CN115671146B

CN115671146B - Plant-derived extracellular vesicles, use thereof and products comprising same

Info

Publication number: CN115671146B
Application number: CN202211281683.3A
Authority: CN
Inventors: 刘冠宏
Original assignee: Shanghai Fumai Biotechnology Co ltd; Shanghai Shengtejia Health Technology Development Co ltd
Current assignee: Shanghai Fumai Biotechnology Co ltd; Shanghai Shengtejia Health Technology Development Co ltd
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2023-09-19
Anticipated expiration: 2041-03-26
Also published as: CN115671146A; CN113209140A; CN113209140B

Abstract

The invention provides a plant-derived extracellular vesicle, uses thereof and products comprising the same. The extracellular vesicles can act as NK cell activators to trigger NK cells to release proteins, such as perforin and/or granzyme, thereby exerting cytotoxicity. The plant-derived extracellular vesicles of the invention overcome the problem of reduced capability of NK cells to attack cancer cells, enhance the capability of NK cells to attack cancer cells and improve the treatment effect of NK cells on cancer.

Description

Plant-derived extracellular vesicles, use thereof and products comprising same

The present invention is a divisional application of the original application of the present invention having the application of the present invention of 2021, 03, 26, 202110327946.9 and the name of "plant-derived extracellular vesicles, uses thereof and products containing the same".

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a plant-derived extracellular vesicle, application thereof and a product containing the same.

Background

On the surface of natural killer cells (natural killer cell, NK), receptor molecules (KIR) that inhibit NK cell activation are expressed. Since the major histocompatibility complex (major histocompatibility complex, MHC) molecules can bind to KIR, cells expressing MHC molecules can signal and thus not be killed by NK cells. Normal cells always express MHC molecules to indicate themselves, so NK cells do not kill normal cells expressing MHC molecules. If "cancer cells" that do not express MHC proteins appear, NK cells will kill these cancer cells.

NK cell therapy aims to apply this property to cancer treatment. Experience has shown, however, that it is very difficult to treat solid tumors with NK cells. One of the reasons is that the normal NK cell culture method does not activate it well or that many cells of the immune system are depleted due to side effects of anticancer drug treatment due to autologous cell transplantation. This is thought to be the reason why NK cells have a greatly reduced ability to attack cancer cells themselves. In addition, NK cell proliferation agents such as IL-2 (interleukin-2) or IL-15 (interleukin-2) are now used in vitro or in vivo to promote proliferation of NK cells, increase the number of NK cells, and maintain the long-term growth of NK cells in order to increase the therapeutic effect of NK cells. However, NK cells may undergo aging during proliferation, and thus the therapeutic effect of NK cells is poor.

Thus, there is a need to explore an agent that can effectively activate NK cells to treat cancer.

Disclosure of Invention

(one) solving the technical problems

Aiming at the defects of the prior art, the invention provides a novel application of plant-derived extracellular vesicles (extracellular vesicle, EV) and patient NK cells in-vitro co-culture, wherein the plant-derived extracellular vesicles can activate the release of NK cell perforin and granzyme, enhance the capability of the NK cells for attacking cancer cells, and overcome the problem of reduced capability of the NK cells for attacking the cancer cells.

(II) technical scheme

In one aspect, the invention provides an NK cell activator comprising a plant-derived extracellular vesicle.

In another aspect, the present invention provides a composition comprising the NK cell activator of the present invention, and an NK cell proliferation-increasing agent.

In another aspect, the invention provides the use of an NK cell activator of the invention or a composition of the invention for the manufacture of a medicament for the treatment of cancer.

In another aspect, the present invention provides a pharmaceutical composition for treating cancer, comprising the NK cell activator of the invention or the composition of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method for treating cancer comprising administering to a subject an NK cell activator, composition or pharmaceutical composition of the invention.

