Chimeric polypeptides, extracellular vesicles containing the same, and their uses

Abstract

The present invention relates to a chimeric polypeptide, a nucleic acid encoding the chimeric polypeptide, and an extracellular vesicle comprising the chimeric polypeptide. The present invention further relates to the use of the extracellular vesicle as a drug for treating various diseases.

Description

[Technical Field]

[0001] The present invention relates to a chimeric polypeptide, a nucleic acid encoding the chimeric polypeptide, and an extracellular vesicle comprising the chimeric polypeptide. The present invention further relates to the use of the extracellular vesicle as a drug for treating various diseases. [Background technology]

[0002] Several target proteins, including type II transmembrane proteins of cells or viruses, proteins derived from the TNF or TNFR superfamily (which are type II transmembrane proteins that are cleaved and release their extracellular portions), proteins derived from the TGF-β superfamily, and proteins derived from the CTRP family, are all known to influence a variety of human and animal conditions, including viral infections, autoimmune diseases, T cell activation, regulation of cellular immune responses, cancer metastasis, and hormonal carcinomas. Therefore, these proteins are promising candidates for drug development for a variety of diseases.

[0003] Despite this promising aspect, the large-scale production of the native functional forms of specific proteins, including post-translational modifications and appropriate highly polymerized complex assemblies, is an extremely difficult task, making it sometimes challenging to find therapeutics available for clinical trials. Coupled with the short half-lives of certain proteins, no production strategy exists to supply high levels of bioactive proteins at a reasonable price.

[0004] Furthermore, all of these proteins become functional only when they acquire their multimerized forms, such as homodimers, heterodimers, trimers, tetramers, or more. Additionally, some of these proteins require membrane immobilization to become functional. Finally, since all of these proteins exhibit their function at their C-terminus, their C-terminal domains must remain accessible to either their receptors or their substrates. Therefore, alternative methods for providing the natively active multimerized forms of the desired proteins are highly desirable.

[0005] In this invention, the inventors have found that specific target proteins can be produced in large quantities when immobilized on extracellular vesicles, some of which can be produced in a highly multimerized form similar to those found in nature, and that extracellular vesicles rich in these proteins can be purified, characterized, and stored in batches that meet the required characteristics. Such standardized materials pave the way for promising strategies to treat a variety of human physiological or pathological conditions (often therapeutically possible or therapeutically targeted) in which the target proteins are involved, such as neurodegenerative diseases, autoimmune diseases, viral infections, T-cell immune responses, NK-cell immune responses, homeostatic imbalance-related diseases, diabetes, obesity and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory diseases, hormonal cancers, and cancer progression. [Overview of the project]

[0006] The present invention can be carried out in any order. i) The amino acid sequence of the target protein selected from type II transmembrane proteins, proteins derived from the TNF or TNFR superfamily, proteins derived from the TGF-β superfamily, proteins derived from the neurotrophin family, and proteins derived from the CTRP family selected from CTRP1, CTRP2, CTRP3, CTRP4, CTRP5, CTRP6, CTRP7, CTRP8, CTRP9, CTRP10, CTRP11, CTRP12, CTRP13, CTRP14 and CTRP15. ii) Optionally, the amino acid sequence of the transmembrane domain of the transmembrane protein, and iii) Amino acid sequence of pilot peptide that interacts with the endosomal transport sorting complex (ESCRT) cellular mechanism and / or amino acid sequence of the submembrane targeting domain Regarding chimeric polypeptides including...

[0007] In some embodiments, components i), ii), and iii) are organized from the C-terminus to the N-terminus in the chimeric polypeptide.

[0008] In some embodiments, the target protein is selected from neuraminidase N2, TRAIL, TNFα, CD30L, CD137L, TGF-β1, BMP7, BMP9, GDNF, BDNF, CTRP3, and OX40L.

[0009] In some embodiments, the submembrane targeting domain is linked to an anchor molecule, and preferably the anchor molecule is a fatty acid.

[0010] In some embodiments, the chimeric polypeptide further comprises at least one linker between the amino acid sequence of the target protein and the amino acid sequence of the transmembrane domain and / or between the amino acid sequence of the transmembrane domain or the pilot peptide and the amino acid sequence of the submembrane targeting domain.

[0011] In some embodiments, the transmembrane domain is selected from the transmembrane domain of a type II transmembrane protein, the transmembrane domain of an influenza virus neuraminidase transmembrane protein, the transmembrane domain of CD40L, and the transmembrane domain of CD8. Preferably, the transmembrane domain of CD40L is as set forth in SEQ ID NO: 17, and the transmembrane domain of CD8 is as set forth in SEQ ID NO: 19. More preferably, the transmembrane domain of CD40L is as set forth in SEQ ID NO: 18, and the transmembrane domain of CD8 is as set forth in SEQ ID NO: 20.

[0012] In some embodiments, the pilot peptide comprises at least one YxxL motif or DYxxL motif and at least one PxxP motif or PPxY motif (wherein "x" represents any amino acid residue, and preferably "x" represents a proline residue).

[0013] In some embodiments, the pilot peptide comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof, where the variant of SEQ ID NO: 47 retains at least three YxxL and / or DYxxL motifs and at least four PxxP motifs (where "x" represents any amino acid residue).

[0014] The present invention also relates to a nucleic acid encoding the chimeric polypeptide of the present invention.

[0015] The present invention provides an extracellular vesicle comprising the chimeric polypeptide of the present invention and / or the nucleic acid of the present invention, preferably · The transmembrane domain of the chimeric polypeptide is fixed to the extracellular vesicle lipid bilayer, and · The target protein of the chimeric polypeptide is exposed on the outer surface of the extracellular vesicle also relates to extracellular vesicles.

[0016] In some embodiments, the extracellular vesicle is an exosome preferably having a diameter in the range of about 30 nm to about 150 nm.

[0017] The present invention also relates to a population of extracellular vesicles of the present invention optionally further comprising a soluble target protein in its cytoplasm.

[0018] In some embodiments, the extracellular vesicles of the present invention or the population of extracellular vesicles of the present invention are purified, preferably ultra-purified.

[0019] The present invention also relates to the nucleic acid of the present invention, the extracellular vesicles of the present invention or the population of extracellular vesicles of the present invention for use as a medicament.

[0020] The present invention also relates to nucleic acids, extracellular vesicles, or populations of extracellular vesicles of the present invention for use in the treatment of diseases, disorders, or conditions selected from neurodegenerative diseases, autoimmune diseases, viral infections, T-cell immune responses, NK-cell immune responses, homeostatic imbalance-related diseases, diabetes mellitus, obesity and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory diseases, hormonal cancers, and cancer progression.

[0021] definition In this invention, the following terms have the meanings set forth below.

[0022] As used herein, the use of “a” and “an” means “at least one” or “one or more” unless the context clearly indicates otherwise. The terms “and” or “or” are generally used to mean “and / or” unless the context clearly indicates otherwise.

[0023] The term "approximately" preceding a number encompasses a range of ±10% or less of the given number. It should be understood that the value referred to by the term "approximately" is also specifically and preferably disclosed.

[0024] "CD8" refers to a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR). In humans, the CD8 transmembrane domain contains the amino acid sequence of SEQ ID NO: 19.

[0025] "CD40 ligand" (also called "CD40L" or "CD154") refers to a transmembrane protein that is a member of the tumor necrosis factor (TNF) superfamily. In humans, the CD40L transmembrane domain contains the amino acid sequence of SEQ ID NO: 17.

[0026] When referring to polypeptides, "chimera" refers to a polypeptide that combines several domains of at least two different types, distinguished by function and / or their cellular localization, and at least two of these domains originate from either different proteins of the same species or different species, or the same protein of different species.

[0027] When "domain" refers to a protein or polypeptide, it refers to a region of the protein or polypeptide having structural and / or functional properties. As used herein, "transmembrane domain" refers to a functional region of a protein or polypeptide that spans the phospholipid bilayer of a biological membrane.

[0028] The term "ESCRT," or "endosomal transport sorting complex," originally refers to a cellular mechanism consisting of five multi-subunit protein complexes that function coordinately in specific endosomes to facilitate the movement of particular cargo from the limiting membrane into budding vesicles within the endosomal lumen. This mechanism is hijacked by several enveloped viruses to budding from the cell membrane (including the plasma membrane).

[0029] "Exosomes" refer to extracellular vesicles produced in the endosomal compartment of eukaryotic cells (Thery et al., 2018. J Extracell Vesicles. 7(1):1535750; Yanez-Mo et al., 2015. J Extracell Vesicles. 4:27066; van Niel et al., 2018. Nat Rev Mol Cell Biol. 19(4):213-228). Typically, exosomes retain CD81, CD63, and CD9 markers on their surface.

[0030] An "expression vector" refers to a vector capable of directing the expression of a target nucleic acid sequence (for example, the nucleic acid according to the present invention) in a suitable host cell, and the vector itself includes a promoter functionally linked to the target nucleic acid sequence and a termination sequence.

[0031] "Extracellular vesicles" refer to any vesicles that are spontaneously released from a cell and consist of a lipid bilayer containing the cytoplasmic fraction of the cell. This expression specifically includes vesicles secreted into the extracellular space, i.e., "exosomes."

[0032] "Identity" or "sequence identity" refers to the number of identical or similar nucleotide or amino acid residues in a comparison between a test sequence and a reference sequence. Sequence identity can be determined by sequence alignment of nucleic acids or amino acid sequences to identify regions of similarity or identity. For the purposes of this specification, sequence identity is generally determined by alignment to identify identical nucleotide or amino acid residues. This alignment may be local or global. Matches, mismatches, and gaps can be identified between the sequences being compared. A gap is a null nucleotide or amino acid residue inserted between residues of an aligned sequence so that identical or similar letters are aligned. Generally, internal gaps and terminal gaps may exist. When using a gap penalty, sequence identity can be determined without penalty for terminal gaps (e.g., no penalty is given for terminal gaps). Alternatively, sequence identity can be determined by:

number

[0033] The terms “isolated” and all variations thereof, as well as “purified” and all variations thereof, are used interchangeably and mean that the molecular entities they refer to (e.g., polypeptides, nucleic acids, extracellular vesicles, etc.) are substantially free from other components (i.e., impurities) that are present in the natural environment in which such molecular entities normally exist. Preferably, isolated or purified molecular entities (e.g., isolated or purified polypeptides, isolated or purified nucleic acids, isolated or purified extracellular vesicles, etc.) are substantially free from other molecular entities related to the cell. “Substantially free” means that the isolated or purified molecular entities constitute more than 50% (i.e., at least 50% pure) of the heterogeneous composition, preferably more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, and more preferably more than 98% or 99%. Purity can be evaluated by a variety of methods known to those skilled in the art, including, but not limited to, chromatography, gel electrophoresis, immunoassays, compositional analysis, and biological assays.

[0034] "Linker" or "spacer" are used interchangeably and refer to an amino acid sequence, typically a synthetic amino acid sequence, that connects or links two peptide or polypeptide sequences to each other. A linker typically connects two peptide or polypeptide sequences by a peptide bond. Linkers are well known in the art and reference is made, for example, to Chen et al., 2013 (Adv Drug Deliv Rev. 65(10):1357-1369) or Klein et al., 2014 (Protein Eng Des Sel. 27(10):325-330), the contents of which are incorporated herein by reference. Examples of suitable linkers include the so-called "GS linker" or "Gly-Ser linker", i.e., an amino acid sequence consisting essentially of glycine (G) and serine (S) residues and typically containing two or more repeats of a peptide motif, although not always. GS linkers are well known and widely used in the art, particularly because of their flexibility. In some embodiments, the GS linker comprises or consists of an amino acid sequence (G x S) y or (SG x ) y (where x is in the range of 1 to 5 or more, such as 1, 2, 3, 4, 5 or more, and y is in the range of 1 to 8 or more, such as 1, 2, 3, 4, 5, 6, 7, 8 or more). In some embodiments, a GS linker having an amino acid sequence (G x S) y or (SG x ) y can further comprise one or several additional G and / or S residues at the N-terminus and / or C-terminus. In some embodiments, the GS linker has an amino acid sequence (GS) y , (GGS) y , (GGGS) y (SEQ ID NO: 48), (GGGGS) y (SEQ ID NO: 49) or (GGGGGS) y(Sequence ID 50) (wherein y is in the range of 1 to 8 or more, such as 1, 2, 3, 4, 5, 6, 7, 8 or more) contains or consists of such. Another example of a suitable linker is the so-called "glycine linker," i.e., an amino acid sequence consisting essentially of glycine (G) residues. Glycine linkers are well known and widely used in the art, particularly due to their flexibility. In some embodiments, the glycine linker is an amino acid sequence (G) z (wherein z is a range of 1 to 10 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)

[0035] "Local alignment" refers to an alignment of two sequences, but only the portion of the sequence that shares similarity or identity. Therefore, local alignment determines whether a subsegment of one sequence exists in another sequence. If there is no similarity, no alignment is returned. Examples of local alignment algorithms include BLAST or the Smith-Waterman algorithm (Smith & Waterman, 1981. Adv Appl Math. 2(4):482-9). For example, 50% sequence identity based on local alignment means that in the alignment of the entire sequences of two sequences to be compared of any length, a region of similarity or identity at a length of 100 nucleotides or amino acid residues has 50% of the same residues in that similarity or identity region.

[0036] A "pilot peptide" refers to a peptide that interacts with the ESCRT protein. The pilot peptide can be delivered to membrane vesicles, particularly exosome-forming vesicles, or to cellular compartments involved in the formation of membrane vesicles, especially exosome-forming vesicles, in eukaryotic cells.

[0037] "Selected from" is used herein, in accordance with common patent application drafting terminology, to introduce a list of elements from which one or more items are selected. Any occurrence of "selected from" herein may be replaced with "selected from a group including or consisting of" without altering its meaning.

