Novel polypeptide

Abstract

The present disclosure provides: a polypeptide that comprises a ubiquitinated enzyme domain, an exosome transfer domain, and a target binding domain; a nucleic acid that encodes the polypeptide; a vector that includes the nucleic acid; a cell that expresses the polypeptide; an exosome that includes the polypeptide; and a pharmaceutical composition that includes the polypeptide, the nucleic acid, the vector, the cell, and / or the exosome.

Description

Novel Polypeptides

[0001] This application claims priority to Japanese Patent Application No. 2024-084722, the entire contents of which are incorporated herein by reference. This application relates to a novel polypeptide.

[0002] Molecularly targeted drugs are playing an increasingly important role in clinical treatment, especially in cancer therapy. However, some target molecules are structurally difficult to access with small molecules, and while inhibiting molecular function is effective in cancer therapy, it is difficult to treat diseases such as neurodegenerative diseases, where the accumulation of abnormal molecules is essential.

[0003] Targeted protein degradation (TPD) technologies, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, specific and nongenetic IAP-dependent protein erasers (SNIPERs), are being actively developed. These technologies utilize a linker between a ubiquitinating enzyme (E3 ligase) and a small molecule compound that binds to the target protein and degrades the target protein via the ubiquitin / proteasome system. However, these technologies have been criticized for their poor cell membrane permeability and poor oral bioavailability, posing concerns for their clinical application. Research has also been conducted on gene therapy using polymeric TPD technologies, known as bioTPD or bioPROTACs, which link target-binding proteins such as antibodies to E3 ligases. However, these technologies face challenges due to the low transfection efficiency and cell tropism of the vectors, making it difficult to achieve comprehensive expression in target cells. Therefore, novel therapeutic technologies that can more efficiently degrade disease target proteins in disease-affected cells are needed.

[0004] The present application aims to provide a polypeptide capable of inducing the degradation of a target protein.

[0005] In one aspect, the present application provides a polypeptide comprising a ubiquitination enzyme domain, an exosome transport domain, and a target binding domain.

[0006] In one aspect, the present application provides a nucleic acid encoding the polypeptide.

[0007] In one aspect, the present application provides a vector comprising the nucleic acid.

[0008] In one aspect, the present application provides a cell expressing the polypeptide.

[0009] In one aspect, the present application provides an exosome comprising the polypeptide.

[0010] In one aspect, the present application provides a pharmaceutical composition comprising the polypeptide, the nucleic acid, the vector, the cell, or the exosome.

[0011] The present application provides a polypeptide capable of inducing degradation of a target protein, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a cell expressing the polypeptide, an exosome containing the polypeptide, and a pharmaceutical composition containing the polypeptide, the nucleic acid, the vector, the cell, or the exosome. The polypeptide of the present application not only induces degradation of the target protein in the cell in which the polypeptide is present, but also diffuses to surrounding cells via exosomes, thereby inducing degradation of the target protein in surrounding cells as well, thereby achieving effective degradation of the target protein.

