3).
실시예
4: 단량체
SIRPα
단백질의 발현 및 정제
본 발명의 일 실시예에 따라 단량체(monomer) SIPRα 단백질(mSIRPα)을 얻기 위해, NH2-Nde I-SIRPα 변이체-Myc-Hind Ⅲ-COOH를 인코딩하는 프라이머를 이용한 PCR 증폭을 통하여 유전자 클론(gene clone)을 제조하였고 상기 유전자 클론은 N-말단 히스티딘 태그와 함께 SIRPα을 발현하기 위해 pET-28a 플라스미드 벡터로 결찰하였다.
이후, 형질전환된 세균 세포는 37℃에서 O.D.600이 0.5가 될 때까지 카나마이신을 포함하는 LB 배지에서 배양하였고 mSIRPα를 생산하기 위하여 0.5 mM 이소프로필 β-D-1-thiogalactopyranoside(IPTG)(Bioneer, 대한민국)를 첨가하여 유도하였다. 20℃에서 18시간 동안 배양한 후에 상기 세포는 6000 g에서 10분 동안 원심분리를 통하여 수득하였고 초음파 분쇄기를 이용하여 균일화한 후 상기 펠렛을 용해 버퍼(1 M Tris-HCl pH 8.0, 150 mM NaCl, 1 mM PMSF)에 재현탁하였으며 Ni-NTA 친화도 크로마토그래피를 통해서 mSIRPα를 정제하였고 항-Myc 항체(1:5000, Abcam, ab9106) 및 HRP-연결된 2차 항체(1:4000, Sigma Aldrich)를 이용한 웨스턴 블랏으로 분석하였다.
그 결과, HEK293T 세포로부터 100 μg의 총 단백질과 SIRPα-엑소좀 2 x 109을 수득하였고 정제된 재조합 SIRPα-Myc 단량체 단백질(mSIRPα)의 웨스턴 블랏 이미지로부터 표준 곡선(standard curve)을 사용하여 엑소좀의 SIRPα의 양을 정량하였으며 상기 측정된 SIRPα-엑소좀의 양은 1 μg exosomes당 5 ng이하로 확인되었다(도 4). 상기 PCR 증폭에 사용된 프라이머에 대한 정보를 하기 표 1에 표시하였다.
프라이머 | 염기서열(5’--> 3’) | 서열번호 |
Forward | AAA CATATG GAA GAG GAG CTG CAG | 67 |
Reverse | AAA AAGCTT TCA ATT CAG ATC CTC TTC | 68 |
실시예
5: 세포 결합 분석
본 발명의 일 실시예에 따른 세포 결합 분석은 HT29 인간 결장 선암종(ATCC), Raji 인간 B 세포 림프종(ATCC) 및 CT26.CL25 마우스 결장암(ATCC) 세포를 10% FBS 및 1% 항생제를 첨가한 RPMI-1640 배양배지에서 배양하였고 37℃ 및 5 % CO2 조건에서 유지하였다. 그 후, HT29 및 Raji 세포는 항-인간 CD47 항체[B6H12.2](Abcam, ab3283)를 첨가하여 배양하였고 암세포 표면에서 CD47 발현을 탐지하기 위하여 CT26.CL25 세포는 항-마우스 CD47 항체(Santa Crus, sc-12731)를 첨가하여 배양하였다. 세포 결합 분석을 위해, HT29, Raji 및 CT26.CL25 세포(1 x 106)는 PBS, 엑소좀 또는 mSIRPα를 첨가하여 4℃에서 30분 동안 배양하였다. 그 후, 상기 세포는 항-Myc 항체(1:400, Abcam, ab9106)를 첨가하여 배양하였고 Alexa fluor 488-결합된 2차 항체(1:800, Jackson ImmunoResearch)를 첨가하여 탐지하였다. 이어서, 상기 세포는 AccuriTM C6 유세포 분석기(BD Biosciences)를 이용하여 측정하였고 FlowJo_V10 소프트웨어(FlowJo)를 이용하여 분석하였다. SIRPα-엑소좀의 CD47에 대한 결합 특이성은 항-인간 CD47 항체(1:100, Abcam, ab3283)를 첨가한 세포를 전-배양하는 방법을 통한 블록 실험으로 분석하였다.
