Patent Application
20210106632
The present invention relates to a novel recombinant plasma membrane-based vesicle, and more specifically, to a novel recombinant plasma membrane-based vesicle for treating cancer.
Cancer refers to a group of diseases associated with abnormal cell growth with a potential for invasion and metastasis to other parts of the body. As of 2015, it is known that there are more than 90 million cancer patients worldwide, with about 14 million new cancer patients occurring every year. Cancer accounts for 15.7% of causes of human deaths. The most frequently occurring cancers are lung cancer, prostate cancer, colon cancer, and stomach cancer in men, and breast cancer, colon cancer, lung cancer, and uterine cervical cancer in women.
For cancer treatment, various therapeutic approaches are attempted, including chemotherapy using various anticancer agents, radiation therapy by irradiation, antibody therapy targeting particular in vivo molecules associated with cancer, etc. However, anticancer agents used in chemotherapy or irradiation have serious side effects because these treatments also affect normal cells and often result in treatment failure or recurrence because cancer cells can acquire resistance to anticancer agents.
Recently, the anti-cancer immunotherapy using the immune system of our body has been showing a surprising effect in clinical treatment. However, due to the complexity of cancer, the anti-cancer immunotherapy has a limitation in that the therapy is only effective in less than about 30% of cancer patients. This is because cancer cells are recognized as “self” by our body's immune cells, and therefore, it is important for cancer cells to be recognized as “non-self” by immune cells, so that the phagocytosis of cancer cells can be induced thereby inducing an amplified immune response.
Exosomes are cell-derived vesicles present in all biological liquids, including blood, urine, and culture media of cell cultures, also called extracellular vesicles or microvesicles. Exosomes are known to have a size between 30 nm and 100 nm, and are secreted from cells when the multivesicular body fuses with a cell membrane, secreted directly through a cell membrane, or are budding directly from a cell membrane. Exosomes are known to play an important role in various processes such as coagulation, intercellular signaling, and metabolic waste management. Exosomes have important advantages as drug carriers compared to liposomes or polymer-type nanoparticles in that exosomes have compositions similar to those of the cells themselves of the human body and in that exosomes are non-immunogenic (Ha et al., Acta Pharm. Sin. B. 6(4): 287-296, 2016). In this regard, various attempts have been made using exosomes so that anticancer drugs (e.g., doxorubicin) are delivered to a tumor tissue (Tian et al., Biomaterials. 35:2383-2390, 2014); paclitaxel and doxorubicin pass through the blood brain barrier and are delivered to the brain (Yang et al., Pharm. Res. 32:2003-2014, 2015); for treatment of Parkinson's disease, catalase passes through the blood brain barrier and is delivered to the brain (Haney et al., J. Control Release. 207: 18-30, 2015); or for treatment of cancer, siRNAs specific to certain genes are delivered (Shtam et al., Cell Commun. Signal. 11: 88, 2013), etc.
An extracellular vesicle refers to a structure in the form of a particle, in which various biomolecules (e.g., proteins, nucleic acid molecules (e.g., RNA), lipids of various functions, etc.) released or secreted from cells into an extracellular environment are enclosed in a cell membrane of a lipid bilayer identical to the cell membrane of the cell from which the various biomolecules are derived. The extracellular vesicle refers to a plasma membrane-based vesicle having an average diameter of 100 nm to 1 μm, which is larger than that of an exosome normally having a size of 30 nm to 100 nm.
A cell-derived nanovesicle, which is a nano-sized vesicle surrounded by a plasma membrane which is a nano-sized cell membrane component formed by artificial methods (e.g., an extrusion process where cells are passed through microfluidic channels, a multi-stage filtration process, etc.), refers to a plasma membrane-based vesicle distinguished from an exosome or extracellular vesicle secreted by a cell and formed naturally.
Meanwhile, vesicular stomatitis virus glycoprotein (VSV-G) is the only virus glycoprotein present in the virion membrane of vesicular stomatitis virus and acts as a protein for adhesion and fusion of the virus into a target cell. The VSV-G protein is a transmembrane protein including two N-linked glycans, which can initiate a membrane fusion event in a low-pH-dependent manner when no other virus protein is present. Since VSV-G proteins can form a complex with a nucleic acid molecule such as DNA, VSV-G proteins have been used as a carrier for direct gene transfer, or have been used effectively in gene therapy by producing more stable and high-titer pseudotyped murine leukemia virus (MLV)-based retrovirus and lentivirus-based vectors. Recently, however, it has been suggested that VSV-G proteins be available for use to deliver various proteins, in addition to genes, to heterologous cells (Mangeot et al., Mol. Ther. 19(9): 1656-1666, 2011).
As a result of the research on the structures and functions of the VSV-G proteins, it was previously confirmed that histidine, which is the 60th, the 162nd, and the 407th amino acid residues based on the mature protein from which the signal sequence was removed, forms a cluster and acts as a pH sensor (Roche et al., Science, 313: 187-191, 2006; Roche et al., Science, 315: 843-848, 2007), and recently, it was reported that when the 162nd amino acid residue, histidine, among the above histidine residues is mutated into arginine (H162R), it induces a membrane fusion at pH 6.8, which is a physiological pH surrounding cancer cells, and promotes the death of cancer cells upon administration of neural stem cells expressing the VSV-G mutant (H162R) (Zhu et al., Mol. Ther. 21 (8) : 1492-1497, 2013).
However, the method disclosed in the related art is a technology difficult to apply to the actual clinical environment in that the supply of neural stem cells is difficult, and that the cancer cell killing effect is not very significant.