[0012] GFP degrading protein (DEG) containing an acylation tag (Xpack, SEQ ID NO: 29) as an exosome transport domain, a substrate recognition subunit (SRS) as a ubiquitination enzyme domain, and a GFP nanobody (GFP nb, SEQ ID NO: 84) as a target binding domain. GFP) structure. The amino acid sequences of the constructed artificial proteins and the sequences of the nucleic acids encoding them are shown in SEQ ID NOS: 88 to 115. EGFP resolution of chimeric proteins. Each artificial protein (using Skp2, FBXO6, FBXO21, FBXO27, FBXO41, FBW1B, FBXW5, FBXW9, FBXW12, WSB1, VHL, CRBN, SOCS1, or SPOP as the ubiquitination enzyme domain) was expressed in a doxycycline-dependent manner in 293T cells constitutively expressing HiBiT-EGFP, and EGFP degradation due to the expression of the artificial proteins was evaluated using the HiBiT assay. DEGs that showed GFP resolution in the screening GFP Validation data for (XP, WSB1). DEGs were detected in 293T cells that constitutively express EGFP. GFP (XP, WSB1) were expressed in a doxycycline-dependent manner, and 24 hours after addition, GFP degradation was confirmed by Western blot analysis. In the figure, "TRE" stands for tetracycline-responsive element, indicating doxycycline dependence. DEG GFP Translocation rate of XP and WSB1 into extracellular vesicles (EVs). HiBiT-tagged DEGs. GFP We created a 293T cell line that expresses (XP, WSB1) in a doxycycline-dependent manner. 48 hours after the addition of doxycycline, cells and culture supernatant were collected. The culture supernatant was treated at 200 g for 5 minutes and 3000 g for 5 minutes, then concentrated by 100 kDa ultrafiltration for simple EV purification. The cells and EVs were each made up to 1 ml with PBS, and the HiBiT signal was quantified in 100 μL aliquots. The ratio was calculated to estimate the transfer rate to EVs. DEG GFP Bystander effect of (XP, WSB1). 293T cells expressing EGFP-KRAS and doxycycline-dependent DEGs. GFP293T cells expressing tetraspanins (XP, WSB1) were co-cultured. Flow cytometry analysis was performed 24 hours after doxycycline addition. Comparison of exosome trafficking tags. The tetraspanins CD9, CD63, and CD81, as well as the acylation tag (Ac), LAMP2, and the negative control EGFP, were tagged with HiBiT to enable tracking. The graph shows the ratio of the HiBiT signal in EVs purified from culture supernatant to the intracellular HiBiT signal. Structures of CD9, CD81, truncated CD9 (sCD9), and truncated CD81 (sCD81). CD9 and CD81 are both four-transmembrane proteins with small extracellular loops (SEL) and large extracellular loops (LEL) as extracellular regions. sCD9 and sCD81 were created by removing and linking most of the SEL and LEL. The sequences in the figure are a portion of the amino acid sequence of sCD9 (positions 33 to 56 of SEQ ID NO: 33) and a portion of the amino acid sequence of sCD81 (positions 33 to 66 of SEQ ID NO: 41). Localization of sCD9. Fluorescence microscopy images showing the expression and localization of EGFP-sCD9-HiBiT and EGFP-CD9-HiBiT, mainly in the intercellular membrane, when transiently expressed in 293T cells. Extracellular migration of sCD9. HiBiT-EGFP-sCD9 and HiBiT-EGFP-CD9 transiently expressed in 293T cells were tracked using the HiBiT tag, and the extracellular migration of sCD9 and CD9 was observed 24 and 48 hours after gene transfer. DEGs using sCD9 GFPConstruction of sCD9 (sCD9, WSB1). Polypeptides combining sCD9, WSB1, and GFP nanobodies were transiently transfected into 293T cells constitutively expressing EGFP-HiBiT in the order shown in the figure, and HiBiT signals were quantified at 48 hours. Performance of modified sCD9 was analyzed using the SLEEQ system. The sequences shown in Table 4 were inserted into the extracellular domain of HiBiT-tagged sCD9, which was transiently expressed in 293T cells. EVs were concentrated by stepwise centrifugation and ultrafiltration to create a solution 10 times more concentrated than the original culture supernatant. This solution was added to 293T cells expressing LgBiT-actin, and the efficiency of EV uptake into the cell and subsequent translocation to the cytoplasm was calculated. The total amount of HiBiT-tagged protein taken up by cells is measured by measuring luminescence when the cells are lysed, and the amount of HiBiT-tagged protein that has escaped endosomes and migrated to the cytoplasm is quantified by measuring luminescence when the cells are not lysed (since LgBiT-actin cannot access endosomes, only HiBiT-tagged protein that has migrated to the cytoplasm is measured as a luminescence signal). Comparing the two values ​​gives the translocation rate. The first "FL" in each label indicates that full-length CD9 is included, and "s" indicates that sCD9 is included. DEG KRAS The structure of (XP, WSB1) (SEQ ID NO: 122). By using the RBD / CRD as the target binding domain, it was designed to target only activated KRAS. DEG expression in HeLa cells was induced in a doxycycline-dependent manner. KRAS Genetic modification was performed using a PiggyBac vector to express XP and WSB1. Cells were treated with doxycycline and / or EGF, or doxycycline, EGF, and MG132, and analyzed by Western blot. KRAS Differences in sensitivity to (XP, WSB1). HiBiT-tagged KRAS WT, G13D (activating mutation), and G13D / K104Q (inactivating mutation) were expressed in doxycycline-dependent DEGs using PiggyBac vectors. KRASThe HiBiT tag in Figure 3C was replaced with an HA tag and verified by Western blotting. KRAS (XP, WSB1) and DEGs KRAS Comparison of KRAS resolution (sCD9, WSB1). DEG KRAS (XP, WSB1) were introduced into pancreatic cancer cell lines (PK-1 or T3M-4) using a PiggyBac vector to allow doxycycline-dependent expression, and cell numbers were measured over time using CellTiter-Glo (Promega). The cell lines used in Figure 3G were treated with doxycycline or DMSO (negative control) and analyzed by Western blot. KRAS (XP, WSB1) and DEGs KRAS Comparison of DEGs (sCD9, WSB1). 