WO2022203548A1 - Method for delivering granzyme b into mammalian cells - Google Patents

Abstract

The invention relates to the field of pharmaceutics, and more particularly to a method for delivering drugs into mammalian cells using a nanocontainer containing granzyme B. The claimed nanocontainer for the targeted transport of granzyme B is capable of binding to the surface of a tumour cell and delivering the cytotoxic agent human granzyme B to trigger apoptosis in the cell. Also proposed is a method for producing a liposomal dosage form for transporting granzyme B into mammalian cells.

Description

METHOD FOR DELIVERY OF GRANZYME B TO MAMMALIAN CELLS

DESCRIPTION

The invention relates to the field of pharmaceuticals, in particular to methods for delivering drugs into a mammalian cell. A method is proposed for delivering granzyme B protein to a mammalian cell to initiate a cytotoxic reaction in it. Variants of nanocontainers for targeted transport of granzyme B are also proposed. The nanocontainer has the ability to bind to the surface of a tumor cell and deliver the cytotoxic agent granzyme B to a person to initiate apoptosis in it, which makes it possible to effectively treat oncological diseases characterized by the expression of targeted antigens.

The invention also relates to the field of passive immunotherapy of oncological diseases, in particular to the use of an immunopreparation, which may be an antibody specifically recognizing an antigen present on a tumor cell, associated with a nanocontainer containing human granzyme B.

Several signaling cascades and proteins are associated with the initiation of cell apoptosis. Initiation of these signaling cascades is possible upon delivery of special activators to the cytoplasm of cells. Accordingly, it is possible to carry out the delivery of special molecules to activate cell death in order to eliminate unwanted cells, which may be tumor cells. The delivery of such molecules can be carried out in various ways using special carriers. Among such carriers, nanocontainers, nanoparticles and CAR-T cells are known.

The claimed invention proposes a new method for delivering granzyme B to a mammalian cell using a container, unlike previously known delivery routes, granzyme B is enclosed inside a nanocontainer capable of penetrating inside the cell, after which granzyme B molecules leave the container. The nanocontainer is then broken down in the lysosome, releasing the contents, including granzyme B molecules, into the lysosome. Depending on the properties of the container, granzyme B molecules can also leave it by diffusion.

At present, many preparations based on nanocontainers have been created.

The basic principle of creating such a drug is to use safe and biodegradable materials from which the nanocontainer wall is built.

The nanocontainer wall may consist of phospholipids, pure carbon, various polymers, cyclodextrins, polyamidoamine dendrimers, metals and their oxides, and

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SUBSTITUTE SHEET (RULE 26) other materials that make it possible to form a structure with sizes from 9 to 1000 nm.

Nanocontainers contain a cavity inside, which can contain various cytostatic agents, including doxorubicin [Green A.E., Rose P.G., 2006]. Such nanocontainers can be used for the treatment of oncological diseases such as ovarian cancer. For a more specific effect on the tumor and to achieve greater drug safety, either whole antibodies or antigen-recognizing antibody fragments can be placed on the surface of the nanocontainer [Matsumura Y., 2003].

There are also known options for creating nanoparticles for the transfer of drugs on their surface. Nanoparticles can be created on the basis of graphene, diamond or gold. The surface of such nanoparticles can be modified to hold drug molecules on it.

Another approach for the therapy of oncological diseases is based on the use of CAR-T cells - lymphocytes, into which constructs are introduced that are designed to express chimeric receptors that bind to a tumor cell [Pehlivan K.S., 2018]. This development increases the likelihood of T-cells and tumor cells approaching and increases the cytotoxic activity of the T-lymphocyte, resulting in tumor cell lysis. Despite the wide variety of drugs based on nanocontainers, and especially CAR-T cells, the use of these drugs does not always allow the patient to be completely cured, which is largely due to the shortcomings of these approaches.

The disadvantages of preparations enclosed in nanocontainers include the limited possibility of packaging cytostatic preparations, which are often poorly soluble compounds. As a result, each individual nanocontainer contains relatively few molecules of the active substance. Since the main purpose of the use of cytostatic drugs is to stop the proliferation and induce the death of tumor cells, the ingestion of a small amount of the drug becomes ineffective due to the binding of a large number of drug molecules to the structural components of the tumor cell, which leads to minor consequences for its vital activity. On the other hand, increasing the concentration of a chemotherapy drug can make the dosage form more toxic to healthy tissues. At the same time, the risk of introducing mutations into normal cells with the subsequent development of new oncological diseases increases. Finally, the use of a chemotherapy drug in a container or in combination with a nanocarrier increases the cost of therapy compared to using the same chemotherapy drug in its conventional form.