또한, 형광현미경 분석을 위해서, HT29 세포(2 x 105)는 상술한 바와 같이, 유리 바닥 4웰 챔버에 파종하였고 항-Myc 항체(1:400, Abcam, ab9106) 및 Alexa fluor 488-결합된 2차 항체(1:800, Jackson ImmunoResearch)를 첨가하여 배양하였다. 잔류 비-특이성 신호(non-specific signals)의 제거 후에 상기 세포는 Hoechst 33258를 이용하여 25℃에서 10분 동안 핵 염색 후에 4 % 파라포름알데하이드를 이용하여 7분간 고정하였다. 엑소좀의 세포 결합 능력은 형광현미경(Nikon Eclipse Ti, Nikon)으로 관찰하였고 LAS AF Lite 소프트웨어(Leica)를 이용하여 분석하였다.
그 결과, 종양 세포를 엑소좀과 함께 배양하여 CD47+ 인간 종양 세포주에서 세포 표면 CD47을 길항하는 SIRPα-엑소좀의 능력을 확인하였다(도 5). 또한, SIRPα-엑소좀은 효과가 SIRPα-엑소좀 농도 의존적으로 확인된 대조군의 엑소좀과 비교하여 HT29 종양 세포 표면의 결합에서 더 높은 CD47에 대한 친화력을 나타내었다. SIRPα-엑소좀의 결합은 항-인간 CD47 항체와의 사전 배양에 의해 감소되어, SIRPα-엑소좀이 종양 세포 표면의 CD47에 특이적으로 결합한다는 것을 나타내었고 인간 Raji Burkitt's 림프종(Raji) 및 마우스 CT26.CL25 결장암 세포에서 SIRPα-엑소좀의 CD47 결합의 유사한 결과를 관찰하였다. 상기 결과는 엑소좀에서 막-연관 SIRPα 변이체의 발현이 암 세포에서 CD47에 결합하는 능력을 가지고 있음을 말해준다(도 6 및 7).
실시예
6: 식균작용 분석
본 발명의 일 실시예에 따라 종양 세포의 길항작용을 가진 CD47이 종양 세포의 대식세포 매개 식균작용(phagocytosis)을 증가시키는 여부를 관찰하기 위해 식균작용 분석을 수행하였다. 구체적으로 체외(In vitro) 식균작용 분석을 위한 골수 유래 대식세포(BMDMs)를 제조하기 위해, BALB/c 마우스를 희생시키고 골수 세포를 다리 뼈(leg bones)로부터 분리하였다. 상기 분리한 골수 세포를 10 % FBS 및 1 % 항생제가 첨가된 RPMI 배지에서 유지시키고 7일 동안 대식세포 콜로니-자극인자(M-CSF)로 분화시켰다. 식균작용은 BMDM과 암 세포를 무-혈청 RPMI 배지에서 4 시간 동안 공 배양하여 분석하였다. 유세포 분석을 위해, 분화된 대식세포(2.5 x 105)는 0.5 μM CellTrackerTM Green으로 염색하였고 엑소좀 또는 mSIRPα 단백질을 암세포와 사전 배양 한 후, BMDM을 4시간 동안 혼합물과 함께 배양하였다. 식균작용의 비율은 AccuriTM C6 유세포 분석기(BD biosciences) 및 FlowJo_V10 소프트웨어(FlowJo)를 사용하여 이중 양성신호(double positive signals)의 백분율로 평가하였다.
또한, 식균작용 지수(phagocytosis index, PI)를 측정하기 위한 형광 현미경 분석을 위해, CellTrackerTM Green(Thermo fisher scientific)으로 염색한 BMDM을 2.5 x 105의 밀도로 35 mm 유리 바닥 디쉬에 파종하고, HT29 세포(1 x 106)의 혼합물을 pHrodo Deep red(Thermo fisher scientific)로 염색하였고 엑소좀을 대식세포로 처리하였다. 4 시간의 공배양 후, 대식세포에 의한 HT29 세포의 탐식(engulfment)은 형광 현미경(Nikon Eclipse Ti, Nikon)에 의해 대식세포에서의 포식세포 형성과 관련된 적색 양성 신호로 분석하였다.