293T cells constitutively expressing HiBiT-KRAS G13D and doxycycline-dependent DEGs. GFP (XP, WSB1), DEG KRAS (XP, WSB1) or DEG KRAS 293T cells expressing sCD9 and WSB1 were co-cultured and harvested 48 hours later. The genes encoding each DEG were linked to the mCherry gene via an IRES linker, so cell lines expressing DEGs in a doxycycline-dependent manner also express mCherry in a doxycycline-dependent manner. The mCherry-negative fraction was sorted by cell sorter at 100,000 cells, and HiBiT signals were measured. DEGs GFP The relative values ​​were plotted using XP and WSB1 as controls. Effects on pancreatic cancer cell lines. Nanoluciferase-labeled pancreatic cancer cell lines (PK-1 or T3M-4) and doxycycline-dependent DEGs were also detected. GFP (XP, WSB1) or DEG KRAS 293T cells expressing sCD9 and WSB1 were co-cultured, and after 120 hours, the cells were harvested, pelleted, and the signal was quantified. KRASGrowth inhibition of pancreatic cancer cell lines (PK-1 or T3M-4) by exosomes containing (sCD9, WSB1). Growth inhibition of pancreatic cancer cell lines via the bystander effect using mesenchymal stem cells. iRFP-labeled pancreatic cancer cell lines (PK-1 or T3M-4) and doxycycline-dependent DEGs. KRAS Mesenchymal stem cells expressing either sCD9 or WSB1 or the same engineered proteins without sCD9 were co-cultured, and after 5 days, the cells were harvested, pelleted, and the signal was quantified. aS (XP, WSB1) (SEQ ID NO: 126) and DEG aS (sCD9, WSB1) (SEQ ID NO: 128) structure. aS Degradation of α-synuclein (aS) by HiBiT. HiBiT-tagged α-synuclein was constitutively expressed in 293T cells, and DEGs were identified in a doxycycline-dependent manner. aS (XP, WSB1) or DEG aS The graph shows the decrease in HiBiT signal. The effect of co-culture. 293T cells constitutively expressing HiBiT-tagged α-synuclein were co-cultured with 293T cells expressing α-synuclein (sCD9, WSB1). The graph shows the decrease in HiBiT signal. aS The aS-expressing cells were co-cultured with 293T cells expressing sCD9 and WSB1, and harvested 48 hours later. The percentage of aS-expressing cells was calculated from the total cell count calculated using CellTiter-glo (Promega) and the number of mCherry-negative cells calculated by flow cytometry, and the aS expression level per cell was calculated as HiBiT / Cell titer. DEGs using AAV vectors aS Degradation of α-synuclein by sCD9 and WSB1. DEGs packaged in AAV2. aS (sCD9, WSB1) were administered to SHSY-5Y cells (a human neuroblastoma cell line), and the reduction of aS was confirmed by ELISA. The bystander effect was verified using a human oligodendroglial cell line (MO3.13 cells). DEG aSSHSY-5Y cells expressing sCD9 and WSB1 were cocultured with MO3.13 cells expressing aS and iRFP to confirm the reduction of aS due to the bystander effect. Examination of exosome translocation rates. Tetraspanins carrying HiBiT-EGFP at their N-terminus were transiently expressed in 293T cells. After 36 hours, the culture supernatant and cell pellet were collected, and the HiBiT signal was compared to calculate the exosome translocation rate. A cell line expressing HiBiT-EGFP under the UBC promoter was created based on 293T cells. The GFP nanobody-tetraspanin-WSB1 was then expressed in these cells in a doxycycline-dependent manner, and the reduction in HiBiT-EGFP expression levels was assessed using the HiBiT assay. A cell line expressing HiBiT-EGFP-KRAS under the EF1 promoter was created based on 293T cells. 1) These cells were co-cultured with 2) cells expressing GFP nanobody-tetraspanin-WSB1 in a doxycycline-dependent manner. HiBiT-EGFP-KRAS per unit cell was estimated and the percentage (%) compared to the negative control value is shown. A cell line expressing HiBiT-EGFP-KRAS under the EF1 promoter was created based on 293T cells. 1) These cells were co-cultured with 2) cells expressing Xpack-GFP nanobody-ubiquitinase in a doxycycline-dependent manner. HiBiT-EGFP-KRAS per unit cell was estimated and the percentage (%) compared to the negative control value is shown. Evaluation of resolution when the exosome transport tag was replaced with sCD9. Using HiBiT-EGFP-expressing 293T cells as the parent cell line, GFP nanobody-sCD9-ubiquitinase was engineered to be inducible by doxycycline, and the HiBiT / cell titer was assessed before and after. 293T cells expressing GFP nanobody-sCD9-ubiquitinase were co-cultured with 293T cells expressing HiBiT-EGFP-KRAS, and the quantitative changes in HiBiT-EGFP-KRAS per cell were calculated. alfaTarget proteins with HiBiT-ALFA tags attached to the N-terminus were constitutively expressed in 293T cells expressing XP and WSB1. The increase or decrease in HiBiT / cell titer before and after the addition of doxycycline is shown. DEGs using sCD9 alfa (sCD9, Skp2) and DEGs alfa Construction of HiBiT-EGFP-ALFA tag (sCD9, WSB1). 293T cells constitutively expressing HiBiT-EGFP-ALFA tag were transiently transfected with polypeptides containing sCD9, Skp2, or WSB1, and nanobodies against the ALFA tag in the order shown in the figure, and HiBiT signals were quantified at 24 hours. Examination of the arrangement of each domain (1). 293T cells constitutively expressing HiBiT-EGFP-ALFA tag were transfected with polypeptides containing the ubiquitination enzyme domain, exosome transport domain, and target-binding domain in the order shown in the figure, and HiBiT signals were quantified at 24 hours. The rate of change in HiBiT / cell titer is shown. Examination of the arrangement of each domain (2). 293T cells constitutively expressing HiBiT-EGFP-ALFA tag were transfected with polypeptides containing the ubiquitination enzyme domain, exosome transport domain, and target-binding domain in the order shown in the figure, and HiBiT signals were quantified at 24 hours. The rate of change in HiBiT / Cell titer is shown.