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SUBSTITUTE SHEET (RULE 26) The production of CAR-T cells, despite the high efficiency in some cases, is a very complex technology that has to be reproduced anew with each new request for therapy. The basis of CAR-T cells is usually the lymphocytes of the patient himself. In cases where the patient has received chemotherapy, the ability to proliferate and cytotoxicity of his T-cells is markedly reduced, which affects the quality of the resulting CAR-T-cells. The use of donor cells can lead to consequences such as graft-versus-host disease caused by partial incompatibility in the repertoire of HLA molecules. The CAR-T cell vaccine does not have a long shelf life as it is a live vaccine. CAR-T cells can be inactivated by the tumor when exposed to tumor PDL1 and PDL2 molecules from the PD1-modified lymphocyte. In addition, the development of an undesirable phenomenon of a “cytokine storm” caused by hyperactivity of CAR-T cells is possible. Although each of these problems has a solution, the removal of CAR-T cells from the body in the event of adverse effects can be difficult, and the overall cost of CAR-T cell therapy, compared with conventional chemotherapy, remains high. An almost insurmountable problem is the production of a large number of CAR-T cells for administration to a patient, since this requires time, which may often not be enough during the progression of the disease. Finally, the use of vectors to deliver genetic material to CAR-T cells can, on the one hand, lead to the immortalization of these cells, and, on the other hand, become a source of genetic material for recombination with viruses that are in a latent state in the patient's genome. As a result, a leukemia-like disease or the emergence of a new type of viral infection may occur.

The main role in the development of cytotoxic reactions of CAR-T cells is played by the granzyme B protein contained in granules in the cytoplasm. This small and highly soluble protein is a protease that activates procaspases 8, 10, 7, and 3, which effectively activates apoptosis [Afonina I.S., 2010]. The appearance of active caspase-3 in the cell leads to its rapid death by apoptosis. Cancer cells that are resistant to chemotherapy can also undergo apoptosis. Granzyme B is quite stable and can be stored for a long time. Thus, the properties of granzyme B allow it to be used as a cytotoxic agent for the treatment of oncological diseases.

To activate apoptosis, granzyme B must enter the cell. During cell lysis, the T-lymphocyte secretes perforin proteins, which form pores in the membrane of the target cell. Through this pore, granzyme B enters the cytoplasm. In the absence of perforin, granzyme B remains in the extracellular space. Granzyme B not

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SUBSTITUTE SHEET (RULE 26) can perforate the cell membrane, while remaining safe for normal cells and ineffective for eliminating tumor cells. The activation of granzyme B also requires the protein cathepsin. Cathepsin is mainly expressed in cells infected with viruses or in tumor cells, but is not active in normal cells. For this reason, granzyme B that enters the cytoplasm of non-tumor or non-infected cells will not be activated and will not trigger apoptosis. Unlike chemotherapy drugs, granzyme B is not a mutagen, as it does not interact with cell DNA. Granzyme B is also unable to carry out cell transformation according to the mechanism observed in hormonal carcinogenesis.

It is noteworthy that in some, only one molecule of granzyme B per cell is enough to trigger apoptosis. In the case of chemotherapy drugs, the number of molecules is in the hundreds or more per cell. Most chemotherapy drugs affect a small range of cancers. Granzyme B is not selective in its action and can activate apoptosis in tumor cells of any origin. Thus, human granzyme B may be both more effective and safer than chemotherapy drugs. Since Granzyme B is highly soluble in neutral pH solutions, any solutions that are harmless to humans with a pH value close to 7.0 can be used.

Granzyme B does not have the ability to independently pass through the lipid membranes of cells, and for its use as a drug, it is necessary to develop a method of delivery.

One known solution for transporting granzyme B into the cell is to create a duplex of granzyme B and a transport peptide. US9724378B2 proposes binding a granzyme B molecule to one of a variety of peptides capable of binding to and passing through the cell membrane. Thus, granzyme B located in the extracellular space can pass through the cell membrane together with the peptide. The advantages of this approach make it possible to create a dosage form of granzyme B that penetrates into the cell and triggers apoptosis. The disadvantage is the non-selectivity of action, as a result of which the duplex of granzyme B and the peptide will penetrate into all cells, including healthy ones, and stimulate their death.

Another well-known solution describes a polymer sphere delivered to a tumor cell by antibodies and releasing granzyme B in close proximity to the tumor cell membrane. Transportation of granzyme B into the cell itself occurs with the help of a peptide that facilitates its penetration through the cell membrane. The main disadvantage is the low stability of the polymer sphere and the risk of penetration of granzyme B into a non-tumor cell. To avoid this,

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SUBSTITUTE SHEET (RULE 26) it is necessary to create such a carrier that would deliver granzyme B directly to the tumor cell.

The idea of the present invention is to create a drug similar in action to CAR-T cells, but at the same time devoid of their disadvantages. Granzyme B-encapsulated nanocontainers and granzyme-targeted molecules are potentially easier to manufacture and store, have no histocompatibility issues with the recipient, and "cytokine storm" side effects are easier to manage. Since such a construct does not have its own genome, there is no problem of "leftover" CAR-T cells, which themselves can cause autoimmune diseases.

The objective of this study is to create a dosage form of granzyme B in a nanocontainer that will allow the delivery of the drug to the cytoplasm of a tumor cell without the need to involve perforin, without the risk of damaging normal cells and stimulating the development of tumor diseases, while being convenient to manufacture, easily stored and introduced into the body. However, the method of production of human granzyme B can be any, including, but not limited to, its isolation from lymphocytes, or production in the form of a recombinant protein in genetically modified bacterial or plant organisms.

The technical result of the invention is the delivery of a granzyme into a cell, in particular, into a tumor cell, followed by activation of apoptosis and the launch of a cytotoxic reaction.