(III) beneficial effects

Compared with the prior art, the invention has the following beneficial effects:

the cytoplasmic granules of NK cells contain proteins such as perforins and granzymes, which play a central role in killing the cytotoxic activity of cancer cells. Perforin is released by injured cells and pierces the cell membrane of cancer cells, allowing the entry of granzymes and related molecules. Granzymes are serine proteases that induce apoptosis in the cytoplasm of target cells, such as cancer cells. The present invention finds that plant-derived extracellular vesicles can be used to stimulate NK cells to release a large number of proteins (e.g. perforins and granzymes), which are the main functional factors of the cytotoxicity of the NK cells. The plant-derived extracellular vesicles provided by the invention overcome the problem of reduced capability of NK cells to attack cancer cells, enhance the capability of NK cells to attack cancer cells, and enhance the treatment effect of NK cells on cancer.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows an increase in the expression level of granzyme of NK cells;

FIG. 2 shows an increase in the expression level of FAS-L by NK cells;

FIG. 3 shows an increase in the expression level of TNF- α by NK cells;

FIG. 4 shows an increase in the secretion amount of IL-2 by NK cells.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Plant-derived extracellular vesicles

The plant-derived extracellular vesicles provided by the present invention can increase the amount of NK cell-releasing proteins (e.g., perforin and granzyme). These proteins are the main functional factors of NK cell cytotoxicity and are weapons of NK cells to attack cancer cells.

The plant-derived extracellular vesicles provided by the invention can also increase the expression level of TNF family molecules (such as FAS-L, TRAIL, TNF-alpha and TWEAK) on the surface of NK cells. TNF family molecules bind to corresponding receptors expressed on target cells, thereby inducing apoptosis.

The plant-derived extracellular vesicles provided by the invention can also increase the secretion of interleukin-2 (IL-2) by NK cells. IL-2 can activate a variety of immune cells, including B cells, NK cells, LAK cells, monocytes, macrophages and oligodendrocytes.

The plant-derived extracellular vesicles provided by the invention enhance the killing effect of NK cells on cancer cells by one or more of the above actions.

The extracellular vesicles described in the present invention are derived from seaweed. In a specific embodiment, the extracellular vesicles are derived from Phaeophyceae (Phaeophyceae) plants. In a more specific embodiment, the extracellular vesicles are derived from plants of the genus water cloud (Ectecarpus). In a more specific embodiment, the extracellular vesicles are derived from water clouds or sea clouds (Nemacystus decipiens). In a more specific embodiment, the extracellular vesicles originate from an adherent alga commonly known as "mozuku". It is a brown algae which has been consumed worldwide since ancient times, is filiform, has a thickness of about 1 to 3.5 mm and a length of 25 to 40 cm. Characterized in that the surface of the leaf body has adhesiveness, and the viscous component thereof

Fucan (fucoidin) content is about 5 to 8 times that of undaria pinnatifida and kelp. In one embodiment of the invention, the extracellular vesicles are extracted from fucans extracted from seaweed.

In one embodiment of the invention, the extracellular vesicles are prepared by the steps of:

extracting fucan from seaweed,

extracting extracellular vesicles from fucans by ultracentrifugation.

In one embodiment of the invention, the extracellular vesicles may be prepared by the steps of:

-extracting refined low molecular weight power fucoidan from Phaeophyceae plants

Centrifuging the pellet followed by taking the supernatant (no pellet is taken),

the supernatant (with green precipitate, removed) is obtained by ultracentrifugation,

-obtaining plant-derived extracellular vesicles in the supernatant.

extracting fucan from seaweed,

dissolving fucan in PBS,

centrifuging at 2,000Xg for 10 minutes at 4 ℃,

taking the supernatant, centrifuging at 35,000rpm for 70 minutes at 4 ℃,

washing the precipitate with PBS and washing the precipitate with PBS,

again centrifuged at 35,000rpm for 70 minutes at 4 ℃,

the supernatant was discarded, and the supernatant containing extracellular vesicles was obtained by centrifuging the PBS liquid containing the suspension at the bottom at 10,000Xg for 10 minutes at 4℃and removing the green pellet.

In the present invention, the numbers referred to in the preparation steps include the range of the numerical value.+ -. 10%. For example, 10 minutes includes a range of 9 minutes to 11 minutes. Also for example 35,000rpm includes a range of 31,500rpm to 38,500 rpm.

In a preferred embodiment, the fucan has a molecular weight of less than or equal to about 500 daltons, such as less than or equal to 400 daltons, less than or equal to 450 daltons, less than or equal to 460 daltons, less than or equal to 470 daltons, less than or equal to 480 daltons, less than or equal to 490 daltons, less than or equal to 495 daltons, less than or equal to 500 daltons, less than or equal to 505 daltons, or less than or equal to 510 daltons.

Extraction of low molecular weight (below about 500 molecular weight) power fucans from seaweed is known in the art, e.g., as published by the university of ninety, professor Bai. Alternatively, commercially available power fucans (e.g., JAN: 4580123711060) may be used. Thus, in a specific embodiment, the plant-derived extracellular vesicles are extracted from fucans.