The objects of the present invention are to solve various problems including the above problems, and an object of the present invention is to provide a recombinant plasma membrane-based vesicle for safe cancer treatment which does not operate under conditions outside the cancer microenvironment while efficiently killing cancer cells on their own without an anticancer agent. In particular, the object of the present invention is to provide a recombinant plasma membrane-based vesicle which enables “xenogenization” so that immune cells can recognize cancer cells as an “enemy”. However, these objects are illustrative, and the scope of the present invention is not limited thereby.
According to an aspect of the invention, there is provided a recombinant plasma membrane-based vesicle, wherein a VSV-G mutant protein in which the 162nd amino acid, histidine, is substituted with arginine is introduced into the membrane.
According to another aspect of the invention, there is provided a pharmaceutical composition for treating cancer containing the recombinant plasma membrane-based vesicle as an active ingredient.
Moreover, according to still another aspect of the invention, there is provided a pharmaceutical composition for treating cancer containing a recombinant plasma membrane-based vesicle, in which a virus-derived fusogenic membrane protein is introduced into the membrane, as an active ingredient.
Moreover, according to still another aspect of the invention, there is provided a recombinant plasma membrane-based vesicle, wherein a VSV-G mutated protein in which the 162nd amino acid, histidine, is substituted with arginine is introduced into the membrane; or the use of a recombinant plasma membrane-based vesicle, in which a virus-derived fusogenic membrane protein is introduced into the membrane, for the preparation of a cancer therapeutic agent.
According to still another aspect of the invention, there is provided a method for treating cancer in a subject with cancer, which includes administering the recombinant plasma membrane-based vesicle or one or more pharmaceutical compositions among those described above to the subject with cancer.
According to an embodiment of the present invention constituted as described above, the present invention can effectively treat cancer without resorting to a complex mechanism such as gene transfer.
Definition of Terms
As used herein, the term “fusogenic membrane protein” refers to a virus-derived membrane protein which plays a key role in inducing the adhesion and membrane fusion of a virus to a host cell for the penetration of the virus into the host cell. A representative example is an envelope glycoprotein derived from the vesicular stomatitis virus (VSV-G).
As used herein, the term “an envelope glycoprotein derived from the vesicular stomatitis virus (VSV-G protein)” refers to the only virus glycoprotein present in the virion membrane of vesicular stomatitis virus and acts as a protein for adhesion and fusion to a heterologous cell of the virus. The VSV-G protein is a transmembrane protein including two N-linked glycans, and can initiate a membrane fusion in a low-pH-dependent manner in the absence of other viral proteins. The VSV-G protein can form a complex with a nucleic acid molecule such as DNA, and thus has been used as a carrier for direct gene transfer or has been used effectively in gene therapy by producing more stable and high-titer pseudotyped murine leukemia virus (MLV)-based retrovirus and lentivirus-based vectors. However, the availability of the VSV-G protein to be used for the delivery of various proteins, in addition to genes, to heterologous cells has recently been proposed (Mangeot et al., Mol. Ther. 19(9): 1656-1666, 2011).
As used herein, the term “plasma membrane-based vesicle” refers to a nano-sized vesicle surrounded by a cell membrane derived from a cell, and includes exosomes produced by secretion or budding from cells, cell-derived nanovesicles prepared by processing extracellular vesicles or cells by an artificial method (e.g., extrusion, etc.), etc.
As used herein, the term “exosome” refers to a cell-derived vesicle present in all biological liquids, including blood, urine, and culture media of cell cultures, and is also called an extracellular vesicle or a microvesicle. Exosomes are known to have a size between 30 nm and 100 nm, and are secreted from cells when the multivesicular body fuses with a cell membrane or secreted directly through a cell membrane. Exosomes are known to play an important role in various processes such as coagulation, intercellular signaling, and metabolic waste management.
As used herein, the term “recombinant exosome” refers to an artificially-produced exosome, which is obtained in such a manner that a transformed host cell is prepared by genetic engineering in which a gene encoding a heterologous protein is transduced into a host cell capable of producing exosomes, and the transformed host cell is cultured and the exosome is obtained from the culture liquid. The recombinant exosome includes a transduced heterologous protein either inside or on exosomal membrane.
As used herein, the term “extracellular vesicle” refers to a structure in the form of a particle, in which various biomolecules (e.g., proteins, nucleic acid molecules (e.g., RNA), lipids of various functions, etc.) released or secreted from cells into an extracellular environment are enclosed in a cell membrane of a lipid bilayer identical to the cell membrane of the cell from which the various biomolecules are derived. An extracellular vesicle refers to a plasma membrane-based vesicle having an average diameter of 100 nm to 1 μm, which is larger than that of an exosome normally having a size of 30 nm to 100 nm.
As used herein, the term “cell-derived nanovesicle”, which is a nano-sized vesicle surrounded by a plasma membrane that is a nano-sized cell membrane component formed by artificial methods (e.g., an extrusion process where cells are passed through microfluidic channels, a multi-stage filtration process, etc.), refers to a plasma membrane-based vesicle distinguished from an exosome or extracellular vesicle secreted by a cell and formed naturally.
As used herein, the term “immunogenic cell death” refers to a kind of cell death caused by cell proliferation inhibitors (e.g., anthracyclines, oxaliplatin, and bortezomib) or by radiation therapy and photodynamic therapy. Unlike normal cell death, the immunogenic cell death, that is, the immunological cell death of cancer cells can induce an effective anticancer immune response via activation of dendritic cells and subsequent activation of a specific T cell response. The material which induces the immunogenic cell death is called an “immunogenic cell death inducer”. The immunogenic cell death and the immunogenic cell death inducers are well summarized in Kroemer et al. (Anna. Rev. Immunol., 31: 51-72, 2013). This document is incorporated herein by reference in its entirety.