[0013] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0014] In this disclosure, amino acid residues are represented by the following abbreviations: Ala or A: alanine, Arg or R: arginine, Asn or N: asparagine, Asp or D: aspartic acid, Cys or C: cysteine, Gln or Q: glutamine, Glu or E: glutamic acid, Gly or G: glycine, His or H: histidine, Ile or I: isoleucine, Leu or L: leucine, Lys or K: lysine, Met or M: methionine, Phe or F: phenylalanine, Pro or P: proline, Ser or S: serine, Thr or T: threonine, Trp or W: tryptophan, Tyr or Y: tyrosine, Val or V: valine.

[0015] Polypeptides Comprising a Ubiquitination Enzyme Domain, an Exosome Transport Domain, and a Target Binding Domain The present application provides polypeptides comprising a ubiquitination enzyme domain, an exosome transport domain, and a target binding domain.

[0016] Ubiquitination enzymes (also known as ubiquitin ligases or E3 ligases) are enzymes that can attach ubiquitin to target proteins. Ubiquitination enzymes (E3s) can ubiquitinate target proteins in cooperation with ubiquitin-activating enzymes (E1s) and ubiquitin-conjugating enzymes (E2s). There are over 600 ubiquitination enzymes (E3s) in humans, which are mainly classified into RING finger, HECT, U-box, and PHD finger types. A representative RING finger ubiquitination enzyme is the Cullin-Ring Ubiquitin Ligase complex (CRL complex), which consists of a RING finger protein, a scaffolding protein called Cullin, an adaptor protein, and a substrate recognition subunit (SRS). Ubiquitination enzymes other than the CRL complex include NEDD4, HECT, βTrCP, and STUB1. The ubiquitination enzyme domain is not particularly limited as long as it can ubiquitinate a target bound to the target-binding domain of the polypeptide of the present application. The ubiquitination enzyme domain may be a ubiquitination enzyme or a part thereof, for example, a component protein of the CRL complex (e.g., SRS) or a part thereof.Examples of ubiquitination enzyme domains include WSB1, Skp2, VHL, SPOP, CRBN, SOCS1, SLMB1, FBXL1, FBXL2, FBXL3, FBXL4, FBXL5, FBXL6, FBXL7, FBXL8, FBXL13, FBXL15, FBXL16, FBXL17, FBXL19, FBXL20, FBXO1, FBXO2, FBXO3, FBXO4, FBXO5, FBXO6, FBXO7, FBXO8, FBXO9, FBXO10, and FBXO11, which are SRSs of the CRL complex. 11, FBXO16, FBXO21, FBXO22, FBXO27, FBXO30, FBXO31, FBXO32, FBXO40, FBXO41, FBXO44, FBW1A, FBW1B, FBXW2, FBXW4, FBXW5, FBXW7, FBXW8, FBXW9, FBXW10, FBXW12, and WSB2; Cullin, a scaffolding protein of the CRL complex, and Skp1, an adaptor protein; NEDD4, HECT, βTrCP, and STUB1; and some of these. In certain embodiments, the ubiquitination enzyme domain comprises Skp2, FBXO6, FBXO21, FBXO27, FBXO41, FBW1B, FBXW5, FBXW9, FBXW12, WSB1, VHL, CRBN, SOCS1, SPOP, or FBXL6, or a portion thereof. In certain embodiments, the ubiquitination enzyme domain comprises Skp2, FBXO6, FBXO21, FBXO27, FBXO41, FBW1B, FBXW5, FBXW9, FBXW12, WSB1, VHL, CRBN, SOCS1, or SPOP, or a portion thereof. In certain embodiments, the ubiquitination enzyme domain comprises Skp2, FBXO6, FBXW9, WSB1, VHL, SOCS1, or SPOP, or a portion thereof. In further embodiments, the ubiquitination enzyme domain comprises WSB1, or a portion thereof. The SRS of a CRL complex has a region that binds to a scaffold protein, such as an F-box domain, and a region that binds to its substrate. When a ubiquitination enzyme domain comprises a portion of the SRS of a CRL complex, the portion of the SRS can comprise the scaffold protein binding region of the SRS so that it can ubiquitinate a target bound to the target binding domain of a polypeptide of the present application.Similarly, one skilled in the art can design a ubiquitination enzyme domain so that it is capable of ubiquitinating a target bound to the target binding domain of a polypeptide of the present application.

[0017] An example of WSB1 is a peptide comprising the amino acid sequence of UniProt accession number Q9Y6I7 (SEQ ID NO: 1). A portion of WSB1 comprises, for example, the amino acid sequence of positions 372 to 421 (SOCS box domain) of SEQ ID NO: 1. As long as it is capable of ubiquitinating a target bound to the target-binding domain, WSB1 or a portion thereof may comprise, for example, an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of positions 372 to 421 of SEQ ID NO: 1. As used herein, "one or more" preferably means 1 to 20, more preferably 1 to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Alternatively, WSB1 or a portion thereof may comprise, for example, an amino acid sequence having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of positions 372 to 421 of SEQ ID NO: 1, or the amino acid sequence of positions 372 to 421 of SEQ ID NO: 1 with T at position 380 substituted with D. A portion of WSB1 may comprise or consist of, for example, the amino acid sequence of positions 372 to 421 of SEQ ID NO: 1 or the amino acid sequence of positions 372 to 421 of SEQ ID NO: 1 with T at position 380 substituted with D.

[0018] In the present application, amino acid substitutions may be conservative or non-conservative. Conservative amino acid substitutions are those generally recognized within the range of conservative substitutions that can be made without changing the physiological activity of the resulting molecule (Watson et al., Molecular Biology of the Gene, etc.). Examples include substitutions between amino acids with similar side chains, such as aspartic acid and glutamic acid (acidic amino acids); lysine, arginine, and histidine (basic amino acids); alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan (nonpolar amino acids); glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine (uncharged polar amino acids); and phenylalanine, tryptophan, and tyrosine (aromatic amino acids). Similarly, they can be classified as glycine, alanine, valine, leucine, isoleucine, serine and threonine (aliphatic amino acids); serine and threonine (aliphatic-hydroxy amino acids); asparagine and glutamine (amide-type amino acids); cysteine ​​and methionine (sulfur-containing amino acids).

[0019] As used herein, "sequence identity" refers to the percentage of identical bases or amino acids at corresponding positions in two or more sequences (nucleotide sequences or amino acid sequences) when the sequences are aligned, taking into account gaps and insertions, to maximize sequence identity. Methods for determining identity are designed to maximize identity between the aligned sequences. Methods for determining identity between two sequences include, but are not limited to, BLASTP, BLASTN, FASTA, etc. Identity between two sequences can also be determined using DNASIS (Hitachi Software Engineering Co., Ltd.) or GENETYX (Genetyx Inc.). Alternatively, for short peptides, identity can be determined simply by comparing the sequences. Those skilled in the art can determine identity between sequences using the methods described above.

[0020] The nucleic acid sequence encoding WSB1 or a portion thereof is not particularly limited as long as it encodes the above-mentioned WSB1 or a portion thereof. For example, the nucleic acid sequence encoding WSB1 or a portion thereof may have a similar structure to the nucleotide sequence set forth in SEQ ID NO: 2 or a portion thereof (e.g., positions 1114 to 1263) at about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% of the nucleotide sequence. or may comprise or consist of a nucleotide sequence having 99% or more sequence identity thereto; or may comprise or consist of the nucleotide sequence set forth in SEQ ID NO: 2 or a portion thereof (e.g., positions 1114 to 1263), or a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 2 or a portion thereof (e.g., positions 1114 to 1263). A nucleic acid sequence encoding WSB1 or a portion thereof may comprise or consist of, for example, the nucleotide sequence in which ACC at positions 1138 to 1140 in SEQ ID NO: 2 has been replaced with GAC, or a portion thereof (e.g., positions 1114 to 1263).

