RU2781640C2 - Method for targeted impact on exosomes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to the field of medicine, namely to a method for in vitro targeted impact on extracellular vesicles with phosphatidylserine exposed on the surface. The specified method includes a stage of introduction of a molecule containing a gamma-carboxyglutamic acid component (hereinafter – GLA-component), wherein the specified GLA-component contains GLA-domain or its active fragment and does not contain an active catalytic domain of GLA-protein, into liquid, which may contain an extracellular vesicle. In this case, the specified GLA-domain or its active fragment are independently selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z, osteocalcin, GLA-matrix protein, GAS6, transthyretin, periostin, GLA 1 enriched in proline, GLA 2 enriched in proline, GLA 3 enriched in proline, and GLA 4 enriched in proline.

EFFECT: present invention provides the use of molecules for detection of extracellular vesicles expressing phosphatidylserine, isolation of extracellular vesicles, targeted impact on extracellular vesicles, and/or loading of vesicles, for example, with therapeutic materials.

26 cl, 30 dwg

Description

CROSS REFERENCES TO RELATED APPLICATIONS

The present invention claims priority under U.S. Provisional Application No. 62/554,530 filed September 5, 2017, U.S. Provisional Application No. 62/554,533 filed September 5, 2017, U.S. Provisional Application No. 62/569,403 filed Oct. 569,411, filed October 6, 2017, U.S. Provisional Application No. 62/584,565, filed November 10, 2017, U.S. Provisional Application No. 62/593,014, filed November 30, 2017, each of which is incorporated herein by reference in its entirety.

SEQUENCE LIST

This application contains a sequence listing submitted electronically via EFS-web, which serves both as a paper copy and as a computer-readable form (CRF), and consists of a file called "ST-CT7-PCT_sequence.txt", created on September 5, 2018, having 9,823 bytes in size and incorporated herein by reference in its entirety.

[0001] The present invention relates to a method for targeting extracellular vesicles, which uses a molecule containing the GLA domain, and extracellular vesicles that are obtained or can be obtained by the method described herein.

BACKGROUND OF THE INVENTION

[0002] Extracellular vesicles (also known as microvesicles or microparticles) have historically been considered a carrier medium for removing waste from cells. However, it has recently been established that, in fact, extracellular vesicles are involved in vital intercellular communication.

[0003] It has been shown that extracellular vesicles can be obtained from almost all mammalian cells, including healthy cells, stem cells, and diseased cells, such as cancer cells. Extracellular vesicles are extremely stable and can exist in virtually all biological fluids, including blood plasma, saliva, urine, bile, synovial fluid, semen, and breast milk.

[0004] It is believed that diseased cells, such as cancer cells, use extracellular vesicles, such as exosomes, to prepare and seed metastatic sites. Cells infected with a pathogen can use extracellular vesicles to spread the infection. In addition, bacterial cells are known to release extracellular vesicles.

[0005] There is great interest in the study, understanding and use of these extracellular vesicles, especially involved in pathological processes. It is assumed that these vesicles can be used in therapy as an alternative to stem cells.

[0006] However, there are certain practical difficulties because vesicles are small and present in vivo at low concentrations . In addition, there are a small number of markers that distinguish extracellular vesicles derived from diseased cells from extracellular vesicles derived from normal cells. Another complication is that these small objects are in vivo in a complex environment, including a mixture of biological molecules, factors, ions, minerals, etc. Therefore, even the isolation and/or monitoring of these extracellular vesicles is a difficult task.

[0007] Vesicle isolation may require seven or more steps, with size typically being the primary parameter used. Because the diameter of extracellular vesicles can be less than 300 nm, and because they have a low refractive index, many currently used methods do not allow detection of extracellular vesicles. To simplify the analysis of extracellular vesicles, a number of miniature systems have been developed using nanotechnologies and microfluidic technologies. These new systems include a micro-NMR device, a nanoplasmonic chip, and a magneto-electrochemical sensor for protein profiling; as well as an integrated liquid cartridge for RNA detection. Flow cytometry is an optical method for detecting extracellular vesicles in suspension. However, the applicability of flow cytometry to the detection of single extracellular vesicles is still limited due to limited sensitivity and potential measurement artifacts such as detection of clusters. Other methods for detecting single extracellular vesicles are atomic force microscopy, nanoparticle tracking analysis, Raman microspectroscopy, tunable resistive pulse detection, and transmission electron microscopy.

[0008] In addition to being able to isolate extracellular vesicles for study and/or diagnosis, it is believed that these extracellular vesicles may be useful as natural vehicles for loading, targeting, and/or treating a number of diseases. However, there are currently serious problems regarding the implementation of the possibility of loading useful substances and fragments for targeted impact:

• into these extracellular vesicles -

например, белков, нуклеиновых кислот (как природных, так и неприродных олигонуклеотидов), низкомолекулярных соединений, ферментов, зондов для определения содержимого внеклеточных везикул для терапевтических, диагностических/прогностических целей и т.д.;

for example, proteins, nucleic acids (both natural and non-natural oligonucleotides), small molecules, enzymes, probes for determining the content of extracellular vesicles for therapeutic, diagnostic/prognostic purposes, etc.;

• and on these extracellular vesicles -

для разработки, изменения или усиления адресного воздействия, дисплея терапевтических белков, мечения и, например, сборки внеклеточных везикул для анализа или манипуляции,

for the development, modification or enhancement of targeted action, display of therapeutic proteins, labeling and, for example, the assembly of extracellular vesicles for analysis or manipulation,

• it has been shown that some miRNAs penetrate into vesicles, but so far there is no reliable mechanism for incorporating the material into the vesicles,

например, повреждение в результате электропорации, введения реагентов для трансфекции может снизить коммерческую применимость внеклеточных везикул,

for example, damage from electroporation, administration of transfection reagents may reduce the commercial utility of extracellular vesicles,

неэффективная трансдукция вирусными векторами, загрузка или выделение с помощью средств, изменяющих внеклеточные везикулы, также могут снизить диагностический или терапевтический потенциал и т.д. (обзор в Vader et al., Adv Drug Delivery Rev 106: 148-156, 2016, Sutaria et al., Pharm Res 34: 1053-1066, 2017, Lu et al., Eur J Pharm and Biopharm 119: 381-395, 2017),

inefficient transduction with viral vectors, loading or isolation with extracellular vesicle modifying agents may also reduce diagnostic or therapeutic potential, etc. (reviewed in Vader et al., Adv Drug Delivery Rev 106: 148-156, 2016, Sutaria et al., Pharm Res 34: 1053-1066, 2017, Lu et al., Eur J Pharm and Biopharm 119: 381-395 , 2017),

в качестве реагентов для повышения проницаемости внеклеточных везикул также предлагали применять сапонины. Однако сапонины обладают сложной биологической активностью, в том числе гемолитической. Фракции сапонинов Quil A и QS-21 используются в качестве вакцинных адъювантов для усиления иммунного ответа на антиген. Поэтому сапонины нельзя использовать непосредственно.

saponins have also been proposed as reagents for increasing the permeability of extracellular vesicles. However, saponins have complex biological activity, including hemolytic. Saponin fractions Quil A and QS-21 are used as vaccine adjuvants to enhance the immune response to antigen. Therefore, saponins cannot be used directly.

[0009] Third, it may be useful to target pathogenic material and signaling molecules in extracellular vesicles to reduce the spread of the pathological process.

[0010] In the present description, the above problems are considered.

[0011] Surprisingly, these extracellular vesicles released from pathogenic cells such as cancer cells, bacterial cells, etc. contain phosphatidylserine exposed on the surface. Exposed phosphatidylserine can, for example, suppress immune responses to "pathogenic" vesicles. Regardless of theory, "pathogenic extracellular vesicles" may be characterized by the presence of exposed phosphatidylserine in contrast to normal healthy extracellular vesicles that do not contain surface-exposed phosphatidylserine.

[0012] Phosphatidylserine can be targeted by GLA components containing a GLA domain without a catalytic domain. These molecules can be used to target vesicles derived from apoptotic cells, eg pathological, diseased/infected cells.

[0013] Even more surprisingly, the present inventors have also demonstrated that GLA components also bind stem cells (eg healthy stem cells). Regardless of theory, these cells can present phosphatidylserine on their surface, which can contribute to the immunosuppressive and inflammatory effects of stem cells.

[0014] Extracellular vesicles derived from cells with surface-exposed phosphatidylserine also contain phosphatidylserine exposed on their outer surface. Thus, GLA components can also be used to target extracellular vesicles from stem cells.

[0015] The GLA domain may be associated with or attached to a payload, such as a label, bead, diagnostic molecule, targeting motif, and/or therapeutic agent. This allows you to isolate, identify, track and/or perform therapeutic intervention on or through these extracellular vesicles.

[0016] Alternatively or in addition, the payload may be a therapeutic, e.g., a drug, a biological therapeutic agent, a polymer, or a toxin, e.g., a therapeutic virus, an oncolytic virus, a viral vector, an antiviral drug, an antibacterial drug, an antiparasitic agent. , an anti-cancer drug, an anti-cancer therapeutic agent, or a chemotherapeutic agent, a virus, or a viral vector (eg, an oncolytic virus).

[0017] Thus, GLA components can be used to fix the payload on the surface of extracellular vesicles through the GLA domain binding phosphatidylserine exposed on the surface.

[0018] In addition, the authors of the present invention have data to suggest that the GLA component of the present invention can carry the payload attached to it inside the extracellular vesicle. Thus, the GLA component can be used to deliver a payload into an extracellular vesicle.

[0019] This is important for therapeutic and/or diagnostic purposes, as known and existing methods and effector/reporter molecules can be repurposed and used for monitoring, isolation, and therapeutic intervention via vesicles.

[0020] Once labeled, the vesicles can be monitored, for example, in vivo , or isolated using known methods, such as flow cytometry, magnetic sorting, and the like.

[0021] In addition, it becomes apparent that the presence of specific types of vesicles can be used as a means of non-invasive diagnosis of certain pathologies.

[0022] In addition, given the suggestion that vesicles are used to seed and promote metastasis in cancer, killing, removing, or targeting these vesicles with a therapeutic agent may be a way to prevent or attenuate metastasis; for example, RNA interference can transport nucleotides into extracellular vesicles to knock out active miRNAs carried in the vesicle.

[0023] Vesicles isolated from infected cells, such as cells infected with a virus, contain cellular material, such as RNA, protein, lipids, and carbohydrates from the infected cell, as well as nucleic acids of viral origin. These vesicles may play a role in infecting healthy cells. Altan-Bonnet N. 2016. Extracellular vesicles are the Trojan horses of viral infection. Curr Opin Microbiol 32:77-81; Schorey JS, Cheng Y, Singh PP, Smith VL. 2015, Exosomes and other extracellular vesicles in host-pathogen interactions; EMBO Rep 16:24-43, Schorey JS, Harding CV. 2016; and Extracellular vesicles and infectious diseases: new complexity to an old story. J Clin Invest 126:1181-9.

[0024] Vesicles, such as exosomes, have several properties that make them ideal for delivering material to cells, including their small size (e.g., allowing passage through the blood-brain barrier), natural ability to fuse with the plasma membrane of cells to deliver their contents , a stable internal environment and the ability to deliver functional molecules to the recipient cell, including nucleic acids (DNA, mRNA and microRNA), lipids and proteins.

[0025] Recent approaches are directed towards the development of extracellular vesicles for therapeutic use. These approaches include changing the contents of the vesicles or manipulating their migratory pathways. In particular, it has been demonstrated that extracellular vesicles can act as therapeutic vesicles for cancer treatment by reducing the invasion, migration and proliferation of tumor cells, increasing sensitivity to chemotherapy, and can cause increased immune responses and cell death.

[0026] In addition, it appears that the vesicles may be suitable for use as a vaccine. Vesicles released from virus-infected cells provide a unique source of properly folded and processed viral material ideal for use as an antigen in vaccination. At the same time, vaccines usually require the presence of an adjuvant to enhance the immune response to the antigenic component.

[0027] Previous attempts to manipulate the contents of extracellular vesicles using classical incubation, electroporation, and transfection approaches have been limited by low transfer efficiency, limited payload size, presence of residual membrane accessory, and limitations on the type of possible payload. Thus, there are still some challenges in realizing the potential of these vesicles. Therefore, there is a need to develop new methods capable of effectively delivering content to and into extracellular vesicles.

[0028] The present invention facilitates the exploitation of the potential of extracellular vesicles, providing for their isolation, use for diagnostic purposes, as targets for therapeutic intervention and for the delivery, for example, by attachment or genetic fusion, of therapeutic agents, including molecules, for example, nucleotides, including RNA and DNA into cells.

[0029] Which, to a greater extent than the expression of phosphatidylserine on the surface of the vesicles, suppresses immune responses to the vesicles. The GLA component (with no attached payload) that binds phosphatidylserine on the surface of the extracellular vesicle unmasks the vesicle from the immune system. Thus, the GLA component of the present invention can be used to improve the visibility of extracellular vesicles to the immune system.

BRIEF DESCRIPTION OF THE INVENTION

[0030] A brief description of the present invention is given in the following paragraphs:

1a. A method for targeting extracellular vesicles with surface-exposed phosphatidylserine, said method comprising the step of administering a molecule containing:

gamma-carboxyglutamic acid component (GLA component), wherein said GLA component contains the GLA domain or its active fragment and does not contain the active catalytic domain of the GLA protein,

into a fluid that may contain extracellular vesicles such as microvesicles, apoptotic bodies and exosomes.

1b. A molecule containing a payload associated with the gamma carboxyglutamic acid (GLA) component,

wherein said GLA component contains a GLA domain or an active fragment thereof and does not contain an active catalytic domain of the GLA protein for use in processing or diagnosing an extracellular vesicle.

1c. A molecule containing a payload associated with the gamma carboxyglutamic acid (GLA) component,

wherein said GLA component contains a GLA domain or an active fragment thereof and does not contain an active catalytic domain of the GLA protein for use in the manufacture of a medicament for treating or diagnosing an extracellular vesicle

2. A method or molecule for use according to paragraph 1a, 1b or 1c, wherein the diameter of said extracellular vesicle is in the range of 10 to 1000 nm.

3. A method or molecule for use according to paragraph 1a, 1b, 1c or 2, wherein the extracellular vesicle is an exosome.

4. A method or molecule for use according to any of paragraphs 1-3, wherein the diameter of said extracellular vesicle (e.g., exosomes) is 1000 nm or less, e.g., 1 nm to 10 μm, e.g., 10 nm to 5 μm , in particular from 10 µm to 1 µm, more specifically from 20 nm to 100 nm, more specifically 30, 40, 50, 60, 70, 80, 90 or 100 nm

5. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 4, wherein the density of said vesicle is in the range of 1 to 1.5 g/ml, e.g. 1 to 1.2 g/ml , in particular from 1.13 to 1.19 g/ml.

6. A method or molecule for use according to any of paragraphs 1a, 1b, 1c to 5, wherein said vesicle contains one or more transmembrane proteins independently selected from Lamp-1, Lamp-2, CD 13, CD86, flotillin , syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, integrin alpha 4, LiCAM, LFA-1, Mac-1 alpha and beta, Vti-IA and B, CD3 epsilon and zeta , CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2/3, HLA-DM (MHC II), immunoglobulins, MHC-I or MHC-II components, TCR-beta, tetraspanins, and a combination of two or more of these proteins.

7. A method or molecule for use in accordance with any of paragraphs 1a, 1b, 1c to 6, wherein said vesicle is released from an unhealthy cell, such as an apoptotic cell, a necrotic cell, a cancer cell, a cell infected with a pathogen (for example, a cell infected with a virus, a bacterial-infected cell, or a parasite-infected cell).

8. A method or molecule for use according to paragraph 7, wherein said cell is a cancer cell.

9. A method or molecule for use according to paragraph 8, wherein said cell is a cancer stem cell.

10. A method or molecule for use according to any of paragraphs 1-6, wherein said extracellular vesicle is derived from a healthy stem cell.

11. A method or molecule for use according to any of paragraphs 1a, 1b, 1c to 10, wherein said fluid includes an ex vivo patient sample, such as a blood sample, fluid taken from a cyst or tumor, a homogenized biopsy, and the like. .

12. A method or molecule for use according to any one of items 1-10, wherein said GLA component is administered in vivo .

13. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 12, wherein the GLA domain or active fragment thereof is independently selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z, osteocalcin, GLA matrix protein, GAS6, transtretin, periostin, proline-rich GLA 1, proline-rich GLA 2, proline-rich GLA 3, and proline-rich GLA 4.

14. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 13, wherein the GLA domain or active fragment thereof is independently selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z and GAS6, e.g. protein S, in particular containing the sequence shown in SEQ ID NO: 1

15. A method or molecule for use according to any of paragraphs 1-11, wherein the GLA domain component further comprises an EGF domain, eg, an EGF calcium binding domain.

16. A method or molecule for use according to paragraph 15, wherein the construct comprises an EGF domain selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z, osteocalcin, GLA matrix protein, GAS6, transtretin , periostin, GLA 1, rich in proline, GLA 2, rich in proline, GLA 3, rich in proline, and GLA 4, rich in proline.

17. A method or molecule for use according to paragraph 16, wherein the construct comprises an EGF domain selected from domains independently selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z and GAS6, for example, the EGF domain of the S protein.

18. A method or molecule for use according to one of paragraphs 1a, 1b or 1c to 17, wherein the GLA component contains the sequence shown in SEQ ID NO: 6, or a derivative thereof, where there is no His tag, in particular the sequence shown in SEQ ID NO: 6.

19. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 14, wherein the GLA domain component further comprises a Kringle domain.

20. The method or molecule for use in accordance with paragraph 19, and the Kringle domain is derived from a protein selected from the group containing activating transcription factor 2 (ATF); factor XII (F12); thrombin (F2); hyaluronan binding protein 2 (HABP2); hepatocyte growth factor (HGF); hepatocyte growth factor activator (HGFAC); protein Kremen 1 (KREMEN1); KREMEN2 ; lipoprotein (a) (LPA); LPAL2; macrophage stimulating protein (MSP or MST1); phosphoinositide-3-kinase interacting protein 1 (PIK3IP1); tissue plasminogen activator (PLAT); urokinase (PLAU); plasmin (PLG); PRSS12; transmembrane tyrosine protein kinase receptor ROR1 (ROR1); and transmembrane tyrosine protein kinase receptor ROR2 (ROR2).