In one embodiment of the invention, the extracellular vesicles are exosomes (exosomes).

NK cell proliferation-increasing agent

Plant-derived extracellular vesicles may be used alone or with NK cell proliferation amplificators to activate NK cells. NK cell proliferation amplificants include IL-2 and/or IL-15.

Accordingly, in a specific embodiment, the present invention provides a pharmaceutical composition comprising an NK cell activator of the present invention, and an NK cell proliferation-expanding agent. In a more specific embodiment, the pharmaceutical composition comprises an NK cell activator and IL-2. In another more specific embodiment, the pharmaceutical composition comprises an NK cell activator and IL-15. In another more specific embodiment, the pharmaceutical composition comprises an NK cell activator, IL-2 and IL-15.

Cancer of the human body

The plant-derived extracellular vesicles provided by the invention can be used for treating cancers.

In a specific embodiment, the cancer may be a hematological tumor or a solid tumor.

In a more specific embodiment, the solid tumor may be ovarian cancer, melanoma, breast cancer, gastric cancer, colorectal cancer, relapsed refractory neuroblastoma, merkel cell carcinoma, rectal cancer, lung cancer, prostate cancer, pancreatic cancer, bladder cancer, cervical cancer, cholangiocarcinoma, gastric sarcoma, glioma, osteosarcoma, or brain cancer.

In a more specific embodiment, the hematological neoplasm may be leukemia, myeloma, or lymphoma.

In a more specific embodiment, the leukemia may be Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia or macrogranular lymphocytic leukemia.

In a more specific embodiment, the myeloma may be asymptomatic myeloma, smoky myeloma (SMM), multiple Myeloma (MM), or light chain myeloma.

In a more specific embodiment, the lymphoma may be non-hodgkin's lymphoma, T-cell lymphoma, and B-cell lymphoma.

Pharmaceutical composition

The invention provides a pharmaceutical composition comprising the NK cell activator of the invention, and a pharmaceutically acceptable carrier.

The invention also provides a pharmaceutical composition comprising the NK cell activator and NK cell proliferation-increasing agent of the invention, and a pharmaceutically acceptable carrier.

In a particular embodiment, the pharmaceutically acceptable carrier may be any one or a combination of at least two of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surfactants, pH regulators, antioxidants, bacteriostats and buffers.

Therapeutic method

The present invention provides a method of treating cancer comprising administering to a subject an NK cell activator of the invention.

In a specific embodiment, the invention provides a method of treating cancer comprising: a therapeutically effective amount of plant-derived extracellular vesicles is administered to a patient. The plant-derived extracellular vesicles activate NK cells in vivo, promote the NK cells to secrete perforin, granzyme, TNF family molecules and IL-2 to express or release, and enhance the killing capacity of the NK cells on cancer cells.

The administration route includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

Combination therapy

The NK cell activators of the present invention may also be used in combination with other anticancer drugs. Thus, any of the above pharmaceutical compositions may also comprise other anticancer agents.

The dosage form of the pharmaceutical composition can be injection, tablet, capsule, granule, suspension, emulsion, solution, lyophilized powder, aerosol or microsphere.

In a specific embodiment, examples of other anticancer agents include cisplatin, thalidomide, oxaliplatin, carboplatin, mitoxantrone, doxorubicin, sunitinib, imatinib, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine, semustine, lomustine, streptozotocin, methotrexate, fluorouracil, fluorouridine, gemcitabine, mercaptopurine, thioguanine, pentastatin, cladribine, fludarabine, vinblastine, taxol, docetaxel, etoposide, teniposide, topotecan, irinotecan, daunorubicin, doxorubicin, bleomycin, mitomycin, demethoxydaunorubicin, epirubicin, buserelin, prednisone, hydroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, estrol, taxifene, fluzamide, fluzoxamine, at least one or a combination of two of the foregoing.

In a specific embodiment, the pharmaceutical composition of the present invention and the anticancer drug may be administered simultaneously or sequentially.

Other routes of administration of anticancer agents include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

Examples

Example 1: preparation of plant-derived extracellular vesicles

1. 1 pack of power fucan (JAN: 4580123711060) was transferred into a 50mL tube.

2. It was sufficiently homogenized by 5 passes using a 20 ml syringe (no needle).

3. 1g aliquots were added to 50mL PBS, respectively.

4. Stir with a stirrer for 1 hour.

5. Standing for 15 minutes.

6. 45mL of the supernatant was centrifuged at 2,000Xg for 10 minutes at 4 ℃.