[0021] Various ubiquitination enzymes or parts thereof can be used as the ubiquitination enzyme domain in the same manner as described for WSB1. The UniProt accession numbers, SEQ ID NOs (amino acid and nucleotide sequences) of the ubiquitination enzymes, and the regions (amino acid and nucleotide sequences) that should be included in the ubiquitination enzyme domain are shown in Table 1.

[0022] The ubiquitination enzyme or a portion thereof has about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11109, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 12109, 12209, 12309, 12409, 12509, 12609, 12709, 12809, 12909, 13009, 13109, 13209, 13309, 13409, 13509, 13609, 13709, 13809, 13909, 13910, 13910, 1392 ...4009, 14109, 14209, 14309, 14409, 14509, 14609, 14709, 148 or may comprise or consist of an amino acid sequence having 97%, 98%, or 99% or more sequence identity with the ubiquitinase; or may comprise or consist of an amino acid sequence set forth in Table 1-1 or Table 1-2, or a portion thereof (e.g., positions X1 to X2 in Table 1-1 or Table 1-2), or an amino acid sequence set forth in Table 1-1 or Table 1-2, or a portion thereof (e.g., positions X1 to X2 in Table 1-1 or Table 1-2) in which one or more amino acids have been substituted, deleted, added, and / or inserted. In one embodiment, the ubiquitinase or a portion thereof comprises or consists of an amino acid sequence set forth in Table 1-1 or Table 1-2, or a portion thereof (e.g., positions X1 to X2 in Table 1-1 or Table 1-2).

[0023] The sequence of a nucleic acid encoding a ubiquitinating enzyme or a portion thereof is not particularly limited as long as it encodes the above-mentioned ubiquitinating enzyme or a portion thereof. Examples of nucleic acid sequences encoding a ubiquitinating enzyme or a portion thereof include, for example, the nucleotide sequence shown in Table 1-1 or Table 1-2 (any of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 173) or a portion thereof (for example, the Y1 to Y2 positions in Table 1-1 or Table 1-2) and a sequence similar to or similar to about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 13 or may comprise or consist of a nucleotide sequence having 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a nucleotide sequence shown in Table 1-1 or Table 1-2, or a portion thereof (e.g., positions Y1 to Y2 in Table 1-1 or Table 1-2), or a nucleotide sequence in which one or more bases have been substituted, deleted, added, and / or inserted in a nucleotide sequence shown in Table 1-1 or Table 1-2, or a portion thereof (e.g., positions Y1 to Y2 in Table 1-1 or Table 1-2). In one embodiment, the sequence of the nucleic acid encoding the ubiquitinating enzyme or a portion thereof comprises or consists of a nucleotide sequence shown in Table 1-1 or Table 1-2, or a portion thereof (e.g., positions Y1 to Y2 in Table 1-1 or Table 1-2).

[0024] The ubiquitination enzyme domain of the present application may be a peptide derived from a human or mouse, or may be a peptide derived from another mammal (e.g., rat, cow, horse, pig, sheep, monkey, dog, cat, etc.) (i.e., an ortholog).

[0025] The exosome transport domain is a domain that enables the transport of the polypeptide of the present application into exosomes. The exosome transport domain is not particularly limited as long as it enables the transport of the polypeptide of the present application into exosomes. The exosome transport domain can be, for example, a membrane protein or a portion thereof. The membrane protein can contain one or more (e.g., one, two, three, four, five, or six) transmembrane regions. In some embodiments, the membrane protein contains one, two, or four transmembrane regions. Examples of membrane proteins include tetraspanin family proteins such as CD9, CD63, CD81, CD82, CD151, TSPAN3, TSPAN6, TSPAN7, TSPAN8, and TSPAN9, and LAMP2. Examples of membrane proteins also include CD19, CD3, CD36, CD38, CD47, TIM218, and STEAP1. Examples of exosome transport domains also include acylation tags. A commercially available acylation tag can be used, for example, XPack (System Biosciences; SEQ ID NO: 29). The sequence of the nucleic acid encoding XPack is shown in SEQ ID NO: 30.

[0026] In one embodiment, the exosome transport domain comprises or consists of a tetraspanin family protein or a portion thereof. Tetraspanin family proteins are four-transmembrane proteins, having an N-terminal intracellular region, four transmembrane regions, an extracellular region between the first and second transmembrane regions from the N-terminus (called the small extracellular loop (SEL)), an intracellular region between the second and third transmembrane regions from the N-terminus (called the intracellular loop (ICL)), an extracellular region between the third and fourth transmembrane regions from the N-terminus (called the large extracellular loop (LEL)), and a C-terminal intracellular region. Herein, some tetraspanin family proteins are also referred to as truncated tetraspanin family proteins, such as truncated CD9 (sCD9), truncated CD63 (sCD63), truncated CD81 (sCD81), truncated CD82 (sCD82), or truncated CD151 (sCD151). As used herein, a "portion" of a protein may consist of a single contiguous amino acid sequence or multiple noncontiguous amino acid sequences in the protein. A truncated tetraspanin family protein can be created, for example, by deleting part or all of the SEL, the second and third transmembrane regions from the N-terminus, the ICL, and the LEL of a tetraspanin family protein, and connecting the first transmembrane region from the N-terminus to the fourth transmembrane region via the extracellular region. Those skilled in the art can identify each region of a tetraspanin family protein using transmembrane region prediction tools or protein domain / motif databases known in the art. In one embodiment, a truncated tetraspanin family protein comprises part or all of the N-terminal intracellular region, the first and fourth transmembrane regions from the N-terminus, and part or all of the C-terminal intracellular region of a tetraspanin family protein, but does not comprise the second and third transmembrane regions from the N-terminus or the intracellular region between these transmembrane regions.Such truncated tetraspanin family proteins may comprise an extracellular region of 10 to 50, 10 to 40, 10 to 30, or 10 to 20 amino acids between the N-terminus and the first and fourth transmembrane regions of the tetraspanin family protein, and this extracellular region may comprise part or all of SEL and / or part or all of LEL. In one embodiment, the truncated tetraspanin family protein comprises part or all of the N-terminal intracellular region, the N-terminus and the first and fourth transmembrane regions, and part or all of the C-terminal intracellular region of the tetraspanin family protein, but does not include the N-terminus and the third transmembrane regions and the intracellular region between these transmembrane regions, and comprises an extracellular region of 10 to 50, 10 to 40, 10 to 30, or 10 to 20 amino acids between the N-terminus and the first and fourth transmembrane regions, and this extracellular region comprises or consists of part of SEL and part of LEL. The length of the truncated tetraspanin family protein can be, but is not limited to, for example, 50 to 200, 60 to 150, or 70 to 100 amino acids.