The proposed method for delivering granzyme B can be used when a nanocontainer is introduced into the bloodstream of cancer patients. Tumor tissue is characterized by the phenomenon of increased vascular permeability, which makes tumor tissue a preferred site for nanocontainers smaller than 600 nm containing granzyme B. Having come into contact with a tumor cell, the nanocontainer fuses with the cell membrane, releasing granzyme B into the cytoplasm. Once in the cytoplasm, granzyme B interacts with procaspases 8, 10, 7, and 3, which activates apoptosis [Afonina I.S., 2010]. Otherwise, the nanocontainer can be captured by the tumor cell membrane and transported inside it by endocytosis. Once in the cytoplasm, the nanocontainer meets the lysosome and breaks down, releasing the contents. Otherwise, after penetration into the cytoplasm, the nanocontainer can passively release granzyme B. In any way, the released granzyme B can activate apoptosis.

To achieve the technical result of the invention, it does not matter which of the currently known variants of nanocontainers to use,

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SUBSTITUTE SHEET (RULE 26) suitable for introduction into the human body. It is possible to assemble different types of nanocontainers or nanocarriers containing granzyme B.

Some of the most available materials for obtaining a nanocontainer and molecules, the use of which will give the nanocontainer additional properties, are listed in Table 1.

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SUBSTITUTE SHEET (RULE 26) Tab. one.

As a material for assembling a nanocontainer, proteins that make up the viral capsid and their analogs can be used. These proteins have the ability to self-assemble at a pH value close to neutral. During self-assembly, a sphere or icosahedron is formed with a shape close to spherical, with a cavity inside. An example would be the capsid of the hepatitis A virus, which is assembled from the proteins VP1, VP2, and VP3. Due to this property, it is possible to produce large quantities of capsid proteins as monomers and mix them with granzyme B at a pH of about 6.0. At these pH values, proteins are in a dissociated form. When the pH is brought to neutral values, self-assembly of capsids will occur, and part of the granzyme B will be captured in

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SUBSTITUTE SHEET (RULE 26) inner cavity. Proteins VP1, VP2 and VP3 can be modified so that molecules for targeted delivery can be attached to their sides located on the outer edges of the capsid. Another well-known example is the canine parvovirus (CPV) protein capsid, which has an affinity for canine and human transferrin receptors [Singh R., 2009]. CPV capsid proteins also have the ability to self-assemble and can be used to deliver small molecules into the cell, including granzyme B.

Another possible way is to create spheres from a hydrophobic polymer of polyethylene vinyl acetate. Thus, a dosage form of epirubicin, packaged in polyethylene vinyl acetate spheres, has already been created [Hassanzadeh F., 2016]. This drug is planned to be used in the treatment of breast cancer. Granzyme B molecules can also get stuck between polymer fibers and be gradually released into the extracellular environment, or into the cell cytoplasm if polyethylene vinyl acetate particles are delivered inside the cell. Transportation into cells is possible due to the attachment of antibodies in the polyethylene vinyl acetate particle that bind to the internalizing cell receptor. Such a receptor can be CD22, CD7, TNFR, and others.

Another possible way is to form nanoparticles from a branched carboxylated poly-P-aminoether [Rui Y., 2019]. Protein particles are held in such a nanoparticle through the formation of hydrogen bonds and salt bridges with polymer threads. Granzyme B can also be retained in this nanoparticle and delivered to the cell cytoplasm. The polymer mostly remains undelivered to the lysosomes, which allows proteins to be released directly into the cytoplasm.

Another possible way is to create a granzyme B nanocarrier, in which the nanocarrier is a copolymer of lactic and glycolic acids. Granzyme B molecules can be attached to this carrier to activate apoptosis and antibodies to be delivered to target cells. In addition, granzyme B molecules can get stuck between the fibers of the copolymer of lactic and glycolic acids and gradually be released from them after penetration into the cytoplasm of cells. Delivery into cells can be carried out using internalizable receptors, to which the antibody from the nanocarrier binds. At present, complexes of the Herceptin antibody and nanoparticles of the copolymer of lactic and glycolic acids are already known [Guo Y., 2018].

Another possible way is to create a nanocontainer from cyclodextrins. Cyclodextrins are cyclic oligosaccharides, the molecules of which are built from six to eight d-glucopyranose units linked

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SUBSTITUTE SHEET (RULE 26) 1,4-glycosidic bond with each other. Cyclodextrins have the shape of a truncated cone with a cavity inside. Along the circumference of the lower base of cyclodextrins are 6-8 primary OH groups, and along the circumference of the upper base - 12-16 secondary hydroxyl groups. Previously, the cytotoxic protein saporin was successfully packaged in a cyclodextrin container [Jiang Y., 2018]. Granzyme B can also be retained in the internal cavity. Attached antibodies can also be used for targeted delivery of cycle o-dextrins with granzyme B.

Another possible way is to create a nanocarrier from fullerenes, in particular from C60 fullerene, graphene macromolecules, carbon nanotubes or nanodiamond molecules [Mendes R., 2013]. Synthesis of fullerene or nanotubes can be carried out in various ways, including flame synthesis, arc discharge synthesis, laser ablation, and chemical vapor deposition. Some of the surface carbons can be oxidized with a mixture of potassium permanganate and sulfuric acid. Granzyme B molecules and antibodies for targeted delivery can be attached to the resulting hydroxyl groups. To date, nanotubes are already used as carriers for monoclonal antibodies and cyclic RGD peptides [Mizejewsk G.J., 1999] [Chakravarty R., 2008]. Nanodiamonds have also been successfully bound to albumin molecules. The resulting nanodiamond-albumin complex was successfully captured by HeLa cells [Tzeng Y.K., 2011].