21. A method or molecule for use in accordance with any of paragraphs 1a, 1b or 1c to 20, wherein the GLA component is associated with the payload.

22. The method or molecule for use in accordance with paragraph 21, wherein the GLA component is conjugated to the payload.

23. A method or molecule for use in accordance with any of paragraphs 1a, 1b or 1c to 22, wherein the GLA component is part of the payload.

24. The method or molecule for use in accordance with paragraph 23, and the payload contains a detectable label.

25. The method or molecule for use according to paragraph 24, wherein said label is selected from a fluorescent molecule (including a fluorescent probe (eg, rhodamine dye, FITC, FAM, CY5), biotin, an enzyme, a label (eg, a HIS tag, FLAG ^-labels , myc-labels), radionuclide (in particular radioactive iodine, radioisotopes, e.g. lighthouse.

26. A method or molecule for use according to any of paragraphs 1a, 1b, or 1c to 25, wherein the payload is a bead, tablet, or label, such as a release bead, such as a magnetic bead.

27. A method or molecule for use in accordance with paragraphs 21-26, wherein the payload is linked to the GLA component via a linker.

28. The method or molecule for use in accordance with paragraph 27, and the linker is cleavable.

29. A method or molecule for use in accordance with any of paragraphs 21-28, wherein the GLA component and payload are diagnostic.

30. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 29, said method comprising the additional step of obtaining an enriched population of extracellular vesicles.

31. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 30, said method comprising the step of isolating extracellular vesicles.

32. A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 31, said method being carried out in vitro .

A method or molecule for use according to any of paragraphs 1a, 1b or 1c to 32, wherein the molecule containing the GLA component is therapeutic.

34. A method or molecule for use in accordance with any of paragraphs 21-25 and 33, wherein the payload comprises a drug, a chemotherapeutic agent, a peptide (including cross-linked peptides), or a biological therapeutic agent, for example, an antiviral drug, an antibacterial drug, antiparasitic agent, anticancer drug, anticancer therapeutic agent.

35. A method or molecule for use in accordance with any of paragraphs 21-25, 33, and 34, wherein the payload comprises a toxin, a polymer (e.g., synthetic or naturally occurring polymers), biologically active proteins (e.g., enzymes, other antibody, or antibody fragments, e.g. intraantibodies), drug (small molecular weight compound (chemical structure), nucleic acids and fragments thereof (e.g. DNA, RNA and fragments thereof), metal chelating agent, nanoparticles or a combination of two or more of them.

36. The method or molecule for use according to paragraph 35, wherein the toxin is selected from auristatin (e.g., MMAE (monomethylauristatin E), MMAF (monomethylauristatin F)), pyrrolebenzodiazepine (PBD), doxorubicin, duocarmycin, maytansinoid (e.g., N2'- deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine (DM3) and N2'-deacetyl-N2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4)), calicheamycin, dolastatin, maytansine, α-amanitin, Pseudomonas exotoxin (PE38), ricin A chain, diphtheria toxin, Pokeweed antiviral protein (PAP) , saporin, gelonin and tubulisin.

37. The method or molecule for use according to any of paragraphs 34-36, wherein the chemotherapeutic agent is selected from temozolomide, epothilones, melphalan, carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine, polypheprozan, ifosfamide, chlorambucil, mechlorethamine, busulfan, cyclophosphamide, карбоплатина, цисплатина, тиотепы, капецитабина, стрептозоцина, бикалутамида, флутамида, нилутамида, ацетата лейпролида, гидрохлорида доксорубицина, сульфата блеомицина, гидрохлорида даунорубицина, дактиномицина, липосомального цитрата даунорубицина, липосомального гидрохлорида доксорубицина, гидрохлорида эпирубицина, гидрохлорида идарубицина, митомицина, доксорубицина, валрубицина, anastrozole, toremifene citrate, cytarabine, fluorouracil, fludarabine, floxuridine, interferon α-2b, plicamycin, mercaptopurine, methotrexate, interferon α-2a, medroxyprogesterone acetate, estramustine sodium phosphate, estradiol, leuprolide acetate, megestrol acetate, diphosphotide acetate, megestrol acetate diethylstilbestrol ata, testolactone, goserelin acetate, etoposide phosphate, vincristine sulfate, etoposide, vinblastine, etoposide, vincristine sulfate, teniposide, trastuzumab, gemtuzumab ozogamicin, rituximab, exemestane, irinotecan hydrochloride, asparaginase, gemcitabine hydrochloride, tomocladabine hydrochloride, altremocladamine hydrochloride, , mitotane, procarbazine hydrochloride, vinorelbine tartrate, sodium pentrostatin, mitoxantrone, pegaspargase, denileukin diftitox, altretinoin, porfimer, bexarotene, paclitaxel, docetaxel, arsenic trioxide, tretinoin, and combinations of two or more of them.

38. The method or molecule for use in accordance with any of paragraphs 34-37, wherein the chemotherapeutic agent is selected from an alkylating agent, an antimetabolite, including thymidylate synthase inhibitors, a taxane, an anthracycline, a microtubule-influencing agent, including plant alkaloids, and combinations of the two or more of them.

39. The method or molecule for use according to paragraph 38, wherein the chemotherapeutic agent is selected from paclitaxel, docetaxel, abraxane, carbazitaxel, derivatives of any of these, and combinations of two or more of any of them.

40. A method or molecule for use in accordance with paragraph 38 or 39, wherein the alkylating agent is selected from a nitrogen mustard analog, a nitrosourea derivative, tetrazine, aziridine, a platinum compound and derivatives thereof, a non-classical alkylating agent, and a combination of two or more of them.

41. The method or molecule for use according to paragraph 40, wherein the platinum compound is selected from cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, lipoplatin, and a combination of two or more of them.

42. A method or molecule for use according to any of paragraphs 38-41, wherein the alkylating agent is an antimetabolite selected from antifolates (e.g., methotrexate and pemetrexed), purine analogs (e.g., thiopurines, e.g., azathiopurine, mercaptopurine, thiopurine, fludarabine (including the phosphate form), pentostatin, and cladribine), pyrimidine analogs (eg, fluoropyrimidines, eg, 5-fluorouracil and its prodrugs, eg, capecitabine [Xeloda®]), floxuridine, gemcitabine, decitabine, raltitrexed hydrochloride (Tomudex), cladribine and 6-azauracil; and combinations of two or more of them.

43. A method or molecule for use according to any of paragraphs 40-42, wherein the anthracycline is selected from daunorubicin (daunomycin), daunorubicin (liposomal), doxorubicin (adriamycin), doxorubicin (liposomal), epirubicin, idarubicin, mitoxantrone, and a combination of two or more of them, in particular, doxorubicin.

44. A method or molecule for use according to any of paragraphs 34-43, wherein the drug is an anticancer drug, for example, selected from a topoisomerase inhibitor, a PARP inhibitor, and a combination or more of them.

45. A method or molecule for use according to any of paragraphs 34-44, wherein the anti-cancer therapeutic agent is a radionuclide, for example, selected from Y-90, P-32, I-131, In-111, Sr-89, Re -186, Sm-153, Sn-117m and combinations of two or more of them.

46. A method or molecule for use according to any one of 1a, 1b or 1c to 25 and 27-45, comprising administering a molecule containing a GLA component and a payload to a patient suffering from cancer.

47. The method or molecule for use in accordance with paragraph 46, and the cancer is an epithelial cancer, for example, colon and rectal cancer, testicular cancer, liver cancer, biliary tract cancer, prostate cancer, pancreatic cancer, breast cancer, cancer ovarian cancer, cervical cancer, uterine cancer, gastric cancer, esophageal cancer, thyroid cancer, kidney cancer, bladder cancer, brain cancer, head and neck cancer, or lung cancer, or alternatively, the cancer may be hemoblastosis, for example , leukemia, lymphoma, myeloma, and chronic myeloproliferative diseases such as AML.

In one embodiment, the method of the present invention does not target the apoptotic body.

[0031] Thus, in one embodiment, molecules in accordance with the present invention are used in the treatment of a pathogen, for example, viral, bacterial, protozoal, parasitic infections, including their intracellular forms.

[0032] The present invention also extends to the use of a GLA component comprising a GLA domain or an active fragment thereof, said GLA component lacking the active catalytic domain of the GLA protein, for intra-vesicle targeting and delivery (including intra-vesicle payload delivery).

[0033] The present invention also extends to the use of a GLA component comprising a GLA domain or an active fragment thereof, wherein said GLA component does not contain the active catalytic domain of the GLA protein, for the manufacture of a drug for targeted and intracellular delivery (including delivery of a payload within a vesicle). , in particular if the payload contains a therapeutic object/molecule).

[0034] Thus, in one aspect, a method is provided for in vitro production/isolation of vesicles from pathogen-infected human cell lines using the method described herein, e.g., Hep-2 cells, A549 cells, Calu-3 cells, HEK cells and Madin Darby kidney cells (MDCK), for example, for use in a vaccine.

[0035] In one embodiment, in vitro generated/isolated vesicles are loaded with a payload, e.g., an oligonucleotide or polynucleotide, e.g., DNA or RNA, e.g., CPG, using a gamma-carboxyglutamic acid component (GLA component) containing a GLA domain, or its active fragment and not containing the active catalytic domain of the GLA protein.

[0036] In one embodiment, in vitro generated/isolated vesicles are loaded with an immunostimulant molecule using a gamma-carboxyglutamic acid component (GLA component) containing the GLA domain or active fragment thereof and not containing the active catalytic domain of the GLA protein.

[0037] An extracellular vesicle mimetic can be produced in vitro by destroying cells by sequential extrusion, see, for example, Jang et al Nano 2013, 7, 7698. These mimetics contain phosphatidylserine on their surface if they are derived from apoptotic or stem cells.

[0038] In one embodiment, the oligonucleotide or polynucleotide is conjugated to a GLA component.

[0039] The present invention also relates to a vaccine composition comprising extracellular vesicles from a cell infected with a pathogen and loaded with an exogenous immunostimulatory molecule, for example, selected from an adjuvant, for example, a TLR9 agonist, for example, CPG, C3b, ICAM-1; and the GLA component of the present invention.

[0040] Vesicles in vaccines can be produced in vivo or in vitro. Vesicles can be generated in vitro in a pathogen-infected human cell line, for example, selected from HEp-2 cells, A549 cells, Calu-3 cells, HEK cells, and Madin Darby kidney (MDCK) cells.

[0041] An exogenous immunostimulatory molecule can be loaded: on the outer surface and/or inside a vesicle "of pathogenic origin".

[0042] The GLA component can be loaded: on the outer surface and/or inside of a vesicle "of pathogenic origin".

[0043] In one embodiment, the immunostimulatory molecule is not linked to the GLA component, i. they are loaded separately, for example, the immunostimulatory molecule can be presented as a plasmid that can be transfected into a vesicle, and the GLA component is presented as a protein.

[0044] Loading the vesicle with both the GLA component and the immunostimulatory molecules provides two separate mechanisms of action in which the GLA component binds phosphatidylserine on the surface of the vesicle, thereby detecting the presence of the vesicle to the immune system. The immunostimulatory molecule then enhances the immune system's response to pathogenic material in the vesicle.

[0045] The exogenous immunostimulatory molecule may be linked to a GLA domain, eg conjugated to a GLA component, or may be a fusion (expression construct) with a GLA component.

[0046] The immunostimulatory molecule associated with the GLA component is advantageous because the GLA component can bind phosphatidylserine on the surface of the vesicle, thereby loading itself as well as the immunostimulatory molecule onto the vesicle. In addition, in some cases, the GLA component can internalize on the vesicle and pull the immunostimulatory molecule along with it.

[0047] In one embodiment, the pathogen is bacterial or viral, such as those listed herein, specifically an influenza virus, such as influenza A, B, C, or D. Influenza A virus has hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18 and neuraminidase subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, for example, a strain selected from influenza A virus: (H1N1) H1N2, H2N1, H911, H3N1, H3N2 and H2N3, novel influenza A virus H1N1 (CDC 2009 H1N1 influenza website).

[0048] The technology according to the present invention may be suitable for obtaining a universal influenza vaccine.

[0049] However, even when used to prepare a seasonal influenza vaccine, the present technology is likely to be more efficient and convenient than culturing currently available vaccines in eggs.

[0050] The present invention also provides "pathogenic" vesicles according to the present invention for treatment, in particular for use as a vaccine.

[0051] In addition, the proposed vesicle "pathogenic origin" in accordance with the present invention for the production of a medicament, in particular, for the production of a vaccine.

[0052] As discussed above, the GLA component can be used to transfer a payload, such as a therapeutic payload, to the interior of an extracellular vesicle. The therapeutic payload can act on the vesicle itself, or be delivered to the cell by the vesicle and act on the cell, or a combination of the two scenarios.

DETAILED DESCRIPTION OF THE INVENTION

[0053] In one embodiment, 1, 2, 3, 4, or 5 payloads are associated with one GLA component.

[0054] A GLA component (also referred to herein as a gamma-carboxyglutamic acid component) refers to a polypeptide containing a GLA domain in the absence of a GLA protein catalytic domain, e.g., an S protein. The polypeptide may further comprise an EGF domain and/or a kringle domain, e.g., protein S. In one embodiment, the GLA component contains 30 to 300 amino acid residues, e.g. , 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 residues. In one embodiment, the weight of the GLA component is in the range of 4.5 to 30 kDa. In one embodiment, the GLA component contains the sequence shown in SEQ ID NO: 1. In one embodiment, the GLA component contains the sequence shown in SEQ ID NO: 6, or a derivative thereof, excluding the His tag.

[0055] GLA (vitamin K-dependent carboxylation/gamma-carboxyglutamic acid) domains herein are protein domains modified by vitamin K-dependent post-translational carboxylation of glutamate residues in the amino acid sequence to produce gamma-carboxyglutamate (Gla). In one embodiment, the implementation of the GLA domain used in the molecules according to the present invention contains from 30 to 45 consecutive residues of the native GLA domain (wild type). In one embodiment, the GLA domain contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 GLA residues.

[0056] In one embodiment, 30% or less of the GLA component is GLA residues.

[0057] In one embodiment, the implementation of the GLA component contains from 1 to 5 disulfide bonds, for example, 1, 2, 3, 4 or 5 disulfide bonds.

[0058] The GLA domain binds calcium by chelating it between two carboxylic acid residues. These residues are part of the region that begins at the N-terminus of the mature form of Gla proteins and ends with a conserved aromatic residue. This leads to retention of the Gla-x(3)-Gla-x-Cys motif, which is located in the middle of the domain and which, apparently, is important for the recognition of the substrate by carboxylase.

[0059] GLA domains are found in a number of proteins, e.g., thrombin, factor VII, factor IX, factor X, protein C, protein S (PrS), protein Z, osteocalcin, matrix GLA protein, GAS6, transthyretin, periostin, GLA 1, rich in proline, GLA 2, rich in proline, GLA 3, rich in proline, and GLA 4, rich in proline.

[0060] As used herein, the term "GLA domain" also covers proteins where 1 to 10 percent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%) amino acids in the native domain GLAs can be substituted and/or deleted provided that the modified domain retains at least 70% (e.g., 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 %) native activity of native (unmodified GLA domain) in a suitable in vitro assay . In one embodiment, the domain is a full length native domain.

[0061] As used herein, the term "EGF domain" refers to a conserved domain of a protein. It contains 30 to 40 amino acid residues and is found in large quantities in mostly animal proteins. In most cases, the EGF-like domain is located in the extracellular domain of membrane-bound proteins or proteins known to be secreted. The EGF-like domain includes 6 cysteine residues. The basic structure of EGF-like domains consists of a double-stranded β-sheet followed by a loop to a short C-terminal double-stranded β-sheet. These two β-sheets are usually referred to as the main (N-terminal) and auxiliary (C-terminal) sheets. EGF-like domains often occur in multiple tandem copies in proteins: these repeats are usually stacked with each other to form a single linear solenoid domain block as a functional unit. In one implementation, the domain used is a full-length native domain.

[0062] As used herein, the term "EGF domain" also covers proteins where 1 to 10 percent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%) amino acids in the native domain EGFs may be substituted and/or deleted provided that the modified domain retains at least 70% (e.g., 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 %) native activity of native (non-modified EGF domain) in a suitable in vitro assay . In one embodiment, the domain is a full length native domain.

[0063] As used herein, the term “Kringle domain” refers to autonomous protein domains that fold into large loops stabilized by 3 disulfide bonds. They are characterized by a structure of three loops and 3 disulfide bridges, the conformation of which is determined by the number of hydrogen bonds and small fragments of the antiparallel beta sheet. They occur in proteins involved in blood coagulation and fibrinolysis, in varying copy numbers, in several plasma proteins, including the prothrombin-type and urokinase-type plasminogen activator, which are serine proteases belonging to the MEROPS S1A family of peptidases.

[0064] As used herein, the term "Kringle domain" also covers proteins where 1 to 10 percent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%) amino acids in the native domain Kringle may be replaced and/or removed provided that the modified domain retains at least 70% (e.g., 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 %) native activity of a native (non-modified Kringle domain) in a suitable in vitro assay . In one implementation, the domain used is a full-length native domain.

[0065] As used herein, an active protein fragment is smaller than the entire native protein (or corresponding domain) and retains at least 50% (e.g., 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the activity of the native full-length domain or protein in the appropriate in vitro assay .

[0066] As used herein, a catalytic domain is a domain (or fragment) located behind an EGF domain in the C-terminal direction, for example, as shown in FIG. 1A.

[0067] As used herein, the term " in vitro " refers to laboratory work performed in a non-human or animal body.

[0068] As used herein, the term " in vivo" refers to operating/testing/treating in a living body, in particular a human, or an animal, in particular a human.

[0069] Extracellular vesicles (EVs) are important mediators of long-range cell-to-cell communication and are involved in a variety of biological processes in both prokaryotic and eukaryotic organisms (reviewed by Arenaccio and Federico, Adv Exp Med Biol 998: 3-19, 2017, Lu et al., Eur J Pharm Biopharm 119: 381-195, 2017, Vader et al., Adv Drug Delivery Rev 106:148-156, 2016, Lefebvre and Lecuyer, Front Microbiol 8: 377, 2017).