[0027] The present application also provides a polypeptide comprising an exosome transport domain, wherein the exosome transport domain comprises or consists of a portion of the tetraspanin family protein (i.e., the truncated tetraspanin family protein). In the polypeptide of this embodiment, the amino acid sequence other than the exosome transport domain and its length are not particularly limited. The total length of the polypeptide may be, for example, 100 to 2,000 or 200 to 1,000 amino acids.

[0028] An example of CD9 is a peptide comprising the amino acid sequence of UniProt accession number P21926 (SEQ ID NO: 31). As long as the polypeptide of the present application can be delivered into exosomes, CD9 may comprise, for example, an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in SEQ ID NO: 31. Alternatively, CD9 may comprise, for example, an amino acid sequence having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 31, as long as the polypeptide of the present application can be delivered into exosomes. CD9 may, for example, comprise or consist of the amino acid sequence from positions 2 to 228 of SEQ ID NO:31.

[0029] The sequence of a nucleic acid encoding CD9 or a portion thereof is not particularly limited, as long as it encodes the above-mentioned CD9 or a portion thereof. The sequence of a nucleic acid encoding CD9 or a portion thereof may, for example, comprise or consist of a nucleotide sequence having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 32; or may comprise or consist of the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in the nucleotide sequence of SEQ ID NO: 32. The sequence of the nucleic acid encoding CD9 or a part thereof may comprise or consist of, for example, the nucleotide sequence from positions 4 to 684 of SEQ ID NO:32.

[0030] sCD9 may comprise, for example, the amino acid sequence of positions 2 to 44 and positions 180 to 220 of UniProt Accession Number P21926 (SEQ ID NO: 33). As long as the polypeptide of the present application can be transferred into exosomes, sCD9 may comprise, for example, an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 33. Alternatively, sCD9 may comprise an amino acid sequence that has about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to, for example, the amino acid sequence of SEQ ID NO: 33, so long as the polypeptide of the present application can be delivered into exosomes. sCD9 can, for example, comprise or consist of the amino acid sequence of SEQ ID NO: 33.

[0031] The sequence of a nucleic acid encoding sCD9 is not particularly limited, as long as it encodes the above-described sCD9. The sequence of a nucleic acid encoding sCD9 may, for example, comprise or consist of a nucleotide sequence having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 34; or it may comprise or consist of the nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in the nucleotide sequence of SEQ ID NO: 34. The sequence of a nucleic acid encoding sCD9 may, for example, comprise or consist of the nucleotide sequence of SEQ ID NO: 34.

[0032] Various tetraspanin family proteins or portions thereof can be used as exosome transport domains in the same manner as described for CD9. Table 2 shows the UniProt accession numbers, SEQ ID NOs (amino acid and nucleotide sequences) of the tetraspanin family proteins, the regions (amino acid and nucleotide sequences) corresponding to the full-length tetraspanin family proteins in the examples, and the regions (amino acid sequences) corresponding to the truncated tetraspanin family proteins in the examples.

[0033] Tetraspanin family proteins or portions thereof may have, for example, at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 31, 35, 39, 43, and 47, or portions thereof. or it may comprise or consist of an amino acid sequence having sequence identity with any of SEQ ID NOs: 31, 35, 39, 43, and 47, or a portion thereof, or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in any of SEQ ID NOs: 31, 35, 39, 43, and 47, or a portion thereof. In one embodiment, the tetraspanin family protein or a portion thereof comprises or consists of an amino acid sequence of any of SEQ ID NOs: 31, 35, 39, 43, and 47, or the amino acid sequence at positions X1 to X2 in Table 2.

[0034] The nucleic acid sequence encoding a tetraspanin family protein or a portion thereof is not particularly limited as long as it encodes the above-mentioned tetraspanin family protein or a portion thereof. The nucleic acid sequence encoding a tetraspanin family protein or a portion thereof may be, for example, any of the base sequences of SEQ ID NOs: 32, 36, 40, 44, and 48 or a portion thereof, or a sequence similar to or similar to about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or or may comprise or consist of a nucleotide sequence having 99% or more sequence identity thereto; or may comprise or consist of any one of the nucleotide sequences of SEQ ID NOs: 32, 36, 40, 44, and 48, or a partial nucleotide sequence thereof, or a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in any one of the nucleotide sequences of SEQ ID NOs: 32, 36, 40, 44, and 48, or a partial nucleotide sequence thereof. In one embodiment, the sequence of the nucleic acid encoding a tetraspanin family protein or a part thereof comprises or consists of any one of the nucleotide sequences of SEQ ID NOs: 32, 36, 40, 44, and 48, or a partial nucleotide sequence thereof.

[0035] Truncated tetraspanin family proteins include, for example, those having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar amino acid sequence to any of SEQ ID NOs: 33, 37, 41, 45, and 49. or may comprise or consist of an amino acid sequence having at least 100% sequence identity with any of SEQ ID NOs: 33, 37, 41, 45, and 49, or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in any of SEQ ID NOs: 33, 37, 41, 45, and 49. In one embodiment, the truncated tetraspanin family protein comprises or consists of the amino acid sequence of any of SEQ ID NOs: 33, 37, 41, 45, and 49.