그 결과, 이중 양성 신호(진한 적색 및 녹색)를 갖는 대식세포의 식균작용은 대조군(PBS-처리 또는 대조군-엑소좀-처리)과 비교하여 SIRPα-엑소좀 처리군에서 농도 의존적으로 증가하는 것으로 나타났고(도 8) SIRPα-엑소좀을 처리한 Raji 및 CT26.CL25 결장암 세포에서도 유사하게 나타났다. 또한, SIRPα-엑소좀의 처리가 종양 세포의 탐식을 증가시키고 이에 따라 PI가 SIRPα-엑소좀 처리군에서 유의하게 증가한 것으로 나타났다(도 9). 따라서 SIRPα-엑소좀에 의한 CD47-SIRPα 상호작용을 차단하는 것은 골수 유래 대식세포(BMDMs)에 의해 다양한 암세포의 식균작용의 증가를 유도한다는 것을 말해준다.
실시예
7: 생체분포(
biodistribution
) 연구
본 발명의 일 실시예에 따라 SIRPα-엑소좀의 생체 분포를 조사하기 위해, 엑소좀을 Cy5.5-NHS로 표지 하였고 Cy5.5-NHS 염료(1 ㎍)를 100 ㎍의 엑소좀에 처리한 후, 실온에서 2 시간 동안 배양하였으며, airfuse 원심분리기(Beckman coulter)를 사용하여 45분 동안 원심분리하였다. 이후 비-결합된 염료를 제거하기 위해 2회 세척을 수행한 후에 표지된 엑소좀 펠릿을 PBS에 재현탁하였고 형광 강도(fluorescence intensity)는 형광 마이크로 플레이트 판독기(Infinite M200 Pro, TECAN)를 사용하여 측정하고 조정하였다. 또한, HT29 종양 보유 BALB/c 누드 마우스에 Cy5.5-표지된 엑소좀(500μg), 유리 염료 및 PBS를 정맥 내(intravenously) 투여 하였고 모든 샘플의 형광 강도는 형광 마이크로 플레이트 판독기로 수득한 데이터에 기초하여 동일한 값으로 조정하였다. 마우스의 생체(In
vivo) 전신 이미징은 IVIS 스펙트럼(Caliper Life Sciences)을 사용하여 여러 시점(5분, 2시간, 4시간, 8시간, 16시간 및 24시간)에서 수행하였다. 아울러, 종양의 형광 강도를 분석하기 위해 Analysis Workstation 소프트웨어(Advanced Research Technologies Inc.)를 사용하여 관심 영역(ROI)에서 입체각(steradian) 당 센티미터 스퀘어 당 총 광자(total photons)를 계산하였고 주입 후 24시간 경과시점에서 마우스를 희생시키고 간, 폐, 비장, 신장 및 심장을 포함한 종양 및 주요 기관을 상기와 동일한 방법으로 절제하고 분석하였다.
그 결과, 절제된 종양의 평균 중량은 종양 성장의 관찰된 퇴화(regression)와 일치하여 대조군보다 SIRPα-엑소좀 처리군에서 현저히 낮게 나타났다(도 10).
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8: 항-종양 효과 분석
본 발명의 일 실시예에 따라 생체내 실험(in
vivo experiments)을 위해 면역-결핍(immuno-deficient) BALB/c 누드 마우스 및 면역 감응(immunocompetent) BALB/c 마우스는 7주령된 시점에서 종양을 이식하고 한국 과학 기술 연구원 (KIST)의 수용시설에서 관리되었다. 그 후, HT29 세포(1 × 107)를 BALB/c 누드 마우스의 왼쪽 다리에 피하 접종하였고 종양을 일주일 동안 성장시킨 다음, 대조군-엑소좀, SIRPα-엑소좀 및 대조군 PBS를 3일마다 5회씩 주입하였다. 그 후, 국소 항-종양 효과의 분석을 위해, 100 μg의 엑소좀을 마우스의 종양 내로 주입하였다. 종양 성장에 대한 SIRPα-엑소 좀의 전신 효과(systemic effect)를 위해, 엑소좀(200 ㎍) 및 PBS를 HT29 종양 보유 마우스에게 3일마다 5회씩 주입하였고 종양이 1000 mm3까지 성장시킨 후 절개하고 무게를 측정하였다. 또한, CT26.CL25 세포(1 × 106)를 면역 감응 BALB/c 마우스의 왼쪽 다리 피하에 이식하였고 평균 크기가 80 mm3인 종양의 안정화를 위해 일주일 경과 후 엑소좀 200 μg, SIRPα-엑소좀 200 μg, mSIRPα 1 μg(SIRPα-엑소좀s 200 μg에서 SIRPα양에 해당) 또는 PBS를 각각 상기 마우스(각각 n = 7 마우스 그룹)의 꼬리 정맥을 통해 주입하였으며 총 5 회 처리가 완료되면 종양을 절개하고 무게를 측정하였다.