[0038] "Subject" refers to a mammal, preferably a human. In some embodiments, the subject may be a "patient," i.e., a warm-blooded animal, preferably a human, who is waiting for or receiving medical care, or who has been, is currently, or will be the subject of medical treatment in the past, or is being observed for the onset of a disease.

[0039] The terms “submembrane targeting domain” or “membrane targeting domain” or “membrane recruitment domain” are interchangeably used to refer to a domain in a cell, particularly a eukaryotic cell (e.g., an exosome-producing cell), that can be fixed to the cell membrane and / or vesicle membrane without inserting itself into the membrane, and such fixation is achieved by one or more anchor molecules and / or by interaction between the submembrane targeting domain and the membrane (e.g., electrostatic interaction). In certain embodiments, the submembrane targeting domain can bind to or interact with the inner surface of the cell membrane (i.e., the cytoplasmic side of the cell membrane) and / or the inner surface of the vesicle membrane (i.e., the luminal side of the vesicle membrane).

[0040] A "therapeutic effective dose" refers to a level or amount of chimeric polypeptide, nucleic acid, extracellular vesicles, a population of extracellular vesicles, a composition, a pharmaceutical composition, a drug, etc., intended to (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or halt the progression, exacerbation, or worsening of one or more symptoms of a disease, disorder, or condition; (3) bring about remission of symptoms of a disease, disorder, or condition; (4) reduce the severity or incidence of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition, without causing serious adverse effects or adverse side effects on the target. A therapeutic effective dose may be administered as a prophylactic measure before the onset of a disease, disorder, or condition. Alternatively or in addition, a therapeutic effective dose may be administered as a therapeutic measure after the onset of a disease, disorder, or condition.

[0041] A "transmembrane protein" refers to a protein that contains at least one transmembrane domain and can be fixed to the phospholipid bilayer of a biological membrane. The transmembrane domain is generally a hydrophobic helical structure and can contain multiple, particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10, or even 20 or more, hydrophobic α-helices. It can also be arranged in a β-sheet, for example, a β-barrel structure typically consisting of 8 to 22 β-strands. Transmembrane proteins can also be classified into types I, II, III, and IV according to the positions of their N-terminus and C-terminus on different sides of the lipid layer. Type I transmembrane proteins are fixed to the lipid membrane by a stop-transfer-anchor sequence, and during synthesis, their N-terminal domain (ER) is targeted to the lumen (and extracellular space if the mature form is on the cell membrane). Types II and III are fixed to a signal-anchor sequence; type II is targeted to the ER lumen by its C-terminal domain, while type III is targeted to the ER lumen by its N-terminal domain. Type IV is further subdivided into IV-A, in which its N-terminal domain targets the cytoplasm, and IV-B, in which its N-terminal domain targets the lumen.

[0042] "To treat" or "treatment" or "alleviation" refers to both therapeutic and preventive measures, the purpose of which is to prevent or slow down (delay the progression of) the targeted pathological condition or disorder. "To treat" or "treatment" may refer to therapeutic measures. "To treat" or "treatment" may refer to preventive treatment. "To treat" or "treatment" may refer to both preventive treatment and therapeutic measures. Those requiring treatment include those already suffering from the disease, as well as those susceptible to the disease or those for whom prevention of the disease is necessary. In some embodiments, "treatment" of a disease, disorder, or condition is considered successful if, after administration of a therapeutic dose of chimeric polypeptide, nucleic acid, extracellular vesicles, populations of extracellular vesicles, compositions, pharmaceutical compositions, drugs, etc., the subject exhibits at least one of the following: some degree of alleviation of one or more symptoms associated with the disease, disorder, or condition being treated, a reduction in morbidity and mortality, and an improvement in quality of life. The above parameters for evaluating treatment success and improvement of the disease can be readily measured by routine procedures familiar to physicians.

[0043] A "vector" refers to a nucleic acid capable of transporting the target nucleic acid to which it is ligated (e.g., the nucleic acid according to the present invention). A vector capable of directing the expression of the target nucleic acid (e.g., the nucleic acid according to the present invention) is called an "expression vector." Generally, expression vectors are in the form of plasmids. In this specification, the terms "plasmid" and "vector" are used interchangeably. However, other forms of expression vectors that perform equivalent functions are also included under the term vector. [Modes for carrying out the invention]

[0044] The present invention can be carried out in any order. i) The amino acid sequence of the target protein selected from type II transmembrane proteins, proteins derived from the TNF or TNFR superfamily, proteins derived from the TGF-β superfamily, and proteins derived from the CTRP family selected from CTRP1, CTRP2, CTRP3, CTRP4, CTRP5, CTRP6, CTRP7, CTRP8, CTRP9, CTRP10, CTRP11, CTRP12, CTRP13, CTRP14 and CTRP15. ii) Optionally, the amino acid sequence of the transmembrane domain of the transmembrane protein, and iii) Amino acid sequence of pilot peptide that interacts with the endosomal transport sorting complex (ESCRT) cellular mechanism and / or amino acid sequence of the submembrane targeting domain Regarding chimeric polypeptides including...

[0045] In some embodiments, components (i), (ii), and (iii) are organized in the chimeric polypeptide from the N-terminus to the C-terminus or from the C-terminus to the N-terminus.

[0046] According to the present invention, the target protein is a therapeutic protein selected from type II transmembrane proteins, proteins derived from the TNF or TNFR superfamily, proteins derived from the TGF-β superfamily, and proteins derived from the CTRP family selected from CTRP1, CTRP2, CTRP3, CTRP4, CTRP5, CTRP6, CTRP7, CTRP8, CTRP9, CTRP10, CTRP11, CTRP12, CTRP13, CTRP14, and CTRP15.

[0047] Type II transmembrane proteins are single-pass transmembrane proteins whose N-terminus is located on the cytoplasmic side of the membrane. They are fixed to the lipid membrane by a signal anchor sequence and target the lumen of the endoplasmic reticulum via their C-terminal domain.

[0048] In some embodiments, the target protein is a type II transmembrane protein.

[0049] Examples of type II transmembrane proteins include, but are not limited to, influenza virus-derived neuraminidase N2, BRI2 (also known as essential membrane protein 2B or ITM2B), proteins from the type II transmembrane serine protease (TTSP) family, Golgi phosphate protein 2 (also known as Golgi membrane protein 1, GOLM1 or Golgi membrane protein GP73, GOLPH2), neprilysin (also known as neutral endopeptidase-24.11, CD10, SFE, EPN, SCA43, CMT2T or NEP), proteins from the asialoglycoprotein receptor (ASGPR) family, and proteins from the Dectin type C lectin-like receptor (CTLR) family (such as killer cell lectin-like receptors (KLRs) that form homo-NKG2D or hetero-dimer NKG2A).

[0050] In some embodiments, the protein of interest is neuraminidase N2 or a variant thereof derived from the influenza A H3N2 strain of influenza virus described in SEQ ID NO: 1.

[0051] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 1 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 1 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0052] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 1 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0053] Tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies of proteins are type II transmembrane proteins that contain a TNF homology domain and form trimers. Members of these superfamilies exist either i) fixed to the plasma membrane, similar to classical type II transmembrane proteins, or ii) released from the cell membrane by extracellular proteolytic cleavage and capable of functioning as cytokines. The TNF family comprises 19 members, and the TNFR family comprises 27 members.

[0054] Examples of proteins derived from the TNF superfamily include, but are not limited to, lymphotoxin α (LTα, also known as TNFβ or TNFSF1B), tumor necrosis factor (TNF, also known as TNFα, Dif, necrosin, or TNFSF1A), lymphotoxin β (LTβ, also known as TNFSF3 or TNFγ), OX40 ligand (OX40L, also known as TNFSF4, CD252, Gp34, or CD134L), and CD40 ligand (TNFS CD40L, also known as F5, CD154, TRAP, Gp39, or T-BAM; Fas ligand (FasL, also known as TNFSF6, CD178, APTL, or CD95L); CD27 ligand (CD27L, also known as TNFSF7 or CD70); CD30 ligand (CD30L, also known as TNFSF8 or CD153); CD137 ligand (CD137L, also known as TNFSF9, 4-1BB ligand, or 4-1BBL); TNF-related apoptosis induction Derivative ligand (TNFSF10, also known as CD253 or APO-2L, TRAIL), nuclear factor κB receptor activator ligand (TNFSF11, CD254, OPGL, TRANCE or ODF, also known as RANKL), TNF-related weak apoptosis inducer (TNFSF12, also known as APO-3L or DR3L, TWEAK), proliferation inducer ligand (TNFSF13, CD256, also known as TALL-2 or TRDL1, APRIL), B cell activator (TNFSF13 B, CD257, BlyS, TALL-1, or TNFSF20 (BAFF), a receptor expressed on T lymphocytes that is homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for binding to herpesvirus entry mediators (TNFSF14, CD258, or HVEML, LIGHT), vascular endothelial growth inhibitor (TNFSF15, TL1, or TL-1A, VEGI), TNF superfamily member 18 (GITRL,Examples include TNFSF18 (also known as AITRL or TL-6) and ectodysprascin A (EDA, also known as ED1-A1 or ED1-A2).

[0055] Examples of proteins derived from the TNF receptor superfamily include, but are not limited to, tumor necrosis factor receptor 1 (TNFR1, also known as TNFRSF1A or CD120a), tumor necrosis factor receptor 2 (TNFR2, also known as TNFRSF1B or CD120b), lymphotoxin β receptor (TNFRSF3, LTβR, also known as LTBR or CD18), OX40 (also known as TNFRSF4 or CD134), CD40 (also known as TNFRSF5 or Bp50), and Fas receptor. Death receptor 3 (also known as TNFRSF6, Apo-1, or CD95), decoy receptor 3 (DCR3, also known as TNFRSF6B, TR6, or M68), CD27 (also known as TNFRSF7, S152, or Tp55), CD30 (also known as TNFRSF8, Ki-1, or TNR8), 4-1BB (also known as TNFRSF9, or CD137), death receptor 4 (DR4, also known as TNFRSF10A, TRAILR1, Apo-2, or CD261), death receptor 5 (TNFRSF10 B, DR5 also known as TRAILR2 or CD262), Decoy receptor 1 (TNFRSF10C, TRAILR3, LIT, TRID or CD263 also known as DCR1), Decoy receptor 2 (TNFRSF10D, TRAILR4, TRUNDD or CD264 also known as DCR2), RANK (also known as TNFRSF11A or CD265), Osteoprotegerin (TNFRSF11B, OPG also known as OCIF or TR1), TWEAK receptor (TNFRSF12A, Fn 14 (also known as CD266), transmembrane activators and CAML interacting factors (TACI, also known as TNFRSF13B, IGAD2, or CD267), BAFF receptor (also known as TNFRSF13B or CD268), herpesvirus entry mediator (HVEM, also known as TNFRSF14, ATAR, TR2, or CD270), nerve growth factor receptor (NGFR, also known as TNFRSF16, p75NTR, or CD271), B cell maturation antigen (BCM, TNFRSF17,Examples include BCMA (also known as CD269 or TNFRSF13A), glucocorticoid-induced TNFR-related receptors (GITR, also known as TNFRSF18, AITR, or CD357), TROY (also known as TNFRSF19, TAJ, or TRADE), death receptor 6 (DR6, also known as TNFRSF21 or CD358), death receptor 3 (DR3, also known as TNFRSF25, Apo-3, TRAMP, LARD, or WS-1), and ectodysprasin A2 receptor (EDA2R, also known as TNFRSF27 or XEDAR).

[0056] In some embodiments, the target protein is a protein derived from the TNF or TNFR superfamily. In some embodiments, the target protein is selected from LTα, TNFα, LTβ, OX40L, CD40L, FasL, CD27L, CD30L, CD137L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, TNFSF18, EDA, TNFR1, TNFR2, LTβR, OX40, CD40, Fas receptor, DCR3, CD27, CD30, 4-1BB, DR4, DR5, DCR1, DCR2, RANK, OPG, TWEAK receptor, TACI, BAFF receptor, HVEM, NGFR, BCMA, GITR, TROY, DR6, DR3, and EDA2R.

[0057] In some embodiments, the target protein is a protein derived from the TNF superfamily. In some embodiments, the target protein is selected from LTα, TNFα, LTβ, OX40L, CD40L, FasL, CD27L, CD30L, CD137L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, TNFSF18, and EDA.

[0058] In some embodiments, the target protein is a protein derived from the TNFR superfamily. In some embodiments, the target protein is selected from TNFR1, TNFR2, LTβR, OX40, CD40, Fas receptor, DCR3, CD27, CD30, 4-1BB, DR4, DR5, DCR1, DCR2, RANK, OPG, TWEAK receptor, TACI, BAFF receptor, HVEM, NGFR, BCMA, GITR, TROY, DR6, DR3, and EDA2R.

[0059] In some embodiments, the target protein is the TRAIL or a variant thereof described in SEQ ID NO: 2. In some embodiments, the target protein is the TRAIL external domain or a variant thereof described in SEQ ID NO: 3. In some embodiments, the target protein is the TRAIL TNF-like domain or a variant thereof described in SEQ ID NO: 4.

[0060] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 2, 3, or 4 or a variant thereof. In some embodiments, the variant of the amino acid sequence of SEQ ID NO: 2, 3, or 4 includes an amino acid sequence that shares at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 2, 3, or 4, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0061] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 2, 3, or 4 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 2, 3, or 4, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0062] In some embodiments, the target protein is TNFα or a variant thereof as described in SEQ ID NO: 5. In some embodiments, the target protein is the TNFα external domain or a variant thereof as described in SEQ ID NO: 6. In some embodiments, the target protein is the TNFα maturation domain or a variant thereof as described in SEQ ID NO: 7.

[0063] Therefore, in some embodiments, the target protein comprises or consists of the amino acid sequence of SEQ ID NO: 5, 6, or 7 or a variant thereof. In some embodiments, the variant of the amino acid sequence of SEQ ID NO: 5, 6, or 7 comprises an amino acid sequence that shares at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 5, 6, or 7, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0064] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 5, 6, or 7 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 5, 6, or 7, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher local sequence identity.