[0036] The nucleic acid sequence encoding a truncated tetraspanin family protein is not particularly limited as long as it encodes the truncated tetraspanin family protein described above. The nucleic acid sequence encoding a truncated tetraspanin family protein may be, for example, any of the nucleotide sequences of SEQ ID NOs: 34, 38, 42, 46, and 50, or a sequence similar to or different from about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156 or may comprise or consist of a nucleotide sequence having 7%, 98%, or 99% or more sequence identity thereto; or may comprise or consist of any of the nucleotide sequences of SEQ ID NOs: 34, 38, 42, 46, and 50, or any of the nucleotide sequences of SEQ ID NOs: 34, 38, 42, 46, and 50 in which one or more nucleotides have been substituted, deleted, added, and / or inserted. In one embodiment, the sequence of the nucleic acid encoding the truncated tetraspanin family protein comprises or consists of, for example, any of the nucleotide sequences of SEQ ID NOs: 34, 38, 42, 46, and 50.

[0037] The exosome transfer domain of the present application may comprise, for example, a cell membrane fusion peptide. Examples of cell membrane fusion peptides include R9, FR, HL6, ZF5.3, INF, GALA, TAT, KL15, SN21, KL15-SN21, INF-TAT, and combinations thereof. The amino acid sequences of the above cell membrane fusion peptides and the sequences of the nucleic acids encoding them are set forth in SEQ ID NOS: 51 to 72. The cell membrane fusion peptide may be, for example, INF or TAT. The cell membrane fusion peptide may be inserted at any position in the exosome transfer domain of the present application, for example, into the extracellular region of the exosome transfer domain of the present application. When the exosome transfer domain of the present application is sCD9, the cell membrane fusion peptide may be inserted, for example, between positions 43 and 44 of SEQ ID NO: 33. When the exosome transfer domain of the present application is sCD9 and contains INF and TAT as cell membrane fusion peptides, the exosome transfer domain of the present application may comprise or consist of, for example, the amino acid sequence set forth in SEQ ID NO: 73. When the exosome transfer domain of the present application is sCD9 and contains INF and TAT as cell membrane fusion peptides, the sequence of the nucleic acid encoding the exosome transfer domain of the present application comprises or consists of, for example, the base sequence set forth in SEQ ID NO: 74.

[0038] The exosome transfer domain of the present application may be a peptide derived from a human or mouse, or may be a peptide derived from another mammal (e.g., rat, cow, horse, pig, sheep, monkey, dog, cat, etc.) (i.e., an ortholog).

[0039] The target binding domain is a domain capable of binding to a protein whose degradation is to be induced by the polypeptide of the present application (i.e., the target of the polypeptide of the present application). The target binding domain is not particularly limited as long as it can bind to the target. The target binding domain can be, for example, a protein or peptide, or a portion thereof, capable of binding to the target. In certain embodiments, the target binding domain is an antibody. As used herein, the term "antibody" also encompasses molecules that contain a portion of an antibody as a component and retain the ability to bind to an antigen. For example, but not limited to, the heavy and light chain variable regions (V H and VL The antibodies of the present application include F(ab')2, Fab', Fab, Fv, disulphide-linked Fv (sdFv), single-chain Fv (scFv), Fab3, diabody, triabody, tetrabody, minibody, bis-scFv, (scFv)2-Fc, intact IgG, nanobody (also referred to as VHH (variable domain of heavy chain of heavy chain antibody)), and polymers thereof. In one embodiment, the target-binding domain is a nanobody or scFv.

[0040] A "nanobody" is an antigen-binding protein based on the variable region domain of an antibody consisting only of a heavy chain. Nanobodies have three complementarity-determining regions (CDRs) and bind to an antigen via these three CDRs. Nanobodies can be obtained by immunizing an animal that produces an antibody consisting only of the heavy chain with an antigen, isolating B cells from the immunized animal, obtaining a cDNA library containing the variable region, incorporating it into a phage display library using M13 phage, and screening with the antigen.

[0041] "scFv" is a heavy chain variable region (V H ) and the light chain variable region (V L ) linked by a peptide linker. The heavy chain variable region and light chain variable region are connected by a peptide linker to promote association between the heavy chain variable region and the light chain variable region while maintaining the antigen specificity of the original antibody by antigen recognition between the heavy chain variable region and the light chain variable region. The peptide linker can be freely modified in structure depending on the state of association between the scFv and the antigen, and a glycine-rich sequence of approximately 15 amino acids can be used, for example. scFv can be obtained by selecting from a phage library in which the antigen-binding site of scFv having a framework sequence has been randomized, using the desired antigen-binding ability as an indicator.

[0042] The variable region of an antibody typically consists of three complementarity determining regions (CDRs) sandwiched between four framework regions (FRs). The amino acid positions assigned to the CDRs and frameworks of an antibody variable region are defined, for example, according to Kabat (see Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md., (1987) and (1991)).

[0043] In the present application, the target is not particularly limited as long as it is a protein that can be degraded by ubiquitination. For example, the target may be a protein associated with a disease. Examples of diseases include, but are not limited to, cancer, neurodegenerative diseases, inflammatory diseases, and metabolic disorders. Cancers include pancreatic cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, head and neck squamous cell carcinoma, thyroid cancer, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, pleurima, male germinoma, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic sarcoma, leiomyosarcoma, and Wilms' tumor. Neurodegenerative diseases include Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Targets include, for example, KRAS, NRAS, HRAS, RHoA, α-synuclein, and TDP43. When the disease is cancer, the target can be, for example, KRAS, NRAS, HRAS, or RHoA. When the disease is a neurodegenerative disease, the target can be, for example, α-synuclein or TDP43.