[0070] Extracellular vesicles are detached from infected (by virus, bacterium or parasite) cells and contain the material of these infectious agents (see review by Schorey et al., EMBO Rep. 16: 24-43, 2015, Schorey and Harding, J. Clin Invest 126: 1181-1189, 2016). This material can range from toxins to virulence factors and infectious virus (both enveloped and non-enveloped; see review by Altan-Bonnet, N, Curr Opin Microbiol 32: 77-81, 2016) and may be a novel means for more efficient viral delivery. populations rather than individual viral particles (Chen YH et al., Cell 160: 619-630, 2015). Therefore, their isolation can provide a rapid diagnostic understanding of the disease.

[0071] Extracellular vesicles is a broad term to describe all secreted membrane vesicles. As used herein, the term includes exosomes, microvesicles (also referred to as microparticles), ectosomes, matrix vesicles, calcifying vesicles, prostomes, oncosomes, retrovirus-like particles, bacterial extracellular vesicles, intraluminal vesicles, and apoptotic bodies.

[0072] In addition, extracellular vesicles containing proteins, RNA and carbohydrates specific for a given infectious agent and cell can also be in situ vaccination systems. In this situation, it is assumed that the use and neutralization of the immunosuppressive molecules of phosphatidylserine at the expense of the GLA domain allows the immune system to detect and remove the relevant antigens and is a new and more effective way to specifically vaccinate against an infectious disease.

[0073] As used herein, extracellular vesicles include microvesicles, apoptotic bodies, and exosomes. The diameter of extracellular vesicles is usually in the range from 10 nm to 5000 nm.

[0074] As used herein, microvesicles refer to vesicles released after formation by budding from the cell membrane, and their diameter, for example, is typically in the range of 100 nm to 1000 nm.

[0075] Exosomes are formed within the multivesicular bodies and are released after the fusion of the multivesicular body with the cell membrane. As a rule, the diameter of exosomes is in the range from 30 to 100 nm.

[0076] As used herein, apoptotic bodies refer to vesicles shedding into the extracellular environment from apoptotic cells. Apoptotic bodies cannot be involved in intracellular communication. Usually the diameter of apoptotic bodies is in the range from 800 nm to 5000 nm.

[0077] See, for example, Modularized Extracellular Vesicles: The Dawn of Prospective Personalized and Precision Medicine, Tao et al Adv. sci. 2018, 5, 1700449; Mesenchymal Stem Cell-derived Extracellular Vesicles: Towards Cell-free Therapeutic Applications Rani et al. www.moleculartherapy.org vol. 23 no. 5, 812-823 May 2015; and Achieving the Promise of Therapeutic Extracelluar Vesicles: The Devil is in the Detail of Therapeutic Loading Sutaria et al , Pharma Res. May 2017; 34(5) 1053-1066, incorporated herein by reference.

[0078] Thus, the present invention provides the use of extracellular vesicles to diagnose, for example, infection with a pathogen, such as a viral infection, a bacterial infection, and/or a parasitic infection.

[0079] Thus, the present invention provides the use of extracellular vesicles for the treatment of, for example, infection by a pathogen, for example, a viral infection, a bacterial infection and/or a parasitic infection, in particular, in the case when the extracellular vesicle is treated to neutralize or eliminate the pathogen material and/or extracellular vesicle is loaded with therapeutic material for delivery to the target cell.

[0080] Thus, the present invention provides the use of extracellular vesicles for diagnosing an infection with a pathogen, eg, a viral infection, a bacterial infection, and/or a parasitic infection.

[0081] Thus, the present invention provides the use of extracellular vesicles for the treatment of, for example, an infection with a pathogen, for example, a viral infection, a bacterial infection and/or a parasitic infection, in particular, in the case when the extracellular vesicle is treated to neutralize or eliminate the pathogen material and/or extracellular vesicle is loaded with therapeutic material for delivery to the cell.

[0082] Thus, the present invention proposes the use of extracellular vesicles for the diagnosis of cancer.

[0083] Thus, the present invention provides the use of extracellular vesicles for the treatment of cancer, in particular when the extracellular vesicle is treated to neutralize or eliminate pathogenic material and/or the extracellular vesicle is loaded with a therapeutic material for delivery to a cell.

[0084] Extracellular vesicles and methods in accordance with the present invention, in particular, extracellular vesicles from stem cells can also be used in the diagnosis and/or treatment of autoimmune diseases.

[0085] In one embodiment, the extracellular vesicles of the present invention are derived from a human cell.

[0086] In one embodiment, the extracellular vesicles are derived from stem cells (particularly healthy stem cells). These vesicles can be used similarly to stem cell therapy.

[0087] In one embodiment, the extracellular vesicles of the present invention are derived from a pathogenic cell, such as a bacterial cell.

[0088] Cells can secrete various types of EV and are classified according to subcellular origin (Colombo et al ., Annu Rev Cell Dev Biol 30: 255-289, 2014). Despite varying origins and sizes, there is no single EV nomenclature due to overlap in vesicle sizes and the absence of subtype-specific markers. As a result, purification, and therefore discrimination between vesicle types, remains a challenging task. For example, the most popular exosome purification protocols that have historically been used in the literature (differential ultracentrifugation, 220 nm filtration (Thery et al., Curr Protoc Cell Bioil, Chapter 3, block 3.22, 2006) and in recently released commercial kits allow co-isolation different types of EVs (see review by Tkach and Thiery, Cell 164: 1226-1232, 2016) Thus, terms such as "exosome" usually refer to a mixed population of small EVs, and therefore, for this invention, we decided to use the general term "EV" for all subtypes of vesicles.

[0089] Typically, extracellular vesicles contain a lipid bilayer containing ceramides, cholesterol, phosphoglycerides, and sphingolipids (Subra et al 2010, Trajkovic et al 2008, Vlassov et al 2012)

[0090] All extracellular vesicles carry surface molecules that provide a targeted effect on recipient cells, where they interact with the signaling pathway and / or deliver their contents to the cytosol in a number of ways (for example, through endocytosis and / or phagocytosis, fusion, etc. ), thereby changing the physiological state of the recipient cell. Because they are natural delivery vehicles for protein, lipids, and genetic material, they represent a unique biovector that is being actively explored for imaging, diagnostic, and/or therapeutic applications in a number of indications (for a review, see Rufino-Ramos et al. , J. Control Release 262: 247-258, 2017, Vader et al., Adv Drug Deliv. Rev 106: 148-156, 2016, Sutaria et al., Pharm Res. 34: 1053-1066, 2017, Ingato et al . ., J. Control Release 241: 174-185, 2016).

[0091] In one embodiment, the extracellular vesicle is an exosome. Exosomes typically range from 30 to 150 nm, eg 30 to 100 nm, and may, for example, have one or more surface markers selected from transferrin, CD9, CD63, CD61, CD81, TSG101, LAMPS, and Alix.

[0092] In one embodiment, the extracellular vesicle is a microvesicle (also referred to as a microparticle) and includes endothelial microparticles. As a rule, the diameter of the microvesicles is in the range from 50 to 2000 nm, and they, for example, can carry one or more of the surface markers selected from VCAMP3 and ARF6. Although circulating endothelial microparticles are found in the blood of healthy individuals, an increase in the number of circulating endothelial microparticles has been found in individuals with certain diseases, including hypertension and cardiovascular disease, as well as preeclampsia and various forms of vasculitis. It has been shown that endothelial microparticles in some of these disease states contain arrays of surface molecules reflecting the state of endothelial dysfunction. Thus, endothelial microparticles can be used as an indicator or indicator of the functional state of the endothelium in a disease, and they can play a key role in the pathogenesis of some diseases, including rheumatoid arthritis.

[0093] In one embodiment, the extracellular vesicle is an ectosome. As a rule, the diameter of ectosomes is in the range from 100 to 1000 nm, for example, from 350 to 400 nm, and they, for example, may bear one or more of the surface markers selected from TyA and C1a.

[0094] In one embodiment, the vesicle is a calcifying extracellular vesicle. These vesicles are released from cells in atherosclerotic plaques. Recently, calcifying EVs derived from macrophages and smooth muscle cells (SMCs) have received increased attention due to their role in vascular calcification. These vesicles are believed to play a mediating role in vascular calcification, which is a major predictor of CV morbidity and mortality.

[0095] In one embodiment, the extracellular vesicle is a matrix vesicle. The matrix vesicles mentioned herein are involved in bone development, with osteoblast-derived vesicles forming hydroxyapatite crystals along collagen fibers in developing bone. They also serve as crystallization nuclei for the formation of microcalcinosis within the fibrous caps of atherosclerotic plaques, leading to plaque instability, rupture and subsequent myocardial infarction and stroke.

[0096] In one embodiment, the extracellular vesicle is a prostasome. The term "prostasome" as used herein refers to vesicles secreted by prostate epithelial cells into seminal fluid. Their diameter is usually in the range from 40 to 500 nm. They have an unusual lipid composition and a dense and highly ordered structure of lipoprotein membranes resembling a lipid raft. The physiological role of the prostasis is to improve sperm motility and protect against attacks from the female immune defense when passing to the egg. Studies have shown that prostate cancer cells and poorly differentiated prostate cells continue to produce and secrete prostates. The high incidence of prostate cancer in older men may be due to protection against the immune system supported by prostomes. Immunoregulatory proteins found in prostomes include: aminopeptidase N (CD13); dipeptidyl peptidase IV (CD26); enkephalinase (neutral endopeptidase, CD10); angiotensin-converting enzyme (ACE, CD143); tissue factor TF (CD142, thromboplastin); hemolysis stimulator (CD55); protectin (CD59, MAC inhibitor); and complement-regulating membrane cofactor protein (CD46). The prostasomes also contain high concentrations of divalent cations: Zn ²⁺ , Ca ²⁺ and Mg ²⁺ .

[0097] In one embodiment, the extracellular vesicle is an oncosome. In this document, the term "oncosome" refers to large extracellular vesicles with a diameter of 1 to 10 microns. In the context of brain tumors, there are EVs released from glioma cells that express EGFRvIII, a mutated form of the receptor. It has been shown that these vesicles are able to transfer the EGFRvIII oncoprotein to the membrane of tumor cells lacking this receptor, thereby spreading the material that promotes tumor formation and causing transformation. Large oncosomes positive for Cav-1 have been shown to distinguish patients with localized prostate cancer from those with castration-resistant and metastatic disease.

[0098] In one embodiment, the extracellular particles are retrovirus-like particles. Their diameter is typically in the range of 75 to 100 nm and they may bear the Gag surface marker.

[0099] In one embodiment, the extracellular vesicle is a bacterial extracellular vesicle. These vesicles typically range in diameter from 10 to 300 nm and may contain PAMPs on their surface.

[0100] In one embodiment, the extracellular vesicle is an intraluminal vesicle. In this document, this term refers to a vesicle located in a cell.

[0101] Reverse fusion is the fusion of internal (intraluminal) vesicles in multivesicular bodies or late endosomes with the endosome boundary membrane. This process is thought to be mediated by lysobiphosphatidic acid (LBPA), phosphatidylinositol-3-phosphate, Alix, and apparently dependent on acidic pH. MHC class 2 and other proteins (CD63 and MPR) use this process to efficiently transport to specific locations in the cytosol and back to the plasma membrane. However, pathogens also use this mechanism to efficiently enter the cell cytosol (eg, VSV, anthrax). Unlike normal cell fusion between endosomes and organelles, reverse fusion requires sheets of exoplasm of the inner vesicles and outer membrane, similar to sperm-egg fusion.

[0102] In one embodiment, the extracellular vesicle is an apoptotic body. As used herein, apoptotic bodies typically range in diameter from 500 to 5000 nm, eg 500 to 4000 nm, and are released by cells undergoing programmed cell death.

Stem cells and markers

[0103] In one embodiment, the extracellular vesicles of the present invention are derived from a stem cell.

[0104] In one embodiment, the stem cells are embryonic stem cells. In one embodiment, the cells are not embryonic stem cells.

[0105] In one embodiment, the stem cell is an adult stem cell, e.g., including progenitor cells, and hematopoietic stem cells, myogenic stem cells, osteoprogenitor stem cells, neural stem cells, mesenchymal stem cells, e.g. satellites, ray glial cells, bone marrow stromal cells, periosteum, pancreatic progenitor cells, endothelial progenitor cells, blast cells, and trophoblastic stem cells.

[0106] In one embodiment, the stem cell is a cancer stem cell.

[0107] In one embodiment, the method relates to mammalian stem cells, such as human stem cells. The stem cells discussed herein are primarily human stem cells. However, a person skilled in the art, if necessary, is able to identify relevant or appropriate population of stem cells for other mammals. For example, SSEA-1 is a marker for mouse embryonic stem cells, human germline cells, and embryonic carcinoma cells; SSEA-3 is a marker for primate embryonic stem cells, human germline embryonic cells, human embryonic stem cells, and embryonic carcinoma cells; SSEA-4 is a marker for primate embryonic stem cells, human germline embryonic cells, human stem cells, embryonic carcinoma cells; CD324 is a marker for human and mouse embryonic stem cells, embryonic cancer cells; CD90 is a marker of human and mouse embryonic stem cells, hematopoietic stem cells, embryonic carcinoma cells; CD117 is a marker for human and mouse embryonic stem cells, hematopoietic progenitor stem cells, neural crest-derived melanocytes, primordial cells, embryonic carcinoma cells; CD326 is a marker for human and mouse embryonic stem cells, embryonic carcinoma cells; CD9 is a human and mouse embryonic stem cell marker; CD24 is a human and mouse embryonic stem cell marker; CD29 is a human and mouse embryonic stem cell marker; CD59 is a human and mouse embryonic stem cell marker; CD133 is a marker for human and mouse embryonic stem cells, embryonic carcinoma cells, hematopoietic stem cells; CD31 is a human and mouse embryonic stem cell marker; TRA-1-60 is a marker for human embryonic stem cells, teracarcinoma, germline embryonic cells, embryonic carcinoma cells; TRA-1-81 is a marker for human embryonic stem cells, teracarcinoma, germline embryonic cells, embryonic carcinoma cells; Frizzled5 is a human and mouse embryonic stem cell marker; stem cell factor (SCF) is a marker of human and mouse embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, embryonic carcinoma cells; and Cripto is a marker for human and mouse embryonic stem cells, cardiomyocytes, and embryonic carcinoma cells.

[0108] Hematopoietic stem cells (HSCs) or hemocytoblasts are stem cells that give rise to all other blood cells through hematopoiesis. They originate from the mesoderm and are located in the red marrow, which is found in the core of most bones.

[0109] The term "cancer stem cells" as used herein refers to tumor-forming cells (i.e., cancer cells occurring within tumors or in hematological malignancies) that have characteristics associated with normal stem cells, specifically, the ability to give rise to all types of cells, occurring in a particular cancer sample. See, for example, Identification and Targeting of Cancer Stem Cells, BioessayS 2009 Oct; 31 (10) 1038-1049, incorporated herein by reference. Cancer stem cells are defined by three distinct properties: i) the selective ability to initiate a tumor and stimulate neoplastic proliferation; ii) the ability to create endless copies of themselves through self-renewal; and iii) the ability to give rise to more mature progeny of cancer cells through differentiation. Cancer stem cells do not necessarily come from healthy stem cells, they can come from a differentiated cell.

[0110] CD34 is also known as the CD34 hematopoietic progenitor antigen and has the function of an intercellular adhesion factor. It can be used as a marker to enrich stem cell populations.

[0111] Stem cells are generally negative for germ-specific surface markers (i.e. Lin-ve), e.g. stem cell is Lin-ve, CD34 +ve, CD38 -ve, CD45RA -ve, CD90- positive. and CD49f _+ve.

[0112] Hematopoietic stem cells can express a surface marker from CD48, CD150, CD244, CD34, CD38, SCA-1, Thy1.1, C-kit, lin, CD135, slam1/CD150, Mac-1. (CD11b), CD4, stem cell factor (SCF) and a combination of two or more of them.

[0113] Osteoprogenitor cells can express a surface marker selected from Gremlin-1, TGF-beta, bFGF, BMP-2, ALPP, MCAM, collagen I, collagen 1 alpha-1, collagen II, RUNX2, decorin, and combinations of two or more of them (for example, all specified markers).

[0114] Osteoblasts or progenitors thereof may express a surface marker selected from Runx2, alkaline phosphatase/ALPP/ALPI, osteocalcin, BAP1, OPN, BAP31, collagen I, SCUBE3, fibronectin, SPARC, IGFBP-3, and a combination of two or more of them.

[0115] Osteocytes or their precursors may express a surface marker selected from:

i) TGF-beta, RANKL, MCSF, sclerostin, DKK, and combinations of two or more of them (for example, all of these markers) and/or

ii) Osterix +ve, CD90 +ve, osteocalcin +ve, collagen I +ve, bone sialoprotein +ve, and combinations of two or more of these (e.g., all of these markers) and/or

iii) alkaline phosphatase/ALPP (placental alkaline phosphatase)/ALPI +ve, collagen I +ve, collagen II +ve, decorin +ve, MCAM/CD146 +ve, MEPE/OF45 +ve, osterix +ve, CD90 +ve, osterix/Sp7 +ve, RUNX2/CBFA1 +ve, thrombopoietin/Tpo +ve, and combinations of two or more of these (e.g., all of these markers)

Myogenic stem cells can express a marker selected from CD56, CD146, VE-cadherin, alpha-smooth muscle actin, FABP3, alpha-7 integrin, desmin, myosin heavy chain, UEA-1 receptor, and combinations of two or more of these (e.g., all specified markers).

[0116] Neuronal stem cells can express a marker selected from:

i) CD133, CD15, CD24 low or -ve, GCTM-2, CD45, CD34, nestin, Sox-2, ABCG2, FGF R4, Frizzled-9, and combinations of two or more of these (e.g., all of these markers), and /or

ii) a CD24 marker expressed at a low level or -ve, and/or

iii) marker combinations CD133 +ve, 5E12 +ve, CD34 -ve, CD45 -ve and CD24 low or -ve

[0117] Mesenchymal stem cells can express a surface marker selected from CD10, CD13, CD73, CD105, CD271, CD140b, CD240, frizzled-9, CD29, CD90, CD146, oct4, SSEA4, STRO-1 stem cell factor (SCF) and combinations of two or more of them.