[0065] In some embodiments, the target protein is CD30L or a variant thereof, as described in SEQ ID NO: 8. In some embodiments, the target protein is the CD30L external domain or a variant thereof, as described in SEQ ID NO: 9.

[0066] Therefore, in some embodiments, the target protein comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9 or a variant thereof. In some embodiments, the variant of the amino acid sequence of SEQ ID NO: 8 or 9 comprises an amino acid sequence that shares at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 8 or 9, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0067] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 8 or 9 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 8 or 9, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0068] In some embodiments, the protein of interest is CD137L or a variant thereof, as described in SEQ ID NO: 10.

[0069] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 10 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 10 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 10, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0070] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 10 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 10, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0071] In some embodiments, the protein of interest is OX40L or a variant thereof, as described in SEQ ID NO: 90.

[0072] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 90 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 90 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 90, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0073] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 90 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 90, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0074] Proteins of the transforming growth factor β (TGF-β) superfamily are synthesized in the form of precursors in which the active protein is located at the C-terminus of the polypeptide and is associated with a propeptide located at the N-terminus of the polypeptide. These proteins require proteolytic cleavage to exhibit biological activity. Proteins of the TGF-β superfamily are active only as homodimers, in which two chains are linked by a single interchain disulfide bond. The TGF-β superfamily is subdivided into subfamilies, namely the TGF-β subfamily, bone morphogenetic proteins, growth and differentiation factors, activin and inhibin subfamilies, left-right determinants, glial cell line-derived neurotrophic factor (GDNF) subfamily, and a wide variety of other members.

[0075] Examples of proteins derived from the TGF-β superfamily include, but are not limited to, TGF-β1 (also known as CED, DPD1, or LAP), TGF-β2 (also known as LDS4 or G-TSF), TGF-β3 (also known as ARVD, ARVD1, RNHF, or LDS5), Bone Morphogenesis Imperative 2 (BMP2, also known as SFSSC or SFSSC1), BMP3 (also known as osteogenin), and BMP4 (MCOPS6, OFC11, or ZY). GDF1 (also known as ME), BMP5, BMP6 (also known as VGR or VGR1), BMP7 (also known as OP-1), BMP8a, BMP8b (also known as OP2), BMP10, BMP11 (also known as GDF11 or VHO), BMP15 (also known as GDF9B, ODG2 or POF4), Growth and Differentiation Factor 1 (GDF1, also known as DORV, DTGA3, RAI, CHTD6, UOG1, LASS1, CERS1 or LAG1), GDF2 (BMP9 or HH GDF3 (also known as T5), GDF3 (also known as KFS3, MCOP7, MCOPCB6, Vg-related gene 2 or Vgr-2), GDF5 (also known as BDA1C, BMP14, CDMP1, LAP-4, LAP4, OS5, SYM1B, SYNS2 or DUPANS), GDF6 (also known as BMP13, CDMP2, KFM, KFS, KFS1, KFSL, LCA17, MCOP4, MCOPCB6, SCDO4, SGM1 or SYNS4), GDF8 (also known as myostatin or MSTN) (also known as), GDF9 (also known as PF14), GDF10 (also known as BMP3B or BIP), GDF15 (also known as MIC-1, MIC1, NAG-1, PDF, PLAB, PTGFB or TGF-PL), Activin A, Activin B, Activin C, Activin D, Inhibin A, Inhibin B, Lefty-Determinant 1 (Lefty-1), Lefty-2, Nodal (also known as HTX5), Artemin (Enovin, EVN),Examples include neublastin (ARTN, also known as NBN), neuthrin (NRTN), persephin (PSPN), glial cell line-derived neurotrophic factor (GDNF), and anti-Müllerian hormone (AMH, also known as Müllerian inhibitory hormone, MIH, MIS, or antim).

[0076] In some embodiments, the target protein is a protein derived from the TGF-β superfamily. In some embodiments, the target protein is selected from TGF-β1, TGF-β2, TGF-β3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF10, GDF11, GDF15, activin A, activin B, activin C, activin D, inhibin A, inhibin B, lefty-1, lefty-2, ARTN, GDNF, NRTN, PSPN, and AMH.

[0077] Examples of proteins derived from the TGF-β subfamily include, but are not limited to, TGF-β1, TGF-β2, and TGF-β3. In some embodiments, the protein of interest is selected from TGF-β1, TGF-β2, and TGF-β3.

[0078] Examples of proteins derived from the bone morphogenetic protein subfamily include, but are not limited to, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15. In some embodiments, the target protein is selected from BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, and BMP15.

[0079] Examples of proteins derived from the growth and differentiation factor subfamily include, but are not limited to, GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF10, GDF11, and GDF15. In some embodiments, the protein of interest is selected from GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF10, GDF11, and GDF15.

[0080] Examples of proteins derived from the activin and inhibin subfamilies include, but are not limited to, activin A, activin B, activin C, activin D, inhibin A, and inhibin B. In some embodiments, the protein of interest is selected from activin A, activin B, activin C, activin D, inhibin A, and inhibin B.

[0081] Examples of proteins derived from the left-right determinant subfamily include, but are not limited to, Lefty-1 and Lefty-2. In some embodiments, the protein of interest is selected from Lefty-1 and Lefty-2.

[0082] Examples of proteins derived from the GDNF subfamily include, but are not limited to, ARTN, GDNF, NRTN, and PSPN. In some embodiments, the protein of interest is selected from ARTN, GDNF, NRTN, and PSPN.

[0083] In some embodiments, the protein of interest is TGF-β1 or a variant thereof, as described in SEQ ID NO: 11.

[0084] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 11 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 11 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 11, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0085] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 11 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 11, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0086] In some embodiments, the protein of interest is BMP7 or a variant thereof, as described in SEQ ID NO: 12.

[0087] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 12 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 12 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 12, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0088] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 12 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 12, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0089] In some embodiments, the protein of interest is BMP9 or a variant thereof, as described in SEQ ID NO: 13.

[0090] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 13 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 13 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 13, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0091] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 13 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 13, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0092] In some embodiments, the protein of interest is GDNF or a variant thereof, as described in SEQ ID NO: 14.

[0093] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 14 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 14 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 14, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0094] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 14 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 14, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0095] Neurotrophins are growth factors that promote the survival, development, and function of neurons. Neurotrophins are synthesized in precursor form, in which the active protein is located at the C-terminus of a polypeptide and is associated with a propeptide located at the N-terminus of the polypeptide. Neurotrophins require proteolytic cleavage to exhibit biological activity.

[0096] Examples of proteins derived from the neurotrophin family include, but are not limited to, nerve growth factor (NGF, also known as NGFB or NGF-β), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). In some embodiments, the protein of interest is selected from NGF, BDNF, NT-3, and NT-4.

[0097] In some embodiments, the protein of interest is BDNF or a variant thereof, as described in SEQ ID NO: 15.

[0098] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 15 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 15 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 15, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0099] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 15 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 15, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0100] The CTRP (C1q / TNF-related) proteins constitute a conserved, structurally related family of 15 members named CTRP1 to CTRP15. All CTRP proteins contain a signal peptide, an N-terminal variable domain with one or more conserved cysteine ​​residues, a collagen-like domain (Col-Gly-XY), and a C-terminal globular C1q / TNF domain. All CTRP proteins can form homotrimers and can also form higher-order multimer 3D structures consisting of multiple trimers.

[0101] In some embodiments, the target protein is a protein derived from the CTRP family. In some embodiments, the target protein is a protein derived from the CTRP family, provided that it is not adiponectin.

[0102] In some embodiments, the target protein is a protein derived from the CTRP family, selected from CTRP1, CTRP2, CTRP3, CTRP4, CTRP5, CTRP6, CTRP7, CTRP8, CTRP9, CTRP10, CTRP11, CTRP12, CTRP13, CTRP14, and CTRP15.

[0103] In some embodiments, the protein of interest is not adiponectin.

[0104] In some embodiments, the protein of interest is CTRP3 or a variant thereof, as described in SEQ ID NO: 16.

[0105] Therefore, in some embodiments, the target protein includes or consists of the amino acid sequence of SEQ ID NO: 16 or variants thereof. In some embodiments, the variants of the amino acid sequence of SEQ ID NO: 16 include amino acid sequences that share at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 16, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0106] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 16 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 16, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0107] In some embodiments, the target protein is (a) A type II transmembrane protein selected from neuraminidase N2, (b) Proteins derived from the TNF or TNFR superfamily selected from LTα, TNFα, LTβ, OX40L, CD40L, FasL, CD27L, CD30L, CD137L, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, TNFSF18, EDA, TNFR1, TNFR2, LTβR, OX40, CD40, Fas receptor, DCR3, CD27, CD30, 4-1BB, DR4, DR5, DCR1, DCR2, RANK, OPG, TWEAK receptor, TACI, BAFF receptor, HVEM, NGFR, BCMA, GITR, TROY, DR6, DR3 and EDA2R, (c) Proteins derived from the TGF-β superfamily selected from TGF-β1, TGF-β2, TGF-β3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF10, GDF11, GDF15, activin A, activin B, activin C, activin D, inhibin A, inhibin B, lefty-1, lefty-2, ARTN, GDNF, NRTN, PSPN, and AMH. (d) A protein from the neurotrophin family selected from NGF, BDNF, NT-3, and NT-4, or (e) Proteins derived from the CTRP family selected from CTRP1, CTRP2, CTRP3, CTRP4, CTRP5, CTRP6, CTRP7, CTRP8, CTRP9, CTRP10, CTRP11, CTRP12, CTRP13, CTRP14, and CTRP15 That is the case.

[0108] In some embodiments, the target protein is selected from neuraminidase N2, TRAIL, TNFα, CD30L, CD137L, TGF-β1, BMP7, BMP9, GDNF, BDNF, and CTRP3.

[0109] In some embodiments, the target protein is selected from neuraminidase N2, TRAIL, TNFα, CD30L, CD137L, TGF-β1, BMP7, BMP9, GDNF, BDNF, CTRP3, and OX40L.

[0110] According to the present invention, the chimeric polypeptide contains the amino acid sequence of the transmembrane domain of a transmembrane protein.

[0111] In some embodiments, the transmembrane domain is the transmembrane domain of a transmembrane protein of any biological origin, including mammals, viruses, and bacteria.

[0112] In some embodiments, the transmembrane domain is a transmembrane domain of a transmembrane protein selected from the group including or consisting of human proteins, non-human animal proteins, pathogen or virulence factor proteins (particularly viral proteins, bacterial proteins, parasitic proteins), or tumor cell proteins.

[0113] In some embodiments, the transmembrane domain is the transmembrane domain of a type I transmembrane protein. The transmembrane domain of the type I transmembrane protein may be used in the chimeric polypeptide described herein, where components i), ii), and iii) are organized from the N-terminus to the C-terminus in the chimeric polypeptide, if present (e.g., construct 1 in Figure 2, Figure 3, or Figure 4).

[0114] In some embodiments, the transmembrane domain is a transmembrane domain of a retroviral transmembrane glycoprotein selected from the group including or comprising bovine leukemia virus (BLV), human immunodeficiency virus (HIV) (including, but not limited to, HIV-1 or HIV-2), human T-cell leukemia virus (HTLV) (including, but not limited to, HTLV-1 or HTLV-2), and Mason Pfizer monkey virus (MPMV).

[0115] In some embodiments, the transmembrane domain is the transmembrane domain of the hemagglutinin transmembrane protein of the influenza virus.

[0116] In some embodiments, the transmembrane domain is the transmembrane domain of CD8.

[0117] In some embodiments, the transmembrane domain is the transmembrane domain of a type II transmembrane protein. The transmembrane domain of the type II transmembrane protein may be used in the chimeric polypeptide described herein, where components i), ii), and iii) are organized from the C-terminus to the N-terminus in the chimeric polypeptide, if present (e.g., constructs 2, 3, 4, and 5 in Figures 2, 3, or 4).

[0118] In some embodiments, the transmembrane domain is the transmembrane domain of the influenza virus neuraminidase transmembrane protein.

[0119] In some embodiments, the transmembrane domain is the transmembrane domain of the CD40 ligand (CD40L).

[0120] In some embodiments, the transmembrane domain is a CD40 ligand (CD40L) or a CD8 transmembrane domain.

[0121] In some embodiments, the transmembrane domain of CD40L includes or consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof. In some embodiments, the transmembrane domain includes or consists of the amino acid sequence of SEQ ID NO: 18 or a variant thereof.

[0122] In some embodiments, the transmembrane domain of CD8 includes or consists of the amino acid sequence of SEQ ID NO: 19 or a variant thereof. In some embodiments, the transmembrane domain includes or consists of the amino acid sequence of SEQ ID NO: 20 or a variant thereof.

[0123] In some embodiments, the amino acid sequence variant of SEQ ID NO: 17, 18, 19, or 20 includes an amino acid sequence that shares at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 17, 18, 19, or 20, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0124] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 17, 18, 19, or 20 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 17, 18, 19, or 20, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher local sequence identity.

[0125] According to the present invention, the chimeric polypeptide contains an amino acid sequence of a peptide that interacts with the endosomal transport sorting complex (ESCRT) cellular mechanism, also known as the "pilot peptide."

[0126] In some embodiments, the pilot peptide can be delivered to membrane vesicles, particularly exosome-forming vesicles or cellular compartments involved in the formation of membrane vesicles, especially exosome-forming vesicles in eukaryotic cells.

[0127] When incorporated into a chimeric polypeptide such as the chimeric polypeptide of the present invention, the pilot peptide enables the delivery of the chimeric polypeptide to membrane vesicles and / or their formation sites, and particularly enables the delivery of the chimeric polypeptide to the membrane of membrane vesicles so that the polypeptide can be secreted by cells associated with membrane vesicles (especially exosomes).