As used herein, the term “anthracycline-type anticancer agent” refers to a cell-cycle nonspecific anticancer agent family used in cancer chemotherapy derived from Streptomyces peucetius var. caesius. The anthracycline-type anticancer agent is used to treat various cancers, including leukemia, lymphoma, breast cancer, stomach cancer, uterine cancer, ovarian cancer, bladder cancer, and lung cancer, and is one of the most effective anticancer agents among the chemotherapy anticancer agents that have been developed. The first found anthracycline-type anticancer agent was daunorubicin, immediately followed by doxorubicin, and subsequently followed by epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, etc., which are developed thereafter. Examples of the mechanism of action of anthracycline-type anticancer agents being proposed include: prevention of the replication of rapidly growing cancer cells by inhibiting DNA and RNA synthesis via intercalation between the bases of DNA/RNA strands; prevention of transcription and replication by inhibiting the relaxation of supercoiled DNA via suppression of the enzyme activity of topoisomerase II; induction of damage to DNA, proteins, and cell membranes through the formation of iron-mediated free oxygen radicals and DNA damage responses; induction of histone eviction from chromatin that deregulates epigenome and transcripts, etc. Recent study has reported that doxorubicin increases Th1 immune responses by activating CD4+ cells (Park et al., Int. Immunopharmacol. 9(13-14): 1530-1539, 2009), and co-administration of dendritic cells with doxorubicin has been reported to show an anticancer activity by inducing immunogenic cell death of osteosarcoma (Kawano et al., Oncol. Lett. 11:2169-2175, 2016).
As used herein, the term “taxane-type anticancer agent (taxanoid anticancer agent or taxane anticancer drug)” refers to a diterpenoid taxane derivative derived from Taxus sp. plants, which is a mitotic inhibitor having a mechanism capable of promoting the assembly of microtubules in cells while inhibiting the disassembly of microtubules therein. Currently commercialized drugs include paclitaxel and docetaxel. Among them, paclitaxel is a taxane-type anticancer agent derived from the periderm of Taxus brevifolia and was approved as a therapeutic agent for refractory ovarian cancer by the US FDA in 1992, whereas docetaxel is a taxane-type anticancer agent derived from Taxus baccata and has a similar efficacy to paclitaxel, is used for treatment of breast cancer, non-cell lung cancer, lymphoma, bladder cancer, etc., and has a higher hydrophilicity than paclitaxel. Recently, taxane-type anticancer agents have also been shown to have a mechanism to promote immunogenic cell death of cancer cells by sensitizing these cancer cells to cytotoxic T lymphocytes.
As used herein, the term “immune checkpoint inhibitor” refers to a type of drug that blocks certain proteins produced by certain types of immune system cells (e.g., T lymphocytes) and some cancer cells, and these proteins suppress immune responses and prevent T lymphocytes from killing cancer cells. Therefore, blocking of these proteins can release the “braking system” of the immune system thereby allowing T lymphocytes to kill cancer cells better. Examples of the “immune checkpoint” well known thus far include PD-1/PD-L1, CTLA-4/B7-1/B7-2, etc. Examples of the PD-1 inhibitors include pembrolizumab (trade name: Keytruda), nivolumab (trade name: Opdivo), etc. Examples of the PD-L1 inhibitors, as a ligand of PD-1, include atezolizumab (trade name: Tecentriq), avelumab (trade name: Bavencio), etc. Meanwhile, ipilimumab (trade name: Yervoy) has been approved by the USFDA as a CTLA-4 inhibitor that inhibits the interaction of CTLA-4/B7-1/B7-2. For the past few years, these drugs have achieved a remarkable success, especially in patients with metastatic melanoma or Hodgkin lymphoma and have shown great potential in clinical trials with respect to different types of cancer patients.
According to an aspect of the present invention, there is provided a recombinant plasma membrane-based vesicle, wherein a VSV-G mutated protein in which the 162nd amino acid, histidine, is substituted with arginine is introduced into the membrane.
The recombinant plasma membrane-based vesicle may be an exosome, an extracellular vesicle, or a cell-derived nanovesicle.
The recombinant plasma membrane-based vesicle may be isolated/purified from a mammalian cell, preferably from a human cell, which is transformed into a gene construct including a polynucleotide encoding the VSV-G mutated protein and thus overexpresses the VSV-G mutated protein.
The recombinant plasma membrane-based vesicle may be obtained from a cell which is transformed to express the VSV-G mutated protein.
According to another aspect of the present invention, there is provided a VSV-G mutated protein in which the 162nd amino acid, histidine, is substituted with arginine is introduced into the membrane, and one or two or more immunogenic cell death inducers are incorporated therein.
The incorporation of a drug into the recombinant plasma membrane-based vesicle can be achieved by culturing cells, which are genetically engineered to produce a recombinant plasma membrane-based vesicle, in a cell culture medium into which the drug is dissolved; or can be produced by reconstituting the recombinant plasma membrane-based vesicles via sonication treatment in a solvent in which the immunogenic cell death inducer is dissolved (Kim et al., Nanomedicine, 12(3): 655-664, 2016). Optionally, when the drug is supported on the plasma membrane-based vesicle, a method in which the isolated plasma membrane-based vesicle can be simply mixed with a drug in an appropriate solvent or medium and then mixed by stirring for an appropriate period of time (Sun et al., Mol. Ther. 18(9): 1606-1614, 2010), or hydrophilic drugs (e.g., nucleic acids) can be incorporated into the plasma membrane-based vesicle by electroporation.
The immunogenic cell death inducer may be an anthracycline-type anticancer agent, a taxane-type anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, cardiac glycoside, a cyclophosphamide-type anticancer agent, a GADD34/PP1 inhibitor, LV-tSMAC, measles virus, bleomycin, mitoxantrone, or oxaliplatin. The cardiac glycoside may be used in combination with a non-immunogenic cell death inducer; the GADD34/PP1 inhibitor may be used in combination with mitomycin; the anthracycline-type anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin; the taxane-type anticancer agent may be paclitaxel or docetaxel; and the anti-EGFR antibody may be cetuximab.
