RU2791356C1 - Tumor extracellular vesicles for treatment of organ failure - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the use of a therapeutic composition containing extracellular vesicles obtained from homologous or xenogeneic tumour cells, suspended in an iso-osmotic saline solution, for the treatment of renal failure.

EFFECT: invention provides an increase in the effectiveness of the treatment of renal failure through using extracellular vesicles derived from tumour cells.

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Description

The invention relates to medicine, namely to regenerative medicine. It describes the possibility of using extracellular vesicles of tumor origin in the treatment of organ failure, in particular renal failure.

Phospholipid extracellular vesicles (exovesicles, EVs) are secreted by all cell types. They are present in cell culture supernatants, as well as in all biological fluids (blood, lymph, saliva, breast milk, cerebrospinal fluid, etc.). EVs are divided into two groups depending on their biogenesis and size: exosomes and microvesicles. Exosomes are formed by budding of the endosomal membrane to form multivesicular bodies. These bodies then fuse with the plasma membrane and are released into the extracellular space as exosomes. Unlike exosomes, microvesicles result from direct outward protrusion of the plasma membrane and their subsequent detachment from the cell. The size of exosomes ranges from 50 to 150 nm, while the size of microvesicles can be much larger (50–1000 nm in diameter). Distinguishing between exosomes and microvesicles is technically difficult due to overlapping sizes, common surface proteins, and lack of specific markers (Elsharkasy et al. 2020). Therefore, in this invention, various types of vesicles will be referred to as exovesicles.

It is firmly established that cell activation, as well as cell apoptosis, stimulate the formation and secretion of EVs, which contain proteins, lipids, DNA, and many types of RNA. The concentration of some biomolecules in EVs suggests that the formation of molecular cargo in EVs does not occur randomly, but is an organized process (Elsharkasy et al., 2020). Biomolecules in EVs are protected by a double layer of phospholipids from rapid degradation by extracellular enzymes. Therefore, EVs can show their functional activity not only in the place of their release, but also in remote places. The negatively charged surface of EVs prevents their nonspecific fusion with cell membranes. Expression of the CD47 immunoglobulin molecule on EVs allows them to evade uptake by phagocytic cells (reviewed by de Jong et al., 2019). Compared to liposomes or polymeric nanoparticles, EVs have a much longer blood half-life. Surface receptors expressed on EVs determine the direction and nature of their migration in the body. For example, EVs secreted by inflammatory immune cells express integrins and other adhesive molecules that ensure their preferential migration to inflammatory foci (Tang et al., 2019).

Several studies have shown the ability of EVs to effectively overcome various biological barriers. For example, EVs have been shown to be able to cross the blood-brain barrier, which is a major barrier to drug delivery to the brain (Chen et. al., 2016).

Due to their natural origin, EVs can cause only minimal reactivity from the immune system. Thousands of blood and plasma transfusions are performed daily, resulting in large numbers of EVs being transferred to patients with no apparent side effects. EVs are not able to reproduce on their own, they do not have mutagenic activity. In general, EVs are characterized by low toxicity. Available data show that even the use of xenogenic EVs does not cause serious side effects (Elsharkasy et al., 2020). From a practical point of view, it is also important that EVs can be stored for a long time at –80°C or in a lyophilized form without loss of their biological activity (Frank et al., 2018).

Mesenchymal stem cells (MSCs) are widely studied and used to treat a wide range of diseases (reviewed by Bruno et al., 2013). In various experimental models illustrating respiratory, renal, hepatic, nervous, musculoskeletal, and cardiovascular diseases, it has been shown that EVs produced by MSCs (MSC-EVs) are able to almost fully reproduce the regenerative effects of MSCs. According to published data, MSC-EVs have pronounced anti-inflammatory, anti-apoptotic, proangiogenic, and immunomodulatory properties similar to those demonstrated by MSCs (reviewed by Zhang et al., 2020).