[0128] Pilot peptides that interact with the ESCRT protein are described in the obtained patents European Patent No. 2268816B1 and U.S. Patent No. 9,546,371B2, the relevant contents of which are incorporated herein by reference.

[0129] In some embodiments, the pilot peptide comprises at least one YxxL motif (wherein "x" represents any amino acid residue). In particular, it may comprise one, two, or three YxxL motifs. One of the YxxL motifs or multiple YxxL motifs of the pilot peptide may be, for example, YINL (SEQ ID NO: 21) or YSHL (SEQ ID NO: 22).

[0130] Alternatively or additionally, the pilot peptide may contain at least one motif equivalent to the YxxL motif, for example, a YxxF motif (wherein "x" represents any amino acid residue). Thus, the YxxF motif or one of the YxxF motifs of the pilot peptide may be, for example, YINF (SEQ ID NO: 23) or YSHF (SEQ ID NO: 24).

[0131] In some embodiments, the pilot peptide includes a DYxxL motif (wherein "x" represents any amino acid residue). The DYxxL motif or one of the DYxxL motifs of the pilot peptide may be, for example, DYINL (SEQ ID NO: 25).

[0132] Alternatively or additionally, the pilot peptide may contain at least one motif equivalent to the DYxxL motif, for example, a DYxxF motif (wherein "x" represents any amino acid residue). Therefore, the DYxxF motif or one of the DYxxF motifs of the pilot peptide may be, for example, DYINF (SEQ ID NO: 26).

[0133] In some embodiments, the pilot peptide further comprises at least one PxxP motif (wherein "x" represents any amino acid residue). In particular, it may comprise one, two, three, or four PxxP motifs.

[0134] In some embodiments, the pilot peptide includes at least one proline-rich motif.

[0135] A "proline-rich motif" refers to an amino acid sequence, typically a synthetic amino acid sequence containing several proline (P) residues. Examples of proline-rich motifs, though not limited to them, include PP, PPP, and PPPP (SEQ ID NO: 89).

[0136] In some embodiments, the pilot peptide comprises at least one PP motif, at least one PPP motif, or at least one PPPP (SEQ ID NO: 89) motif.

[0137] In some embodiments, the pilot peptide comprises at least one PPxY motif (wherein "x" represents any amino acid residue, preferably "x" represents a proline (P) residue).

[0138] In some embodiments, one of the PxxP motifs of the PxxP motif or pilot peptide may be, for example, PSAP (SEQ ID NO: 27) or PTAP (SEQ ID NO: 28).

[0139] In some embodiments, the pilot peptide comprises at least one YxxL motif or DYxxL motif and at least one PxxP motif.

[0140] In some embodiments, the pilot peptide comprises or consists of amino acid sequences having one, two, or three YxxL and / or DYxxL motifs and one, two, three, or four PxxP motifs.

[0141] In some embodiments, the pilot peptide comprises or consists of amino acid sequences having three YxxL and / or DYxxL motifs and four PxxP motifs.

[0142] In some embodiments, a YxxL motif, or at least one of two YxxL motifs if there are two or more, is located downstream, or at the C-terminal, of one or more PxxP motifs.

[0143] Examples of proteins having pilot peptides containing at least one YxxL motif include cellular proteins and viral proteins. In particular, these viral proteins are enveloped virus or herpesvirus proteins, such as the transmembrane glycoprotein of the Epstein-Barr virus LMP2-A protein, which contains at least two YxxL motifs.

[0144] In some embodiments, the pilot peptide is a pilot peptide of a retroviral transmembrane glycoprotein. In some embodiments, the pilot peptide may be a pilot peptide of a retroviral transmembrane glycoprotein selected from the group including or comprising bovine leukemia virus (BLV), human immunodeficiency virus (HIV) (including, but not limited to, HIV-1 or HIV-2), human T-cell leukemia virus (HTLV) (including, but not limited to, HTLV-1 or HTLV-2), and Mason Pfizer monkey virus (MPMV).

[0145] In some embodiments, the pilot peptide has the following amino acid sequence: ·PxxPxxxxPxxPxSxYxxLxPxxPExYxxLxPxxPDYxxL (Sequence ID 29) ·PxxPx n PxxPx n SxYxxLx n PxxPEx n YxxLx n PxxPDYxxL (Sequence ID 30), ·PxxPxxxxPxxPxSxYxxLxPxxPExYxxLxPxxPDYxxLxxxx (Sequence ID 31), and ·PxxPx n PxxPx n SxYxxLx n PxxPEx n YxxLx n PxxPDYxxLxxxx (Sequence ID 32) (In the formula, "x" and "x nEach of these contains one of the following: any amino acid residue and any one or more amino acid residues.

[0146] In some embodiments, the pilot peptide has the following amino acid sequence: ·PxxPxxxxxxxxxxxxYxxL (Sequence ID 33), ·PxxPxxxxxxxxxxxDYxxL (Sequence ID 34), · PxxPxxYxxxxxxxxxYxxL (Sequence ID 35) ·PxxPxxYxxxxxxxxDYxxL (Sequence ID 36) · PxxPExYxxLxPxxPDYxxL (Sequence ID 37) ·PxxPx n YxxL (sequence number 38), ·PxxPx n DYxxL (Sequence ID 39), ·PxxPx n Yx n YxxL (Sequence ID 40), ·PxxPx n Yx n DYxxL (Sequence ID 41), ·PxxPEx n YxxLx n PxxPDYxxL (sequence number 42), ·PxxPxxxxPxxPxxxYxxLxPxxPExYxxLxPxxPDYxxL (Sequence ID 43), ·PxxPx n PxxPx n YxxLx n PxxPEx n YxxLx n PxxPDYxxL (Sequence ID 44), ·PxxPxxxxPxxPxxxYxxLxPxxPExYxxLxPxxPDYxxLxxxx (Sequence ID 45), and ·PxxPx n PxxPx n YxxLx n PxxPEx n YxxLx nPxxPDYxxLxxxx (Sequence ID 46) (In the formula, "x" and "x n Each of these contains one of the following: any amino acid residue and any one or more amino acid residues.

[0147] In particular, "n" may be 1 or greater and less than 50. In particular, "n" may have any value between 1 and 20.

[0148] In some embodiments, the pilot peptide contains 6 to 100 amino acid residues, particularly 20 to 80, 30 to 70, or 40 to 60 amino acid residues, for example, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

[0149] In some embodiments, the pilot peptide comprises or consists of the amino acid sequence APHFPEISFPPKPDSDYQALLPSAPEIYSHLSPTKPDYINLRPAP (SEQ ID NO: 47) or its variants.

[0150] The amino acid sequence variant of SEQ ID NO: 47 may include an amino acid sequence that shares at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 47, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0151] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 47 may include an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 47, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more local sequence identity.

[0152] Preferably, the amino acid sequence variant of SEQ ID NO: 47 retains at least one, two, or three YxxL or DYxxL motifs and one, two, three, or four PxxP motifs.

[0153] More preferably, the amino acid sequence variant of SEQ ID NO: 47 retains three YxxL and / or DYxxL motifs and four PxxP motifs.

[0154] In some embodiments, the chimeric polypeptide further comprises at least one linker.

[0155] In some embodiments, at least one linker connects the amino acid sequence of the target protein to the amino acid sequence of the transmembrane domain.

[0156] In some embodiments, at least one linker connects the amino acid sequence of the transmembrane domain to the amino acid sequence of the submembrane targeting domain. In some embodiments, at least one linker connects the amino acid sequence of the pilot peptide to the amino acid sequence of the submembrane targeting domain.

[0157] In some embodiments, at least one linker is not detachable. In some embodiments, at least one linker is detachable.

[0158] In some embodiments, at least one linker is a Gly-Ser linker. Examples of Gly / Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, and G4S linkers, including iterations and combinations thereof.

[0159] In some embodiments, at least one linker is (GGGS) n The array (sequence number 48) (wherein n is a positive integer in the range of 1 to 10, preferably 1 to 5) contains or consists of such an array.

[0160] In some embodiments, at least one linker is (GGGSGGGGS) n The array (sequence number 51) (wherein n is a positive integer in the range of 1 to 10, preferably 1 to 5) contains or consists of such an array.

[0161] In some embodiments, at least one linker connecting the amino acid sequence of the protein of interest and the amino acid sequence of the transmembrane domain comprises the sequence SGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGGS (SEQ ID NO: 52). The linker may be used in the chimeric polypeptide described herein, where components i), ii), and iii) are organized from N-terminus to C-terminus in the chimeric polypeptide, if present (e.g., construct 1 in Figures 2, 3, and 4).

[0162] In some embodiments, at least one linker connecting the amino acid sequence of the protein of interest to the amino acid sequence of the transmembrane domain comprises the sequence GGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSG (SEQ ID NO: 53). The linker may be used in the chimeric polypeptide described herein, where components i), ii), and iii) are organized from the C-terminus to the N-terminus in the chimeric polypeptide, if present (e.g., constructs 3, 4, and 5 in Figures 2, 3, and 4).

[0163] In some embodiments, if the chimeric polypeptide contains two or more linkers, the two or more linkers may be the same or different.

[0164] In some embodiments, the chimeric polypeptide includes an amino acid sequence of a submembrane targeting domain.

[0165] In some embodiments, the chimeric polypeptide includes an amino acid sequence of a pilot peptide that interacts with the endosomal transport sorting complex (ESCRT) cellular mechanism and an amino acid sequence of a submembrane targeting domain.

[0166] In some embodiments, the submembrane targeting domain is added to the chimeric polypeptide described herein, where components i), ii), and iii) are organized from the C-terminus to the N-terminus in the chimeric polypeptide, if present (e.g., constructs 4 and 5 in Figures 2, 3, and 4).

[0167] In some embodiments, the submembrane targeting domain is sufficient to enable the chimeric polypeptide to be immobilized to the lipid bilayer of the cell membrane or vesicle membrane, preferably by one or more anchor molecules and / or by interactions such as electrostatic interactions.

[0168] Therefore, the submembrane targeting domain, due to its presence in the chimeric polypeptide, enables the chimeric polypeptide to be immobilized on the cell membrane or vesicle membrane when expressed in cells without insertion into the membrane.

[0169] In some embodiments, the submembrane targeting domain gives the chimeric polypeptide the property of binding to the inner surface of the cell membrane (i.e., the cytoplasmic side of the cell membrane) and / or the inner surface of the vesicle membrane (i.e., the luminal side of the vesicle membrane).

[0170] In some embodiments, the chimeric polypeptide further comprises a submembrane targeting domain, preferably which is linked to an anchor molecule.

[0171] An "anchor molecule" refers to any molecule that can be inserted into at least one layer of the lipid bilayer of a cell membrane or vesicle membrane. In particular, anchor molecules are lipids or lipid-containing molecules. Therefore, the submembrane targeting domain is said to be "attached to lipids."

[0172] In some embodiments, the anchor molecule comprises or consists of one or more lipids or lipid-containing molecules, wherein the lipids include hydrophobic carbon chains that allow them to be encapsulated in the lipid bilayer of a cell membrane or vesicle membrane.

[0173] In some embodiments, the lipids are fatty acids, including but not limited to myristic acid, palmitic acid, and isoprenoids (e.g., geranyl-geranyl and farnesyl).

[0174] In some embodiments, the anchor molecule is covalently linked to the submembrane targeting domain.

[0175] In some embodiments, the anchor molecule is linked to the submembrane targeting domain by a glycine (e.g., myristic acid), cysteine, or serine amino acid residue in the submembrane targeting domain. This linkage may be via an amide or thioester bond.

[0176] In some embodiments, the submembrane targeting domain is either the submembrane targeting domain of an extrinsic membrane protein or a variant of the submembrane targeting domain of an extrinsic membrane protein.

[0177] In some embodiments, the submembrane targeting domain includes or comprises consensus sequences that enable the attachment (e.g., by acylation or prenylation) of fatty acids and in particular myristic acid, palmitic acid, or isoprenoids (e.g., geranyl-geranyl and farnesyl).

[0178] In some embodiments, the submembrane targeting domain is (M)G-X1-X2-X3-X4 (In the formula, X1, X2, and X3 represent any amino acid residue independently of each other. X4 represents an amino acid selected from a serine (S) residue or a cysteine ​​(C) residue, and (M) represents methionine, an initiator that, when located at the N-terminus of this chimeric polypeptide, can be removed in vivo by post-translational processing. It contains or consists of a consensus array.

[0179] In some embodiments, • X1 is selected from C, S and L, and / or • X2 is selected from S, I, V, M and L, and / or • X3 is selected from K, Q, H, F, C and S, and / or • The X4 can be selected from S or C.

[0180] In some embodiments, if the chimeric polypeptide described herein includes a submembrane targeting domain linked to an anchor molecule, it is preferably located at the N-terminal position of the chimeric polypeptide.

[0181] In some embodiments, the submembrane targeting domain may further comprise several basic amino acid residues, particularly several amino acid residues selected from K, R, and H. “Several” means at least two and preferably at least three or more. These basic amino acid residues may be involved in interactions with lipids of the cell membrane or vesicle membrane, particularly choline and its derivatives (e.g., phosphatidylcholine), thereby increasing the affinity between the submembrane targeting domain and these membranes.

[0182] In some embodiments, the basic amino acid residues may be located within and / or outside the consensus sequence (M)G-X1-X2-X3-X4.

[0183] Therefore, in some embodiments, the submembrane targeting domain is ·below: • (M)GXXKS (Sequence No. 55) ·CKXK, and ·CKXKXXXXRRR (Sequence ID 56) (In the formula, X represents any amino acid residue, and (M) represents methionine, an initiator that, when located at the N-terminus of this chimeric polypeptide, can be removed in vivo by post-translational processing. It contains or consists of amino acid sequences selected from among them, or It is a variant of one of these sequences that retains the ability of a submembrane targeting domain to be fixed to the lipid bilayer of the cell membrane or vesicle membrane.