A stem cell derived from adipose tissue may express a surface marker selected from:

i) K15, CD34, nestin, follistatin, p63, alpha 6 integrin, tanascin C, EGFR, IGFR, frizzled factors, and a combination of two or more of these, and/or

i) iCD44, ICAM/CD54, CD34, members of the integrin family, and combinations of two or more of them.

[0118] A stem cell from ovarian and fallopian tube epithelial stem cells may express a surface marker selected from Gremlin 1, Lrig1, Lgr5, Bmi1, Tert, HopX, and combinations of two or more of these.

[0119] Embryonic stem cells may contain one or more surface markers selected from CD24, CD29, CD31, CD59, CD90, CD117, CD133, CD324, CD326, SSEA-3, SSEA-4, TRA-1-60, TRA -1-81, frizzled5, stem cell factor, crypto (TDGF-1).

[0120] Extracellular vesicles from stem cells can be identified based on markers of the cell from which they originated.

Other definitions

[0121] the Term "payload" in this document refers to a molecule associated with the GLA domain, in particular for delivery to the vesicle. The payload may include a drug, a toxin, a chemotherapeutic agent, a polymer, a biologically active protein, a peptide (e.g., a stable peptide), a polynucleotide (e.g., DNA and RNA, including microRNA, shRNA, RNAi, etc., including molecular beacons), radionuclides, metal chelating agent, oncolytic viruses, viral vectors, labels and/or reporter groups. As used herein, the term "linked" refers to any method of linking the payload to the GLA domain, including fusion protein formation (e.g., using an amide bond) or chemical conjugation (e.g., using maleimide chemistry, click chemistry, and etc.). The term "payload" can also be used to refer to material delivered into the extracellular vesicle, said material not being bound to the GLA component.

[0122] The GLA domain is a unique discovery and delivery platform that uses exposed phosphatidylserine (PS) to target and access a selected cell. Phosphatidylserine is the main signal for the recognition and uptake of apoptotic cells. Apoptosis is an evolutionarily conserved and highly regulated mode of cell death (Poon IK et al., Nat Rev Immunol 14: 166-180, 2014). The engulfment of apoptotic cells is known as efferocytosis, which serves to immediately and efficiently remove apoptotic cells before loss of membrane integrity and release of inflammatory content, and thus counterbalances the deleterious inflammatory effects of apoptosis. PS expression allows inhibition of TLR- and cytokine-induced signaling cascades and DC immunogenic maturation (Poon IK et al., Nat Rev Immunol 14: 166-180, 2014, Birge RB Celll Death Differ 23: 962-978, 2016). Therefore, under physiological conditions, externalized phosphatidylserine functions as a dominant and evolutionarily conserved immunosuppressive signal, promoting tolerance and preventing local and systemic activation of the immune system. In pathological settings, the innate immunosuppressive effect of phosphatidylserine is exploited by numerous viruses, other microorganisms, and parasites to facilitate infection and, in many cases, establish latency (Birge RB et al., Cell Death Differ 23: 962-978, 2016, Amara A and Mercer J , Nat Rev Microbiol 13: 461-469, 2015, Moller-Tank, S and Maury W Virology 468-470: 565-580, 2014). Phosphatidylserine is downregulated in the tumor microenvironment, counteracting the development of antitumor immunity, and exosomes expressing phosphatidylserine are currently being explored as an early diagnostic tool in cancer control (Birge RB et al., Cell Death Diff. 23:962-978, 2016 , Lea et al., Oncotarget 8: 14395-14407, 2017, Li X and Wang X, Mol Cancer 16: 92, 2017). Regardless of theoretical concepts, the authors of the present invention believe that not all molecules of phosphatidylserine are equivalent from a biological point of view. The inventors believe that phosphatidylserine exposed to the TMEM16F enzyme is involved in immune suppression and is "visible" to molecules in accordance with the present invention.

[0123] The term "oncolytic virus" as used herein refers to a virus that:

• preferentially infects and destroys cancer cells, or

• selectively replicates in cancer cells (for example, because its replication depends on a gene that is activated in cancer cells, such as p53.

[0124] The term "viral vector" as used herein refers to a replication-deficient virus, typically encoding a transgene.

[0125] As used herein, the term in vitro refers to laboratory work performed outside of the human or animal body.

[0126] As used herein, the term " in vivo" refers to operation/testing/treatment in a living organism, particularly a human or animal.

[0127] As used herein, the term "molecule" is used in its broadest sense and includes synthetic chemical molecules as well as macromolecules, e.g., proteins, polymers (natural or otherwise), ribonucleic acid molecules, labels, etc.

[0128] A molecular beacon is an oligonucleotide hybridization probe capable of reporting the presence of specific nucleic acids in homogeneous solutions. It is a hairpin-shaped molecule with an internally quenched fluorophore that is restored to fluorescence upon binding to a target nucleotide sequence.

[0129] The term "crosslinked peptide" as used herein refers to several tandem peptides held by a synthetic bracket designed to enhance the pharmacological efficacy of the peptides.

[0130] The term "drug" as used herein, unless the context indicates otherwise, is intended to refer to a small chemical entity, for example, synthesized by organic synthesis, in particular, a molecule approved or licensed, or in the process of obtaining a license for therapeutic use especially on people. As used herein, the term "drug" includes an antiviral compound, an antibiotic, and an anticancer therapeutic agent.

[0131] As used herein, the term "antiviral compound" (antiviral agent) refers to a class of drugs used specifically for the treatment of viral infections, including broad-spectrum antiviral agents, as well as "narrow" spectrum antiviral agents specific to a particular virus or a particular family of viruses.

[0132] As used herein, the term "antibiotic" refers to a drug or agent that inhibits the growth of bacteria or kills bacteria. The terms "antibacterial agent" and "antibiotic" are used interchangeably in this document, unless the context indicates otherwise.

[0133] As used herein, the term "antiparasitic agent" refers to a drug or agent that inhibits the growth of a parasite, kills a parasite, or removes parasites from a host organism.

[0134] "Anti-cancer therapeutic agent" is a broad term that includes anti-cancer drugs, chemotherapy, radiation therapy, immuno-oncological therapy, and so on.

[0135] As used herein, an anticancer drug generally refers to a small molecule cancer therapy.

[0136] As used herein, the term "chemotherapy" generally refers to a cytotoxic agent and includes anticancer agents.

[0137] A biological therapeutic agent (also referred to as a biopharmaceutical or biological agent) is a therapeutic product having a biological "origin", for example, recombinant proteins and fragments, including antibody molecules, including antibodies, binding antibody fragments and polyspecific antibody molecules , polynucleotides, therapeutic viruses, oncolytic viruses, viral vectors, and complex combinations of such materials. Biologically active protein is a subgroup of biological therapeutic agents and includes recombinant proteins and their active fragments (including antibody molecules).

[0138] In the present application, antibody molecules include a complete antibody containing full-length heavy and light chains or fragments thereof, and a molecule containing any of these fragments, for example, Fab, modified Fab, Fab', modified Fab', F(ab') 2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv, intrabodies, bi-, tri- or quadrivalent antibodies, bis-scFv, diabodies, tribodies, tetrabodies and epitope-binding fragments of any of them (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and manufacturing these antibody fragments are well known in the art (see, for example, Verma et al ., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International Patent Applications WO2005/003169, WO2005/003170 and WO2005/003171. Polyvalent antibodies may be specific to several substances, for example, may be bispecific or monospecific (see, for example, WO 92/22853 and WO05/113605). Bispecific and multispecific antibody variants are specifically addressed in this example because the goal is to neutralize two independent target proteins. The variable regions of the antibodies described herein can be configured to produce a single antibody variant capable of binding to and neutralizing two target antigens.

[0139] Antibodies and their binding fragments, in particular, small fragments of antibodies, for example, domain antibodies, VHHs, single chain Fv (scFv), double chain scFv, dsFv and intrabodies can be delivered into the cell using the technology of the present invention.

[0140] In one embodiment, the antibody molecule is a human antibody or a humanized antibody.

[0141] A toxin is a poisonous substance, especially one of natural origin, such as a protein. Many toxins, such as calicheamicin, are used in the treatment of cancer. In addition, chemotherapeutic agents can be considered toxic (or toxins). Thus, the definition of a toxin overlaps with other definitions in this document. At the same time, neurotoxins, such as snake venom, are toxins, but not chemotherapeutic agents. However, those skilled in the art are familiar with these technical definitions and are able to understand the meaning of the context of the present invention.

[0142] As used herein, the term "diagnostic agent" is a tool used in analysis or imaging to diagnose or monitor or understand the status of a disease. The diagnostic agent usually contains a reporter molecule, such as a label, etc., which can be visualized, measured or controlled in some way.

[0143] Radionuclides suitable for use in the present invention include thallium-201, technetium-99m, iodine-123, iodine-131, iodine-125, fluorine-18, and oxygen-15.

[0144] The term "abnormal cell" or "pathogenic cell" as used herein refers to a cell that differs from a normal healthy cell, in particular by mutation or activation of a marker or markers, such as an abnormality associated with a predisposition to or development of a condition or diseases; with a condition or disease, for example, a pre-cancerous cell, a cancer cell, a pathogen-infected cell, a sickle cell cell, and the like.

[0145] As used herein, apoptosis is a cell death pathway that is a normal and controlled part of an organism's growth. Cell death by apoptosis is less harmful to surrounding tissue than cell death mechanisms such as necrosis.

[0146] As used herein, the term "necrosis" is the death of cells from disease or injury. It releases cytokines and factors into the surrounding tissue that can damage surrounding cells. Gangrene is an example of necrotic cell death.

Chemotherapeutic agents

[0147] In this document, the terms "chemotherapeutic agent", "chemotherapy" or "cytotoxic agent" are used interchangeably, unless otherwise indicated.

[0148] As used herein, the term "chemotherapy" is intended to refer to specific anticancer chemicals or drugs that "selectively" destroy malignant cells and tissues, e.g., alkylating agents, antimetabolites, including thymidylate synthase inhibitors, anthracyclines, agents that affect on microtubules, including plant alkaloids, topoisomerase inhibitors, PARP inhibitors and other anticancer agents. "Selectively" in this context is used in a broad sense, since, of course, many of these agents have serious side effects.

[0149] The preferred dosage may be selected by the practitioner depending on the nature of the cancer being treated.

[0150] Examples of alkylating agents that can be used in the process of the present invention include nitrogen mustard analog alkylating agents, nitrosourea derivatives, tetrazines, aziridines, platinum preparations and their derivatives, and non-classical alkylating agents.

[0151] An example of a platinum-containing chemotherapeutic agent (also referred to as platinum) is cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin (a liposomal variant of cisplatin), specifically cisplatin, carboplatin, and oxaliplatin.

[0152] The dose of cisplatin is from about 20 to about 270 mg/m ² depending on the specific cancer. Often the dose is in the range of about 70 to about 100 mg/m ² .

[0153] Nitrogen mustard analogs include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, and busulfan.

[0154] Nitrosourea derivatives include N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine, and streptozotocin. Tetrazines include dacarbazine, mitozolomide, and temozolomide.

[0155] Aziridines include thiotepa, mitomycin diaziquone (AZQ).

[0156] Examples of antimetabolites that can be used in the method of the present invention include antifolates (eg, methotrexate and pemetrexed), purine analogs (eg, thiopurines, eg, azathiopurine, mercaptopurine, thiopurine, fludarabine (including the phosphate form), pentostatin, and cladribine ), pyrimidine analogs (eg, fluoropyrimidines, eg, 5-fluorouracil and its prodrugs, eg, capecitabine [Xeloda®]), floxuridine, gemcitabine, cytarabine, decitabine, raltitrexed (Tomudex) hydrochloride, cladribine, and 6-azauracil.

Examples of anthracyclines that can be used in the method of the present invention include daunorubicin (daunomycin), daunorubicin (liposomal), doxorubicin (adriamycin), doxorubicin (liposomal), epirubicin, idarubicin, valrubicin currently only used to treat bladder cancer, and mitoxantrone, an anthracycline analog, in particular doxorubicin.

[0157] Examples of agents that affect microtubules that can be used in the method according to the present invention include vinca alkaloids and taxanes.

[0158] Vinca alkaloids include all-natural chemicals, such as vincristine and vinblastine, as well as semi-synthetic vinca alkaloids, such as vinorelbine, vindesine, and vinflunine.

[0159] Taxanes include paclitaxel, docetaxel, abraxane, carbazitaxel, and derivatives thereof. As used herein, taxane derivatives include modified taxanes, eg taxol, eg in micellar formulations; derivatives also include chemical derivatives in which chemical synthesis is used to modify the starting material, which is a taxane.

[0160] Topoisomerase inhibitors that can be used in the method of the present invention include type I topoisomerase inhibitors, type II topoisomerase inhibitors, and topoisomerase type II poisons. Type I inhibitors include topotecan, irinotecan, indothecan, and indimithecan. Type II inhibitors include genistein and ICRF 193, which has the following structure:

[0161] Type II poisons include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, and fluoroquinolones.

[0162] In one embodiment, the chemotherapeutic agent is a PARP inhibitor.

Viruses suitable for use as a payload in the present invention

[0163] In one embodiment, the virus used in the present invention is an enveloped virus, e.g., selected from herpesvirus (e.g., herpes simplex virus 1), poxvirus (e.g., vaccinia virus), hepadnavirus, flavivirus, togavirus, coronavirus, virus hepatitis D, orthomyxovirus, paramyxovirus (eg, measles virus or Newcastle disease virus), rhabdovirus, bunyavirus, filovirus, and Rhabdoviridae (eg, vesicular stomatitis virus, Indiana strain (VSV).

[0164] In one embodiment, the virus used in the present invention is a non-enveloped virus, e.g., selected from Adenoviridae (e.g., adenovirus), Papilomaviridae, Picornaviridae (e.g., Coxsackie virus or Seneca Valley virus (e.g., senecavirus)), reovirus .

[0165] In one embodiment, the virus is an adenovirus, e.g., a human adenovirus, e.g., selected from a group B virus (in particular, Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad51, or a chimeric virus on their based, for example, Enadenotucirev), group C virus (in particular Ad1, 2, 5, 6 or a chimeric virus based on them), group D virus (in particular Ad8, Ad10, Ad13, Ad15, Ad17, Ad19, Ad20, Ad22, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39, Ad42, Ad43, Ad44, Ad45, A46, Ad47, Ad48, Ad49, Ad50 or a chimeric virus based on them), group E virus (in particular, Ad4) , a virus of group F (in particular, Ad40, Ad41 or a chimeric virus based on them) and a chimeric virus based on two or more viruses of group B, C, D, E or F.

[0166] The vast majority of viruses contain well-described proteins associated with recognition and uptake by target cells. Their tropism to redirect or provide more selective targeting of oncolytic viruses to tumors can be modified using the methods described in a review by Verheije and Rottier, Adv. Virology 2012: 798526, 2012.

[0167] Additional viral cell surface proteins that are not involved in the targeting of the native virus may contain targeting motifs artificially introduced into them (for example, the minor virion envelope protein IX Ad, Salisch et al., PLoS One 12: e0174728, 2017 ).

[0168] Enveloped viruses contain an outer membrane (envelope) that covers the capsid of the virus. The envelope usually comes from parts of the host cell membranes (phospholipids and proteins) and, in addition, includes some viral proteins. Glycoproteins on the surface of the envelope serve for identification and bind to receptor sites on the host membrane. The virus envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter the cell and infect the host.

[0169] Various oncolytic viruses are described in WO2014/13834, incorporated herein by reference.

[0170] Herpes simplex virus (HSV) enters cells using four major glycoproteins - gD, gH/gL, gB, activated in a cascaded manner by binding gD to one of its receptors, nectin 1 and HVEM. HSV retargeting was achieved by introducing ligands and scFv into the gC and/or gD or gH protein (Campadelli-Fiume, G et al., Rev in Med Virol 21: 213-226, 2011, Gatta, V PLoS Pathog 11: e1004907, 2015) . Oncolytic vectors based on herpes simplex virus type 1 have been developed for clinical use. These viruses are capable of replication and contain mutations in genes affecting viral replication, neuropathogenicity, and immune evasion, and include, for example, first-generation viruses such as NV1020 (R7020), dls ptk, d18.36tk, hrR3, R3616, 1716 , second generation viruses, e.g. G207 (MGH-1), 3616UB, SUP, NV1023, third generation viruses, e.g. G47Δ, transcription expression vectors, e.g. G92A, d 12.CALP, Myb34.5, transgene expression vectors, for example, rRP450, and other viruses, for example, Talimogene laherparepvec (T-Vec). It is believed that HSV-1 vectors can be used in the treatment of a wide range of solid tumors, for example, glioma, melanoma, breast, prostate, colon, ovarian and pancreatic cancer. HSV-1 virus infects a wide range of cell types and species, it is cytolytic in nature, the replicative life cycle of the virus leads to the destruction of the host cell, it has a well-characterized and large genome (152K), but contains many non-essential genes, which provides up to 30 kb of space for the introduction of therapeutic genes. As a rule, HSV viruses do not mutate in the thymidine kinase gene for safety reasons. Talimogene laherparepvec is an oncolytic herpesvirus approved for use in the treatment of melanoma. Other herpesvirus-based viruses include G207, SEPREHVIR (HSV-1716) developed by Virttu Biologics, HSV-1 mutant R3616, HSV-1 mutant 1716, NV1020 (R7020), R3616 mutant (with RL1 deleted), KM100 mutant with insertions in UL48 ( encodes the transactivator envelope protein pUL48 [VP16]) and the RL2 , G92A, mutant genes, Myb34.5 and rQNestin34.5.