Another possible way is to create a nanocarrier from metals or their oxides, in particular, the creation of gold particles. Protein molecules can be attached to the surface of a gold nanoparticle due to the formation of covalent and non-covalent bonds [Tian L., 2017]. Because granzyme B is a protein, it can also be attached to the surface of the gold particle. In addition to granzyme B, antibodies for targeted delivery can also be attached.

Another possible way is to form a nanocontainer, which is a liposome. The wall of the liposome may, for example, be composed of lipids. Liposomes can be prepared by evaporating the lipid-containing solvent and coating the resulting lipid film with a granzyme B solution. The liposomes can then be sonicated, extruded, or otherwise processed to obtain their optimum size. The surface of liposomes can be modified with polyethylene glycol to increase their stability and create a binding site for antibodies intended for targeted delivery. An example of successful protein packaging in a liposome is Lipovaca Influenzal Vaccine, an influenza vaccine. In this preparation, in the cavity of the liposome

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SUBSTITUTE SHEET (RULE 26) the influenza virus proteins hemagglutinin and neuraminidase are retained.

Immunoliposome constructs for the delivery of doxorubicin have also been successfully created [Sokolova D.V., 2011]. Packaging of granzyme B into liposomes may be one of the most convenient ways to create a nanocontainer for transporting this protein.

The liposomes delivered to the cytoplasm of cells often fuse with the lysosome, and the content of the liposomes is degraded by the low pH and the complex of enzymes contained in the liposomes. Granzyme B can quickly become unstable in the lysosome and must therefore be protected. This is possible by creating pH-sensitive liposomes, the wall of which responds to a decrease in pH. The liposome, which contains, for example, N-acylphosphatidylethanolamine, fuses with the lysosome membrane when it enters the lysosome, and the contents of the liposome are released into the cytoplasm of the cell. Polyethylene glycol also imparts similar properties to the liposome. If substances from the group of unsaturated phosphatidylethanolamines, such as diacetylene-phosphatidyl-ethanolamine, palmitoyl-oleoyl-phosphatidyl-ethanolamine and dioleoyl-phosphatidyl-ethanolamine, are embedded in the wall of the liposome, such a liposome, if it enters the lysosome, can destroy both its own membrane, and the lysosome membrane. In this case, both granzyme B and the enzymes contained in the lysosome will be released into the cytoplasm of the cell. The release of lysosomal enzymes will not prevent granzyme B from performing its main task.

Another way to create a container is based on the use of oligonucleotides. Although oligonucleotides are linear molecules, they are able to bind to each other due to the complementary interaction of nitrogenous bases. Part if one oligonucleotide is complementary to only part of the other oligonucleotide, unbound strands will remain. Due to this, the formation of branched structures is possible. The structure of several oligonucleotides can be chosen in such a way that they will form three-dimensional structures with a cavity inside. Sequential addition of such oligonucleotides to a solution of granzyme B can lead to the capture of the latter into the cavity. Antibodies can be attached to the surface of the resulting granzyme B container for targeted delivery to the desired cells. Oligonucleotides are convenient in that they are capable of self-assembly at temperatures below 50°C. At lower temperatures, the structures of oligonucleotides are stable. Once in the cytoplasm of the cell, the nucleotide wall of the container will be attacked by nucleases and release the contents.

Nanocontainers and nanoparticles for the delivery of human granzyme B into the tumor cell cytoplasm have not yet been developed as

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SUBSTITUTE SHEET (RULE 26) medicinal product and have never been produced. In one of the works, vesicles with granzyme B packed in them were created as a model for studying the ability of granzyme B to spontaneously pass through the membrane [Besenicar M.R., 2008]. In a paper where granzyme B is presented as a promising drug for tumor therapy, it is proposed to create a chimeric granzyme combined with other proteins [Hlongwane

R., 2018].

World experience shows that packaging of granzyme B into liposomes can be carried out quite simply. Previously, experiments were carried out to create liposomes with proteolytic enzymes packaged in them. So, in 1988, Alkhalaf and colleagues made a liposomal proteinase, which was used to accelerate the ripening of cheese [el Soda M., 1988]. The procedure involved preparing a solution of 400 mg of proteinase in 20 ml of Tris buffer pH 7.0. This solution was used to capture the enzyme in liposomes. A thin lipid film was produced, which was dissolved in 40 ml of diethyl ether, and mixed with the proteinase solution, shaken, and the ether was evaporated at +30°C with occasional manual shaking. To separate liposomes from unbound protein, centrifugation at 100,000 g was used according to the protocol proposed by Piard et al. [Piard J.-C., 1986]. Since proteinase and granzyme B are similar in terms of size, mass and solubility, the proposed method can be used to create a liposomal form of granzyme B. However, new equipment has been developed recently to facilitate the construction of liposomes. In this regard, a new protocol for the assembly of the liposomal form of granzyme B was developed.