[0184] In some embodiments, the submembrane targeting domain is derived from proteins of the Src family of proteins. Examples of such proteins, but not limited to, include the Src, Yes, Lyn, Fyn, Lck, Blk, Fgr, Hck, and Yrk proteins (Resh, 1994. Cell. 76(3):411-413), and more specifically, the N-terminal region of one of these proteins, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 N-terminal amino acid residues of one of these proteins.

[0185] In some embodiments, the submembrane targeting domain is derived from a c-Src or v-Src protein, preferably c-Src.

[0186] Alternatively, the submembrane targeting domain may be derived from other acylated proteins, such as viral capsid proteins, including, but not limited to, human immunodeficiency virus (HIV) MA protein or filovirus protein.

[0187] In some embodiments, the submembrane targeting domain is derived from the Src protein.

[0188] In some embodiments, the submembrane targeting domain is derived from the Src protein and has the following amino acid sequence: (M)GSSKSKPKDPSQRRR (Sequence ID 57) (M)GSSKSKPKDPSQRRRKSR (Sequence No. 58) ·(M)GSSKSKPKDPSQRRRKSRGPGG (Sequence No. 59), or • Any variant of these sequences that retains the ability of a submembrane targeting domain to be fixed to the lipid bilayer of the cell membrane or vesicle membrane. (In the formula, (M) represents methionine, an initiator that, when located at the N-terminus of the chimeric polypeptide, can be removed in vivo by post-translational processing.) It includes one of these or consists of them.

[0189] In some embodiments, the submembrane targeting domain is derived from the Src protein and includes or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof.

[0190] In some embodiments, the amino acid sequence variant of SEQ ID NO: 57, 58, or 59 includes an amino acid sequence that shares at least 70% overall sequence identity with the amino acid sequence of SEQ ID NO: 57, 58, or 59, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more overall sequence identity.

[0191] In addition or alternatively, the amino acid sequence variant of SEQ ID NO: 57, 58, or 59 includes an amino acid sequence that shares at least 70% local sequence identity with the amino acid sequence of SEQ ID NO: 57, 58, or 59, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher local sequence identity.

[0192] In some embodiments, the submembrane targeting domain is derived from a Src protein and comprises or consists of the amino acid sequence of SEQ ID NO: 57, 58, or 59 or a variant thereof, preferably SEQ ID NO: 58, and further comprises one or more anchor molecules as defined above, particularly myristic acid (in the form of a myristyl moiety) linked to a glycine residue at position 2.

[0193] In some embodiments, the submembrane targeting domain is derived from the Src protein and comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof, and further comprises one or more anchor molecules as defined above, in particular myristic acid (in the form of a myristyl moiety) linked to a glycine residue at position 2.

[0194] In some embodiments, if the chimeric polypeptide includes a submembrane targeting domain, this submembrane targeting domain may be linked to the rest of the chimeric polypeptide by at least one linker.

[0195] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a type II transmembrane protein or a protein derived from the TNF or TNFR superfamily as defined herein. It includes or consists of (e.g., structure 1 in Figure 1).

[0196] In some embodiments, the chimeric polypeptide is formed from the C-terminus to the N-terminus. • The amino acid sequence of the target protein, which is a type II transmembrane protein or a protein derived from the TNF or TNFR superfamily as defined herein, and • Amino acid sequence of the pilot peptide as defined herein It includes or consists of (e.g., structure 2 in Figure 1).

[0197] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a type II transmembrane protein or a protein derived from the TNF or TNFR superfamily as defined herein. Optionally, linker, and • Amino acid sequence of the submembrane targeting domain as defined herein It includes or consists of (e.g., structure 3 in Figure 1).

[0198] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a type II transmembrane protein or a protein derived from the TNF or TNFR superfamily as defined herein. • The amino acid sequence of the pilot peptide defined herein, Optionally, linker, and • Amino acid sequence of the submembrane targeting domain as defined herein It includes or consists of (e.g., structure 4 in Figure 1).

[0199] In some embodiments, the chimeric polypeptide is N-terminus to C-terminus, • The amino acid sequence of the target protein, which is a protein derived from the TNF or TNFR superfamily, the TGF-β superfamily, the neurotrophin family, or the CTRP family as defined herein. Optional linker, • The amino acid sequence of the transmembrane domain of the transmembrane protein as defined herein, and • Amino acid sequence of the pilot peptide as defined herein This includes or consists of (e.g., Structure 1 in Figures 2, 3, and 4).

[0200] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a protein derived from the TNF or TNFR superfamily, the TGF-β superfamily, the neurotrophin family, or the CTRP family as defined herein. Optionally, linker, and • Amino acid sequence of the transmembrane domain of the transmembrane protein as defined herein This includes or consists of (e.g., structure 2 in Figures 2, 3, and 4).

[0201] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a protein derived from the TNF or TNFR superfamily, the TGF-β superfamily, the neurotrophin family, or the CTRP family as defined herein. Optional linker, • Amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein, • Amino acid sequence of the pilot peptide as defined herein This includes or consists of (e.g., structure 3 in Figures 2, 3, and 4).

[0202] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a protein derived from the TNF or TNFR superfamily, the TGF-β superfamily, the neurotrophin family, or the CTRP family as defined herein. Optional linker, • Amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein, Optionally, linker, and • Amino acid sequence of the submembrane targeting domain as defined herein This includes or consists of (e.g., structure 4 in Figures 2, 3, and 4).

[0203] In some embodiments, the chimeric polypeptide is C-terminus to N-terminus, • The amino acid sequence of the target protein, which is a protein derived from the TNF or TNFR superfamily, the TGF-β superfamily, the neurotrophin family, or the CTRP family as defined herein. Optional linker, • Amino acid sequence of the transmembrane domain of a transmembrane protein as defined herein, • The amino acid sequence of the pilot peptide defined herein, Optionally, linker, and • Amino acid sequence of the submembrane targeting domain as defined herein This includes or consists of (e.g., structure 5 in Figures 2, 3, and 4).

[0204] Optionally, one or more linkers may be added between the components of the chimeric polypeptide of the present invention.

[0205] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of neuraminidase N2 described in SEQ ID NO: 1, with the amino acid sequence described in SEQ ID NO: 61 linked at its N-terminus (e.g., construct 2 in Figure 1). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 68.

[0206] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of TRAIL described in SEQ ID NO: 2, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 61 (e.g., construct 2 in Figure 1). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 69.

[0207] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of the TRAIL external domain described in SEQ ID NO: 3, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 2). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 76.

[0208] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of the TRAIL TNF-like domain described in SEQ ID NO: 4, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 2). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 77.

[0209] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of TNFα described in SEQ ID NO: 5, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 61 (e.g., construct 2 in Figure 1). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 70.

[0210] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of the TNFα external domain described in SEQ ID NO: 6, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 2). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 78.

[0211] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of the TNFα maturation domain described in SEQ ID NO: 7, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 2). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 79.

[0212] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of CD30L described in SEQ ID NO: 8, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 61 (e.g., construct 2 in Figure 1). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 71.

[0213] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of the CD30L external domain described in SEQ ID NO: 9, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 2). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 80.

[0214] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of CD137L described in SEQ ID NO: 10, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 61 (e.g., construct 2 in Figure 1). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 72.

[0215] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of CD137L described in SEQ ID NO: 10, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 62 (e.g., construct 3 in Figure 1). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 73.

[0216] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of CD137L described in SEQ ID NO: 10, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 63 (e.g., construct 4 in Figure 1). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 74.

[0217] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of OX40L described in SEQ ID NO: 90, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 61 (e.g., construct 2 in Figure 1). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 91.

[0218] In some embodiments, the chimeric polypeptide contains or consists of the TGF-β1 amino acid sequence described in SEQ ID NO: 11, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 3). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 81.

[0219] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of BMP7 described in SEQ ID NO: 12, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 3). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 82.

[0220] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of BMP9 described in SEQ ID NO: 13, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 3). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 83.

[0221] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of GDNF described in SEQ ID NO: 14, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 3). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 84.

[0222] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of BDNF described in SEQ ID NO: 15, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 3). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 85.

[0223] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of CTRP3 described in SEQ ID NO: 16, which is linked at its C-terminus to the amino acid sequence described in SEQ ID NO: 64 (e.g., construct 1 in Figure 4). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 75.

[0224] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of CTRP3 described in SEQ ID NO: 16, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 65 (e.g., construct 3 in Figure 4). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 86.

[0225] In some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence of CTRP3 described in SEQ ID NO: 16, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 66 (e.g., construct 4 in Figure 4). Thus, in some embodiments, the chimeric polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 87.

[0226] In some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence of CTRP3 described in SEQ ID NO: 16, which is linked at its N-terminus to the amino acid sequence described in SEQ ID NO: 67 (e.g., construct 5 in Figure 4). Thus, in some embodiments, the chimeric polypeptide contains or consists of the amino acid sequence described in SEQ ID NO: 88.

[0227] The present invention also relates to nucleic acids encoding the chimeric polypeptides of the present invention as defined herein.

[0228] In some embodiments, the nucleic acid encoding the chimeric polypeptide of the present invention is associated with lipid nanoparticles or a viral vector. When associated with lipid nanoparticles, the nucleic acid can be transfected into a target, and when associated with a viral vector, the nucleic acid can be transduced into a target. In some embodiments, when the nucleic acid is associated with lipid nanoparticles and transfected into a target, or when it is associated with a viral vector and transduced into a target, the nucleic acid enables the production of the chimeric polypeptide in situ. In some embodiments, when the chimeric polypeptide is produced from the nucleic acid in situ, it is directly associated with an extracellular vesicle of the target.

[0229] Another object of the present invention is an expression vector comprising a nucleic acid encoding the chimeric polypeptide of the present invention.

[0230] In some embodiments, the expression vector preferably includes a sequence encoding the chimeric polypeptide of the present invention, which is operably linked to a regulatory element.

[0231] Examples of regulatory elements include, but are not limited to, promoters, Kozak consensus start sequences, polyadenylation signals, and termination sequences (i.e., stop codons). Regulatory elements are particularly well-suited for nucleic acid expression in cells such as bacteria, yeast, insect cells, mammalian cells, or human cells.

[0232] In some embodiments, the expression vector according to the present invention is monocistronic.

[0233] "Monocistronic" means that a single nucleic acid encoding a single protein is expressed in a single expression vector.

[0234] In some embodiments, the expression vector according to the present invention is polycistronic.

[0235] "Polycistronic" means that at least two nucleic acids, each encoding a single protein, are expressed in a single expression vector.

[0236] Another object of the present invention is a cell containing a nucleic acid encoding the chimeric polypeptide of the present invention or an expression vector containing a nucleic acid encoding the chimeric polypeptide of the present invention.

[0237] The present invention further relates to extracellular vesicles (EVs) comprising chimeric polypeptides as defined herein.

[0238] The present invention also relates to extracellular vesicles (EVs) comprising nucleic acids encoding the chimeric polypeptide of the present invention as defined herein.

[0239] The present invention also relates to extracellular vesicles (EVs) comprising chimeric polypeptides as defined herein and / or nucleic acids encoding the chimeric polypeptides of the present invention as defined herein.

[0240] In some embodiments, the extracellular vesicles retain on their outer surface the protein of interest contained in the chimeric polypeptide. In some embodiments, the transmembrane domain of the chimeric polypeptide is fixed to the extracellular vesicle lipid bilayer.

[0241] In some embodiments, the extracellular vesicles retain on their outer surface the nucleic acid encoding the chimeric polypeptide as defined above in this specification.

[0242] As used herein, the expression "retain on its outer surface" means that the protein of interest contained in the chimeric polypeptide is partially or wholly exposed on the outside of the extracellular vesicle. This configuration enables oligomer formation between the protein of interest contained in the chimeric polypeptide and either another protein of interest of an adjacent chimeric polypeptide or a soluble protein of interest within the same extracellular vesicle.

[0243] In some embodiments, the extracellular vesicles are small extracellular vesicles.

[0244] In some embodiments, the extracellular vesicles are exosomes.

[0245] In some embodiments, exosomes have a diameter in the range of about 30 nm to about 150 nm, preferably about 30 nm to about 120 nm, more preferably about 40 nm to about 80 nm. In some embodiments, exosomes have a diameter in the range of about 30 nm to about 120 nm. In some embodiments, exosomes have a diameter in the range of about 30 nm to about 150 nm.

[0246] A further object of the present invention is a population of extracellular vesicles as defined above in this specification.

[0247] In some embodiments, the population of extracellular vesicles is monodispersed in an aqueous solution, preferably a 0.9% NaCl aqueous solution and / or PBS.

[0248] "Monodisperse" means that the size of extracellular vesicles in a population of extracellular vesicles is substantially uniform. "Substantially uniform" means that the extracellular vesicles have a narrow size distribution that is approximately average in size. In some embodiments, these extracellular vesicles in a 0.9% NaCl aqueous solution and / or PBS have sizes exhibiting a standard deviation of less than 100% from their average size, such as 75%, 50%, 40%, 30%, 20%, less than 10%, or less than 5%.

[0249] In some embodiments, the extracellular vesicle population further comprises the cytoplasmically soluble target protein, i.e., the free form of the target protein, in other words, the target protein not included in the chimeric polypeptide according to the present invention.

[0250] In some embodiments, the extracellular vesicle population further comprises soluble nucleic acids encoding the chimeric polypeptide as defined herein, i.e., nucleic acids that are not attached to the membrane of the extracellular vesicle in its cytoplasm.

[0251] In some embodiments, the population of extracellular vesicles further contains additional molecules of interest in its cytoplasm. In some embodiments, these molecules of interest are nucleic acid molecules or small therapeutic molecules.

[0252] A further object of the present invention is a method for obtaining an extracellular vesicle or population of extracellular vesicles containing a chimeric polypeptide as defined herein or a nucleic acid encoding the chimeric polypeptide.