그 결과, Cy5.5 염료로 표지된 SIRPα-엑소좀은 종양 부위에 연속적으로 축적되었고 이는 종양 세포에서 과발현된 CD47 과의 상호작용뿐만 아니라 나노 입자와 같은 엑소좀의 침투성 및 보유 효과가 향상되었음을 말해준다(도 11). 또한 체외(ex
vivo) 이미지 분석 결과 마우스의 간과 신장에서 Cy5.5로 표지된 엑소좀이 축적되어 전체 동물 이미징 분석 결과를 뒷받침하였고(도 12), SIRPα-엑소좀으로 처리한 마우스는 대조군과 비교하여 종양 성장이 다소 감소하였으나 유의적인 차이를 나타내지는 않았다. 한편, 면역-결핍(immuno-deficient) 및 면역 감응(immunocompetent) 마우스를 이용하여 SIRPα-엑소좀 주입에 따른 항-종양효과를 관찰한 결과, HT29 종양 보유 BALB/c 면역-결핍 마우스에 SIRPα-엑소좀을 투여할 경우 종양의 성장을 약간 감소시키나 유의한 수준이 아니었던 반면, CT26.CL25 종양 보유 면역 감응 BALB/c 마우스에 SIRPα-엑소좀을 투여할 경우에는 매우 우수한 항-종양효과를 나타내었다. 이는 본 발명의 SIRPα-엑소좀의 투여함으로써 면역기능을 경유한 SIRPα-엑소좀의 현저한 항-종양 효과를 입증하는 것이다(도 13 및 14). 한편, 재조합 SiRPα 단백질(mSIRPα)을 투여할 경우에는 암세포의 CD47에 효율적으로 결합할 수 있음에도 불구하고 종양의 식균작용을 제대로 유도하지는 못하였다(도 15).
결론적으로 본 발명의 일 실시예에 따라 제조한 재조합 엑소좀(SIRPα-엑소좀)은 CD47-SIRPα 상호작용을 차단함에 따라서 대식세포와 수지상세포의 식균작용을 증가시켜 시험관내 조건은 물론 동물모델 실험에서 매우 현저한 항-종양 효과를 나타내었으므로 암치료를 위한 신규 항암제로 활용가능하다.
본 발명은 실시예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술 분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허 청구범위의 기술적 사상에 의하여 정해져야 할 것이다.
Claims (14)
- 탐식작용 촉진 단백질이 엑소좀(exosome) 표면에 제시된, 재조합 엑소좀.
- 제1항에 있어서,상기 탐식작용 촉진 단백질은 수용체 티로신 카이네이즈의 막통과 도메인의 N-말단에 연결된 융합단백질인, 재조합 엑소좀.
- 제2항에 있어서,상기 수용체 티로신 카이네이즈는 PDGFR(platelent-derived growth factor receptor), EGFR(epidermal growth factor receptor), FGFR(fibroblast growth factor receptor), VEGFR(vascular endothelial growth factor receptor), HGFR(hepatocyte growth factor receptor), Trk(tropomyosin receptor kinase), IR(insulin receptor), LTK(Leukocyte receptor tyrosine kinase), 안지오포이에틴 수용체(angiopoietin receptor), ROR(receptor tyrosine kinase-like orphan receptors), DDR(discoidin domain receptor), RETR(rearranged during transfection receptor), PTK(tyrosine-protein kinase-like), RYK(related to receptor tyrosine kinase), 또는 MuSK(muscle-specific kinase)인, 재조합 엑소좀.
- 제1항에 있어서,상기 탐식작용 촉진 단백질은 SIRP 또는 상기 SIRP의 CD47 결합도메인을 포함하는 단편, Surfactant protein A, Surfactant protein D 또는 항-CD47 항체인, 재조합 엑소좀.