According to still another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer containing the recombinant plasma membrane-based vesicle as an active ingredient.
According to still another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer containing a recombinant plasma membrane-based vesicle, wherein a virus-derived fusogenic membrane protein is introduced into the membrane, as an active ingredient.
In the above pharmaceutical composition, the virus-derived fusogenic membrane protein may be a vesicular stomatitis virus (VSV)-G protein, gibbon ape leukemia virus (GALV.fus), influenza virus hemagglutinin, F protein of respiratory syncytial virus, HIV virus gp120 or gp41, E protein of flavivirus, E1 protein of alphavirus, baculovirus gp64, hepatitis C virus gp31 or gp70, H protein or F protein of measles virus, or ebola virus gp1 or gp2; and the VSV-G protein may be a wild-type VSV-G protein or a pH-sensitive mutant VSV-G protein, in which the 162nd amino acid, histidine, is substituted with arginine.
The above pharmaceutical composition may further contain one or two or more anticancer compounds.
The anticancer compound may be an immunogenic cell death inducer or an immune checkpoint inhibitor; the immunogenic cell death inducer may be an anthracycline-type anticancer agent, a taxane-type anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, cardiac glycoside, a cyclophosphamide-type anticancer agent, a GADD34/PP1 inhibitor, LV-tSMAC, measles virus, bleomycin, mitoxantrone, or oxaliplatin. The cardiac glycoside may be used in combination with a non-immunogenic cell death inducer; the GADD34/PP1 inhibitor may be used in combination with mitomycin; the anthracycline-type anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin; the taxane-type anticancer agent may be paclitaxel or docetaxel; and the anti-EGFR antibody may be cetuximab. Meanwhile, the immune checkpoint inhibitor may be a PD-1/PD-L1 interaction inhibitor or a CTLA-4/B7-1/B7-2 interaction inhibitor; the PD-1/PD-L1 interaction inhibitor may be an antibody targeting PD-1 or PDL1, or a functional fragment of the antibody, or a single chain-based antibody analog; the CTLA-4/B7-1/B7-2 interaction inhibitor may be an antibody targeting CTLA-4, B7-1, or B7-2, or a functional fragment of the antibody, or a single chain-based antibody analog; the antibody targeting PD-1 or PDL1 may be pembrolizumab, nivolumab, atezolizumab, or avelumab; and the CTLA-4/B7-1/B7-2 interaction inhibitor may be ipilimumab. Meanwhile, the functional fragment of the antibody may be Fab, F(ab′)2, or Fab′; and the single chain-based antibody analog may be scFv, sdAb, diabody, monobody, variable lymphocyte receptor (VLR), nanobody, or llama heavy chain antibody fragment (VHH).
Meanwhile, in the above composition, the anticancer compound may be enclosed inside the plasma membrane-based vesicle, may be simply mixed and formulated, may be packaged separately and mixed immediately before use, or may be administered simultaneously or at staggered time intervals. However, when the immune checkpoint inhibitor is an antibody, a functional fragment of an antibody, or a single chain-based antibody analog, the immune checkpoint inhibitor may be enclosed inside the exosome, but may be co-administered with a recombinant plasma membrane-based vesicle derived from the cells of the invention, or may be presented on the membrane surface of the recombinant plasma membrane-based vesicle to exert the functions. When the antibody, a functional fragment of the antibody, or a single chain-based antibody analog is presented on the membrane surface of the recombinant plasma membrane-based vesicle, the recombinant plasma membrane-based vesicle, in which an immune checkpoint inhibitor according to an embodiment of the present invention is presented on the membrane surface, can be prepared in such a manner that a gene encoding the recombinant plasma membrane-based vesicle is transduced into host cells and expressed to be presented on the membrane surface of the host cells using genetic recombination technology in the same way as the virus-derived fusogenic membrane protein used in the present invention, and then, nano-sized vesicles, which are secreted or budding from these host cells or prepared by artificially processing the cells, are obtained. In this case, the antibody, a functional fragment of the antibody, or a single chain-based antibody analog may be genetically recombined with a separate transmembrane domain or anchoring domain in order to be presented on the cell membrane surface. Optionally, it is also possible to use at first IgM or IgD (i.e., a non-secreting membrane-bound antibody), instead of genetic recombination to have the transmembrane domain or membrane-anchoring domain.
In the case of the composition, the recombinant plasma membrane-based vesicle and the immunogenic cell death inducer may be provided in the form of a pre-mixed composition, may be packaged separately, and mixed and administered immediately prior to use, or administered separately at regular time intervals.
The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations, but is preferably in a parenteral formulation. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrates, surfactants, etc. are commonly used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc. and these solid preparations are prepared by mixing at least one excipient (e.g., starch, calcium carbonate, sucrose or lactose, gelatin, etc.) in one or more compounds. In addition to simple excipients, lubricants (e.g., magnesium stearate, talc, etc.) may also be used. Liquid formulations for oral administration include suspensions, liquid medicine for internal use, emulsions, syrups, etc. and various excipients (e.g., wetting agents, sweeteners, fragrances, preservatives, etc.) may be included, in addition to commonly used simple diluents (e.g., water and liquid paraffin). Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable esters (e.g., ethyl oleate), etc. may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. may be used as a base for suppositories.
The pharmaceutical composition may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.
The pharmaceutical composition of the present invention may be administered orally or parenterally. When the pharmaceutical composition is administered parenterally, it is possible to administer the pharmaceutical composition via various routes (e.g., intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, transdermal absorption, etc.).
The composition of the present invention is administered in a pharmaceutically effective amount.
As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment. Effective dosage levels may depend upon factors including the subject's type, the severity of the disease, the subject's age, sex, drug activity, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, drugs 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, and more preferably at a dose of 1 mg/kg to 500 mg/kg. Meanwhile, the dose may be appropriately adjusted according to the age, sex, and conditions of the patient.