[0253] General means and methods for obtaining extracellular vesicles or populations of extracellular vesicles are well known in the art. See, for example, Whitford & Guterstam, 2019. Future Med Chem. 11(10):1225-1236; Taylor & Shah, 2015. Methods. 87:3-10; Desplantes et al., 2017. Sci Rep. 7(1):1032.

[0254] In some embodiments, the method for obtaining extracellular vesicles or populations of extracellular vesicles includes the step of producing extracellular vesicles or populations of extracellular vesicles as defined herein.

[0255] In some embodiments, the step of producing the extracellular vesicles or populations thereof includes transfecting cells with nucleic acids encoding the chimeric polypeptide as defined herein.

[0256] In some embodiments, the cells are HEK293 T cells or cells derived from induced cell lines. In some embodiments, the cells are adipocytes. In some embodiments, the cells are immune cells, including, but not limited to, mast cells, lymphocytes (e.g., T cells or B cells), and dendritic cells. In some embodiments, the cells are stem cells, including, but not limited to, embryonic stem cells, adult stem cells (e.g., hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, or neural crest stem cells), cancer stem cells, induced pluripotent stem cells (iPSCs), and induced stem cells (iSCs).

[0257] In some embodiments, the method for obtaining extracellular vesicles or populations of extracellular vesicles further comprises the step of culturing transfected cells for a sufficient time to enable the production of extracellular vesicles in a medium that does not contain extracellular vesicles (i.e., serum-free medium, medium supplemented with extracellular vesicle-depleted serum, or medium supplemented with extracellular vesicle-depleted platelet lysate).

[0258] In some embodiments, the method for obtaining extracellular vesicles or a population of extracellular vesicles further comprises a step of purifying the extracellular vesicles or population of extracellular vesicles.

[0259] In some embodiments, the step of purifying the extracellular vesicles or population of extracellular vesicles includes clarification of the culture supernatant of the transfected cells (e.g., by centrifugation or deep filtration), filtration, ultrafiltration, diafiltration, size exclusion purification, and / or ion exchange chromatography. Other methods for purifying the extracellular vesicles or population of extracellular vesicles include, but are not limited to, ultracentrifugation, tangential flow filtration (TFF), and BE-SEC chromatography.

[0260] In some embodiments, the extracellular vesicles or populations of extracellular vesicles of the present invention are purified. Thus, the present invention also relates to the purified extracellular vesicles or populations of extracellular vesicles.

[0261] Methods for purification are well known to those skilled in the art and are not limited to those described herein.

[0262] In some embodiments, the extracellular vesicles or population of extracellular vesicles of the present invention are purified by ultracentrifugation to obtain semi-purified extracellular vesicles or population of extracellular vesicles. Therefore, the present invention also relates to semi-purified extracellular vesicles or populations of extracellular vesicles.

[0263] The semi-purified extracellular vesicles or populations of extracellular vesicles used herein include extracellular vesicles or populations of extracellular vesicles, proteins fixed to the membrane of the extracellular vesicles or populations of extracellular vesicles or closely associated with the extracellular vesicles or populations of extracellular vesicles, and a crown of proteins associated with the extracellular vesicles or populations of extracellular vesicles (see Figure 5B).

[0264] In some embodiments, the extracellular vesicles or populations of extracellular vesicles of the present invention are purified by tangential flow filtration and chromatography, particularly by SEC and BE-SEC chromatography, to obtain ultra-purified extracellular vesicles or populations of extracellular vesicles. Thus, the present invention also relates to ultra-purified extracellular vesicles or populations of extracellular vesicles.

[0265] As used herein, the ultra-purified extracellular vesicles or population of extracellular vesicles comprise the extracellular vesicles or population of extracellular vesicles and proteins that are immobilized on the membrane of the extracellular vesicles or population of extracellular vesicles or are closely associated with the extracellular vesicles or population of extracellular vesicles (see Figure 5C).

[0266] The present invention further relates to a composition comprising, consisting of, or consisting essentially of the chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle or population of extracellular vesicles as defined hereinabove.

[0267] As used herein with respect to the composition, "consisting essentially of" means that the chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle or population of extracellular vesicles is the only therapeutic agent, i.e., the agent having biological activity, in the composition.

[0268] In some embodiments, the composition further comprises the soluble protein of interest as defined hereinabove, i.e., the free-form protein of interest.

[0269] In some embodiments, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient.

[0270] Thus, another object of the present invention is a pharmaceutical composition comprising, consisting essentially of, or consisting of the chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle or population of extracellular vesicles as defined hereinabove and at least one pharmaceutically acceptable excipient.

[0271] The term "pharmaceutically acceptable excipients" includes all solvents, diluents, dispersions, coatings, antimicrobial agents, antifungal agents, isotonic agents, and absorption retarders. Such excipients do not cause adverse reactions, allergic reactions, or other adverse reactions when administered to animals, preferably humans. Formulations for human administration must meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the FDA or EMA.

[0272] Pharmaceutically acceptable excipients that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable oils, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0273] In some embodiments, the pharmaceutical composition further comprises the soluble protein of interest as already defined above, i.e., the free form of the protein of interest.

[0274] In some embodiments, the pharmaceutical composition further comprises a soluble nucleic acid encoding the chimeric polypeptide.

[0275] The present invention further relates to drugs comprising, essentially consisting of, chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles, or populations of extracellular vesicles as defined herein.

[0276] In some embodiments, the drug further comprises, i.e., the soluble protein of the target, as already defined above, and the free form of the protein of the target.

[0277] In some embodiments, the drug further comprises a soluble nucleic acid encoding the chimeric polypeptide.

[0278] In some embodiments, the composition, the pharmaceutical composition, or the drug comprises purified extracellular vesicles or a population of extracellular vesicles as defined herein.

[0279] The present invention further relates to a kit of parts comprising, in a first part, an extracellular vesicle or population of extracellular vesicles as defined herein, and in a second part, a soluble protein of interest already defined herein, i.e., a free form of the protein of interest or a soluble nucleic acid encoding the chimeric polypeptide.

[0280] In some embodiments, the two parts of this kit of parts are intended for simultaneous use or for continuous use in any order.

[0281] The present invention further relates to chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles or populations of extracellular vesicles, compositions, pharmaceutical compositions, drugs or kits of parts as defined herein, for use as drugs or pharmaceuticals.

[0282] The present invention further relates to chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles or populations of extracellular vesicles, compositions, pharmaceutical compositions, drugs or kits of parts as defined herein, for use in the treatment of diseases, disorders or conditions.

[0283] The present invention further relates to chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles or populations of extracellular vesicles, compositions, pharmaceutical compositions, drugs or kits of parts as defined herein, for treating or for use in treating diseases, disorders or conditions in subjects requiring them.

[0284] The present invention further relates to the use of chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles or populations of extracellular vesicles, compositions, pharmaceutical compositions, drugs or kits of parts as defined herein, in the manufacture of drugs for treating diseases, disorders or conditions in subjects requiring them.

[0285] The present invention further relates to the use of chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles or populations of extracellular vesicles, compositions, pharmaceutical compositions, drugs or kits of parts as defined herein, for treating diseases, disorders or conditions in subjects requiring such treatment.

[0286] The present invention further relates to a method for treating a disease, disorder or condition in a subject requiring such treatment, comprising or comprising administering to the subject a chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle or population of extracellular vesicles, population of extracellular vesicles, composition, pharmaceutical composition, drug or kit of parts as defined herein.

[0287] The present invention further relates to a method for treating a disease, disorder or condition in a subject requiring such treatment, comprising or comprising administering to the subject a therapeutically effective amount of a chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle or population of extracellular vesicles, population of extracellular vesicles, composition, pharmaceutical composition, drug or kit of parts as defined herein.

[0288] In some embodiments, the disease, disorder, or condition is selected from neurodegenerative diseases, autoimmune diseases, viral infections, T-cell immune responses, NK-cell immune responses, homeostatic imbalance-related disorders, diabetes mellitus, obesity and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory diseases, hormonal cancers, and cancer progression.

[0289] Examples of neurodegenerative diseases, though not limited to these, include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia Friedreich, Huntington's disease, Lewy body dementia, Parkinson's disease, spinal muscular atrophy, multiple sclerosis (MS), multiple system atrophy, and prion diseases.

[0290] As used herein, the term “autoimmune disease” refers to a disease in which the immune system triggers an immune response (e.g., a B-cell response or a T-cell response) against an antigen that is part of a normal host (i.e., an autoantigen), resulting in tissue damage. In autoimmune diseases, the host’s immune system fails to recognize a particular antigen as “self,” and an immune response is triggered against host tissues that express that antigen.

[0291] Examples of autoimmune diseases envisioned in the present invention include, but are not limited to, rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjögren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenic purpura, Goodpasture syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, acquired hemophilia, thrombotic thrombocytopenic purpura, uveitis, and IgG4-related autoimmune diseases.

[0292] As used herein, the term “viral infection” refers to any disease or condition caused by a virus.

[0293] Examples of viral infections include, but are not limited to, the common cold caused by rhinovirus, influenza caused by influenza virus, COVID-19 caused by SARS-CoV-2 virus, lung infections caused by respiratory syncytial virus (RSV), gastroenteritis caused by norovirus, rotavirus, or astrovirus, hepatitis caused by hepatitis virus, hemorrhagic fever caused by Ebola virus, yellow fever caused by arbovirus, dengue fever caused by dengue virus, acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV), cervical cancer caused by human papillomavirus (HPV), genital herpes caused by herpesvirus (HSV), chickenpox caused by varicella-zoster virus, measles caused by measles virus, rubella caused by RuV virus, congenital viral infections caused by cytomegalovirus (CMV) or Zika virus, and neurological infections caused by West Nile virus, poliovirus, or RABV virus.

[0294] As used herein, the term “inflammatory disease” refers to a wide variety of disorders and conditions characterized by inflammation. Symptoms of inflammatory diseases may include chronic pain, swelling, redness, joint and muscle stiffness, and loss of function and movement in the affected area. Inflammatory diseases exhibit the following pathophysiological characteristics: • Inflammatory response to substances of unknown origin that affect various tissues and organs. • Responses in response to genetic mutations in the response characteristics of immune cells such as antigen-presenting cells, B and T lymphocytes, • Production of autoantibodies (spontaneous and pathogenic autoantibodies) against exogenous or endogenous antigens. • Production of antigen-specific inflammatory cells such as lymphocytes and T cells, • Production and deposition of abnormal proteins and other inflammatory products in various tissues, which trigger further inflammatory and immune responses. These responses include inflammation of blood vessels in the surrounding tissue (i.e., vasculitis), and / or • Production of pro-inflammatory and anti-inflammatory mediators This refers to the many different states that can possess this characteristic.

[0295] As used herein, the term “cancer” has its general meaning in the art and refers in particular to diseases caused by the uncontrolled division of abnormal cells. The term “cancer” encompasses solid tumors and hematological malignancies, and includes both primary and metastatic cancers.

[0296] Examples of cancer include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidneys, liver, lungs, nasopharynx, neck, ovaries, prostate, pancreas, skin, stomach, testes, tongue, or uterus.

[0297] As used herein, the term “hormonal cancer” refers to cancer that is hormone-dependent or hormone-sensitive. Examples of hormonal cancers include, but are not limited to, breast cancer, prostate cancer, endometrial cancer, ovarian cancer, testicular cancer, and thyroid cancer.

[0298] Examples of cardiovascular diseases include, but are not limited to, myocardial infarction, cerebral infarction, acute myocardial infarction, ischemia, coronary heart disease, acute coronary syndrome, stroke, aneurysm, stable or exertional angina, cardiomyopathy, hypertensive heart disease, heart failure (chronic and acute), cor pulmonale, arrhythmias, inflammatory heart diseases such as endocarditis, myocarditis, peripheral artery disease, SIRS-related myocardial and vascular dysfunction, and atherosclerosis.

[0299] The present invention also relates to extracellular vesicles or groups thereof that hold a target protein exposed on their outer surface, for use in the treatment of diseases, disorders or conditions selected from neurodegenerative diseases, autoimmune diseases, viral infections, T-cell immune responses, NK-cell immune responses, homeostasis, diabetes, obesity and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory diseases, hormonal cancers and cancer progression.

[0300] In some embodiments, the extracellular vesicle is partially or entirely coated with the recombinant protein of interest.

[0301] The “recombinant protein of interest” refers to an exogenous protein of interest that is not endogenously produced by the cell. Extracellular vesicles partially or entirely coated with the recombinant protein of interest can be obtained by contacting the extracellular vesicles with the protein of interest either in cellulo (e.g., transfecting an extracellular vesicle-producing cell with a nucleic acid encoding the protein of interest, thereby causing the cell to produce the exogenous protein of interest) or ex cellulo (e.g., providing the protein of interest in a form that has been previously produced in a suitable recombinant expression system and further purified).

[0302] The present invention also relates to extracellular vesicles or populations thereof that hold nucleic acids exposed on their outer surface, encoding the chimeric polypeptide of the present invention as defined herein, for use in the treatment of diseases, disorders or conditions selected from neurodegenerative diseases, autoimmune diseases, viral infections, T-cell immune responses, NK-cell immune responses, homeostasis, diabetes, obesity and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory diseases, hormonal cancers and cancer progression.

[0303] In some embodiments, the extracellular vesicle is partially or entirely coated with nucleic acid.

[0304] "Coated" means that the target protein or nucleic acid encoding the chimeric polypeptide is exposed on the outer surface of the extracellular vesicle and bound thereby by any suitable type of interaction (but not limited to electrostatic interactions, protein-protein interactions, protein-lipid interactions, etc.) with the external components of the vesicle.

[0305] In some embodiments, the components of a chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, drug, or kit of parts as defined herein are formulated for administration to a subject requiring it.

[0306] In some embodiments, administration to the subject may be parenteral, by inhalation spray, rectally, nasally, or via implanted reservoir. The term “administration” includes, in particular, subcutaneous, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intrathecal, intrahepatic, intrafocal, and intracranial injection or infusion techniques.