- 제4항에 있어서,상기 SIRP는 SIRPα, SIRPγ 또는 이들의 고친화성 변이체인, 재조합 엑소좀.
- 제5항에 있어서,상기 SIRP는 서열번호 1 내지 61 중 어느 하나의 아미노산 서열로 구성되는, 재조합 엑소좀.
- 제1항에 있어서,상기 엑소좀은 내부에 항암제를 포함하는, 재조합 엑소좀.
- 제7항에 있어서,상기 항암제는 항암 단백질 또는 항암 화합물인, 재조합 엑소좀.
- 제8항에 있어서,상기 항암 단백질은 아스파라기네이즈(asparaginase), 단백질 독소, 암항원에 특이적인 항체 또는 상기 항체의 단편, 종양억제 유전자(tumor suppressor gene) 또는 항혈관생성인자(antiangiogenic factor)인, 재조합 엑소좀.
- 제8항에 있어서,상기 항암 화합물은, 메토트렉세이트(methotrexate), 피리미딘 유사체(pyrimidine analogs), 히드록시우레아(hydroxy urea), 퓨린 유사체(purine analogs), 알킬화제(alkylating agents), 면역원성 세포사멸 유도제, 유사분열 억제제(mitotic inhibitors), 신생혈관억제제, 삽입성 물질(ntercalating agents) 또는 방사성 핵종(radionuclides)인, 재조합 엑소좀.
- 제10항에 있어서,상기 면역원성 세포사멸 유도제는 안트라사이클린 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside) + 비-면역원성 세포사멸 유도제, 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제 + 마토마이신, LV-tSMAC, Measles 바이러스, 또는 옥살리플라틴인, 재조합 엑소좀.
- 제11항에 있어서,상기 안트라사이클린 계열 항암제는 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin), 또는 발루비신(valrubicin)인, 재조합 엑소좀.
- 치료적으로 유효한 양의 제1항 내지 제12항 중 어느 한 항의 재조합 엑소좀 및 약학적으로 허용가능한 담체를 포함하는, 항암용 약학적 조성물.
- 제13항에 있어서,하나 이상의 항암제를 추가로 포함하는 항암용 약학적 조성물.
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CN201780081782.8A CN110248645B (zh) | 2016-12-29 | 2017-12-29 | 基于外来体的抗癌剂 |
JP2019534796A JP6857736B2 (ja) | 2016-12-29 | 2017-12-29 | 新規なエクソソーム系抗癌剤 |
EP17888530.7A EP3563835A4 (en) | 2016-12-29 | 2017-12-29 | NEW EXOSOME-BASED ANTI-CANCER AGENT |
US17/712,155 US11952412B2 (en) | 2016-12-29 | 2022-04-03 | Exosome-based anticancer agent |
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KR102521664B1 (ko) * | 2019-09-02 | 2023-04-14 | 경북대학교 산학협력단 | Il-2 표면 발현 세포외 소포체를 유효성분으로 포함하는 암 질환 예방 또는 치료용 조성물 |
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KR102462745B1 (ko) * | 2021-04-29 | 2022-11-04 | 재단법인 아산사회복지재단 | 형광 상관 분광법을 이용한 세포외소포체에 표지된 형광 염료의 정량 분석 방법 및 이의 용도 |
KR20230021248A (ko) * | 2021-08-05 | 2023-02-14 | 경북대학교 산학협력단 | T 세포의 세포외 소포체 및 항암제를 유효성분으로 포함하는 병용 투여용 약학적 조성물 |
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KR20240045411A (ko) * | 2022-09-29 | 2024-04-08 | (주)카이노스메드 | Faf1 엑소좀을 고수율로 생산하는 세포주 및 이의 제조방법 |
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US20220251170A1 (en) | 2022-08-11 |
KR20180078173A (ko) | 2018-07-09 |
JP6857736B2 (ja) | 2021-04-14 |
KR102129067B1 (ko) | 2020-07-08 |
US11952412B2 (en) | 2024-04-09 |
CN110248645B (zh) | 2022-04-26 |
EP3563835A1 (en) | 2019-11-06 |
US20200148746A1 (en) | 2020-05-14 |
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