The pharmaceutical compositions of the present invention may be administered either as an individual therapeutic agent or in combination with other anticancer agents, and may be administered sequentially or simultaneously with conventional anticancer agents, and may be administered single or multiple doses. Considering all the above factors, it is important to administer the amount that can obtain the maximum effect in a minimal amount without side effects, and the amount may be easily determined by those skilled in the art.
In addition, according to still another aspect of the present invention, there is provided a recombinant plasma membrane-based vesicle, wherein a VSV-G mutated protein in which the 162nd amino acid, histidine, is substituted with arginine is introduced into the membrane; or the use of a recombinant plasma membrane-based vesicle, in which a virus-derived fusogenic membrane protein is introduced into the membrane, for the preparation of a cancer therapeutic agent.
According to still another aspect of the present invention, there is provided a method for treating cancer in a subject with cancer, which includes administering the recombinant plasma membrane-based vesicle or one or more pharmaceutical compositions among those described above to the subject with cancer.
In the above methods for treating cancer, the subject may be a human or a non-human mammal.
The inventors have prepared a recombinant exosome which presents the VGV-G H162R mutated protein on the surface (see
In addition, the present inventors have conducted various analyses to determine the anticancer mechanism of the recombinant plasma membrane-based vesicle of the present invention, and as a result, have confirmed that the recombinant exosome according to an embodiment of the present invention not only can increase cancer-specific antigen presentation of dendritic cells (
Furthermore, to determine whether the recombinant exosome according to an embodiment of the present invention is dependent on T cell immunity, the present inventors have conducted animal experiments using nude mice lacking T-cell immunity and as a result, have confirmed that the anti-cancer effect disappeared (see
Furthermore, the present inventors have confirmed by experiments that the recombinant exosome including a wild-type VSV-G protein, not a pH-sensitive VSV-G protein, also shows an anticancer activity by inducing T cell-specific immune responses although the effects are slightly less effective. Although it is not possible to present a viral-derived fusogenic membrane protein on the surface of cancer cells in a cancer microenvironment through the fusion of cancer cells with the recombinant exosome, the VSV-G protein itself can act as a TLR4 agonist, and thus it was confirmed that the T cell-specific immune response induced by the recombinant exosome containing the wild-type VSV-G protein in the membrane is the effect induced by activating phagocytes via binding to TLR4 receptors on phagocytes. Therefore, it can be expected that other virus-derived fusogenic membrane proteins in addition to the VSV-G protein may also be able to exhibit an effect similar to that of the wtVSVG exosome or the mVSVG exosome used in the present invention. In fact, it has been reported that a virus-derived fusogenic membrane protein (e.g., the GALV.fus protein derived from gibbon ape leukemia virus) enhances the anticancer effects of oncolytic herpes simplex virus (Fu et al., Mol. Ther. 7(6): 748-754, 2003).
The above results were achieved without using other anticancer agents, and it is expected that the co-administration of the recombinant exosome according to an embodiment of the present invention with other immunogenic cell death inducer(s) (e.g., doxorubicin) will show strong a synergistic effect. Therefore, the recombinant exosome according to an embodiment of the present invention is expected to be very useful for the development of a new anticancer drug that exhibits a strong anticancer activity while minimizing the side effects of the conventional anticancer agents.
In addition, although the anticancer effect was confirmed by using a recombinant exosome in an embodiment of the present invention, the plasma membrane-based vesicles derived from cells (e.g., extracellular vesicles and cell-derived nanovesicles), which are similar in structure to the recombinant exosome, are also expected to exhibit the same function as the recombinant exosome according to an embodiment of the present invention, when the host cell is transformed to include the virus-derived fusogenic membrane protein in the membrane, and then the plasma membrane-based vesicles are prepared from the transformed host cell by a conventional method (i.e., genetic recombination).
Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to these Examples and Experimental Examples disclosed below, but can be implemented in various forms, and the following Examples are provided to make the disclosure of the present invention complete, and to fully convey the scope of the present invention to those skilled in the art.
The present inventors, to prepare a recombinant exosome including a VSV-G H162R mutated protein, first prepared a gene construct encoding a H162R mutated protein, in which histidine, the 162nd amino acid residue of VSV-G (the 178th based on the protein disclosed in GenBank No. CAC47944), is substituted with arginine. Specifically, for this purpose, site-directed mutagenesis was performed using the plasmid DNA (pCMV-VSV-G Envelope Vector, RV-110, Cell Biolabs, abbreviated as “VSV-G construct” hereinafter), which includes the polynucleotide (SEQ ID NO: 2) encoding the wild-type VSV-G protein (SEQ ID NO: 1), as a template along with a forward primer (5′-ATA TGC CCC ACT GTC CGC AAC TCT ACA ACC TGG-3′) of SEQ ID NO: 3 and a reverse primer (3′-TAT ACG GGG TGA CAG GCG TTG AGA TGT TGG ACC-5′) of SEQ ID NO: 4 (the bold codes correspond to the mutated amino acid arginine).
The present inventors transfected the pCMV-VSV-G H162R plasmid vector (abbreviated as “VSV-G H162R construct” hereinafter,
Then, to confirm whether the VSV-G H162R mutated protein is included in the exosome, part of the recovered exosome was disrupted and Western blot analysis was performed using an anti-VSV-G antibody (Abcam, ab50549) and an anti-Alix antibody (exosome marker, Santacruz, sc99010) (
Subsequently, the present inventors, while photographing the recovered recombinant exosome under a transmission electron microscope (
The present inventors prepared, as a Comparative Example, a gene construct to express a wild-type VSV-G protein in which the 162nd amino acid was not mutated, and then prepared a recombinant exosome (wtVSVG-Exo) in which the wild-type VSV-G protein is present in the membrane using the method of Example 2.