7. The supernatant (without taking the precipitate) was taken.

8. Through a 0.22um filter.

9. Centrifuge at 35,000rpm for 70 minutes at 4 ℃.

10. The pellet was washed with PBS.

11. Again centrifuged at 35,000rpm at 4℃for 70 minutes.

12. The supernatant was discarded and the bottom suspension containing PBS liquid was transferred to a 1.5mL tube.

13. Centrifuge at 10,000Xg for 10 min at 4℃with green precipitate removed.

14. The supernatant was transferred to a new tube to obtain plant-derived extracellular vesicles.

Example 2: effect of plant-derived extracellular vesicles in enhancing NK cell granzyme content

First, the amount of granzyme B of human NK cells (Lonza, poietics. TM. Human NK cells; product code: 2W-501) to which no extracellular vesicles were added was quantified, and set to 1.0. Then, at a concentration of 100 extracellular vesicles per human NK cells, human NK cells were incubated with the plant-derived extracellular vesicles prepared in example 1 for 48 hours, and then relative values of the amount of granzyme B were calculated. A control group was additionally provided. Human NK cells were incubated with DC cell (dendritic) derived extracellular vesicles at a concentration of 100 extracellular vesicles per human NK cell for 48 hours, and then relative values of the amount of granzyme B were calculated. Methods for preparing extracellular vesicles of DC cell origin are known in the art. The amount of Granzyme B was quantified by ELISA using Granzyme B ELISA development kit (human alkaline phosphatase) (product code: 3485-1A-6, cosmo Bio Inc.) following the protocol of the attached specification. The experimental results are plotted in figure 1.

As shown in FIG. 1, the expression of granzyme B in NK cells was increased by more than 8-fold upon addition of plant-derived extracellular vesicles. Whereas the expression of granzyme B in NK cells increased more than 5-fold following addition of DC cell-derived extracellular vesicles (data not shown).

Example 3: action of plant-derived extracellular vesicles to enhance FAS-L expression levels in NK cells

FAS-L is a TNF family molecule expressed on the surface of NK cells that induces cell death by binding to FAS-L receptors expressed on target cells (e.g., cancer cells). Thus, it was quantitatively analyzed whether plant-derived extracellular vesicles increased the expression level of FAS-L in human NK cells. For the quantification of FAS-L, human soluble FasL ligand was quantified by colorimetry using ELISA kit (FasL, soluble ELISA kit; cosmo Bio product code: ALX-850-246-KI 01). The method follows the attached specification.

First, the FAS-L amount of human NK cells to which no extracellular vesicles had been added was quantified with a kit, and this value was set to 1.0. Then, at a concentration of 100 extracellular vesicles per human NK cells, human NK cells were incubated with the plant-derived extracellular vesicles prepared in example 1 for 72 hours, and then the relative value of FasL amounts was calculated. A control group was additionally provided. Human NK cells were incubated with DC cell-derived extracellular vesicles at a concentration of 100 extracellular vesicles/human NK cells for 72 hours, and then the relative value of FasL amount was calculated. The experimental results are plotted in figure 2.

As shown in FIG. 2, the expression of FAS-L in NK cells was increased 3-fold after addition of plant-derived extracellular vesicles. After addition of DC cell-derived extracellular vesicles, FAS-L expression in NK cells was increased 1.9-fold (data not shown).

Example 4: action of plant-derived extracellular vesicles to enhance TNF-alpha expression levels in NK cells

Similar to FAS-L, TNF- α, which is a TNF family molecule, binds to TNF- α receptors expressed on target cells (e.g., cancer cells), thereby inducing cell death. Thus, it was quantitatively analyzed whether extracellular vesicles increased the expression level of TNF- α in human NK cells. TNF- α was quantified by a sandwich method on a 96-well plate coated with a capture antibody by ELISA kit (human TNF- α assay ELISA kit; cosmo Bio product code: KE 00068). The method follows the attached specification when implemented.

First, the amount of TNF-. Alpha.in human NK cells without extracellular vesicles was quantified with a kit and set to 1.0. Then, at a concentration of 100 extracellular vesicles per human NK cells, human NK cells were incubated with the plant-derived extracellular vesicles prepared in example 1 for 48 hours, and then the relative values of TNF- α amounts were calculated. A control group was additionally provided. Human NK cells were incubated with DC cell-derived extracellular vesicles at a concentration of 100 extracellular vesicles/human NK cells for 48 hours, and then the relative value of TNF-. Alpha.amount was calculated. The experimental results are plotted in figure 3.