[0171] Poxvirus - vaccinia virus, for example, modified vaccinia virus Ankara (MVA) can be used (Galmiche MC et al., J Gen Virol 78: 3019-3027, 1997); The MVA can be replaced with a p14 fusion molecule carrying an inserted scFv against the tumor-associated MUC-1 antigen (Paul, S. et al., Viral Immunol. 20: 664-671, 2007). See also review by Liang L et al., Viruses 6: 3787-3808, 2014, Hsiao JC et al., J Virol 73: 8750-8761, 1999, review by Chen TL and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T et al., J Biochem 144: 287-294, 2008. JX-594, developed by Jennerex, is a vaccinia virus with a thymidine kinase deletion and the addition of GMCSF. GL-ONC1 is an attenuated vaccinia virus (Lister strain) that causes regression and elimination of a wide range of solid tumors in preclinical mouse models.

[0172] Paramyxovirus (eg, measles virus or Newcastle disease).

[0173] Measles virus (MeV) is a single-stranded (minus-stranded) enveloped (non-segmented) RNA virus of the genus Morbillivirus, family Paramyxoviridae. The measles virus contains two envelope glycoproteins: the hemagglutinin attachment protein (H) and the fusion protein (F). Attachment, entry and subsequent intercellular fusion are mediated through 2 measles virus receptors, CD46 and the lymphocyte activation signaling molecule (SLAM). See, for example, a review by Msaouel P et al., Methods Mol Biol 797: 141-162, 2012, Robinson S. and Galanis, E. Expert Opin Biol Ther. 17: 353-363, 2017, Aref S et al., Viruses 8. Pii:E294, 2016); (reviewed by Chen TL and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T et al., J Biochem 144: 287-294, 2008) and (Russell SJ and Peng KW, Curr Topic Microbiol. Immunol 330: 213-241, 2009, Robinson S and Galanis, E Expert Opin Biol Ther 17: 353-363, 2017, Aref S et al., Viruses 8. Pii: E294, 2016). The measles virus encoding human sodium iodide thyroid symporter or MB-NIS is an attenuated oncolytic strain of the Edmonston (Ed) measles virus. Radioactive iodine imaging provides a new way to monitor NIS gene expression.

[0174] In addition, Newcastle disease virus can be used.

[0175] Adenoviridae. Adenoviruses are among the most widely studied viruses used as oncolytic agents. A number of peptides and proteins have been inserted into the viral proteins associated with the virion in order to modify the native tropism of the virus (reviewed by Verheije MH and Rottier PJM Adv Virol 2012: 798526, 2012). However, they all depend on the assembly of the virus in the kernel, which creates significant problems.

[0176] Other non-enveloped viruses include Coxsackievirus, poliovirus, and reovirus. See, for example, review by Altan-Bonnet, N, Curr Opin Microbiol 32: 77-81, 2016 and Chen YH et al., Cell 160: 619-630, 2015, review by Chen TL and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T et al., J Biochem 144: 287-294, 2008 and review by Verheije MH and Rottier PJM Adv Virol 2012: 798526, 2012).

[0177] There are many adenoviruses, for example, Ad5-YCD/mutTKSR39rep-hIL12, which has started to be used for the treatment of prostate cancer, CGTG-102 (Ad5/3-D24-GMCSF), created by Oncos Therapeutics, for example, for the treatment of soft tissue sarcomas , Oncorine (H101), CG0070, Enadenotucirev (EnAd) WO2005/118825, OvAd1 and OvAd2 described in WO2008/080003, ONCOS-102, for example, for the treatment of unresectable malignant pleural mesothelioma, and DNX-2401, for example, for the treatment of glioma.

[0178] Cavatak is the trade name for a wild-type Coxsackie A21 virus preparation that can be used to treat malignant melanoma. Seneca Valley virus (NTX-010) and (SVV-001), for example, for the treatment of small cell lung cancer and neuroblastoma.

[0179] Reovirus - Reolysin® (pelareorep; wild-type reovirus; Dearing serotype 3; Oncolytics Biotech), for example, for the treatment of various cancers and cell proliferation disorders.

[0180] Vesicular stomatitis virus (VSV). VSV is another enveloped virus being investigated as an oncolytic agent. See, for example, Betancourt D et al., J Virol 89: 11786-11800, 2015) and Hastie E and Grdzelishvili VZ J Gen Virol 93: 2529-2545, 2012).

Proteins encoded by the virus

[0181] In one embodiment, the virus or vector used in the method of the present invention contains a transgene, e.g., a transgene that replaces defective genetic material in a cell to confer a new or enhanced function to the cell, increase the sensitivity of the cell to treatment, block function in the cell or expression of a therapeutic protein or peptide. In one embodiment, the virus used as a payload according to the present invention contains a transgene or transgenes, for example, encoding an agent independently selected from an RNAi sequence, protein, polypeptide or peptide (for example, an antibody molecule or its binding fragment, chemokine, cytokine, immunomodulator, fluorescent label or enzyme).

[0182] This includes, but is not limited to, unique formats that have shown promise in preclinical studies but have not been shown to be efficient and cost-effective delivery vehicles, such as peptides, intrabodies, and alternative scaffolds (reviewed by Boldicke T, Protein Sci 26: 925-945, 2017 , Marschall and Dubel, Comput Struct Biotechnol J 14: 304-308, 2016, Miersch and Sidhu F1000Res 5.pii.F1000 Faculty Rev. 1947, 2016, Peptides, Tsomaia Eur J Med Chem 94:459-470, 2015, Marschall ALJ et al, Mabs 7: 1010-1035, 2015, AlDeghaither D et al., J Clin Pharmacol 55: S4-S20, 2015))), including agents that have a therapeutic effect on tumor cells, tumor stem cells, endothelium, associated with the tumor, and the stroma associated with the tumor. Of particular interest are molecules that can serve multiple functions, such as being used as therapeutics, biomarkers, and/or diagnostics. The herpes simplex virus thymidine kinase (HSV-TK) gene is a well established prodrug converting enzyme for which a clinically approved prodrug (ganciclovir-GCV) has been developed, see eg Holder et al., Cancer Res. 53: 3475-3485, 1993, Touraine RL et al., Gene Therapy 5: 1705-1711, 1998),

[0183] In addition, thymidine kinase protein expression can also be used to visualize and track virotherapy activity during a course of treatment. Positron emission tomography and single photon emission computed tomography are methods commonly used to detect, monitor and treat cancer; both of these methods can be used to detect thymidine kinase protein expression upon administration of an appropriate thymidine kinase substrate (Wang JQ et al., Bioorg Med Chem 13: 549-556, 2005, Tjuvajev JG et al, J Nucl Med 43: 1072-1083, 2002). Alternatively, the NIS gene can be used and investigated as an agent for diagnostic and therapeutic purposes in the application of oncolytic viruses, much like TK (Miller A and Russell S Expert Opin Biol Ther 16: 15-32, 2016, Ravera S et al. , Annu Rev Physiol 79: 261-289, 2017, Portulano et al., Endocr Rev. 35: 106-149, 2014).

[0184] In one embodiment, antibodies that interact with and inhibit RAS or RAS signaling pathway proteins are encoded in the virus of the present invention, for example, as a fusion protein with a GLA component. The RAS genes make up a multigene family that includes HRAS , NRAS , and KRAS . See, for example, Bos JL, Cancer Res. 49: 4682-4689, 1989; and Cetin M et al., J Mol Biol. 429:562-573, 2017.

Tags

[0185] In one embodiment, the payload contains a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme, a dye, or a ligand.

[0186] In the present invention, a label is any group that can be detected using an assay. Non-limiting examples of reporter molecules include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands such as biotin. The term "tag" in this document also includes tags such as His tags, Flag tags, and the like. The tags include biotin, which is a substrate for avidin.

[0187] Labels can be linked to GLA components by conjugation or attachment. The label may be the only payload or used in addition to another entity, such as a therapeutic payload.

[0188] Label conjugates are generally preferred for use as diagnostic agents. Diagnostic agents generally fall into two classes: those used for in vitro diagnostics and those used for in vivo diagnostic protocols, commonly referred to as "guided imaging". Many suitable imaging agents are known in the art, as well as methods for attaching them to peptides and polypeptides (see, for example, US Pat. Imaging groups used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR detectables, and x-ray imaging agents.

[0189] In the case of paramagnetic ions, ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II) can be mentioned as an example. , neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), and gadolinium is particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0190] In the case of radioactive isotopes for therapeutic and/or diagnostic use, mention may be made of astatine ²¹¹ , ¹⁴ carbon, ⁵¹ chromium, ³⁶ chlorine, ⁵⁷ cobalt, ⁵⁸ cobalt, copper ⁶⁷ , ¹⁵² Eu, gallium ⁶⁷ , ³ hydrogen, iodine ¹²³ , iodine ¹²⁵ , iodine ¹³¹ , indium ¹¹¹ , ⁵⁹ iron, ³² phosphorus, rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , ⁷⁵ selenium, ³⁵ sulfur, technetium ^99m and/or yttrium ⁹⁰ . ¹²⁵ I is suitable for use in certain implementations, and technetium ^99m and/or indium ¹¹¹ are particularly suitable due to their low energy and suitability for long range detection. Radiolabeled peptides and polypeptides can be prepared according to methods well known in the art. For example, peptides and polypeptides can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent, eg sodium hypochlorite, or an enzymatic oxidizing agent, eg lactoperoxidase. Peptides can be labeled with technetium ^99m by ligand exchange, for example by reducing pertechnetium with tin solution, chelating the reduced technetium on a Sephadex column, and loading the peptide onto the column. Alternatively, direct labeling methods can be used, eg by incubating the pertechnate, the reducing agent, eg SNCl ₂ , the buffer solution, eg sodium potassium phthalate solution, and the peptide. Intermediate functional groups that are often used to bind radioactive isotopes that exist as metal ions to a peptide are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0191] Fluorescent labels suitable for payload applications include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-P6G, BODIPY-TMR, BODIPY-TRX, Cascad Blue, Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renografin, ROX, TAMRA, TET, tetramethylrhodamine and/or Texas Red.

[0192] Another type of payload suitable for in vitro use is where the peptide is linked to a secondary binding ligand and/or an enzyme (enzyme label) that forms a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, hydrogen peroxidase (horseradish), or glucose oxidase. Suitable secondary binding ligands are biotin compounds and avidin and streptavidin. The use of such labels is well known to those skilled in the art and is described, for example, in US Pat. Nos. 3,817,837; 3,850,752;

[0193] Other attachment methods are known in the art for linking a peptide to its "conjugation partner". Some attachment methods include using a metal chelate using, for example, an organic chelating agent, such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycuryl-3 attached to an antibody (US Pat. Nos. 4,472,509 and 4,938,948). Peptides or polypeptides can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanate.

[0194] In one embodiment, the label can stain or label the stem cell nucleus.

Combination Therapy

[0195] In one embodiment, a combination of chemotherapeutic agents, eg, a platinum-containing drug, and 5-FU or a prodrug thereof, eg, cisplatin or oxaliplatin and capecitabine or gemcitabine, eg, FOLFOX, is used.

[0196] In one embodiment, the implementation of a chemotherapeutic agent includes a combination of chemotherapeutic agents, in particular, cytotoxic chemotherapeutic agents.

[0197] In one embodiment, the chemotherapeutic combination includes a drug containing platinum, for example, cisplatin and fluorouracil or capecitabine.

[0198] In one embodiment, the chemotherapy combination comprises capecitabine and oxaliplatin (Xelox).

[0199] In one embodiment, the chemotherapeutic agent is a combination of folinic acid and 5-FU, optionally in combination with oxaliplatin.

[0200] In one embodiment, the chemotherapeutic agent is a combination of folinic acid, 5-FU, and irinotecan (FOLFIRI), optionally in combination with oxaliplatin (FOLFIRINOX). The regimen consists of: irinotecan (180 mg/m ² iv over 90 minutes) along with folinic acid (400 mg/m ² [or 2x250 mg/m ² ] iv over 120 minutes); then fluorouracil (400-500 mg/m ² IV bolus), then fluorouracil (2400-3000 mg/m ² IV for 46 hours). This cycle is usually repeated every two weeks. The dosages shown above may vary from cycle to cycle.

[0201] In one embodiment, the chemotherapeutic combination comprises a microtubule inhibitor, e.g., vincristine sulfate, epothilone A, N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide (AWT-751), chemotherapeutic a taxol derivative such as paclitaxel, abraxane or docetaxel, or a combination thereof.

[0202] In one embodiment, an mTor inhibitor is used in the chemotherapeutic combination. Examples of mTor inhibitors include: everolimus (RAD001), WYE-354, KU-0063794, papamycin (sirolimus), temsirolimus, deforolimus (MK-8669), AZD8055 and BEZ235 (NVP-BEZ235).

[0203] In one embodiment, a MEK inhibitor is used in the combination therapy. Examples of MEK inhibitors include: AS703026, CI-1040 (PD184352), AZD6244 (selumetinib), PD318088, PD0325901, AZD8330, PD98059, U0126-EtOH, BIX 02189 or BIX 02188.

[0204] In one embodiment, an AKT inhibitor is used in the chemotherapeutic combination. Examples of AKT inhibitors include: MK-2206 and AT7867.

[0205] In one embodiment, an aurora kinase inhibitor is used in combination. Examples of aurora kinase inhibitors include: Aurora A I inhibitor, VX-680, AZD1152-HQPA (baracertib), SNS-314 mesylate, PHA-680632, ZM-447439, CCT129202, and hesperadin.

[0206] In one embodiment, the combination therapy uses a p38 inhibitor, such as that described in WO2010/038086, such as N- [4-({4-[3-(3- tert -butyl-1- p -tolyl- 1H -pyrazol-5-yl)ureido]naphthalene-1-yloxy}methyl)pyridin-2-yl]-2-methoxyacetamide.

[0207] In one embodiment, a Bcl-2 inhibitor is used in combination. Examples of Bcl-2 inhibitors include: obatoclax mesylate, ABT-737, ABT-263 (navitoclax) and TW-37.

[0208] In one embodiment, the chemotherapeutic combination contains an antimetabolite, such as capecitabine (xeloda), fludarabine phosphate, fludarabine (fludara), decitabine, raltitrexed (tomudex), gemcitabine hydrochloride, and cladribine.

[0209] In one embodiment, the chemotherapeutic combination contains ganciclovir, which may help control immune responses and/or tumor vascularization.

[0210] In one embodiment, the chemotherapeutic agent comprises a PARP inhibitor.

[0211] In one embodiment, the combination therapy comprises an inhibitor of cancer metabolism that specifically inhibits the activity of the DHODH enzyme.

In one embodiment, one or more of the treatments used in the method of the present invention are metronomic, that is, continuous or frequent low doses of anti-cancer drugs, often given concomitantly with other treatments.

[0213] In one embodiment, multiple cycles of treatment (e.g., chemotherapy) are provided, e.g., 2, 3, 4, 5, 6, 7, 8.

[0214] In one embodiment, chemotherapy is administered on a 28 day cycle.