A number of studies have focused on the use of MSCs in the treatment of renal failure. Caused by various causes, acute renal failure (ARF) often leads to chronic renal failure (CRF). About 10-15% of the population suffer from this disease to varying degrees. CRF is often associated with cardiovascular disease and/or diabetes. Experiments on rats have shown that MSC-EVs can improve the function of an injured kidney and inhibit T-cell proliferation in vitro. They improved function and stimulated morphological recovery of the kidney in experimental models of CKD, diabetic nephropathy, and renal fibrosis. It is assumed that the therapeutic effect of MSC-EVs is mediated, at least in part, by the microRNA they contain. Favorable clinical effects of MSC-EV on the damaged kidney may also be associated with exovesicular β, which stimulates the functional activity of regulatory T cells (Zhang et al., 2020). MSC-EVs can also significantly reduce serum markers of kidney failure (urea, creatinine, and transaminases) (Kilpinen et al., 2013).

It is known that mitochondria are not viable in the extracellular space. EVs are able to transport mitochondria and maintain their viability by maintaining the intracellular environment inside them. The possibility of transfection of exovesicular mitochondria into the target cell has been convincingly proven. It has been established that transfected mitochondria can enhance energy production in cells and, possibly, due to this, contribute to the restoration of a damaged organ (Ikeda et al., 2021). The exovesicular transfer of functionally complete mitochondria into senescent cells, aimed at replacing defective mitochondria, forms a new approach to the prevention and treatment of age-related diseases.

A method of treating a patient suffering from a disease or condition caused by stem cell dysfunction or accelerated aging is known, which includes administering to the patient a composition containing extracellular vesicles obtained from mesenchymal stem cells of a healthier or younger subject than the patient (patent WO2017189842 A1, IPC A61K35/28 , published 02.11.2017). It is shown here that MSC-EVs can “rejuvenate” cells at the biochemical and genetic levels. This effect, at least in part, may be due to the transfer of functionally active mitochondria into target cells. Such a transfer could provide "energetic rejuvenation" of target cells.

A known method of treatment using a composition based on microvesicles containing specific miRNAs that can function as intercellular regulators of cell reprogramming or transdifferentiation, as well as stimulators of tissue regeneration, remodeling and reconstruction (WO2012020307 A2, IPC A61K35 / 22, publ. 16.02.2012 ).

MSC-EVs can be used as drug carriers into target cells (WO2016123556 A1, IPC C12P21/00, published 08/04/2016). For example, erythropoietin-treated (i.e., erythropoietin-loaded) MSC-EVs were found to be more effective in protecting the kidneys from fibrosis-related damage compared to untreated MSC-EVs (Wang et al., 2015).

Xenogenic MSC-EVs can also affect the growth and functional activity of target cells (Saleh et al., 2019). Hence, it can be assumed that therapeutic EVs can be obtained not only from human cells, but also from animal cells.

The objective of the invention is to create on the basis of EV an affordable and inexpensive drug that provides effective restoration of damaged organs and tissues of the body.

The technical result of the invention is to increase the effectiveness of the treatment of renal failure due to the use of EVs obtained from tumor cells.

The problem is solved and the technical result is achieved by using a therapeutic composition containing extracellular vesicles obtained from homologous or xenogenic tumor cells, suspended in saline, for the treatment of renal failure.

According to the invention, extracellular vesicles can be obtained from various types of tumors.

According to the invention, the therapeutic composition can be used for the treatment of renal insufficiency.

According to the invention, the therapeutic composition can be used to transfer biomolecules to target cells.

According to the invention, the treatment composition can be used to transfer mitochondria to target cells.

Based on the experimental studies carried out by the authors, it was found that tumor exovesicles (T-EVs) have regenerative properties comparable to those of MSCs and MSC-EVs. This means that tumor-derived EVs are capable of concentrating biomolecules that can stimulate the regenerative activity of various types of normal cells. Thus, these O-EVs can become a universal remedy for the treatment of organ failure caused by disease or injury.