For the loading of doxorubicin (DOX) (i.e., an immunogenic cell death inducer) to the recombinant exosome prepared in Example 2 above, purified exosomes (about 1011 exosomes) were first mixed with DOX in 1 mL PBS. Then, the DOX-recombinant exosome mixture was sonicated using the Model 505 Sonic Dismembrator with a 0.25-inch tip under the following settings: 20% amplitude, 6 cycles of 30 seconds on/off for three minutes with a two-minute cooling period between each cycle. After sonication, the Exo-DOX solution was incubated at 37° C. for 60 minutes to recover the exosomal membranes. Excess free drugs are removed by size exclusion chromatography using a NAP-10 Sephadex G25 column (GE Healthcare, Buckinghamshire, UK).
The present inventors attempted to determine whether the recombinant exosome prepared in Example 2 above can be fused with cancer cells in actual in vitro conditions.
For this purpose, specifically, the present inventors added 5×105 cells each of the 4T1-Luc mouse breast cancer cell line, the EL4-Ova lymphoma cell line, and the CT26.CL25 colon cancer cell line to a 1 ml of fusion buffer (1.8 mM NAH2PO4, 8.4 mM Na2HPO4, 10 mM HEPES, 10 mM MES, 2.5 mM NaCl, the pH is adjusted to 7.4, 6.8, or 5.5 with HCl), performed a fusion at 37° C. for 10 minutes along with 50 μg of mVSVG-Exo or wtVSVG-Exo (the exosome prepared using the pCMV-wild-type VSVG plasmid vector), washed the resultant with a culture medium, and then stabilized in the culture medium at 37° C. for hour. In this case, the pH of the fusion buffer was divided into groups of pH 7.4, pH 6.8, or pH 5.5. Then, the degree of fusion of mVSVG-Exo or wtVSVG-Exo on the surface of cancer cells was evaluated through the anti-VSVG antibody staining using a flow cytometer. Experimental results revealed that it was possible for the wtVSVG-Exo to fuse with the surface of the cancer cell membrane, i.e., editing of the cancer cell membrane, at only pH 5.5 for all cancer cells, whereas the mVSVG was able to fuse with the surface of the cancer cell membrane not only at pH 5.5 but also at pH 6.8 of a tumor microenvironment (
Meanwhile, a low-density lipoprotein receptor (LDLR) is most well known as a receptor of a cell membrane, to which VSVG binds to fuse with the cell membrane. As such, the present inventors examined the degree of LDLR expression by Western blot analysis using an anti-LDLR antibody against various cells including cancer cells, with which the recombinant exosome according to an embodiment of the present invention fuses. As a result, as seen in
Then, the present inventors attempted to confirm whether the mVSVG-Exo promotes the fusion between cancer cells after being fused to the surface of cancer cells. For this purpose, specifically, the present inventors stained 5×105 cells each of the 4T1-Luc (i.e., breast cancer cell line), the EL4-Ova (i.e., lymphoma cell line), and the CT26.CL25 (i.e., colon cancer cell line) with Deep red (1 μM) or Green CMFDA (1 μM), and treated with mVSVG-Exo as described in Experimental Example 1 above to perform a fusion under the conditions of pH 7.4 or pH 6.8. Then, 24 hours after seeding each of the resultants in a 35 mm culture dish, the degree of cancer cell fusion (Green CMFDA Signal, Deep red signal double positive cell) was examined by flow cytometry. As a result, as seen in
Then, the present inventors examined whether the mVSVG-Exo itself induces death of cancer cells. For this purpose, specifically, the present inventors performed fusions for the 4T1-Luc (i.e., breast cancer cell line), the EL4-Ova (i.e., lymphoma cell line), and the CT26.CL25 (i.e., colon cancer cell line) by treating each of them with the mVSVG-Exo, the control group exosome (Con-Exo), or a buffer (treatment only with a fusion buffer without an exosome) using a fusion buffer at pH 7.4 or pH 6.8, as described in Experimental Example 1 above. Thereafter, 5×103 cells were seeded in a 96-well plate with a normal medium in each group, and after 24 hours, cell viability was measured by CCK analysis. As a result, as seen in
The present inventors performed a phagocytosis analysis to confirm whether the fusogenic exosome according to an embodiment of the present invention promotes phagocytosis of cancer cells by macrophages and dendritic cells.
Specifically, the present inventors performed fusions for the 4T1-Luc (i.e., breast cancer cell line), the EL4-Ova (i.e., lymphoma cell line), and the CT26.CL25 (i.e., colon cancer cell line) by treating each of them with the mVSVG-Exo, the control group exosome (Con-Exo), or a buffer (treatment only with a fusion buffer without an exosome) using a fusion buffer at pH 7.4 or pH 6.8, as described in Experimental Example 1 above. After editing the membranes of the cancer cells, the cancer cells were stained with pH rodo SE 120 ng/ml. The bone marrow-derived macrophages and bone marrow-derived dendritic cells stained with Green CMFDA (1 μM) and the cancer cells stained with pH rodo SE were co-cultured at a 1:2 ratio for 2 hours. Then, the degree of phagocytosis of cancer cells by macrophages and dendritic cells was confirmed using a fluorescence microscope. As a result of the experiments as seen in
This suggests that the fusogenic exosome of the present invention exhibits an anti-cancer activity based on multiple functions of performing anti-cancer actions by promoting not only fusion of cancer cells but also phagocytosis on cancer cells by macrophages and dendritic cells.