[0215] The GLA components of the present invention can be adapted to treat one or more of the following infections by targeting extracellular vesicles derived from cells infected with pathogens: Acinetobacter infections ( Acinetobacter baumannii ), actinomycosis ( Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus ), African sleeping sickness, also known as African trypanosomiasis ( Trypanosoma bruce i ) AIDS - acquired immunodeficiency syndrome (HIV (human immunodeficiency virus)), amoebiasis ( Entamoeba histolytica ), anaplasmosis ( Anaplasma species ), angiostrongylidosis ( Angiostrongylus ), anisakiasis ( Anisakis ), anthrax ( Bacillus anthracis ), infection with Arcanobacterium haemolyticum ( Arcanobacterium haemolyticum ), Argentine hemorrhagic fever ( Junin virus ), ascariasis ( Ascaris lumbricoides ), aspergillosis ( Aspergillus species), astrovirus infection (family Astroviridae ), babesiosis (species s Babesia ), Bacillus cereus infections (Bacillus cereus), bacterial pneumonia (various bacteria), bacterial vaginosis (bacterial vaginosis microbiota), Bacteroides ( Bacteroides ) infections, balantidiasis ( Balantidium coli ), bartonellosis ( Bartonella ), Baylisascaris ( Baylisascaris ) infections, BK virus infection (BK virus), black piedra ( Piedraia hortae ), blastocystosis ( Blastocystis ), blastomycosis ( Blastomyces dermatitidi s), Bolivian hemorrhagic fever (Machupo virus), botulism and infant botulism ( Clostridium botulinum ; note: botulism is not an infection of Clostridium botulinum, it is caused by ingestion of botulinum toxin), Brazilian hemorrhagic fever (Sabia virus), brucellosis ( Brucella ), bubonic plague ( Enterobacteriaceae ), Burkholderia infection (Burkholderia), Buruli ulcer ( Mycobacterium ulcerans ) , calicivirus infection ( Caliciviridae (Norovirus and Sapovirus)), campylobacteriosis ( Campylobacter ), candidiasis, also known as thrush ( Candida ), capillariasis (an intestinal disease caused by Capillaria philippinensis, a liver disease caused by Capillaria hepatica , and a lung disease caused by Capillaria aerophila ), Carrión fever (Bartonella bacilliformis), cat-scratch disease ( Bartonella henselae ), cellulitis (usually group A Streptococcus and Staphylococcus ), Chagas disease, also known as American trypanosomiasis ( Trypanosoma cruzi ), chancre (Haemophilus ducreyi), chicken pox ( varicella zoster virus), chikungunya fever (Alphavirus), chlamydia ( Chlamydia trachomatis ), Chlamydophila pneumoniae infection, also known as TWAR ( Chlamydophila pneumoniae ), cholera ( Vibrio cholerae ), chromoblastomycosis ( Fonsecaea pedrosoi ), chytridiomycosis ( Batrachochytrium dendrabatidis ), clonorchiasis (Clonorchis sinensis), colitis caused by Clostridium difficile ( Clostridium difficile ), coccidioidomycosis ( Coccidioides immitis and Coccidioides posadasii ), Colorado tick fever (Colorado tick fever virus), colds/acute viral nasopharyngitis/acute viral upper respiratory infection (usually rhinoviruses and coronaviruses), Congo hemorrhagic fever Crimea (Congo-Crimea hemorrhagic fever virus), cryptococcosis ( Cryptococcus neoformans ), cryptosporidiosis ( Cryptosporidium ), skin form of larva migrans syndrome (usually Ancylostoma braziliense and a number of other parasites), cyclosporiasis (Cyclospora cayetanensis), cysticercosis (Taenia solium), cytomegalovirus infection (Cytomegalovirus), dengue fever (dengue viruses, e.g. DEN-1, DEN-2, DEN-3 and DEN-4), dienthamoebiasis (Dientamoeba fragilis), diphtheria (Corynebacterium diphtheriae), diphyllobothriasis (Dilobothrium), dracunculiasis (Dracunculus medinensis ), Ebola hemorrhagic fever (Ebolavirus), echinococcosis (Echinococcus), ehrlichiosis (Ehrlichia), Enterobacteriaceae (carbapenem-resistant Enterobacteriaceae ), enterobiasis (Enterobius vermicularis), Enterococcus infection ( Enterococcus ), enterovirus infection (Enterovirus), epidemic typhus (Rickettsia prowazekii), erythema infectiosum (parvovirus B19), roseola infantum (human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7)), fascioliasis ( Fasciola hepatica and Fasciola gigantica ), fasciolopsiasis ( Fasciolopsis buski ), filariasis (Filarioidea), food poisoning caused by Clostridium perfringens ( Clostridium perfringens ), free-living amoeba infections (various pathogens), Fusobacterium (Fusobacterium) infections, ha callous gangrene (usually Clostridium, for example, perfringens), geotrichosis (Geotrichum candidum), giardiasis ( Giardia lamblia ), glanders ( Burkholderia mallei ), gnathostomiasis ( Gnathostoma spinigerum and Gnathostoma hispidum ), gonorrhea ( Neisseria gonorrhoeae ), inguinal granuloma (Klebsiella granulomatis) , group A streptococcus (Streptococcus pyogenes) infections, group B streptococcus ( Streptococcus agalactiae ) infections, Haemophilus influenzae ( Haemophilus influenzae ) infections, enteroviral vesicular stomatitis (enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71)), hantavirus pulmonary syndrome ( Sin Nombre virus), disease caused by the Heartland virus (Heartland virus), Helicobacter pylori infection ( Helicobacter pylori ), hemolytic uremic syndrome ( Escherichia coli , e.g. O157:H7, O111 and O104:H4), hemorrhagic fever with renal syndrome ( family Bunyaviridae), hepatitis A (hepatitis A virus), hepatitis B (hepatitis B virus), hepatitis C (hepa hepatitis C), hepatitis D (hepatitis D virus), hepatitis E (hepatitis E virus), herpes simplex (herpes simplex virus 1 and 2 (HSV-1 and HSV-2), histoplasmosis (Histoplasma capsulatum), hookworm ( Ancylostoma duodenale and Necator americanus ), human bocavirus infections (human bocavirus), human ehrlichiosis caused by Ehrlichia ewingii ( Ehrlichia ewingii ), human granulocytic anaplasmosis ( Anaplasma phagocytophilum ), human metapneumovirus infection (human metapneumovirus), human monocytic ehrlichiosis (Ehrlichia chaffeensis), human papillomavirus infections (human papillomavirus), human parainfluenza virus infections (human parainfluenza viruses), hymenolepiasis ( Hymenolepis nana and Hymenolepis diminuta ), infectious mononucleosis caused by Epstein-Barr virus (Epstein-Barr virus), influenza ( Orthomyxoviridae ), isosporosis ( Isospora belli ), diseases Kawasaki, keratitis (various pathogens), Kingella kingae ( Kingella kingae ) infections, Lassa fever (vir with Lass), legionellosis, also known as Legionnaires' disease (Legionella pneumophila), legionellosis, also known as Pontiac fever (Legionella pneumophila), leishmaniasis ( Leishmania ), leprosy ( Mycobacterium leprae and Mycobacterium lepromatosis ), leptospirosis ( Leptospira ), listeriosis (Listeria monocytogenes), Lyme disease ( Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii ), lymphatic filariasis ( Wuchereria bancrofti and Brugia malayi ), lymphocytic choriomeningitis (lymphocytic choriomeningitis virus), malaria ( Plasmodium ), Marburg hemorrhagic fever (Marburg virus), measles ( measles), Middle East respiratory syndrome (Middle East respiratory syndrome coronavirus), melioidosis ( Burkholderia pseudomallei ), meningitis (various pathogens), meningococcal disease (Neisseria meningitidis), metagonimiasis (usually Metagonimus yokagawai ), microsporidiosis ( Microsporidia phylum ), molluscum contagiosum Moll yuska), monkeypox (monkeypox virus), infectious parotitis (infectious mumps virus), rat typhus (Rickettsia typhi), mycoplasma pneumonia ( Mycoplasma pneumoniae ), mycetoma (actinomycetoma) and fungal eumycetoma), myiasis (larvae of parasitic Diptera flies ), conjunctivitis of newborns (most often Chlamydia trachomatis and Neisseria gonorrhoeae ), norovirus infection (norovirus), nocardiosis ( Nocardia , for example, N. asteroides ), onchocerciasis (Onchocerca volvulus), opisthorchiasis ( Opisthorchis viverrini and Opisthorchis felineus ), paracoccidioidomycosis ( Paracoccidioides brasiliensis ), paragonimiasis ( Paragonimus , e.g. westermani ), pasteurellosis ( Pasteurella ), pelvic inflammatory disease (various pathogens), whooping cough ( Bordetella pertussis ), plague ( Yersinia pestis ), pneumococcal infection (S treptococcus pneumoniae ), pneumocystis pneumonia ( Pneumocystis jirovecii ), pneumonia (various pathogens), poliomyelitis (poliovirus), infections and Prevotella (Prevotella), primary amoebic meningoencephalitis (usually Naegleria fowleri ), progressive multifocal leukoencephalopathy (JC virus), psittacosis ( Chlamydophila psittaci ), Q fever ( Coxiella burnetii ), rabies (rabies virus), relapsing fever ( Borrelia , for example, B. hermsii and B. recurrentis ), Respiratory syncytial virus infections (Respiratory syncytial virus), Rhinosporidiosis ( Rhinosporidium seeberi ), Rhinovirus infection (Rhinovirus), Rickettsia infection ( Rickettsia spp.), Vesicular rickettsiosis (Rickettsia akari), Rift Valley fever (virus Rift Valley fever), Rocky Mountain spotted fever ( Rickettsia rickettsii ), rotavirus infection (rotavirus), rubella (rubella virus), salmonellosis ( Salmonella ), severe acute respiratory syndrome (SARS coronavirus), schistosomiasis ( Schistosoma ), sepsis (various pathogens) , shigellosis ( Shigella ), herpes zoster (varicella-zoster virus), smallpox (Variol a major or Variola minor), sporotrichosis ( Sporothrix schenckii ), staphylococcal food poisoning ( Staphylococcus ), staphylococcal infection ( Staphylococcus ), strongyloidiasis (Strongyloides stercoralis), subacute sclerosing panencephalitis (measles virus), syphilis ( Treponema pallidum ), teniasis ( Taenia ), tetanus ( Clostridium tetani ), ringworm of the beard and mustache (usually Trichophyton ), ringworm ( Trichophyton tonsurans ), ringworm of the trunk (usually Trichophyton ), inguinal ringworm (usually Epidermophyton floccosum , Trichophyton rubrum and Trichophyton mentagrophytes ), ringworm of the hands ( Trichophyton rubrum ), black lichen (usually Hortaea werneckii ), tinea pedis (usually Trichophyton ), dermatophytic onychomycosis (usually Trichophyton ), pityriasis versicolor (Malassezia), toxocariasis ( Toxocara canis or Toxocara cati ), trachoma ( Chlamydia trachomatis ), toxoplasmosis ( Toxoplasma gondii ), trichinosis ( Trichinella spiralis ), tricho monosis ( Trichomonas vaginalis ), trichuriasis ( Trichuris trichiura ), tuberculosis (usually Mycobacterium tuberculosis ), tularemia ( Francisella tularensis ), typhoid fever ( Salmonella enterica subsp. enterica, serovar typhi ), typhus (Rickettsia), Ureaplasma urealyticum infection ( Ureaplasma urealyticum ), coccidioidal granuloma ( Coccidioides immitis or Coccidioides posadasii ), Venezuelan equine encephalitis (Venezuelan equine encephalitis virus), Venezuelan hemorrhagic fever (Guanarito virus), Vibrio infections vulnificus (Vibrio vulnificus), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) enteritis, viral pneumonia (various viruses), West Nile fever (West Nile virus), white piedra (Trichosporon beigelii), Yersinia pseudotuberculosis (Yersinia pseudotuberculosis), yersiniosis ( Yersinia enterocolitica ), yellow fever (yellow fever virus) and zygomycosis (Zygomycetes).

Intracellular pathogens

[0216] Intracellular pathogens can be among the most difficult pathogens to treat, since the presence of the pathogen inside the cell provides it with some level of protection in the cellular environment.

[0217] Pathogens can be viruses, bacteria, fungi, protozoa, etc. Molecules according to the present invention are particularly suitable for the treatment of intracellular pathogens, in particular those described herein.

[0218] Known intracellular bacteria include Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Salmonell typhi, Brucella, Legionella, Mycobacterium (e.g., Mycobacterium tuberculosis), Nocardia, Rhodococcus equi, Yersinia, Neisseria meninggitidis .

[0219] One or more of the antibiotics is selected from erythromycin, doxycycline, azithromycin, rifampicin, streptomycin, gentamicin, doxycycline, ciprofloxacin, ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol, TMP-SMZ (trimethoprim-sulfamethoxazole), levofloxacin, moxifloxacin, tetraclaritcyclromycin , glycylcyclines, ethambutol, rifabutin, imipenem, cefotaxime, amikacin, vancomycin, minocycline, penicillin G, ampicillin, fluoroquinolones, azotreon, and combinations of two or more of them.

[0220] Mycobacterium tuberculosis infection is notoriously difficult to treat. In one independent aspect, the present invention provides a molecule for the treatment of latent and/or active tuberculosis, in which one or more of the drugs for the treatment of tuberculosis is conjugated as a payload to the GLA component described herein.

[0221] Molecules according to the present invention can significantly increase the effectiveness of modern drugs by delivering them into the cell where mycobacteria are located.

[0222] Treatment of tuberculosis depends on a number of factors, including whether tuberculosis is latent or active, whether the patient is an adult, a child, a pregnant woman, HIV positive, or a combination of the above. The following details apply to all aspects of the present invention, for example, the kind of molecules obtained and the way they are used to treat patients.

[0223] Treatment of latent tuberculosis in HIV-infected patients and children aged 2 to 11 is daily isoniazid for 6-9 months. Pregnant patients may receive treatment twice a week instead of daily.

[0224] Thus, in one embodiment, in a molecule according to the present invention, the GLA component is associated with a payload comprising isoniazid or its equivalent.

In the treatment of latent tuberculosis in patients aged 12 years and older without complicating factors, isoniazid and rifapentine can be administered once a week for 3 months. Alternatively, rifampin can be given daily for 4 months.

[0225] Thus, in one embodiment, the payload further comprises rifapentine.

[0226] Alternatively, a molecule can be proposed in which the GLA domain described herein is associated with a payload containing rifapentine. For use in treatment, a combination of molecules according to the present invention can be provided.

[0227] First-line drugs for the treatment of active tuberculosis are often selected from isoniazid, rifampin, ethambutol, pyrazinamide, and a combination of two or more of these.

[0228] Thus, a molecule in accordance with the present invention is provided, containing the GLA component described herein and associated with a payload containing rifampin.

[0229] In addition, a molecule in accordance with the present invention is provided, containing the GLA component described herein and associated with a payload containing ethambutol.

[0230228] In an additional embodiment, a molecule according to the present invention is provided, comprising the GLA component described herein and associated with a pyrazinamide-containing payload.

[0231] It is expressly provided that the payload used in the treatment of tuberculosis using extracellular vesicles described in this document may contain two or more, for example, three or four drugs, for example: isoniazid and rifamycin, isoniazid and pyraxinamide, isoniazid and ethambutol, rifamycin and pyraxinamide, rifamycin and ethambutol, pyraxinamide and ethambutol, isoniazid and rifamycin and pyraxinamide, isoniazid and rifamycin and ethambutol, and isoniazid and rifamycin and pyraxinamide and ethambutol.

[0232] Viral pathogens include influenza virus, human immunodeficiency virus, dengue, West Nile virus, variola virus, respiratory syncytial virus, Korean hemorrhagic fever virus, varicella-zoster virus, herpes simplex virus 1 or 2, Epstein-Barr virus, virus Marburg, hantavirus, yellow fever virus, hepatitis A, B, C or E viruses, Ebola virus, human papillomavirus, rhinovirus, coxsackievirus, poliovirus, measles virus, rubella virus, rabies virus, Newcastle disease virus, rotavirus, HIV (e.g. , HTLV-1 and -2).

[0233] Antivirals can be linked to the GLA component and independently selected from one or more of: abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, Atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudin, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fomivirsen, fosamprenavir, foscarnet, sfosonet, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, type II interferon, interferon III type I, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxidine, metizazon, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, PEG interferon alfa-2, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, raltegravir , reverse transcriptase inhibitor, ribavirin, rimantadine, ritonavir, pyramidin, ca quinavir, stavudine, synergistic enhancer (antiretroviral), tea tree oil, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0234] It is well known in the art that antivirals can be used in combination, for example, to increase efficacy.

[0235] Thus, in one embodiment, a payload molecule of the present invention is provided for the treatment of protozoal diseases, eg, malaria, African sleeping sickness, and the like.

[0236] Known parasitic protozoa include parasites similar to Plasmodium, such as the causative agent of malaria. For the treatment of malaria, drugs are used, for example, quinine and related agents, choloroquine, amodiaquine, pyrimethamine, proguanil, sulfonamides, mefloquine, atovachone, primaquine, aremizinin and its derivatives, halofantrine, doxycycline, clindamycin, sulfadiazine, and a combination of two or more of them.

[0237] In one embodiment, the implementation of the proposed molecule according to the present invention in the form of a pharmaceutical composition containing an excipient, diluent and/or carrier. In one embodiment, the composition is a parenteral composition.

[0238] "Parenteral formulation" means a formulation intended for delivery other than through the gastrointestinal tract. Typical routes of parenteral delivery include injection, implantation or infusion.

[0239] In one embodiment, the parenteral formulation is in the form of an injection. Injection includes intravenous, subcutaneous, intracranial, intrathecal, intratumoral, or intramuscular injection. "Injection" in this document means the introduction of liquid into the body through a syringe.

[0239] In one embodiment, the parenteral formulation is in the form of an infusion.

[0241] "Infusion" as used herein means the administration of fluids at a slow rate using a drip, infusion pump, syringe pump, or equivalent. In one embodiment, the infusion is administered over a period of time ranging from 1.5 minutes to 120 minutes, such as about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16 , 17, 18, 19 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 65, 80, 85, 90, 95, 100, 105, 110 or 115 minutes.

[0242] In one embodiment, the formulation is for intravenous (IV) administration. This route is particularly effective as it provides rapid access to most organs and tissues and is particularly useful for the treatment of metastases, such as established metastases, especially those located in highly vascularized areas such as the liver and lungs.

[0243] Therapeutic formulations are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other parenteral formulation suitable for human administration, and may be prepared as a pre-filled device such as a syringe or vial, especially as a single dose.

[0244] As discussed above, such a formulation typically contains a pharmaceutically acceptable diluent or carrier, for example, a virus-compatible non-toxic isotonic carrier in which the virus is stable for the desired period of time.

[0245] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper flow can be maintained, for example, by using a dispersant or surfactant, such as lecithin, or a nonionic surfactant, such as polysorbate 80 or 40. The presence of a surfactant can help maintain the desired particle size in dispersions. Examples of isotonic agents include carbohydrates, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition.

[0246] Thus, in an embodiment, there is provided a molecule in accordance with the present invention, wherein the GLA component described herein is associated with a payload containing one or more antimalarial drugs.

[0247] It is understood that "comprising" in the context of this application means "including".

[0248] Where technically feasible, embodiments of the present invention may be combined.

[0249] This document describes implementation options that contain certain characteristics/elements. The present invention also extends to individual embodiments consisting or essentially consisting of these features/elements.

[0250] Technical references, such as patents and applications, are incorporated herein by reference.

[0251] The prior art is part of the technical description of the present application and can be used as the basis for amendments, since the discussion in it is not limited to the discussion of the prior art and also includes a discussion of technical problems encountered in the field and the application of the present invention.

[0252] Any implementations specifically and expressly set forth herein may form the basis of a disclaimer, alone or in combination with one or more additional implementations.

[0253] This application claims priority of US patent applications with serial numbers: 62/554530, 62/569403, 62/554533, 62/569411, 62/584565 and 62/593014. Each of these applications is incorporated herein by reference. These applications can be used as the basis for amending the present application.

[0254] The following is a description of the present invention with reference to the following examples, which are purely illustrative and should not be construed as limiting the essence of the present invention in any way.

EXAMPLES

Figure 1A-D shows different representations of the structures of the GLA protein.

Figure 1E shows an embodiment of the GLA component in accordance with the present invention.

2 shows protein S (PrS) and annexin staining of breast cancer cell lines treated with peroxide to induce apoptosis. A , human MDA-231 cells treated with peroxide and stained with FITC-PrS. B , untreated MDA-231 cells, stained similarly. A. C , treated MDA-231 cells, stained with annexin. D , human MCF-7 cells treated with peroxide and stained with PrS. E , mouse MET-1 cells treated similarly to D. F , mouse 4T1 cells treated similarly to D.

Figure 3 shows the overlapping but not overlapping localization of PrS and annexin in cells. A , mouse 4T1 cells treated with peroxide and stained with Cy5-PrS ( RED ) and FITC-annexin ( GREEN ). Light arrow - jointly localized signals; red arrows, cells stained with PrS rather than annexin; green arrow - cells relatively more brightly stained with annexin and less brightly with PrS, indicating a clear pattern of binding (insets show staining with PrS and annexin separately). B , treated 4T1 cells stained with FITC-PrS and Cy5-annexin. Green arrows are cells stained with PrS, not annexin. C , Cy5-annexin staining of treated 4T1 cells pre-incubated with 1000-fold excess of cold annexin.

Figure 4 shows COS-1 apoptotic cells stained with PrS and annexin. Cells were treated with t-BHP as described and stained with FITC-annexin (left) and Cy5-PrS (right). Arrows point to subcellular structures that are believed to be apoptotic bodies.