As shown in FIG. 3, TNF- α expression in NK cells was increased 3.4-fold upon addition of plant-derived extracellular vesicles. After addition of DC cell-derived extracellular vesicles, TNF- α expression in NK cells was increased 2.3-fold (data not shown).

Example 5: plant-derived extracellular vesicles enhance IL-2 secretion in NK cells

Interleukin 2 (IL-2) is the primary immunomodulatory cytokine produced by T cells in response to antigen stimulation and mitogen activation. Signaling through the IL-2 receptor pathway is important for T cell proliferation and provides other necessary functions for normal immune responses. IL-2 signals through the IL-2 receptor complex. IL-2 also activates various immune cells including B cells, NK cells, LAK cells, monocytes, macrophages and oligodendrocytes. IL-2 is the primary cytokine widely used in therapeutic prescriptions. Thus, it was quantitatively analyzed whether plant-derived extracellular vesicles increased IL-2 expression levels of human NK cells. For the quantification of IL-2, human soluble IL-2 was quantified by sandwich method on a 96-well plate coated with a capture antibody using ELISA kit (human IL-2 assay ELISA kit; cosmoBio product code: KE 00017). The method follows the attached specification.

First, the amount of IL-2 in human NK cells to which no extracellular vesicles had been added was quantified with a kit and set to 1.0. Then, at a concentration of 100 extracellular vesicles per human NK cells, the human NK cells were incubated with the plant-derived extracellular vesicles prepared in example 1 for 48 hours, and then the relative value of the amount of IL-2 was calculated. A control group was additionally provided. Human NK cells were incubated with DC cell-derived extracellular vesicles at a concentration of 100 extracellular vesicles/human NK cells for 48 hours, and then the relative value of the amount of IL-2 was calculated. The experimental results are plotted in fig. 4.

As shown in FIG. 4, the amount of IL-2 secreted by NK cells was increased 2.9-fold after addition of plant-derived extracellular vesicles. After addition, the amount of IL-2 secreted by NK cells was increased 1.8-fold (data not shown).

Discussion of the invention

Compared to the control group without any extracellular vesicles added and the control group with DC cell-derived extracellular vesicles added, the plant-derived extracellular vesicles of the present invention can significantly increase the release amount of NK cell proteins (including perforin and granzyme), and the expression amount of TNF family molecules and IL-2. Therefore, NK cells are effectively activated, so that the problem of reduced capability of attacking cancer cells by the NK cells is solved, the capability of attacking the cancer cells by the NK cells is enhanced, the treatment effect of the NK cells on the cancer is enhanced, and the cancer cells can be specifically inhibited.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition comprising an NK cell activator, and an NK cell proliferation-increasing agent,

the NK cell proliferation amplification agent comprises IL-2 and/or IL-15;

the NK cell activator comprises an extracellular vesicle derived from seaweed prepared by the steps of:

dissolving fucoidan extracted from seaweed in PBS,

centrifuging at 2,000Xg for 10 minutes at 4 ℃,

taking the supernatant, centrifuging at 35,000rpm for 70 minutes at 4 ℃,

washing the precipitate with PBS and washing the precipitate with PBS,

again centrifuged at 35,000rpm for 70 minutes at 4 ℃,

2. Use of the composition of claim 1 for the manufacture of a medicament for the treatment of cancer.

3. A pharmaceutical composition for treating cancer, comprising the composition of claim 1, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is in the form of an injection, a tablet, a capsule, a granule, a suspension, an emulsion, a lyophilized powder, an aerosol, or a microsphere;

the pharmaceutical composition also includes other anticancer drugs; the other anticancer drug is selected from cisplatin, thalidomide, oxaliplatin, carboplatin, mitoxantrone, doxorubicin, sunitinib, imatinib, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine, semustine, lomustine, streptozotocin, methotrexate, fluorouracil, floxuridine, gemcitabine, mercaptopurine, thioguanine, penstatin, cladribine, fludarabine, vinca alkaloid, vincristine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, irinotecan, daunorubicin, bleomycin, mitomycin, demethoxydaunorubicin, epirubicin, buserelin, prednisone, hydroxyprogesterone caproate, medroxyprogesterone acetate, diethylstilbestrol, ethinyl alcohol, moxifene, anastrozole, fluzamide, bortezomib, and combinations of at least one or two of any of them.