As one of the embodiments of the invention, the packaging of granzyme B into liposomes by the Bengham method is shown. The method is based on obtaining a thin lipid film, which is formed by removing the solvent from the previously obtained organic lipid solution by distillation under vacuum on a rotary evaporator. The dissolution of all precisely measured components of the bilipid layer is carried out in non-polar organic solvents, in which they dissolve well. If the mass of all components is carefully weighed, then the volume of the solvent is not so important for the final result, since the solvent is completely evaporated during operation. The liposomes obtained as a result of such an assembly can be easily sorted by size and purified from granzyme B not included in them.

In one of the possible variants of the assembly of liposomal granzyme B, weighings of a mixture of lipids (egg or soy phosphatidylcholine, cholesterol, mPEG2ooo-DSPE and pNP-PEG3000-DOPE in the range of molar ratios (6-7):(2-4):(0, 005- 0.014): (0-0.01) in 2 ml of solvent (chloroform, either in 95% ethyl alcohol, or deionized water)

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SUBSTITUTE SHEET (RULE 26) was loaded into a round bottom flask of a BLfCHI Rotavapor R-200 rotary evaporator with a BiiCHI Heating Bath B-490 water bath (BLJCHI Labortechnik AG, Switzerland) or a Laborota 4003 control rotary evaporator (Heidolph Instruments GmbH, Germany). A film was formed in a rotary evaporator by evaporating the solvent at 37 ± 1 °C using egg lecithin (Avanti Polar Lipids, Ins; USA) or 40 ± 1 °C when obtaining liposomes from soy lecithin (Avanti Polar Lipids, Ins; USA) in vacuum conditions until a translucent lipid film is formed. The resulting film was dried under vacuum (120 mbar) to constant weight. Then the film was hydrated with granzyme B dissolved in PBS buffer or in deionized water at a concentration of 10 mg/mL. The rotor speed was 35-40 rpm.

The resulting dispersions were successively passed through nylon membrane filters (Pall Corporation, USA or Pall Eurasia LLC, Russia) with a pore diameter of 1.2 µm (1 time), 0.45 µm (1 time) and 0.22 µm (1- Five times). Analysis of the average diameter of the obtained liposomes and assessment of their size distribution was performed using the method of correlation light scattering spectroscopy (dynamic laser light scattering) on a Nicomp 380 Submicron Particle Sizer device. To do this, 10 μl of liposomal dispersion was added to a cuvette for a nanosizer (Nicomp 380 Submicron Particle Sizer (Particle Sizing Systems, USA)) and deionized water (FS 42-2619-98) was added to 1 ml, after which the cuvette was placed in the device and measurement began. . All computational operations were performed on a computer in automatic mode using the Nicomp PSS WC370 MFC Application 1.0.001 program.

The chromatographic method can be used to purify liposomes. To do this, 1 ml of water for injection was applied to a chromatographic column (NAP-5 (Amersham Biosciences, Sweden)) filled with Sephadex (G-25 Medium (Amersham Biosciences, Sweden)). After the passage of water, about 1 ml of the dispersion was slowly applied to the dry surface of the column. At the exit from the column, 2 fractions were obtained: fraction I - liposomal granzyme B (cloudy colored liquid), fraction II - granzyme B not included in liposomes (transparent colored solution). The change of fractions was fixed visually.

To determine the content of granzyme B in liposomes, one can use the method of spectrophotometry on a Cary 100 instrument (Varian, Inc., Australia) using a working standard sample (RS) of granzyme B at a wavelength of 279 ± 2 nm.

Measurement of the optical density of alcoholic solutions of liposomes with granzyme B and CO granzyme B can be carried out relative to 95% ethanol in cuvettes with a thickness

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SUBSTITUTE SHEET (RULE 26) optical layer 10 mm. The content of granzyme B (X, mg) was calculated by formula (1).

X = (D*a*C)/(D ₀ *Co)(l) where D is the optical density of the sample solution; Do is the optical density of CO granzyme B; C is the dilution value of the sample; Co - the value of the dilution of CO granzyme B; a - SS sample of granzyme B, mg.

The efficiency of incorporating granzyme B into liposomes (B, %) was calculated using the formula

(2).

B = (Di*Ci*Vi)/(D*C*V) (2) where Di is the optical density of the fraction solution with purified liposomal granzyme B; D is the optical density of the initial liposomal dispersion solution; Ci is the dilution value of the fraction with purified liposomal granzyme B; C - dilution value of the original liposomal dispersion; Vi - fraction volume with purified liposomal granzyme B, ml; V is the volume of the initial liposomal dispersion applied to the column, ml.

The delivery of granzyme B to the tumor cell can be made more efficient by modifying liposomes. Modifications make it possible to give them the ability to selectively bind to the surface of tumor cells and penetrate into them. This can be achieved by attaching to the surface of liposomes structures that bind to antigens located on the surface of predominantly tumor cells. Among these antigens may be proteins CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD 10, CDlla, CDl lc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD28, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD44, CD45, CD51, CD52, CD56, CD62L, CD70, CD79b, CD80, CD95, CD117, CD125, CD137, CD138, CD 147, CD242, CD319 , glycophorin A, immunoglobulin light and/or heavy chain antigens, T-cell receptor, ACVR2B, VEGF, VEGFR, VEGFR2, PCSK9, HLA-DR, BCMA, BAFF, EGFR, FGFR2, EpCAM, RANKL, GD2, SLAMF7, CCR4, CEA, DPP4, RTN4, PDGFRa, Notch 1, HER2, B7-H3, Nectin-4, ICAM-1, CTLA4, PD-L1, PD-L2, SOST, CSF1R, MUC1, VWF, DLL4, MUC5AC, RANKL, DR5 , AXL, HER3, NKG2A, MCP-1, PDCP1, PD l, MCAM, PTK7, IGHE, TFPI, TfR, GCGR, CCR2, PCSK9, PRAME and others. Structures that can bind these antigens can be antibodies, including human, murine, chimeric, humanized, rat, camel and other