Figure 5 shows differential staining of extracellular vesicles with PrS and annexin. Extracellular vesicles were obtained from 4T1 cells and stained with FITC-PrS (GREEN) and Cy5-annexin (RED). Arrows indicate vesicles stained with annexin only (RED arrow), PrS only (GREEN arrow), and both proteins (light arrow).

The figure 6 shows the subcellular localization of PrS and annexin. A , B , apoptotic 4T1 cells stained with FITC-PrS (GREEN arrows) and Cy5-annexin (RED arrows); light arrows - joint localization. C , possible apoptotic bodies.

The figure 7 shows the internalization of PrS for 5 minutes. Apoptotic 4T1 cells were stained with FITC-PrS (green) and Cy5-annexin (red) and visualized for 10 minutes after protein addition. A , the merged image. B , Hoescht nuclei staining only.

Figure 8 shows BLI images of 4T1 tumors in mice.

Figure 9 SPECT imaging of the effect of doxorubicin on 4T1 tumors using radiolabeled PrS and annexin. Mice with 4T1 breast cancer tumors were imaged using 99mTc PrS (A and B) or annexin (C and D) before (A and C) and 24 hours after the addition of doxorubicin (B and D).

Figure 10 shows SPECT imaging of mice treated with cyclohexamide. Five mice per panel are shown before ( A and C) and 24 h after ( B and D ) treatment. Mice were visualized using ^99m Tc PrS ( A and B ) or annexin ( C and D ), with arrows showing enhanced liver signal.

The figure 11 shows the localization of Cy5 PrS in the infected spleen. CD1 mice were infected with the bioluminescent Listeria bacterium and visualized 2 days after infection. Mice were injected with Cy5 PrS 30 min before sacrifice, the spleens were removed and frozen. Moderately infected ( A ) and control uninfected ( C ) mice are shown. Sections of infected ( B ) and uninfected ( D ) spleens of each mouse along the Cy5 channel are shown, combined with phase contrast images.

The figure 12 shows the localization of Cy5 PRS in tumors treated with doxorubicin. Mice with implanted 4T1 breast cancer tumors were treated with doxorubicin (images on the right) or left untreated (images on the left). After 24 hours, mice were intravenously injected with Cy5 PrS and sacrificed after 30 minutes. Tumors were removed, frozen and sectioned for fluorescence microscopy. Merged Cy5/phase contrast images from four different mice are shown.

Figure 13 shows TSC differentiation. TSCs were cultured in the presence (left) or in the absence (right) of growth factors. Arrows in the right panel indicate giant cells characteristic of differentiation.

Figure 14 shows PrS staining of trophoblast stem cells and differentiated trophoblasts. Trophoblast stem cells (left) were differentiated into trophoblast giant cells (right) by removal of growth factors. Cells were stained with Cy5-PrS and visualized.

The figure 15 shows the differentiation of MSCs. MSCs were processed as described in the text to differentiate into adipocytes (upper images) or osteoblasts (lower images). Differentiated cells in each case showed the expected morphology.

Figure 16 shows MSCs stained with PrS (green), annexin (red) and Hoechst (blue). Cells were visualized within 10 minutes after adding the dye mixture.

Figure 17 shows TSC stained with PrS (green, lightest area), annexin (red, bright areas around the cell membrane) and Hoechst (blue). Cells were visualized within 5 minutes after adding the dye mixture.

Figure 18 shows differential staining of TSC vesicles. TSC was stained as in Figure 17 . A group of cells secreted large vesicles stained with annexin (red) but not with PrS (green).

Figure 19 shows PrS staining of C17.2 neuronal progenitor cells. Cells were stained with PrS-FITC and visualized using standard (non-confocal) microscopy.

The figure 20 shows the internalization of PrS in TSC at 4C. FITC-PrS (green) and Cy5-annexin (red) were added to TSC at 4°C and visualized using confocal microscopy.

Figure 21 shows mouse bone marrow cells negative for sprout markers and stained using the SCA-1/c kit. At this point in the analysis, cells were not stained with either PI (propidium iodide; to detect dead cells) or PrS. Lack of staining for hematopoietic lineage markers (left panel) and c-kit and SCA1 staining (right panel) defines the HSC population shown in green (lightest areas).

Figure 22 PrS staining of long-lived HSCs. HSCs were isolated as in Figure 1 and stained with FITC-PrS. The character of SLAM was determined using Cy7 (x-axis).

Figure 23 PrS staining of short-lived HSCs. HSCs were isolated as in Figure 1 and stained with FITC-PrS. The character of SLAM was determined using Cy7 (x-axis).

Figure 24 shows the internalization of PrS into long-lived HSCs. HSCs were obtained as described, stained with PrS and examined using confocal microscopy. Green (lightest areas) - FITC-PrS; blue - staining of Hoescht nuclei; red - PI. Note that PI staining is absent in the nuclei, indicating that the cells are alive.

Figure 25 shows an example of a dead HSC with PI in the core.

Figure 26 GLA-mediated delivery is non-toxic to cells

The present application also includes sequences 1-6 in the corresponding sequence listing.

This project initiated the testing of labeled recombinant PrS as an in vivo imaging agent for SPECT (single photon computed tomography). Surprisingly, this molecule was found to be rapidly internalized by apoptotic cells. This unexpected discovery led to further study of this phenomenon, after which it was found that PrS is also internalized by several types of non-apoptotic stem cells.

PrS is the GLA domain of the S protein and the EGF domain of the S protein shown in SEQ ID NO: 6.

Ways

To ensure fluorescence, Cy5 and FITC were conjugated using Amersham (GE Heathcare) and Molecular Probes (Invitrogen) labeling kits, respectively, according to the manufacturer's instructions. Both sets contained columns to remove unconjugated fluorophore. Initially, 0.77 mg PrS (fraction 2) in 1 ml and 0.77 mg of annexin in 1 ml were labeled with FITC to test the specificity of binding to apoptotic cells. For co-localization and competition studies, 0.68 mg PrS (fraction 3) in 1 ml and 0.68 mg annexin were labeled with Cy5. For confocal microscopy, 0.76 mg PrS from the second batch was labeled with FITC and the previously labeled Cy5-conjugated annexin was used. It should be noted that the exact labeling efficiency was not determined, and the recovery from the columns was assumed to be 85% according to the labeling kit manufacturers' instructions. Thus, the relative intensity of staining of the two proteins may in any case reflect these unaccounted for factors. Cells were initially stained for 30 minutes, but subsequently found to be less than 5 minutes sufficient. To test the specificity of PrS to apoptotic cells, four breast cancer cell lines were initially used; Human MDA-231 and MCF7 and mouse 4T1 and MET-1. In addition, COS-1 monkey kidney cells were subsequently used. Apoptosis was induced with hydrogen peroxide or tert-butyl hydroperoxide (t-BHP). Cells were seeded in 24-well plates at 6×10 ⁴ cells per well or Eppendorf slides at 1×10 ⁴ cells per well, and apoptosis was induced the next day using 2 mM H ₂ O ₂ or t-BHP at time points from 30 min to 2 hours. After induction, wells were washed with annexin binding buffer (AB; Santa Cruz Biotech) and stained with labeled protein. Based on past experience and literature data, 5.5 μg/ml annexin protein was used for staining. This amount was corrected for an equimolar addition of PrS based on the molecular weight of annexin being 36 kDa and recombinant PrS being 30 kDa based on the provided gel images. Cells were stained for 15 min. Hoechst 33342 stain was used for nucleic acid visualization. The wells were then washed with AB and observed using an EVOS fluorescence microscope for viability. For confocal microscopy, a Leica SP8 microscope at the Cell Imaging Science Center at Stanford University was used. The wells were then washed with AB and observed using a Leica sp8 microscope. Hoechst 33342 dye was used to visualize the nuclei. For trophoblastic stem cell (TSC) toxicity studies, PrS was added and viability tested with trypan blue using the Nexcelom Cellometer.

To test the ability of labeled proteins to detect tumors, 5×10 ⁴ 4T1-luc cells were implanted in groups of 5 male BALB/c mice in the left axillary fat pad. Mice were imaged using bioluminescent imaging (BLI) in vivo every day to monitor tumor growth, starting 1 week after implantation. Mice were then treated on day 11 post-implantation with intraperitoneal (ip) administration of doxorubicin at a dose of 13 mg/kg body weight, and BLI was performed the next day. Control mice with tumors were not treated with doxorubicin. At 48 hours post-treatment, mice were imaged 1 hour after intravenous indicator injection (anesthesia 1.3 g/kg ip urethane) using an A-SPECT single head gamma camera (Gamma Medica) with a 1 mm pinhole collimator , for 128 steps on a 128×128 imaging matrix, 15 seconds per step, 2.7 cm ROR; RH = upper chest/neck. The administered dose of each protein was 160 μl (800 μCi). The animals were then sacrificed and biodistribution assessed. In the cyclohexamide treatment experiment, groups of 5 young (7 week old) male Swiss Webster mice were anesthetized (1.3 g/kg ip urethane) and intravenously injected with 50 mg/kg cycloheximide. 1 hour 45 minutes after the administration of cycloheximide, an indicator was administered (PrS = 180 μl/1.2 mCi per dose; annexin V = 170 μl/1.05 mCi per dose). 45 minutes after tracer administration, mice were imaged to 10-minute whole body static images using a single head collimator with parallel apertures (128×128 array) on an A-SPECT gamma camera.

To test the specific localization of fluorescent PrS in areas of apoptosis due to infection in live animals, CD1 mice were intravenously injected with the bioluminescent bacteria Listeria monocytogenes . This bacterial pathogen infects many organs, including the spleen, which undergoes extensive apoptosis of monocytes and granulocytes. At certain times after infection, the spleen is the main site of bacterial replication, so BLI signals from bacteria in the spleen can be correlated with the location of apoptotic probes. Mice were infected and visualized every day. When spleen signals were detected (day 2 after infection with 2×10 ⁵ cfu bacteria in 8-week-old CD1 mice), mice were injected with 300 mg/kg body weight of Cy5-PrS, after 30 minutes the animals were sacrificed, the spleens were removed, they were frozen in OCT and received sections for fluorescence microscopy. Uninfected control mice were used.

Flow cytometry was performed. Freshly prepared FITC-PrS prepared as described above was used. This laboratory routinely isolated mouse hematopoietic stem cells (HSCs). Cells were isolated from bone marrow of healthy mice by staining with c-Kit+, negative for cell sprout markers. To further explore the characteristics of the cells, they were also stained with SLAM markers. These markers stain self-renewing and differentiating cells, while unstained HSCs can only differentiate. Subsequent staining with FITC-PrS revealed the percentage of positive SLAM-staining cells shown in the results. Cells were then sorted by FITC and examined by confocal microscopy using a Hoechst 33342 for nuclear imaging.

results

To assess the specificity of PrS binding in the context of apoptosis in cell culture, the inventors used several human and mouse breast cancer cell lines. Apoptosis was induced with peroxide as described above and FITC-PrS binding was evaluated. Examples of these experiments are shown in figure 2 . Untreated cells showed minimal binding as shown in panel B of Figure 2 . Peroxide concentrations and incubation times were chosen to treat a small fraction of the cells, since at higher concentrations and/or longer incubation times, cells detached and staining and microscopy were not possible. In addition, the presence of a large number of untreated cells served as an internal negative control in each field. FITC-annexin showed specificity for apoptosis similar to PrS, being an internal positive control. The two proteins were then tested for co-localization and competitive binding. PrS and annexin labeled with both FITC and Cy5 were prepared for co-localization. 4T1 cells were treated with peroxide and stained with Cy5- and FITC-labeled PrS and annexin using both combinations of fluorophores. The cells were then visualized under an EVOS fluorescence microscope. The results are shown in figure 3 . Under test conditions, all brightly stained cells showed staining with both proteins. However, when using Cy5 or FITC, PrS appeared to stain (albeit weakly) some cells that did not stain with annexin ( Figure 3 ). The relative intensity of staining of different cells with each protein sometimes differed between the two probes, i.e. sometimes annexin stained two cells with the same intensity but PrS did not, and vice versa ( Fig. 3A , green arrow and inset). Thus, although both probes tended to stain the same cells, they did not appear to differ significantly. In a competitive assay, increasing excess amounts of unlabeled annexin were pre-incubated with apoptotic 4T1 cells for 15 minutes and then the cells were stained with Cy5-PrS. Surprisingly, PrS staining was not blocked even by 1000-fold excess of annexin (maximum excess tested, Fig. 3C ), although these proteins are thought to bind to the same PS-exposing target molecule. Co-staining of annexin and PrS has been observed with many cell types. Although the two proteins usually stained the same cells with each cell type, other differences became apparent. In particular, some objects smaller than cells were stained differently ( Fig. 4 ). These entities, which were present in increased numbers after peroxide treatment, were interpreted as apoptotic bodies; membrane-bound cell fragments resulting from the fragmentation of apoptotic cells. As shown in Figure 4 , PrS stained these entities while annexin did not, although some of these entities stained with both proteins. This observation was unexpected. To further explore the differential staining of subcellular entities, extracellular vesicles (EVs) were generated from mouse 4T1 tumor cells using a standard centrifugation protocol. The two proteins also stained these vesicles differently ( Figure 5 ), which may have biological and therapeutic implications.

EVs, in particular exosomes, microvesicles (MVs), and apoptotic bodies (ABs), are thought to play a key role in cell-to-cell communication through the transfer of biomolecules between cells. The biogenesis of these types of EVs is different, and they are formed from the endosomal (exosome) or plasma membrane (MV) or are products of programmed cell death (AB). All mammalian cells are believed to secrete EV. Each type of EV can transfer molecular weight to both neighboring and distant cells, influencing cell behaviors such as those involved in tumor development and progression. EVs may, in fact, be involved in almost all hallmarks of cancer, including maintenance of proliferative signaling pathways, evasion of growth suppression, resistance to cell death, reprogramming of energy metabolism, loss of genome stability, and development of the tumor microenvironment. They are also involved in the induction of angiogenesis, control of invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune control. Immune cells also appear to communicate via EV and can recognize EV as signals from tumor cells, infected tissues, and wounds. A deeper understanding of the biology of EVs and their contribution to the manifestation of the hallmarks of cancer is leading to new possibilities for the diagnosis and treatment of cancer. The development of additional EV surface markers is important for the development of this area, and PrS may be such a determinant.

Following these studies using fluorescence microscopy, the subcellular localization of PrS and annexin staining was assessed using confocal microscopy. Mouse 4T1 cells (without Luc-GFP reporters) were plated on 8-chamber glass slides at 1×10 ⁴ cells per chamber and apoptosis was induced by exposure to 2 mM H ₂ O ₂ or t-BHP (for 2 hours) the next day. The cells were then washed and stained for 15 minutes with PrS and annexin. The Hoechst 33342 dye was used to stain the nucleic acid. In all cases, the brightest stained cells were stained with both probes. However, in many cells, labeled PrS was observed in the cytoplasm, but labeled annexin was not ( Fig. 6 ). Although annexin was internalized and appeared in the vesicles of some cells, internalized annexin was not observed in the same cell along with surface-localized PrS. These results were unexpected since both proteins were expected to bind PS.

At the same time, it is clear that these two proteins reacted differently to the tested inhibitors. To further study the internalization of PrS, a time dependence experiment was performed. Apoptotic 4T1 cells were stained for 5 minutes with Cy5-Annexin and FITC-PrS and observed for 5 minutes after probe addition. PrS appeared in the cytoplasm of these cells immediately, indicating internalization within 5 minutes ( Fig. 7 ). Time-dependence images also showed that PrS and annexin did not always stain the same cells in the same way at early time points. The cells in figure 7 appear to be in various stages of apoptosis, as the cell on the left shows an uncondensed nucleus surrounded by an apparently intact nuclear membrane, while the right cell shows strong staining, often indicative of chromatin condensation that occurs at later stages of apoptosis. This staining pattern may indicate that PrS is bound during apoptosis earlier than annexin. Although this is only a guess at the moment, this preference explains many of the differences between these proteins that have been observed so far. For example, staining some cells with PrS rather than annexin, such as in Figures 3A and B, may be associated with PrS binding earlier in apoptosis. Several experiments were performed to study the localization of PrS in live animals. These studies used chemical and infectious induction of apoptosis in vivo, as well as the localization of PrS in tumors treated with doxorubicin, which is known to induce apoptosis. SPECT imaging using HYNIC-labeled PrS and annexin was performed in animals with implanted 4T1luc mammary tumors treated with doxorubicin. Because 4T1 tumors were labeled with luciferase, they could be imaged in mice using bioluminescent imaging in vivo (BLI). One of the images obtained in this experiment is shown in Figure 8 . This method can be used to evaluate tumor implantation and track progression in individual animals over time. PrS and annexin labeled with ^99m Tc were then used for SPECT imaging of doxorubicin-treated and control animals. An example of the results is shown in figure 9 . Images of the head and chest of two animals showed non-specific accumulation of the PrS probe in the salivary gland and a low signal-to-noise ratio when using this probe. Therefore, the display threshold in the PrS images shown was lowered in order to more effectively detect the background signal, which resulted in an overestimation of the coloring of the images. The low signal-to-noise ratio is probably due to the suboptimal labeling of only 1 mg of protein with HYNIC, and also due to the impossibility of conducting controlled studies of the HYNIC label: protein ratio.

In addition, SPECT imaging was performed on mice treated with cyclohexamide, which induces apoptosis in the liver ( Fig. 10 ). Each panel of Figure 10 shows whole body images of 5 mice. As with many radioactively labeled probes, a background signal is seen in the kidneys. Treatment of mice with cyclohexamide increased the SPECT signal of annexin in the liver. PrS again showed a low signal compared to annexin. Annexin was able to detect liver apoptosis in cyclohexamide-treated mice, while PrS showed only a slight increase in drug-induced liver signal. Cy5-PrS was administered to mice infected with bacteria that elicit apoptotic responses and tumor-bearing mice to test for PrS localization in apoptotic tissues and treated tumors, regardless of SPECT imaging and associated complications of HYNIC labeling. Listeria monocytogenes , a bacterial pathogen labeled with luciferase and well characterized for BLI, was used for infection. The characteristic BLI signals in the spleen provide excellent co-localization studies. CD1 mice were infected as described above and visualized by BLI on day 2 post-infection. The mice were then injected with Cy5-PrS and euthanized 30 minutes later, and the spleens were removed for sectioning and fluorescence microscopy ( Fig. 11 ). In all cases, spleen sections from infected mice showed much higher Cy5 fluorescence signals than control sections. In figure 11 , the infected mouse showed a small number of photons, indicating that the infection in this animal has not yet progressed very far. On this day, in many mice, the signal intensity of the spleen was 10 times higher. However, the Cy5 channel fluorescence was still very strong compared to the uninfected control shown. This result may reflect the current innate immune response to infection, as granulocytes and macrophages have been shown to be the main source of the annexin signal in these animals (these cells are programmed to apoptosis to limit tissue destruction).