VSV-G protein is known as a TLR4 agonist. As such, the present inventors examined whether the recombinant exosome according to an embodiment of the present invention promotes the maturation of dendritic cells through the TLR4 pathway. For this purpose, specifically, bone marrow-derived dendritic cells (1×106 cells) were seeded in a 6-well culture dish and treated with 500 ng of mVSVG-Exo or 500 ng of Con-Exo for 24 hours. Then, the cells were collected and centrifuged, stained using an anti-CD40 and an anti-CD86 antibodies that reflect dendritic cell maturation, and then the maturation of dendritic cells was evaluated by flow cytometry. As a result of the experiments as seen in
The present inventors examined whether the recombinant exosome according to an embodiment of the present invention can inhibit the growth of cancer cells under in vivo conditions from the results of Experimental Examples 1 to 4 above.
Specifically, the present inventors inoculated cancer cells to 7-week-old Balb/c wild-type mice (females, n=21), in which cancer was induced by subcutaneous inoculation (day 0) on the back, and injected 100 μg of the recombinant exosome (mVSVG-Exo) obtained from Example 2 above or the control group exosome (Con-Exo) into the tumor on the 5th day and the 6th day after the inoculation. In this case, only PBS was administered to the control group. Then, the size of the cancer tissue and the weight of the experimental animals were examined until the 16th day after the cancer cell inoculation at 3 day intervals (
Moreover, the present inventors subcutaneously inoculated EL4-Ova cancer cells (1×106 cells) on the left side of the back of C57BL/6 wild-type mice (day 0) to induce cancer, and then, administered wtVSVG-Exo (100 μg), mVSVG-Exo (100 μg), Con-Exo (100 μg), which are comparative groups, or PBS into the tumor by injection on the 6th day and the 7th day after the cancer cell inoculation. Cancer size and weight of experimental animals were measured at 3 day intervals (
As a result of the experiments as seen in
The present inventors examined the expression level of VSVG proteins on the surface of tumor cells to determine whether the in vivo anticancer effect of the recombinant exosome according to an embodiment of the present invention shown in Experimental Example 5 is due to the mVSV-G protein introduced into the exosome. For this purpose, specifically, the present inventors subcutaneously injected EL4-Ova cancer cells (1×106 cells) into the left side of the back of C57BL/6 wild-type mice to induce cancer, and when the tumor size reached 100 mm3 after the cancer cell injection, administered wtVSVG-Exo (100 μg), mVSVG-Exo (100 μg), Con-Exo (100 μg), or PBS into the tumor by injection. Two hours after the administration, cancer tissues were extracted therefrom and converted into single cells, and flow cytometry accompanied with staining using an anti-VSVG antibody was performed (
These results confirm that the in vivo anticancer activity of the recombinant exosome according to an embodiment of the present invention is caused by the transfer of the VSVG protein into the cancer cell membrane, the VSV-G protein being present in the exosomal membrane, by the fusion of cancer cells and the recombinant exosome in a tumor microenvironment.
7-1: Examination of Effects on Dendritic Cells
The present inventors examined whether the recombinant exosome according to an embodiment of the present invention exhibits an anticancer activity by activating the functions of dendritic cells. For this purpose, specifically, the present inventors subcutaneously injected EL4-Ova cancer cells (1×106 cells) on the left side of the back of C57BL/6 wild-type mice (day 0) to induce cancer, and then, administered wtVSVG-Exo (100 μg), mVSVG-Exo (100 μg), Con-Exo (100 μg), or PBS into the tumor on the 6th day (the day of cancer cell injection is set as day 0) and the 7th day of cancer cell injection. On the 18th day after the cancer cell injection, mice were sacrificed to extract cancer tissue and tumor-draining lymph nodes. Then, to analyze the degree of cancer antigen presentation of dendritic cells, the tumor-draining lymph nodes which were converted into single cells were stained with an anti-CD11c antibody (i.e., a marker for dendritic cells) and an anti-H2Kb-Ova antibody (which can measure the loading of ovalbumin (i.e., a cancer-specific antigen) on MHC-1), and then analyzed by flow cytometry (
7-2: Examination of Effects of CD8 T Cells on Infiltration into Cancer Tissue
The present inventors examined the effects of the recombinant exosome according to an embodiment of the present invention on the infiltration of CD8 T cells into cancer tissue. For this purpose, specifically, the cancer tissue extracted from the experimental animal of Experimental Example 7-1 was embedded in the OCT compound and then frozen sections were prepared, which were stained with an anti-CD8 antibody and the degree of CD8 T cell invasion in cancer tissue was analyzed by a fluorescence microscope (
7-3: Analysis of Cross-Prime Ability
The present inventors examined whether the recombinant exosome according to an embodiment of the present invention has a cross-prime ability to CD8 T cells. For this purpose, specifically, the present inventors subcutaneously injected EL4-Ova cancer cells (1×106 cells) on the left side of the back of C57BL/6 wild-type mice (day 0) to induce cancer, and then, administered wtVSVG-Exo (100 μg), mVSVG-Exo (100 μg), Con-Exo (100 μg), or PBS into the tumor on the 6th day (the day of cancer cell injection is set as day 0) and the 7th day of cancer cell injection. On the 18th day after the cancer cell injection, mice were sacrificed to extract cancer tissue and spleen tissues. Then, to evaluate the cross-prime ability of macrophages and dendritic cells, dendritic cells (CD11c-positive cells) and macrophages (F4/80-positive cells) were isolated from single-celled cancer tissues using F4/80 or CD11c magnetic particles. Then, OT-1 CD8 T cells were isolated from the spleens of OT-1 transgenic mice having OT-1 CD8 T cells capable of recognizing ovalbumin loaded on MHC1 using a CD8 T cell column, and the dendritic cells or macrophages, which were isolated from cancer tissues, and the OT-1 CD8 T cells were co-cultured in a culture medium at a 1:5 ratio for 3 days, respectively. After 3 days, the medium was recovered, and the expression level of INF-γ for each group was analyzed by ELISA assay using an anti-IFN-γ antibody (