[0307] In some embodiments, the components of a chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle, population of extracellular vesicles, composition, pharmaceutical composition, drug, or kit of parts as defined herein can be administered to a subject requiring it in a therapeutically effective amount.

[0308] However, naturally, the total daily dose of any chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle, group of extracellular vesicles, composition, pharmaceutical composition, drug, or kit of parts components as defined herein shall be determined by the attending physician within the bounds of appropriate medical judgment.

[0309] In particular, the specific therapeutically effective dose level for any particular patient is determined by a variety of factors, including the disease being treated and its severity, the activity of the chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle, extracellular vesicle population, composition, pharmaceutical composition, drug, or kit of parts component used as defined herein, the age, weight, overall health status, sex, and diet of the subject, the timing of administration, route of administration, and excretion rate of the chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle, extracellular vesicle population, composition, pharmaceutical composition, drug, or kit of parts component used as defined herein, the duration of treatment, drugs used in combination with or concurrently with the chimeric polypeptide, nucleic acid, expression vector, cell, extracellular vesicle, extracellular vesicle population, composition, pharmaceutical composition, drug, or kit of parts component used as defined herein, and similar factors well known in the medical field. The total dose required for each treatment may be administered in multiple doses or single doses.

[0310] The present invention also relates to the in vitro or in vivo use of the chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles, or populations of extracellular vesicles of the present invention in non-therapeutic methods.

[0311] In some embodiments, the chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles, or populations of extracellular vesicles of the present invention are used in vitro or in vivo to evaluate the function or biological activity of a target protein in various biological processes.

[0312] In some embodiments, the chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles, or populations of extracellular vesicles of the present invention are used in conjunction with in vitro or in vivo assays to determine the effect of a target protein in a given biological process.

[0313] In some embodiments, the chimeric polypeptides, nucleic acids, expression vectors, cells, extracellular vesicles, or populations of extracellular vesicles of the present invention are used in conjunction with in vitro or in vivo assays for developing therapeutic molecules.

[0314] In some embodiments, the therapeutic molecule is a small molecule, an antibody, an agonist, or an antagonist. [Brief explanation of the drawing]

[0315] [Figure 1] This is a schematic diagram showing four exemplary constructs of a chimeric polypeptide containing a type II transmembrane protein as the target protein according to the present invention. [Figure 2] This is a schematic diagram showing five exemplary constructs of a chimeric polypeptide containing TNF or a protein derived from the TNF receptor superfamily as the target protein according to the present invention. [Figure 3] This is a schematic diagram showing five exemplary constructs of a chimeric polypeptide containing a TGF-β superfamily-derived protein as the target protein according to the present invention. [Figure 4] This is a schematic diagram showing five exemplary constructs of a chimeric polypeptide containing a protein derived from the CTRP family as the target protein according to the present invention. [Figure 5] These are schematic diagrams of extracellular proteins (EVs) and their environment in culture medium (Figure 5A), semi-purified EVs (Figure 5B), and ultra-purified EVs (Figure 5C). Figure 5A: EVs in culture medium are associated with proteins and impurities. Figure 5B: Semi-purified EVs by ultracentrifugation are associated with the crown of associated proteins. Figure 5C: Ultra-purified EVs by TFF (tangential flow filtration) and chromatography are associated with proteins immobilized on or closely associated with the membrane. [Figure 6]Figures 6A–6E are combinations of photographs of immunotransfer analysis and graphs showing the expression of CD137L constructs in extracellular vesicles. To express and select the constructs in extracellular vesicles, HEK293T cells were transfected with DNA encoding different CD137L constructs: (i) DNA encoding the wild-type CD137L protein (encoding a polypeptide with SEQ ID NO: 10), (ii) DNA encoding CD137L construct 1 containing a pilot peptide fused upstream of CD137L (encoding a polypeptide with SEQ ID NO: 72), (iii) DNA encoding CD137L construct 2 containing a Src peptide fused upstream of CD137L (encoding a polypeptide with SEQ ID NO: 73), or (iv) DNA encoding CD137L construct 3 containing both the Src peptide and the pilot peptide fused upstream of CD137L (encoding a polypeptide with SEQ ID NO: 74). Extracellular vesicles produced by cells transfected with each of the DNA constructs and the control DNA were purified, lysed, and their protein contents were separated by polyacrylamide gel electrophoresis and analyzed by immunotransfer (WB). Figure 6A shows the proteins of each extracellular vesicle lysate analyzed using Mini-Protean TGX Stain Free Gels (4-15%) (BioRad). Figure 6B shows the WB probed with anti-Alix antibody. Figures 6C-6D show the same Western blots probed with anti-CD137L antibody at exposure times of 85 seconds (Figure 6C) or 139 seconds (Figure 6D). Figure 6E is a histogram showing the amount of each CD137L construct relative to the amount of Alix in each lane of the Western blot shown (ratio of the number of pixels in the CD137L construct band to the number of pixels in the Alix band). [Figure 7]Figures 7A and 7B are combinations of graphs showing the median diameter and expression of CD81 in extracellular vesicles containing the CD137L protein. Figure 7A shows the concentration and size distribution of extracellular vesicles containing the CD137L protein. Figure 7B shows ELISA detection of CD81 on extracellular vesicles produced by untransfected cells (EV standard) and on extracellular vesicles containing CD137L. [Figure 8] These are images of immunotransfer analysis showing the expression of the OX40L construct in cells and extracellular vesicles. HEK293T cells were stably transfected with DNA encoding the OX40L construct, which includes a pilot peptide fused upstream of OX40L (SEQ ID NO: 91), to express the fusion protein, which was then sorted together with the extracellular vesicles. Cell pellets were prepared, and extracellular vesicles were purified from conditioned medium. Cells and extracellular vesicles were lysed under either reducing or non-reducing (natural) conditions as instructed, and then subjected to or not subjected to deglycosylation using PNGase. After separation of the lysates by polyacrylamide gel electrophoresis, immunotransfer analysis (WB) was performed. The WB was probed with an anti-pilot peptide rabbit polyclonal antibody. [Table 1] JPEG2026522289000003.jpg207159JPEG2026522289000004.jpg211159JPEG20265222890000 05.jpg211159JPEG2026522289000006.jpg215159JPEG2026522289000007.jpg213159JPEG202 6522289000008.jpg204159JPEG2026522289000009.jpg216159JPEG2026522289000010.jpg2 14159JPEG2026522289000011.jpg211159JPEG2026522289000012.jpg213159JPEG2026522289 000013.jpg205159JPEG2026522289000014.jpg206159JPEG2026522289000015.jpg211159JP EG2026522289000016.jpg208159JPEG2026522289000017.jpg210159JPEG2026522289000018. jpg208159JPEG2026522289000019.jpg205159JPEG2026522289000020.jpg210159JPEG202652 2289000021.jpg214159JPEG2026522289000022.jpg210159JPEG2026522289000023.jpg32159 [Examples]

[0316] The present invention is further illustrated by the following examples. Example 1: Production of semi-purified extracellular vesicles containing the target chimeric protein. material and method Production of target proteins and extracellular vesicles that hold target proteins in mammalian cells

[0317] Extracellular vesicles were produced in HEK293T cells obtained from the American Cell Culture and Preservation Center (ATCC). The cells were cultured at 37°C in a 5% CO2 humidified incubator in DMEM supplemented with 5% thermoinactivated fetal bovine serum (iFBS), 2 mM GlutaMAX, and 5 μg / mL gentamicin. HEK293T cells were routinely tested and found to be negative using the MycoAlert® mycoplasma detection kit (Lonza Nottingham).

[0318] Nucleic acid sequences encoding target chimeric proteins that target exosomes (SEQ ID NOs. 68-88) were inserted into eukaryotic cell expression vectors under the control of the CMV / HTLV chimeric promoter. If necessary, a resistance gene encoding zeocin was added downstream of the CMV IRES sequence simultaneously with the target protein-encoding nucleic acid sequence to enable co-expression of zeocin resistance and establishment of stable transfected cell lines. These nucleic acid sequences were transfected into HEK293T cells using PEI. If necessary, select stable transfected cell lines were obtained in the presence of 500 μg / mL of zeocin over 15 days.

[0319] For large-scale exosome production, transfected HEK293T cells were seeded in 1 L of complete medium in a cell chamber consisting of 10 trays. After 24 hours, the cultures were supplied to a medium supplemented with extracellular vesicle-free iFBS and incubated for a further 48 hours.

[0320] Purification of target protein-extracellular vesicles and extracellular vesicles The cell culture supernatant was collected from HEK293T cells transfected with cell culture medium, and the target protein-extracellular vesicles were isolated as described above (Taylor & Shah, 2015. Methods. 87:3-10; Desplantes et al., 2017. Sci Rep. 7(1):1032; Corso G. et al., 2017. Scientific Reports. 7:11561. DOI:10.1038 / s41598-017-10646-x). Briefly, the cell culture supernatant was clarified by two consecutive centrifugations (1300 rpm for 10 minutes and 4000 rpm for 15 minutes, both at 4°C), and then filtered through a 0.22 μm membrane filter. The supernatant was then concentrated by ultrafiltration and diafiltration and packed into either a size exclusion chromatography (SEC) column or a BE-SEC column (CL2-B, Sephacryl S1000, or Captocore, GE Healthcare). Fractions containing extracellular vesicle biomarkers (CD81 and CD63) were identified by ELISA. Extracellular vesicle fractions containing the target proteins identified by Western blotting were concentrated as needed and used for analysis and injection.

[0321] SDS-PAGE, Western blotting, and antibodies The protein concentration of the target protein-extracellular vesicles was measured using a BCA assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific). The target protein-extracellular vesicle preparations were lysed, separated by SDS-PAGE on a 4-15% acrylamide gel (4-15% Mini-PROTEAN® TGX Stain-Free® Gel Kit, Bio-Rad), and then transferred to a PVDF membrane. For Western blotting under non-reducing conditions, a loading buffer without DTT was used.

[0322] Immunodetection of the target protein was performed using a primary antibody against either the target protein or the anti-Ciloa pilot peptide (PP) (our proprietary antibody produced in rabbits).

[0323] Specific extracellular vesicle markers were detected by immunoassay using primary antibodies against one of the following: CD81 (Genetex reference number #GTX101766), CD63 (Genetex reference number #GTX132953), Alix (Proteintech reference number #12422-1-AP), or syntenin (Fisher Scientific reference number #11326573).

[0324] Next, the membrane was incubated with the corresponding secondary HRP-labeled antibody (donkey anti-mouse, anti-rabbit, or anti-goat HRP, Jackson ImmunoResearch reference numbers #715-035-150, 711-035-152, or 715-038-147).

[0325] The signal was detected using a high-sensitivity chemiluminescence detection kit (Super Signal West Pico Plus; ThermoFischer Scientific reference number #34580), and the membrane was imaged using the ChemiDoc Imaging System (Bio-Rad).

[0326] Using these primary antibodies and their respective secondary antibodies, the target protein was also detected on the surface of extracellular vesicles by ELISA.

[0327] Target protein and extracellular vesicle marker-specific IgG ELISA The extracellular vesicle surface contents of the target protein and CD81 and CD63-specific surface markers were determined by ELISA using some of the antibodies mentioned above, as well as anti-CD81 (Ancell reference number #ANC-302-020) or anti-CD63 (Agro-Bio reference number #S12086).

[0328] In short, MaxiSorp ELISA plates (Nunc) were coated overnight at 4°C with 100 μL of serial 1 / 2 dilutions (starting with 1 μg) of the target protein-extracellular vesicles in 50 mM sodium carbonate / sodium bicarbonate (pH 9.6) buffer per well. The coated plates were washed three times with 200 μL of 1×PBS and saturated with 200 μL of 3% BSA in 1×PBS per well at 37°C for 1 hour. The plates were washed three times with 1×PBS and then incubated with the primary antibody dilution (1:500 for adiponectin or 1:10000 for extracellular vesicle-specific markers) in 3% BSA and 5% FBS at 37°C for 2 hours. Next, the plates were washed three times with 200 μL of 1×PBS per well and incubated with 100 μL of the corresponding secondary HRP-labeled antibody per well (as specified in the Western blot above), diluted 1:10000 in 3% BSA in 1×PBS. After incubation with the secondary antibody, the plates were washed five times with 200 μL of 1×PBS per well and developed with 100 μL of TMB (Bio-Rad reference numbers #R8 / R9) per well for 30 minutes. The reaction was stopped by adding 50 μL of stop solution (2N sulfuric acid) per well.

[0329] The absorbance at 450 nm was read using a ClarioStar Plus plate reader (BMG Labtech). The reciprocal antibody titer was defined as the dilution at which the OD at 450 nm was three times that of the background.

[0330] Example 2: Production of ultra-purified extracellular vesicles containing the target chimeric protein. material and method Production of target proteins and extracellular vesicles that hold target proteins in mammalian cells Extracellular vesicles containing the target chimeric proteins (SEQ ID NOs. 68-88) were produced as described above.

[0331] Production of the target protein on ultra-purified EV from culture medium. Cell culture media stably expressing DNA constructs containing different target proteins (SEQ ID NOs. 68-88) or control cells were concentrated and purified using TFF and BE-SEC chromatography. Extracts from ultra-purified extracellular proteins (EVs) and producing cells were subjected to SDS-PAGE separation under reducing or non-reducing conditions and analyzed by Western blotting, followed by color development using primary antibodies against the target proteins described herein, and then secondary HRP-labeled antibodies.

[0332] Characterization of highly refined EVs Cell culture media stably expressing DNA constructs containing different target proteins (SEQ ID NOs. 68-88) or control cells were concentrated and purified using TFF and BE-SEC chromatography. Extracts from ultra-purified EV and producing cells were subjected to SDS-PAGE separation under reducing or non-reducing conditions, analyzed by Western blotting, and colored using primary antibodies against anti-Alix (EV marker) and target proteins, followed by secondary HRP-labeled antibodies.