Viable mitochondria were found in the O-EV by confocal microscopy. Therefore, like MSC-EVs, O-EVs can transfer mitochondria into target cells and, thus, carry out their “energy rejuvenation”. This property of O-EV can probably be used to treat age-related mitochondrial diseases such as Alzheimer's disease and Parkinson's disease, as well as primary mitochondrial diseases such as Kearns-Syre syndrome and Melas syndrome, which are currently considered incurable.

O-EV can be obtained not only from human cells, but also from animal cells. By analogy with MSC-EVs, O-EVs can be used as universal vehicles for the delivery of biomolecules of exogenous factors to target cells.

In contrast to the labor-intensive protocol for generating MSCs, which requires the presence of a donor, tumor cells can be easily propagated outside the body. The process for obtaining drug compositions based on O-EV can be adapted for large-scale production. Exovesicular preparations can be stored frozen or lyophilized for a long time. These formulations can be standardized based on physical, chemical and biological properties. Thus, relatively inexpensive drugs based on O-EVs can be widely used to treat common, socially significant, severe diseases, including those characterized by a high mortality rate.

The following are descriptions of experiments that were performed on male CBA mice aged 4 to 6 months.

Acute renal failure (ARF) was induced by a single intramuscular injection of 50% glycerol at a dose of 8.6 mg/kg to mice. Chronic renal failure (CRF) was induced by three intramuscular injections of glycerol.

To generate MSCs, bone marrow was taken from the femur and tibia of intact CBA mice and suspended using a glass homogenizer. The resulting cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, and antibiotics. Cells not attached to the plastic surface were removed during the scheduled medium change starting on day 3. MSCs attached to the plastic had a spindle-shaped fibroblast-like shape. They formed a continuous monolayer by the 4th week of cultivation. MSCs were harvested using 0.25% versene-trypsin solution. Cells were washed twice with cold serum-free medium before further use.

Mouse tumor cell lines (L929 sarcoma, Lewis lung carcinoma (LLC) and B16 melanoma) were maintained and expanded in vitro in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and antibiotics.

To obtain EV MSCs, L929, LLC or B16 cells (1 × 10 ⁶ /ml) were subjected to apoptosis by culturing them in a serum-free medium for 48 hours. After cultivation, cell debris was removed by centrifugation at 2000 g for 15 min, and the resulting supernatant was subjected to centrifugation to precipitate EV at 14000 g for 60 min at 4°C. The pellet was resuspended in saline. The size of EVs expressing annexin V was determined by flow cytometry on a CytoFlex cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) using TruCOUNT (TC) calibration nanoparticles, according to the manufacturer's instructions. The EV concentration was determined by the amount of protein measured by the Bradford method.

In the ARF model, MSCs (2 × 10 ⁶ /mouse) or EVs (30–50 μg/mouse) were administered to mice intravenously (into the retroorbital sinus) one day after the administration of glycerol. Four groups of animals with AKI (10 mice per group) were formed:

1. The control group received no treatment.

2. The group received MSCs.

3. The group received MSC-EV.

4. The group received O-EV secreted by L929 cells (L929-EV).

In the CRF model, MSCs (2 × 10 ⁶ /mouse) or EVs (30-50 µg/mouse) were administered intravenously to mice 3 weeks after their last injection of glycerol. Six groups of mice with CRF were formed:

1. The control group received no treatment.

2. The group received MSCs.

3. The group received MSC-EV.

4. The group received L929-EV.

5. The group received LLC-EV.

6. The group received B16-EV.

The experiments were completed 11 days after the introduction of MSCs or EVs.

For histological examination, the kidneys of mice were fixed in a 4% formalin solution, then they were dehydrated and embedded in paraffin. Sections 4–5 µm thick were made using a rotary microtome (Microm HM 340E; Carl Zeiss, Germany) and stained with hematoxylin/eosin, Sirius red, or Mallory's trichrome. Light microscopy and microphotography were performed using an Axioskop 40 light microscope (Carl Zeiss, Germany). Morphometric analysis of the histological structure of the kidneys was performed on paraffin sections by determining the following parameters: the diameters of the superficial renal glomeruli; the diameters of the collecting tubules of the kidneys and the size of the cells in the middle third of the medullary region. The measurements were carried out in one field of view (eyepiece lens 10×25; objective lens 63×). Statistical analysis of morphometric data was performed using one-way analysis of variance.