The localization of fluorescent PrS was then tested in doxorubicin-treated 4T1 tumors. Mice with implanted tumors were treated with doxorubicin as described above and Cy5-PrS was intravenously administered 30 min prior to sacrifice and removal of tumors for sectioning and fluorescence microscopy. The results are shown in figure 12 . Areas of intense staining were observed in treated animals, while more modest signal was observed in sections of untreated tumors. Although some of the untreated tumors did have small areas of signal above background, signals similar in intensity to the treated tumors were not observed in any of the untreated sections.

Stem cells differ in phenotype from differentiated cells and can express PS without apoptosis, avoiding the induction of an immune response. Trophoblast stem cells (TSC) differentiate in culture into several types of trophoblasts. TSC is obtained from mouse uterine scrapings grown in the presence of fibroblast growth factor, activin and heparin. When these factors are removed from the environment, TSCs spontaneously differentiate into giant cells ( Fig. 13 ). TSCs were stained with PrS, while differentiated trophoblasts derived from these cells in culture were not stained ( Fig. 14 ). In addition, PrS was determined to be internalized into stem cells without inducing apoptosis. This result confirms the observations made on tumor cell lines with induced apoptosis. Without apoptosis induction, minimal staining was observed in tumor cells. Mesenchymal stem cells (MSCs) and TSCs were used to test for internalization into stem cells. MSC was obtained from mouse bone marrow. Mice bone marrow was washed and cultured for 6 days in the absence of growth factors. During this incubation, MSCs and hematopoietic stem cells (HSCs) replicated while fibroblasts adhered but did not increase in number for several generations. After 6 days the monolayer became visible. During reseeding with trypsinization, adhesive MSCs were preserved, while HSCs growing in suspension were lost. Fibroblasts were not supported due to the lack of growth factors and were also not preserved. Thus, this simple procedure made it possible to obtain an almost homogeneous population of MSCs. To confirm the identity of these cells, the cultures were separately treated with dexamethasone and glycerophosphate (to induce differentiation into osteoblasts) or dexamethasone and indomethacin (to induce differentiation into adipocytes). The results are shown in figure 15 . In response to the above treatment, corresponding cells appeared among the differentiated cells. Adipocytes contained large fat vesicles, while osteoblasts were dark cells with characteristic intracellular collagen and mineralization.

To assess the subcellular staining pattern, undifferentiated MSCs were stained with PrS and annexin, as well as with Hoechst nuclear staining reagent, and observed using confocal microscopy. The results of the observations are shown in Figure 16 . PrS was quickly internalized. In the case of MSCs, approximately 1 in 20 cells stained with PrS, which is consistent with previous data, but the exact percentage of staining was not determined. The morphology of MSCs is heterogeneous, and the cells secrete a large amount of a substance into the medium, some of which adheres to the surface of the glass slide, resulting in a background on some images. However, the data clearly showed internalized PrS 5 minutes after addition and annexin on the surface. TSCs were also stained and visualized similarly to MSCs. The observations also confirm internalization into these cells, which also occurred within 5 minutes of protein addition. The results are shown in figure 17 . TSCs are morphologically quite variable and can be multinucleated in the absence of differentiation, as seen in the figure. As in the case of MSCs, these primary cells release large amounts of material into the medium, some of which the inventors have identified as extracellular vesicles (previous data). This material also made visualization difficult. Some EVs stained with annexin rather than PrS and this phenomenon can be observed in TSC in Figure 18 . In this image of a TSC cluster, vesicles secreted by cells are stained with annexin, not with PrS that has internalized. These patterns raise interesting questions regarding the specificity and targets of PrS and annexin binding. Both of these proteins are believed to bind PS. However, differential binding to EV, as well as a different pattern of subcellular localization, indicates that their binding mechanisms are not exactly the same. Further research is needed to establish the basis for this difference, which may be important. In addition, PrS staining of the C17.2 neuronal progenitor cell line ( Figure 19 ), which is a transformed cell line capable of differentiating in vitro into astrocytes and other neuronal cells, was observed. Approximately 5% of these transformed cells stained, although this percentage is an estimate. It is noteworthy that penetration into TSC occurred even when the cells were cooled to 4°C ( Fig. 20 ). However, it should be noted that the chamber could not be continuously cooled once placed on the microscope. However, the temperature could not rise much during the 5 minutes of the imaging procedure. Although this result is interesting, it needs to be explicitly reproduced under more controlled conditions. If this result is confirmed, the mechanism should indeed be quite interesting.

The inventors have succeeded in staining hematopoietic stem cells (HSCs) with PrS. Using flow cytometry, the inventors determined that the HSCs stained with PrS and observed the internalization of PrS in these cells using confocal microscopy. HSCs were identified and isolated using fluorescent activated cell sorting (FACS). Cells were identified in the bone marrow as negative for SCA/c-kit-positive cell lineage markers ( Fig. 21 ). They were then stained with FITC-PrS. According to the staining pattern with the SLAM marker, two populations of HSCs can be identified - short-lived and long-lived. The SLAM (lymphocyte activation signaling molecule) markers CD48, CD150, CD229, and CD244 differentially stained HSCs with different patterns, so positive SLAM staining for a specific pattern is indicative of the ability to both self-renew and differentiate, while HSCs that stain negatively for the SLAM pattern , could only differentiate. PrS stained a subpopulation of long-lived HSCs ( Fig. 22 ) as well as short-lived HSCs ( Fig. 23 ). The cells showed negative staining with propidium iodide (PI), which means that they are all living cells. This result confirms previous experiments demonstrating that a subpopulation of stem cells stained with PrS without inducing apoptosis.

We then proceeded to test the internalization of PrS in HSCs. This experiment was complicated by many factors. Perhaps the most difficult was the survival of HSCs in culture, which died in large numbers in the medium during the night. Therefore, it was necessary to time the experiment in such a way as to perform the analysis using flow cytometry and confocal microscopy on the same day. In addition, the cells were non-adherent, making microscopy difficult. To increase the efficiency of microscopy, cells were resuspended in a small drop of medium. Finally, it was necessary to ensure that the PrS-stained cells, analyzed by microscopy, remained viable. Many HSCs died during analysis and isolation. Therefore, in addition to the Hoescht nuclear stain, PI was added and scans were performed using a different channel. The presence of PI-bright nuclei indicated dead cells. Despite these and time constraints, the inventors were able to perform the experiment and confirmed the internalization of PrS in live HSCs ( FIG. 24 ). Cell viability was confirmed by the absence of nuclear staining with PI. However, some cells were dead or dying, as shown in Figure 25 . Despite the complexity and duration of the experiment shown, the results demonstrated the presence of internalization.

Finally, Figure 26 shows preliminary toxicity studies on TSC; it was found that after 30 minutes at a concentration of 135 μg/ml, the viability decreased only to a minimal extent (from 78% to 74%) compared with PBS. Considering that at this level 10% of the culture volume was PrS-containing solution, this result supported the qualitative findings that PrS is essentially non-toxic to stem cells, and the slight toxicity observed may well be due to contamination of the preparation itself. Lower concentrations of PrS did not affect viability. The highest level of protein tested was over 1,000 times the concentration used for staining. Although full toxicity studies, which were not formally part of this project, would require much more extensive testing, PrS exhibited very little toxicity in these experiments.

Free information

[0275] The above results showed that PrS was rapidly internalized by a variety of PrS-expressing cells, including stem cells from many species, indicating that PrS has unique characteristics that can be manipulated to develop a therapeutic agent. In addition, the difference in specificity between PrS and annexin, such as shown in Figures 3 and 7, indicates differences in the binding mechanism of the two proteins. The fact that annexin is a tetramer and PrS a monomer cannot explain these differences, and these data indicate that some other component on the cell surface may be involved in PrS binding. The binding mechanism, specificity and internalization of PrS, and the possibility of modular manipulation provide many possibilities.

EXAMPLE 2

[0276] Stem cells differ in phenotype from differentiated cells and can express PS without apoptosis, avoiding the induction of an immune response. Stem cells were stained with a molecule carrying a GLA domain according to the present invention and containing a fluorescent label as a payload, without inducing apoptosis.

[0277] Trophoblast stem cells (FIG. 14) differentiating into several types of trophoblast cells in the placenta were stained with protein S, while differentiated trophoblasts derived from these cells in culture were not stained with it. This staining made it possible to distinguish between in vivo differentiated stem cells and in vitro differentiated cells.

[0278] These molecules of the present invention can be used to target cells in vivo or in ex vivo samples.

conclusions

[0279] Extracellular vesicles represent an excellent opportunity for the diagnosis and treatment of many diseases. The present inventors have demonstrated that the Gla domain recognizes the expression of phosphatidylserine. This property can be used to: detect extracellular vesicles expressing phosphatidylserine, isolate extracellular vesicles, target extracellular vesicles, and/or load the vesicles with, for example, therapeutic materials. These results are unexpected since extracellular vesicles are small subcellular entities.

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Ala Asn Thr Phe Leu Xaa Xaa Val Arg Lys Gly Asn Leu Xaa Arg Xaa

1 5 10 15

Cys Val Xaa Xaa Thr Cys Ser Tyr Xaa Xaa Ala Phe Xaa Ala Leu Xaa

20 25 30

Ser Ser Thr Ala Thr Asp Val Phe Trp Ala Lys Tyr Thr

35 40 45

<210> 4

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<220>

<221> miscellaneous

<222> (6)..(7)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (14)..(14)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (16)..(16)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (19)..(20)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (25)..(26)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (29)..(29)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (35)..(35)

<223> X - gamma-carboxyglutamic acid residue

<400> 4

Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa

1 5 10 15

Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe Lys

20 25 30

Asp Ala Xaa Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser

35 40 45

<210> 5

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<220>

<221> miscellaneous

<222> (6)..(7)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (14)..(14)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (16)..(16)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (19)..(20)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (25)..(26)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (29)..(29)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (32)..(32)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (35)..(35)

<223> X - gamma-carboxyglutamic acid residue

<400> 5

Ala Asn Ala Phe Leu Xaa Xaa Leu Arg Gln Gly Ser Leu Xaa Arg Xaa

1 5 10 15

Cys Lys Xaa Xaa Gln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe Xaa

20 25 30

Asp Ala Xaa Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser

35 40 45

<210> 6

<211> 250

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<220>

<221> miscellaneous

<222> (6)..(7)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (14)..(14)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (16)..(16)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (19)..(20)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (25)..(26)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (29)..(29)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (32)..(32)

<223> X - gamma-carboxyglutamic acid residue

<220>

<221> miscellaneous

<222> (36)..(36)

<223> X - gamma-carboxyglutamic acid residue

<400> 6

Ala Asn Ser Leu Leu Xaa Xaa Thr Lys Gln Gly Asn Leu Xaa Arg Xaa

1 5 10 15

Cys Ile Xaa Xaa Leu Cys Asn Lys Xaa Xaa Ala Arg Xaa Val Phe Xaa

20 25 30

Asn Asp Pro Xaa Thr Asp Tyr Phe Tyr Pro Lys Tyr Leu Val Cys Leu

35 40 45

Arg Ser Phe Gln Thr Gly Leu Phe Thr Ala Ala Arg Gln Ser Thr Asn

50 55 60

Ala Tyr Pro Asp Leu Arg Ser Cys Val Asn Ala Ile Pro Asp Gln Cys

65 70 75 80

Ser Pro Leu Pro Cys Asn Glu Asp Gly Tyr Met Ser Cys Lys Asp Gly

85 90 95

Lys Ala Ser Phe Thr Cys Thr Cys Lys Pro Gly Trp Gln Gly Glu Lys

100 105 110

Cys Glu Phe Asp Ile Asn Glu Cys Lys Asp Pro Ser Asn Ile Asn Gly

115 120 125

Gly Cys Ser Gln Ile Cys Asp Asn Thr Pro Gly Ser Tyr His Cys Ser

130 135 140

Cys Lys Asn Gly Phe Val Met Leu Ser Asn Lys Lys Asp Cys Lys Asp

145 150 155 160

Val Asp Glu Cys Ser Leu Lys Pro Ser Ile Cys Gly Thr Ala Val Cys

165 170 175

Lys Asn Ile Pro Gly Asp Phe Glu Cys Glu Cys Pro Glu Gly Tyr Arg

180 185 190

Tyr Asn Leu Lys Ser Lys Ser Cys Glu Asp Ile Asp Glu Cys Ser Glu

195 200 205

Asn Met Cys Ala Gln Leu Cys Val Asn Tyr Pro Gly Gly Tyr Thr Cys

210 215 220

Tyr Cys Asp Gly Lys Lys Gly Phe Lys Leu Ala Gln Asp Gln Lys Ser

225 230 235 240

Cys Glu Ser Arg His His His His His His His

245 250

<---

Claims (29)

1. An in vitro method for targeting extracellular vesicles with surface-exposed phosphatidylserine, said method comprising the step of administering a molecule containing: gamma-carboxyglutamic acid component (GLA component), wherein said GLA component contains the GLA domain or its active fragment and does not contain the active catalytic domain of the GLA protein, into a fluid that may contain an extracellular vesicle; wherein said GLA domain or active fragment thereof is independently selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z, osteocalcin, GLA matrix protein, GAS6, transthyretin, periostin, GLA 1, proline rich, GLA 2 rich in proline, GLA 3 rich in proline, and GLA 4 rich in proline. 2. The in vitro method according to claim 1, characterized in that the extracellular vesicle is an exosome. 3. The in vitro method according to claim 2, characterized in that the diameter of the exosome is from 30 to 100 nm. 4. The in vitro method according to any one of paragraphs. 1-3, characterized in that the density of the vesicle is from 1 to 1.5 g/ml. 5. The in vitro method according to any one of paragraphs. 1-4, characterized in that the vesicle contains one or more transmembrane proteins independently selected from Lamp-1, Lamp-2, CD 13, CD86, flotillin, syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44 , CD45, ICAM-1, integrin alpha 4, LiCAM, LFA-1, Mac-1 alpha and beta, Vti-IA and B, CD3 epsilon and zeta, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4 , FcR, GluR2/3, HLA-DM (MHC II), immunoglobulins, MHC-I or MHC-II components, TCR-beta, tetraspanins, and combinations of two or more of these proteins. 6. The in vitro method according to any one of paragraphs. 1-5, characterized in that said vesicle is released from an unhealthy cell. 7. The method according to claim 6, wherein the cell is a cancer cell. 8. The method according to claim 7, characterized in that the cell is a cancer stem cell. 9. The in vitro method according to any one of paragraphs. 1-8, characterized in that said fluid includes a sample obtained from a patient, ex vivo. 10. The in vitro method according to any one of paragraphs. 1-9, characterized in that the GLA domain or its active fragment originates from the S protein, for example, contains the sequence shown in SEQ ID NO: 1. 11. The in vitro method according to any one of paragraphs. 1-10, characterized in that the GLA component additionally contains an EGF domain. 12. The method according to claim 11, characterized in that the construct contains an EGF domain selected from thrombin, factor VII, factor IX, factor X, protein C, protein S, protein Z, osteocalcin, GLA matrix protein, GAS6, transthyretin, periostin , GLA 1, rich in proline, GLA 2, rich in proline, GLA 3, rich in proline, and GLA 4, rich in proline. 13. The in vitro method according to claim 12, characterized in that the EGF domain originates from the S protein. 14. The in vitro method according to any one of paragraphs. 1-13, characterized in that the GLA component contains the sequence shown in SEQ ID NO: 6, or its derivative without a His-tag. 15. The in vitro method according to any one of paragraphs. 1-14, characterized in that the GLA domain component further comprises a Kringle domain. 16. The in vitro method according to claim 15, characterized in that the Kringle domain comes from a protein selected from the group containing transcription activating factor 2 (ATF); factor XII (F12); thrombin (F2); hyaluronan binding protein 2 (HABP2); hepatocyte growth factor (HGF); hepatocyte growth factor activator (HGFAC); protein Kremen 1 (KREMEN1); KREMEN2; lipoprotein (a) (LPA); LPAL2; macrophage stimulating protein (MSP or MST1); phosphoinositide-3-kinase interacting protein 1 (PIK3IP1); tissue plasminogen activator (PLAT); urokinase (PLAU); plasmin (PLG); PRSS12; transmembrane tyrosine protein kinase receptor ROR1 (ROR1); and the transmembrane tyrosine protein kinase receptor ROR2 (ROR2). 17. The in vitro method according to any one of paragraphs. 1-16, characterized in that the GLA component is associated with the payload. 18. The in vitro method according to claim 17, characterized in that the GLA component is conjugated to the payload. 19. The in vitro method according to any one of paragraphs. 1-18, characterized in that the GLA component is part of a hybrid protein. 20. The in vitro method according to any one of paragraphs. 1-19, characterized in that the payload contains a detectable label. 21. The in vitro method according to claim 20, characterized in that said label is selected from a fluorescent protein, biotin, enzyme, label, radionuclide, luminescent label or compound that can be detected using NMR or EPR spectroscopy. 22. The in vitro method according to any one of paragraphs. 1-21, characterized in that the payload is a granule, tablet or label. 23. The in vitro method according to any one of paragraphs. 17-22, characterized in that the payload is connected to the GLA component via a linker. 24. The in vitro method according to claim 23, wherein the linker is cleavable. 25. The in vitro method according to any one of paragraphs. 1-24, characterized in that the method includes an additional step of obtaining an enriched population of vesicles. 26. The in vitro method according to any one of paragraphs. 1-25, characterized in that the method includes the step of isolating vesicles.

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