7-4: Examination of Immune Memory Ability
The present inventors examined whether the recombinant exosome according to an embodiment of the present invention can trigger cancer-specific immune responses by remembering cancer antigens when the same cancer recurs after cancer treatment is completed. For this purpose, the spleen tissues extracted from the experimental animals of Experimental Example 7-3 above were converted into single cells, and 5×106 of the single cells were seeded in a 12 well culture dish along with a culture medium (1 ml) and treated with PBS or ovalbumin (10 μg/ml), which is a cancer-specific antigen, for 24 hours. Then, the medium was recovered and the expression of IFN-γ in the medium was analyzed for each group by performing ELISA assay using an anti-IFN-γ antibody (
7-5: Identification of Immune Cells Associated with Anticancer Activity of Recombinant Exosome
The present inventors attempted to examine what immune cells the recombinant exosome according to an embodiment of the present invention is dependent on. First, to confirm whether anti-cancer effects can also be exhibited in nude mice lacking T-cell immunity due to enhanced phagocytosis of phagocytes, the present inventors subcutaneously injected EL4-Ova cancer cells (1×106 cells) on the left side of the back of nude mice to induce cancer, and then, administered mVSVG-Exo (100 μg), Con-Exo (100 μg), or PBS into the tumor by injection on the 6th day (the day of cancer cell injection is set as day 0) and the 7th day after the cancer cell injection. Cancer size was measured at 3 day intervals, and on the 18th day after the cancer cell injection, the mice were sacrificed and cancer tissues were extracted therefrom and weighed (
Then, to confirm what kind of T cell immunity among T cell immunities the effect of mVSVG-Exo is dependent on, the present inventors subcutaneously injected EL4-Ova cancer cells (1×106 cells) on the left side of the back of C57BL/6 wild-type mice to induce cancer, and then, administered mVSVG-Exo (100 μg), Con-Exo (100 μg), or PBS into the tumor by injection on the 6th day (the day of cancer cell injection is set as day 0) and the 7th day after the cancer cell injection. Then, to remove CD8 T cells, 150 μg of an anti-CD8 neutralizing antibody was intraperitoneally administered at 3 day intervals starting from one day before the cancer cell injection. Cancer size was measured at 3 day intervals, and on the 18th day after the cancer cell injection, the mice were sacrificed and cancer tissues were extracted therefrom and weighed (
Lastly, the present inventors subcutaneously injected EL4-Ova cancer cells (1×106 cells) on the left side of the back of BATF3 knockout mice lacking CD103 and CD8 dendritic cells, which play the most important role in forming T Cell immunity, to induce cancer, and then, administered mVSVG-Exo (100 μg), Con-Exo (100 μg), or PBS into the tumor by injection on the 6th day (the day of cancer cell injection is set as day 0) and the 7th day after the cancer cell injection. Cancer size was measured at 3 day intervals, and on the 18th day after the cancer cell injection, the mice were sacrificed and cancer tissues were extracted therefrom and weighed (
The above results suggest that the recombinant exosome including the VSV-G H162R mutated protein according to an embodiment of the present invention promotes a specific anticancer immune effect at the pH of a microenvironment of cancer tissue. Such an anticancer effect of the recombinant exosome including the VSV-G H162R mutated protein was achieved without the aid of other anticancer agents, and a significant synergistic effect is expected when other anticancer agents, especially immunogenic cell death inducers, are enclosed therein or co-administered with the same. In particular, when the anticancer agents are incorporated into the recombinant exosome according to an embodiment of the present invention having a membrane structure, the recombinant exosome can deliver the anticancer agents incorporated inside to the inside of cancer cells by a cancer cell-specific fusion, and thus, has an advantage in that the side effects that may occur in the anticancer agents by the actions on normal cells, can be minimized.
Therefore, the recombinant plasma membrane-based vesicle including the recombinant exosome according to an embodiment of the present invention not only has an anti-cancer activity itself, but is also expected to have a stronger anticancer activity in combination with other anticancer agents, and thus, can be very useful for the development of a novel anticancer agent that has a strong anticancer effect while minimizing side effects.
SEQ ID NO: 1 is the amino acid sequence of a wild-type VSV-G protein.
SEQ ID NO: 2 is the nucleic acid sequence of a polynucleotide encoding the wild-type VSV-G protein.
SEQ ID NO: 3 is the nucleic acid sequence of a forward primer used to clone a polynucleotide encoding the wild-type VSV-G protein.
SEQ ID NO: 4 is the nucleic acid sequence of a reverse primer used to clone a polynucleotide encoding the wild-type VSV-G protein.
SEQ ID NO: 5 is the amino acid sequence of the H162R mutated VSV-G protein according to an embodiment of the present invention.
SEQ ID NO: 6 is the nucleic acid sequence of a polynucleotide encoding the H162R mutated VSV-G protein.
SEQ ID NO: 7 is the amino acid sequence in the vicinity of the 162nd amino acid, histidine, of the wild-type VSV-G protein.
SEQ ID NO: 8 is the amino acid sequence in the vicinity of the 162nd amino acid, arginine, of the H162R mutated VSV-G protein.
SEQ ID NO: 9 is the nucleic acid sequence of a polynucleotide encoding a peptide in the vicinity of the 162nd amino acid, histidine, of the wild-type VSV-G protein.
SEQ ID NO: 10 is the nucleic acid sequence of a polynucleotide encoding a peptide in the vicinity of the 162nd amino acid, arginine, of the H162R mutated VSV-G protein.
Although the present invention has been described with reference to Examples and Experimental Examples, these are merely illustrative and will be understood by those skilled in the art that various modifications and other equivalent Examples and Experimental Examples are possible therefrom. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0126202 | Sep 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/011504 | 9/28/2018 | WO | 00 |