[0333] The presence of the EV marker (CD81) and the target protein on the surface of EVs was detected by ELISA. Different types of EVs were immobilized on ELISA plates at dilutions of 1 to 1 / 128 (here, 1 = 50 μl of pure EV) and detected with anti-CD81 antibody or antibody against the target protein, followed by secondary anti-HRP antibody.

[0334] Example 3: Expression of the chimeric polypeptide material and method Cell culture and production of extracellular vesicles HEK293T cells were cultured at 37°C in a 5% CO2 humidified incubator in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% thermoinactivated fetal bovine serum (iFBS), 2 mM GlutaMAX, and 5 μg / mL gentamicin.

[0335] HEK293T cells seeded in complete medium in culture flasks were transfected with polyethyleneimine using DNA encoding different protein constructs. Zeosin (Invivogen) selective pressure was applied to establish stable cell lines. The absence of mycoplasma was evaluated using the MycoStrip® kit (Invivogen). For EV production, cells were cultured for 48 hours in either a T150 flask or a 10-layer cell factory in exosome-free medium.

[0336] Purification of extracellular vesicles The conditioned cell medium was collected and clarified by two consecutive centrifugations (300 g for 10 minutes and 3,000 g for 15 minutes, both at 4°C). The clarified medium was filtered through a 0.22 μm membrane filter, and then i) for small volumes, ultracentrifugation at an average of 110,000 g for 2 hours (TLA100.1 rotor, Beckman Coulter), followed by direct exosome (EV) pellet solubilization in Laemmli sample buffer for Western blot (WB) analysis, or ii) for large volumes, by filtration, diafiltration, and concentration by TFF. The concentrated EV was finally purified by size exclusion chromatography (SEC). Finally, the fraction containing pure EV was pooled, and the EV was sterile filtered through a 0.22 μm filter and stored at 4°C. EV purified using this latter protocol can be subjected to both WB analysis or ELISA.

[0337] Size distribution and particle numbering of extracellular vesicles The size distribution and particle number of EVs were obtained by nanoflow cytometry using a NanoAnalyzer instrument (NanoFCM).

[0338] SDS-PAGE, Western blotting (WB), and antibodies To prepare the cell extract, the cells were harvested, centrifuged at 300g for 10 minutes at 4°C, washed with 1x phosphate-buffered saline (PBS), and dissolved in 1% IGEPAL (Sigma).

[0339] Protein concentrations in cell extracts and EV batches were determined using a BCA assay (Thermo Scientific). For SDS-PAGE, 5 μg of pure EV or 10 μg of cell extract was dissolved in Laemmli sample buffer and heated at 95°C for 5 minutes. EV and cell extract preparations were separated by SDS-PAGE on a 4–15% gradient polyacrylamide gel (Bio-Rad), and the proteins were subsequently transferred to a PVDF membrane. Immunodetection of the proteins was performed using primary antibodies specifically recognizing either Alix (mouse monoclonal antibody #12422-1-AP, Proteintech), CD137L (mouse monoclonal antibody #GTX117355, GeneTex), or a pilot peptide (rabbit, proprietary antibody). The membranes were then incubated with the corresponding secondary horseradish peroxidase (HRP) labeled antibodies (donkey anti-mouse HRP or donkey anti-rabbit HRP, Jackson ImmunoResearch). The signal was detected using a high-sensitivity chemiluminescence detection kit (Thermo Scientific), and the film was imaged using the ChemiDoc Imaging System (Bio-Rad).

[0340] Anti-CD81 ELISA Serial dilutions of pure EV (1 μg to 1 ng) were coated onto 96-well ELISA plates overnight at 4°C. After saturating with 3% BSA in PBS at 37°C for 1 hour, anti-CD81 antibody was added and incubated at 37°C for 2 hours. The plates were then washed three times and incubated with secondary HRP-labeled anti-mouse IgG (donkey antibody, Jackson ImmunoResearch) at 37°C for 1 hour. After five washes, the chromogenic peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the plates were incubated in the dark at room temperature for 30 minutes. The reaction was stopped by adding sulfuric acid. Optical density (OD) was measured at 450 nm using a CLARIOstar Plus plate reader (BMG Labtech).

[0341] result Expression of the CD137L construct HEK293T cells were transfected with DNA encoding different protein constructs: (i) DNA encoding the wild-type CD137L protein (encoding a polypeptide with SEQ ID NO: 10), (ii) DNA encoding CD137L construct 1 containing a pilot peptide fused upstream of CD137L (encoding a polypeptide with SEQ ID NO: 72), (iii) DNA encoding CD137L construct 2 containing a Src peptide fused upstream of CD137L (encoding a polypeptide with SEQ ID NO: 73), or (iv) DNA encoding CD137L construct 3 containing both the Src peptide and the pilot peptide fused upstream of CD137L (encoding a polypeptide with SEQ ID NO: 74). Extracellular vesicles produced by cells transfected with each of these DNA constructs and control DNA were purified, lysed, and their protein contents were separated by polyacrylamide gel electrophoresis and analyzed by immunotransfer (WB).

[0342] Figure 6A shows the proteins in each extracellular vesicle lysate analyzed using Mini-Protean TGX Stain Free Gels (4-15%) (BioRad), indicating that similar amounts of extracellular vesicles were loaded into each gel lane.

[0343] Figure 6B shows a Western blot probed with an anti-Alix antibody (i.e., Alix is ​​an extracellular vesicle-specific marker). Similar intensity Alix bands in each lane confirm that similar amounts of extracellular vesicles were loaded into each gel lane.

[0344] Figures 6C and 6D represent the same Western blot probed with anti-CD137L antibody. In Figure 6C, the exposure time was 85 seconds. Figure 6D shows a longer exposure time of 139 seconds to visualize faint bands corresponding to Src-CD137L construct 2 (SEQ ID NO: 73) and Src-PP-CD137L construct 3 (SEQ ID NO: 74). The bands are present at apparent molecular weights of 27kDa, 30kDa, 33kDa, and 36kDa, corresponding to the expected molecular weights of wild-type CD137L protein (SEQ ID NO: 10), Src-CD137L construct 2 (SEQ ID NO: 73), PP-CD137L construct 1 (SEQ ID NO: 72), and Src-PP-CD137L construct 3 (SEQ ID NO: 74), respectively.

[0345] Figure 6E shows a histogram of the amount of each CD137L construct relative to the amount of Alix in each lane of the shown WB (ratio of the number of pixels in the CD137L construct band / the number of pixels in the Alix band). As shown in Figure 6E, some wild-type CD137L proteins (SEQ ID NO: 10) spontaneously target extracellular vesicles. CD137L construct 1 (SEQ ID NO: 72), corresponding to a pilot peptide fused upstream of CD137L, shows increased targeting to extracellular vesicles compared to the wild-type CD137L protein. In contrast, CD137L construct 2 (SEQ ID NO: 73), corresponding to a Src peptide fused upstream of CD137L, and CD137L construct 3 (SEQ ID NO: 74), corresponding to both the Src peptide and the pilot peptide fused upstream of CD137L, show a significant decrease in targeting to extracellular vesicles compared to the wild-type CD137L protein.

[0346] These results indicate that the pilot peptide increases the amount of CD137L targeting to extracellular vesicles, but the addition of the Src peptide inhibits CD137L targeting to extracellular vesicles.

[0347] Characterization of extracellular vesicles that retain the CD137L protein. HEK293T cells were transfected with DNA encoding CD137L construct 1 (encoding a polypeptide with sequence number 72) containing a pilot peptide fused upstream of CD137L. Extracellular vesicles produced by the transfected cells were purified and analyzed for CD81 by nanoflow cytometry and ELISA.

[0348] Figure 7A shows the concentration and size distribution of extracellular vesicles containing the CD137L protein. The concentration and size distribution of CD137L-EVs were obtained by analyzing the extracellular vesicles containing the CD137L protein using a Nanoanalyzer instrument (NanoFcm brand). As shown in Figure 7A, the extracellular vesicles containing the CD137L protein have a median diameter of 72 nm.

[0349] Furthermore, Figure 7B shows the detection of the CD81 marker (i.e., a marker specific to extracellular vesicles) by ELISA. As shown in Figure 7B, extracellular vesicles containing the CD137L protein show the same amount of CD81 marker when compared to extracellular vesicles obtained from untransfected cells (EV standard).

[0350] Expression of OX40L constructs HEK293T cells were stably transfected with DNA encoding an OX40L construct containing a pilot peptide fused upstream of OX40L (encoding the polypeptide of SEQ ID NO: 91) to express the fusion protein, which was then sorted by extracellular vesicles. Cell pellets were prepared, and extracellular vesicles were purified from conditioned medium. Cells and extracellular vesicles were lysed under either reducing or non-reducing (natural) conditions, and then subjected to or not subjected to deglycosylation using PNGase. The lysates were separated by polyacrylamide gel electrophoresis, and immunotransfer analysis (WB) was performed. The WB was probed with an anti-pilot peptide rabbit polyclonal antibody.

[0351] As shown in Figure 8, the OX40L construct is expressed in cells, which has an apparent molecular weight of 37 kDa, which decreases to 27 kDa after deglycosylation by PNGase digestion (Figure 8 - Cellular PP-OX40L lane and Cellular PP-OX40L + PNGase lane). The glycosylated 37 kDa form is sorted by extracellular vesicles, as indicated by the lower molecular weight protein after PNGase digestion (Figure 8 - EV PP-OX40L lane and EV PP-OX40L + PNGase lane). The OX40L construct from extracellular vesicles treated under native conditions reveals the presence of a higher molecular weight species exhibiting its native multimerization (Figure 8 - EV PP-OX40L lane).

Claims

1. In any order, i) The amino acid sequence of the target protein selected from type II transmembrane proteins, proteins derived from the TNF or TNFR superfamily, proteins derived from the TGF-β superfamily, proteins derived from the neurotrophin family, and proteins derived from the CTRP family selected from CTRP1, CTRP2, CTRP3, CTRP4, CTRP5, CTRP6, CTRP7, CTRP8, CTRP9, CTRP10, CTRP11, CTRP12, CTRP13, CTRP14 and CTRP15. ii) Optionally, the amino acid sequence of the transmembrane domain of a transmembrane protein, and iii) Amino acid sequence of pilot peptide interacting with the endosomal transport sorting complex (ESCRT) cellular mechanism and / or amino acid sequence of the submembrane targeting domain A chimeric polypeptide containing [a specific component].

2. The chimeric polypeptide according to claim 1, wherein the components i), ii) and iii) are organized from the C-terminus to the N-terminus in the chimeric polypeptide.

3. The chimeric polypeptide according to claim 1 or 2, wherein the target protein is selected from neuraminidase N2, TRAIL, TNFα, CD30L, CD137L, TGF-β1, BMP7, BMP9, GDNF, BDNF, CTRP3, and OX40L.

4. The chimeric polypeptide according to any one of claims 1 to 3, wherein the submembrane targeting domain is linked to an anchor molecule, and preferably the anchor molecule is a fatty acid.

5. The chimeric polypeptide according to any one of claims 1 to 4, further comprising at least one linker between the amino acid sequence of the target protein and the amino acid sequence of the transmembrane domain and / or between the amino acid sequence of the transmembrane domain or the amino acid sequence of the pilot peptide and the amino acid sequence of the submembrane targeting domain.

6. The transmembrane domain is selected from the transmembrane domain of a type II transmembrane protein, the transmembrane domain of an influenza virus neuraminidase transmembrane protein, the transmembrane domain of CD40L, and the transmembrane domain of CD8, preferably the transmembrane domain of CD40L is as described in SEQ ID NO: 17, and the transmembrane domain of CD8 is as described in SEQ ID NO: 19, more preferably the transmembrane domain of CD40L is as described in SEQ ID NO: 18, and the transmembrane domain of CD8 is as described in SEQ ID NO: 20, the chimeric polypeptide according to any one of claims 1 to 5.

7. The chimeric polypeptide according to any one of claims 1 to 6, wherein the pilot peptide comprises at least one YxxL motif or DYxxL motif and at least one PxxP motif or PPxY motif (wherein "x" represents any amino acid residue, preferably "x" represents a proline residue).

8. The chimeric polypeptide according to any one of claims 1 to 7, wherein the pilot peptide comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof, wherein the variant of SEQ ID NO: 47 holds at least three YxxL and / or DYxxL motifs and at least four PxxP motifs (wherein "x" represents any amino acid residue).

9. A nucleic acid encoding a chimeric polypeptide according to any one of claims 1 to 8.

10. Preferably, - The transmembrane domain of the chimeric polypeptide is immobilized on the extracellular vesicle lipid bilayer, The target protein of the chimeric polypeptide is exposed on the outer surface of the extracellular vesicle. Extracellular vesicles comprising the chimeric polypeptide according to any one of claims 1 to 8 and / or the nucleic acid according to claim 9.

11. The extracellular vesicle according to claim 10, wherein the extracellular vesicle is preferably an exosome having a diameter in the range of about 30 nm to about 150 nm.

12. A group of extracellular vesicles according to claim 10 or 11, further optionally comprising a soluble protein of interest within its cytoplasm.

13. A collection of extracellular vesicles according to claim 10 or 11, or extracellular vesicles according to claim 12, which have been purified, preferably ultra-purified.

14. A nucleic acid according to claim 9 for use as a drug, an extracellular vesicle according to claim 10, 11, or 13, or a group of extracellular vesicles according to claim 12 or 13.

15. A nucleic acid according to claim 9, an extracellular vesicle according to claim 10, 11, or 13, or a population of extracellular vesicles according to claim 12 or 13, for use in the treatment of a disease, disorder, or condition selected from neurodegenerative diseases, autoimmune diseases, viral infections, T-cell immune responses, NK-cell immune responses, homeostatic imbalance-related diseases, diabetes mellitus, obesity and related metabolic diseases, insulin resistance, cardiovascular diseases, inflammatory diseases, hormonal cancers and cancer progression.

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