Blood creatinine levels were measured using the BioVision, Creatinine (Mouse) ELISA Kit rev 03/18, Catalog Number E4369-100 (Abcam). The determination of FABP1 (fatty acid binding protein-1) in the blood was carried out using the Mouse/Rat FABP1/L-FABP Immunoassay Quantikine® ELISA test system, RnDSystems Catalog Number RFBP10.

Statistical analysis of the data was performed using the Graph Prism 8 program. The significance of differences was assessed using Student's t-test. Differences were considered significant at p <0.05.

The results of the experiments are presented in the images of Fig.1-6, where Fig.1 shows the size of the EV (A - standard particles labeled with annexin V-FITC without EV; B - MSC-EV; C-B16-EV; D-LLC- EV, and E-L929 EV); figure 2 shows the morphological structure of the renal tissue in control mice with CKD (glomeruli, proximal and distal tubules a, collecting tubules and loops of Heney in the medulla b, c); figure 3 shows the morphological structure of the renal tissue in mice with CKD treated with MSC (glomeruli, proximal and distal tubules a, collecting tubules and structures of Heney's loop at the border between the cortex and medullary region b); figure 4 shows the morphological structure of kidney tissue in mice with CKD treated with B16-EV (glomeruli, proximal and distal tubules a, collecting tubules and loop structures of Heney in the middle part of the medulla b); figure 5 shows the morphological structure of kidney tissue in mice with CRF treated with LLC-EV (glomeruli and proximal tubules a, collecting (Bellini) ducts of the papilla b); figure 6 shows the diameter (μm) of the collecting ducts in mice with CRF.

As shown in figure 1, the size of the vast majority of EVs exceeded 100 nm. Thus, most of the EVs used in the described experiments can be classified as microvesicles.

Example 1 Tumor EVs improve kidney function in a model of acute renal failure.

In experiments, serum creatinine was used as a marker of kidney damage, the determination of which is routinely used to calculate the glomerular filtration rate. In mice with AKI, the creatinine level increased from 2.244 to 3.114 ng/ml. Table 1 shows the concentration of creatinine (ng/ml) in the blood serum of mice with acute renal failure. It was found that administration of MSC or MSC-EV to mice with AKI significantly reduced their blood creatinine levels, thus indicating an improvement in renal excretory function. However, the largest decrease in creatine levels was observed in mice treated with L929-EV.

Table 1.

OPN (control) Treatment 3.114 MSC MSK-EV L929-EV 2.676* 2.409* 2.104*

Note: * P< 0.05 compared to control.

The improvement in kidney function caused by therapeutic effects was accompanied by corresponding changes in the morphological structure of the kidneys. However, most of these changes in the ARF model were not obvious. Therefore, comparative studies of the effects of tumor EVs on the functional and morphological characteristics of the kidney were carried out in the CRF model, in which regenerative processes are given more time.

Example 2. Tumor EVs improve the functional and morphological characteristics of the kidneys in the CRF model.

Two functional quantitative parameters were investigated in the CKD model: creatinine and FABP1 (fatty acid binding protein-1). An increase in the production of fat-binding FABP1 is believed to be an early marker of kidney damage. In our experiments, the development of chronic renal failure led to a significant increase in the serum level of FABP1 from 0.8 to 1.085 ng/ml. Table 2 shows the concentration of creatinine and FABP1 (ng/ml) in the blood serum of mice with CRF. As shown in Table 2 , administration of MSCs or MSC-EVs to mice resulted in a decrease in blood levels of both creatinine and FABP1 to almost normal values. A similar effect was also noted in cases of application of tumor (L929, LLC or B16) EVs.

Table 2.