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SUBSTITUTE SHEET (RULE 26) antibodies or their fragments containing antigen-recognizing sites, or human proteins capable of binding to the listed antigens according to the ligand-receptor principles. Such a dosage form, the so-called immunoliposomal form, has a significantly higher affinity for the tumor cell, and the probability of successful penetration of granzyme B into the cytoplasm increases.

The immunoliposomal form of granzyme B can be used to treat diseases such as liver hemangioma, rectal cancer, skin melanoma, uveal melanoma, uterine tumors, prostate tumors, larynx cancer, gastric cancer, skin cancer, bone cancer, lung cancer, brain cancer, pituitary tumors , bladder cancer, liver cancer, esophageal cancer, kidney cancer, ovarian cyst, prostate cancer, breast sarcoma, chondrosarcoma, salivary gland cancer, cervical cancer, thyroid cancer, testicular cancer, ovarian cancer, acute myeloid leukemia, acute lymphoid leukemia, chronic B-cell lymphoid leukemia, chronic T-cell lymphoid leukemia, hairy cell leukemia, multiple myeloma, chronic myeloid leukemia, Burkitt's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, splenic marginal zone cell lymphoma, zone cell lymphoma mantle, T-cell lymphomas of the skin, myelodysplastic syndrome and others.

The immunoliposomal form of granzyme B can be prepared according to a similar protocol for the creation of the liposomal form of granzyme B at the stages from the formation of the lipid film to its hydration with a solution of PBS or water. In the composition of the lipid composition, instead of PNP-PEG3000-DOPE, the pNp-PEG3ooo-Hpid component in comparable molar ratios should be used. At the loading stage, a solution of granzyme B in 10 sM HEPES buffer pH 8.2-8.4 is added to the resulting empty liposomes. After that, the pH of the dispersion is adjusted to 8.5-8.6 with 3M NaOH solution. Then incubation is carried out in a water bath at a temperature of +45°C for 1.5 hours. After that, a solution of the necessary monoclonal antibodies is added to the dispersion in a molar ratio of antibody/pNp-PEOzooo-Hpry 1:40. This is followed by incubation for 12 hours at +4°C with constant stirring. Purification of liposomal dispersion from granzyme B not included in granzyme B is carried out by column chromatography according to the protocol proposed for the creation of liposomal granzyme B. Quantitative determination of the content of granzyme B inside immunoliposomes and evaluation of the efficiency of its encapsulation is carried out according to protocols similar to those proposed for the creation of liposomal granzyme B.

Liposomes and immunoliposomes are able to deliver granzyme B to the target cell, fuse with its membrane and release the contents inside. Thus granzyme B

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SUBSTITUTE SHEET (RULE 26) enters the cytoplasm, where it can activate apoptosis. The action of immunoliposomes is potentially more selective. The release of granzyme B from the liposomal form is possible only in the case of direct destruction of liposomes or fusion of liposomes with the tumor cell membrane. In a living person, conditions are relatively stable, and the liposomal form of granzyme B will be virtually immune to spontaneous degradation.

The resulting drug potentially has significant cytotoxic activity, and the possibility of using the antibody for targeted delivery allows it to be used in almost any oncological and oncohematological diseases. Each individual component currently used in the creation of liposomes and immunoliposomes is non-toxic. Combinations of these components with each other are also non-toxic. Unlike chemotherapy drugs, the resulting drug will not have a mutagenic effect. Liposomes and immunoliposomes with granzyme B are easy to dose when administered to patients. The drug is easy to store in a soluble and lyophilized form, since granzyme B is a stable protein. Due to this, liposomal granzyme B can be accumulated in large quantities and stored, used when needed, which is not achievable in the case of CAR-T cells.

Modifications of the primary structure of granzyme B are also possible to enhance its properties. The cytotoxicity of granzyme B, delivered to the tumor cell in one way or another, can be increased by making changes to the protein molecule. Tumor cells can express protease inhibitor 9 (IP-9), which blocks the functions of granzyme B. Granzyme B used in the dosage form can be synthesized by replacing lysine, located at position 27 of the protein, with alanine [Sun J., 2011] and arginine at position 201 of the protein to lysine or alanine [Losasso V., 2012].

In cases where tumor cells express cathepsin at a low level, or do not express it at all, granzyme B that has entered their cytoplasm may remain in an inactive form and will not be able to trigger apoptosis. To increase the effectiveness of the drug, you can use the mutant granzyme B, which does not require cathepsin activation from the very beginning. For this, glycine and glutamic acid, located at positions 19 and 20 in the immature protein, must be removed in the structure of granzyme B. On the other hand, before granzyme B can be activated by the cathepsin protein even before packaging into liposomes.