Parameter CRF (control) Treatment MSC MSK-EV L929-EV LLC-EV B16-EV Creatinine 4.818 3.182* 2.767* 2.264* 2.101* 2.095* FABP1 1.085 0.639* 0.780* 0.785* 0.791* 0.781*

Note: * P< 0.05 compared to control.

On histological sections, the development of CRF led to the expansion of the collecting ducts of the kidney (Fig. 2a), cell hypertrophy of the collecting duct (Bellini duct) (Fig. 2b), degeneration of Hentle's loop cells in the middle and lower parts of the medullary region (Fig. 2c ). At the same time , there were no pronounced dystrophic changes in the renal glomeruli in mice that received MSC or MSC-EV. These changes were also not observed in mice treated with O-EV. The pariental and visceral layers of the renal corpuscle (Schumlansky-Bowman's capsule) appeared to be well preserved, and the urinary (Bowman's) space was clearly visible (Figs. 3-5). In general, treatment of mice with CRF with O-EV normalized the morphological structure of the damaged kidney (Fig. 6).

The presented data unequivocally testify to the high ability of tumor EVs to stimulate regenerative processes in normal tissues. This ability seems to be inherent in different tumors, regardless of their origin.

There are concerns about the possible oncogenic properties of O-EV. It is known that O-EVs can enhance tumor growth (Gregory et al., 2018). However, O-EVs do not have mutagenic activity that can directly induce carcinogenesis. O-EVs seem to be able to accelerate the growth of cells that have become tumorigenic as a result of prior carcinogenesis. Our observations for 6 months of mice treated with O-EV did not reveal an increased incidence of cancer in them. However, the safety of repeated long-term administration of O-EV in the treatment of chronic diseases, including age-related disorders, requires further careful study. On the other hand, concerns about the use of O-EV for the treatment of life-threatening conditions should fade into the background, since the therapeutic benefit in such cases outweighs the risks of potential complications. This applies not only to patients with renal insufficiency, but also to patients with severe damage to other organs.

Thus, the invention describes a universal approach to the treatment of diseases associated with damage to internal organs, such as acute renal failure or CKD. This approach is based on the use of EVs of tumor origin. Compared to MSC-derived EVs, tumor EVs are much easier and cheaper to obtain in industrial quantities and it is easier to standardize their biological activity. As drugs, EVs of tumor origin can be used to treat various degenerative diseases in humans and animals.

Literature

1. Elsharkasy OM, Nordin JZ, Hagey DW, de Jong OG, Schiffelers RM, Andaloussi SE, Vader P. Extracellular vesicles as drug delivery systems: Why and how? Adv Drug Deliv Rev. 2020;159:332-343. doi: 10.1016/j.addr.2020.04.004. Epub 2020 Apr 16. PMID: 32305351.

2. de Jong OG, Kooijmans SAA, Murphy DE, Jiang L, Evers MJW, Sluijter JPG, Vader P, Schiffelers RM. Drug delivery with extracellular vesicles: from imagination to innovation. Acc Chem Res. 2019 Jul 16;52(7):1761-1770. doi: 10.1021/acs.accounts.9b00109. Epub 2019 Jun 5. PMID: 31181910; PMCID: PMC6639984.

3. Tang TT, Lv LL, Lan HY, Liu BC. Extracellular vesicles: opportunities and challenges for the treatment of renal diseases. front Physiol. 2019 Mar 19;10:226. doi: 10.3389/fphys.2019.00226. PMID: 30941051; PMCID: PMC6433711.

4. Chen CC, Liu L, Ma F, Wong CW, Guo XE, Chacko JV, Farhoodi HP, Zhang SX, Zimak J, Ségaliny A, Riazifar M, Pham V, Digman MA, Pone EJ, Zhao W. Elucidation of exosome migration across the blood-brain barrier model in vitro. Cell Mol Bioeng. 2016 Dec;9(4):509-529. doi: 10.1007/s12195-016-0458-3. Epub 2016 Jul 7. PMID: 28392840; PMCID: PMC5382965.