Granzyme B can be produced in a variety of ways. Thus, it can be isolated from natural killer or T-killer granules. Granzyme B can also be produced in recombinant form in bacterial cells. Dedicated from

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SUBSTITUTE SHEET (RULE 26) bacteria, recombinant granzyme B must be refolded, for which detergent-free refolding buffer solutions are suitable.

For convenience, an active cathepsin-independent form of recombinant granzyme B can be created immediately. For this, sequences for encoding 6 histidine residues and a site with the DDDDK sequence for cleavage of a protein recognized, for example, by enterokinase, are introduced into the structure of the gene to be expressed in bacteria.

Currently, granzyme B may be commercially available, but can be produced under laboratory conditions in a bacterial strain.

The invention is illustrated by the following examples.

Example 1 Assembly of Liposomal Granzyme B.

Liposomal granzyme B was assembled according to our proposed protocol. To form the film, the reagents egg phosphatidylcholine, cholesterol, and mPEG2ooo-DSPE were used in molar ratios of 7:2:0.005, dissolved in deionized water. The film was hydrated with granzyme B dissolved in 2 ml of deionized water. The average diameter of the resulting liposomes was 0.22 µl. Purity - about 98%. The incorporation efficiency was 24% of the initial weight of the granzyme B protein used.

Example 2 Assembly of Liposomal Granzyme B Using pNP-PEG3ooo-

DOPE.

Liposomal granzyme B was assembled according to our proposed protocol. To form the film, the reagents egg phosphatidylcholine, cholesterol, mPEG2ooo-DSPE and pNP-PEG3ooo-DOPE in molar ratios of 7:3:0.014:0.01, dissolved in deionized water, were used. The film was hydrated with granzyme B dissolved in 2 ml of deionized water. The average diameter of the resulting liposomes was 0.22 µl. Purity - about 99%. The incorporation efficiency was 25% of the initial weight of the granzyme B protein used.

Example 3 Assembly of immunoliposomal granzyme B with antibody 5D3.

Assembly of immunoliposomal granzyme B was carried out according to our proposed protocol for liposomal granzyme with the addition to create

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SUBSTITUTE SHEET (RULE 26) immunoliposomes. Soy phosphatidylcholine, cholesterol, mPEG2ooo-DSPE, pNp-PEG3ooo-lipid and in molar ratios of 6:4:0.014:0.01 dissolved in chloroform were used to form the film. The film was hydrated with granzyme B dissolved in 10 mM HEPES buffer pH 8.2. Monoclonal humanized 5D3 antigen-recognizing antibodies were added to the liposome dispersion.

PRAME. The average diameter of the resulting liposomes was 0.22 µl. Purity - about 99%.

The incorporation efficiency was 13% of the initial weight of the granzyme B protein used.

Example 4 Assembly of immunoliposomal granzyme B with anti-CD117 antibody.

Assembly of immunoliposomal granzyme B was carried out according to our proposed protocol for liposomal granzyme with an addition to create immunoliposomes. The film was formed using reagents soy phosphatidylcholine, cholesterol, mPEG2ooo-DSPE and pNp-PEG3ooo-lipid in molar ratios of 7:3:0.01:0.014, dissolved in 95% ethanol. The film was hydrated with granzyme B dissolved in 10 mM HEPES buffer pH 8.4. Monoclonal mouse antibodies ICO-406 recognizing the CD117 antigen were added to the liposome dispersion. The average diameter of the resulting liposomes was 0.22 µl. Purity - about 98%. The incorporation efficiency was 14% of the initial weight of the granzyme B protein used.

Example 5 Cytotoxicity of immunoliposomal granzyme B against mel melanoma cells Cat.

C0117-expressing tumor cells of the mel Kot line (metastatic melanoma) in the amount of 50*10 ³ in 180 μl of RPMI medium containing 10% fetal calf serum were planted in the wells of a 96-well plate. The cells were incubated for 24 hours at +37°C in an atmosphere containing 5% CO2. After that, a series of dilutions of immunoliposomal granzyme B conjugated with an antibody against CD 117 antigen were added to the wells in a volume of 20 μl per well. The dilutions were 2-fold starting from a concentration of 5 mg/ml of granzyme enclosed in liposomes. Empty immunoliposomes were used as controls, which were assembled without the addition of granzyme B. There were 9 dilutions in total, three repetitions each. Cells with immunoliposomal granzyme B were incubated for 24 h at +37°C in an atmosphere containing 5% CO2. To assess cytotoxicity, an MTT test was performed according to the standard method. It was found that the cells of the mel Kot line died after

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SUBSTITUTE SHEET (RULE 26) incubation with immunoliposomal granzyme B. The higher the concentration of immunoliposomal granzyme B, the greater the proportion of cells died. When incubated with empty liposomes, mel Koh cells did not die (Fig. 1).

Example 6 Cytotoxicity of immunoliposomal granzyme B against SK-BR-3 breast cancer cells.