5. Milasan A, Farhat M, Martel C. Extracellular vesicles as potential prognostic markers of lymphatic dysfunction. front Physiol. 2020 May 25;11:476. doi: 10.3389/fphys.2020.00476. PMID: 32523544; PMCID: PMC7261898.

6. Frank J, Richter M, de Rossi C, Lehr CM, Fuhrmann K, Fuhrmann G. Extracellular vesicles protect glucuronidase model enzymes during freeze-drying. sci rep. 2018 Aug 17;8(1):12377. doi: 10.1038/s41598-018-30786-y. Erratum in: Sci Rep. 2019 Oct 25;9(1):15702. PMID: 30120298; PMCID: PMC6098026.

7. Bruno S, Collino F, Tetta C, Camussi G. Dissecting paracrine effectors for mesenchymal stem cells. Adv Biochem Eng Biotechnol. 2013;129:137-52. doi: 10.1007/10_2012_149. PMID: 22968371.

8. Zhang B, Tian X, Hao J, Xu G, Zhang W. Mesenchymal stem cell-derived extracellular vesicles in tissue regeneration. Cell Transplant. 2020 Jan-Dec;29:963689720908500. doi: 10.1177/0963689720908500. PMID: 32207341; PMCID: PMC7444208.

9. Kilpinen L, Impola U, Sankkila L, Ritamo I, Aatonen M, Kilpinen S, Tuimala J, Valmu L, Levijoki J, Finckenberg P, Siljander P, Kankuri E, Mervaala E, Laitinen S. Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning. J Extracell Vesicles. 2013 Dec 10;2. doi: 10.3402/jev.v2i0.21927. PMID: 24349659; PMCID: PMC3860334.

10. Ikeda G, Santoso MR, Tada Y, Li AM, Vaskova E, Jung JH, O'Brien C, Egan E, Ye J, Yang PC. Mitochondria-rich Extracellular vesicles from autologous stem cell-derived cardiomyocytes restore energetics of ischemic myocardium. J Am Call Cardiol. 2021 Mar 2;77(8):1073-1088. doi: 10.1016/j.jacc.2020.12.060. PMID: 33632482.

11. Wang Y, Lu X, He J, Zhao W. Influence of erythropoietin on microvesicles derived from mesenchymal stem cells protecting renal function of chronic kidney disease. Stem Cell Res Ther. 2015 May 22;6(1):100. doi: 10.1186/s13287-015-0095-0. PMID: 25998259; PMCID: PMC4469245.

12. Saleh AF , Lázaro-Ibáñez E , Forsgard MA , Shatnyeva O , Osteikoetxea X , Karlsson F , Heath N , Ingelsten M , Rose J , Harris J , Mairesse M , Bates SM , Clausen M , Etal D , Leonard E , Fellows MD , Dekker N , Edmunds N . Extracellular vesicles induce minimal hepatotoxicity and immunogenicity. Nanoscale. 2019 Apr 4;11(14):6990-7001. doi: 10.1039/c8nr08720b. PMID: 30916672.

13. Gregory CD, Dransfield I. Apoptotic tumor cell-derived extracellular vesicles as important regulators of the onco-regenerative niche. Front Immunol. 2018 May 23;9:1111. doi: 10.3389/fimmu.2018.01111. PMID: 29875772; PMCID: PMC5974173.

Claims (5)

1. The use of a therapeutic composition containing extracellular vesicles derived from homologous or xenogeneic tumor cells suspended in an iso-osmotic saline solution for the treatment of renal insufficiency. 2. The use of a therapeutic composition according to claim 1, characterized in that the extracellular vesicles are obtained from various tumors. 3. The use of a therapeutic composition according to claim 1, 2, characterized in that it is intended for the treatment of acute or chronic insufficiency. 4. The use of a therapeutic composition according to claim 1, 2, characterized in that it is intended for the transfer of biomolecules to target cells. 5. The use of a therapeutic composition according to claim 1, 2, characterized in that it is intended for the transfer of mitochondria into target cells.

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