PRAME-expressing tumor cells SK-BR-3 (breast cancer) in an amount of 50*10 ³ in 180 μl of RPMI medium containing 10% fetal calf serum were planted in the wells of a 96-well plate. The cells were incubated for 24 hours at +37°C in an atmosphere containing 5% CO2. After that, a series of dilutions of immunoliposomal granzyme B conjugated with an antibody against the PRAME antigen were added to the wells in a volume of 20 μl per well. The dilutions were 2-fold starting from a concentration of 5 mg/ml of granzyme enclosed in liposomes. Empty immunoliposomes were used as controls, which were assembled without the addition of granzyme B. There were 9 dilutions in total, three repetitions each. Cells with immuliposomal granzyme B were incubated for 24 h at +37°C in an atmosphere containing 5% CO2. To assess cytotoxicity, an MTT test was performed according to the standard method. It was found that SK-BR-3 cells died after incubation with immunoliposomal granzyme B. The higher the concentration of immunoliposomal granzyme B, the greater the proportion of cells died. When incubated with empty liposomes, SK-BR-3 cells did not die (FIG. 2).

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SUBSTITUTE SHEET (RULE 26)

Claims

CLAIM

1. A method for delivering granzyme to a mammalian tumor cell using a container, characterized in that granzyme B is enclosed inside a nanocontainer capable of penetrating inside the cell, after which granzyme B molecules leave the container and enter the cytoplasm.

2. the method according to claim 1, wherein the container enters the cell by endocytosis.

3. the method of claim 1, wherein the container is broken down in the lysosome, releasing granzyme B.

4. The method of claim 1, wherein the granzyme B molecules leave the container by diffusion.

5. The method according to claim 1, wherein the granzyme B, which has entered the cell, initiates the apoptosis reaction.

6. the method according to claim 1, where granzyme B, which has penetrated into the cell, initiates a cytotoxic reaction.

7. The method of claim 1 wherein granzyme B has N-terminal domain modifications.

8. The method according to claim 1, in which granzyme B has mutations that reduce its sensitivity to protease inhibitors.

9. Nanocontainer for delivering granzyme B to a mammalian cell, characterized in that it penetrates the cell through the cell membrane, releasing granzyme B inside the cell.

10. Nanocontainer according to claim 9, which is capable of being destroyed in lysosomes.

11. Nanocontainer with granzyme B according to item 9, capable of penetrating into the cell through endocytosis.

12. Nanocontainer with granzyme B according to item 9 capable of merging with the cell membrane.

13. Nanocontainer with granzyme B according to item 9, capable of independently passing through the cell membrane.

14. Nanocontainer with granzyme B according to paragraph 9 with dimensions from 1 to 999 nm, preferably up to 600 nm.

15. A nanocontainer with granzyme B according to claim 9, having a surface on which targeted delivery molecules can be placed that can bind to antigens located on the surface of predominantly tumor cells.

16. Nanocontainer with granzyme B according to item 13, which, using molecules for targeted delivery, interacts with antigens on the cell surface selected from the list: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CDl la, CDl lc, CD13, CD14, CD15, CD 16, CD 18, CD 19, CD20, CD21, CD22, CD23, CD25, CD28, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD44, CD45, CD51, CD52, CD56 , CD62L, CD70, CD79b, CD80, CD95, CD117, CD125, CD137, CD138, CD147, CD242, CD319, glycophorin A, lung antigens

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SUBSTITUTE SHEET (RULE 26) and/or immunoglobulin heavy chain, T-cell receptor, ACVR2B, VEGF, VEGFR,

VEGFR2, PCSK9, HLA-DR, BCMA, BAFF, EGFR, FGFR2, EpCAM, RANKL, GD2, SLAMF7,

CCR4, CEA, DPP4, RTN4, PDGFRa, Notch 1, HER2, B7-H3, Nectin-4, ICAM-1, CTLA4, PD-

Ll, PD-L2, SOST, CSF1R, MUC1, VWF, DLL4, MUC5AC, RANKL, DR5, AXL, HER3,

NKG2A, MCP-1, PDCP1, PD-1, MCAM, PTK7, IGHE, TFPI, TfR, GCGR, CCR2, PCSK9,

PRAME.

17. A nanocontainer with granzyme B according to item 13, on which molecules for targeted delivery that bind antigens on the surface of a tumor cell are represented by structures selected from the list: antibodies, antigen-binding fragments, molecules containing antigen-recognizing sites, proteins capable of binding with antigens according to ligand-receptor principles.

18. Nanocontainer with granzyme B according to item 9, the walls of which are made of at least one type of material selected from the list: peptide molecules, oligonucleotides, polyethylene vinyl acetate, branched carboxylated poly-b-amino ester, copolymers of lactic and glycolic acids, cyclodextrins, carbon, gold , iron oxide, lipids.

19. Nanocontainer with granzyme B according to item 16, the walls of which additionally contain antibodies, proteins, ligands for membrane receptors, polyethylene glycol.

20. Granzyme B nanocontainer according to item 16, in which the container wall contains molecules selected from the list: phosphatidylcholine, cholesterol, mPEG2ooo-DSPE, pNp-PEG3ooo-lipid, proteins, peptides, polyethylene vinyl acetate, lactic acid, glycolic acid, o-dextrins cycle , allotropic modifications of carbon, gold.

21. A method for producing liposomes loaded with granzyme B protein, including obtaining a lipid bilayer dissolved in chloroform, dispersing it with a proteinase solution, shaking and evaporating, characterized in that to obtain a lipid bilayer, the components dissolved in a non-polar solvent are taken in the following ranges of molar ratios: phosphatidylcholine (6 to 7): cholesterol (2 to 4): mPEG2ooo-DSPE (0.005 to 0.014), and optionally: PNP-PEG3000-DOPE (0.0001 to 0.01).

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