[0044] In one embodiment, the target is KRAS, and the target-binding domain binds to KRAS. KRAS is a protein belonging to the RAS family. Examples of KRAS amino acid sequences include the amino acid sequence of UniProt accession number P01116 (SEQ ID NO: 75). Examples of nucleic acid sequences encoding KRAS include the nucleotide sequence set forth in SEQ ID NO: 76. Mutations in KRAS are observed in approximately 20% of human cancers, particularly in over 90% of pancreatic cancers. While KRAS is a highly potent target, there are currently no effective inhibitors, and pancreatic cancer remains a representative cancer type with a poor prognosis. KRAS is a G protein whose activity is controlled by cycling between an activated form bound to GTP and an inactivated form bound to GDP. However, specific mutations found in cancers maintain an activated state, which experimentally has been shown to be a prerequisite for carcinogenesis in pancreatic cancer and necessary for tumor maintenance. Examples of such activated KRAS include the G12C, G12D, and G13D mutants of KRAS, each consisting of the amino acid sequence set forth in SEQ ID NO: 75. Proteins that recognize activated KRAS include the downstream effector protein RAF, and previous reports have shown that the Ras-binding domain (RBD) and cysteine-rich domain (CRD) of this protein are required for recognizing activated KRAS. Thus, in one embodiment, the target is activated KRAS, and the target-binding domain comprises the RBD domain and / or the CRD domain. A target-binding domain comprising the RBD domain and the CRD domain may, for example, comprise or consist of the amino acid sequence set forth in SEQ ID NO: 77. As long as the target-binding domain can bind to the target of the polypeptide of the present application, it may also comprise or consist of the amino acid sequence of SEQ ID NO: 77, in which one or more amino acids have been substituted, deleted, added, and / or inserted.Alternatively, the target-binding domain may comprise or consist of an amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, for example, the amino acid sequence of SEQ ID NO: 77. The sequence of the nucleic acid encoding the target-binding domain comprising the RBD domain and the CRD domain may comprise or consist of, for example, the base sequence set forth in SEQ ID NO: 78. The sequence of the nucleic acid encoding the target-binding domain comprising the RBD domain and the CRD domain may comprise or consist of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 78, so long as the target-binding domain is capable of binding to the target of the polypeptide of the present application. Alternatively, the target-binding domain may comprise or consist of a nucleotide sequence that has, for example, about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 78, so long as the target-binding domain is capable of binding to the target of the polypeptide of the present application.

[0045] In another embodiment, the target is α-synuclein, and the target-binding domain binds to α-synuclein. α-Synuclein forms Lewy bodies, which are aggregates in Parkinson's disease and multiple system atrophy, and is thought to be central to the pathogenesis. α-Synuclein accumulates in neurons in Parkinson's disease, and in oligodendroglia, which are supporting cells, in multiple system atrophy. α-Synuclein exists intracellularly in various sizes, from monomers to polymers, but most are monomers, and some are thought to form fibrils and precursors to exhibit cytotoxicity. When the target is α-synuclein, the target-binding domain can be an antibody that binds to α-synuclein, for example, a nanobody that binds to α-synuclein. Commercially available nanobodies that bind to α-synuclein are available. For example, NbSyn87, which recognizes the N-terminus (J Mol Biol. 2013 Jul 24;425(14):2397-2411), and PFFN, which specifically recognizes fibrils (Nat Commun. 2022 Jul 19;13(1):4060), are known. The amino acid sequence and nucleic acid sequence of NbSyn87 are shown in SEQ ID NOs:79 and 80, respectively. The target-binding domain may, for example, comprise or consist of the amino acid sequence set forth in SEQ ID NO:79. As long as the target-binding domain is capable of binding to the target of the polypeptide of the present application, it may also comprise or consist of the amino acid sequence of SEQ ID NO:79 in which one or more amino acids have been substituted, deleted, added, and / or inserted. Alternatively, the target-binding domain may comprise or consist of an amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 79, for example, as long as it is capable of binding to the target of the polypeptide of the present application. The sequence of the nucleic acid encoding the target-binding domain may comprise or consist of the nucleotide sequence set forth in SEQ ID NO: 80, for example.The sequence of the nucleic acid encoding the target-binding domain may comprise or consist of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 80, so long as the target-binding domain is capable of binding to the target of the polypeptide of the present application. Alternatively, the target-binding domain may comprise or consist of a nucleotide sequence that has, for example, about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 80, so long as the target-binding domain is capable of binding to the target of the polypeptide of the present application.

[0046] The polypeptide of the present application may have a peptide linker between each of the ubiquitination enzyme domain, the exosome transport domain, and the target binding domain. The amino acid length and amino acid composition of the peptide linker are not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The amino acid length of the peptide linker is, for example, 1 to 200 amino acids, 1 to 100 amino acids, 2 to 50 amino acids, 2 to 30 amino acids, or 2 to 15 amino acids. Examples of the peptide linker include Gly-Ser, Gly-Gly-Gly-Gly-Gly-Ser (i.e., Gly4Ser; SEQ ID NO: 81), Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser (i.e., (Gly4Ser)2; SEQ ID NO: 82), and (Gly4Ser). x (wherein x is an integer greater than or equal to 3, e.g., 3); EAAAK (SEQ ID NO: 83).

[0047] In the polypeptide of the present application, the order in which the ubiquitination enzyme domain, exosome transport domain, and target-binding domain are linked is not particularly limited. The polypeptide of the present application may, for example, comprise the ubiquitination enzyme domain, exosome transport domain, and target-binding domain in the following order from the N-terminus: ubiquitination enzyme domain, exosome transport domain, and target-binding domain; ubiquitination enzyme domain, target-binding domain, and exosome transport domain; exosome transport domain, ubiquitination enzyme domain, and target-binding domain; exosome transport domain, target-binding domain, and ubiquitination enzyme domain; target-binding domain, ubiquitination enzyme domain, and exosome transport domain; or target-binding domain, exosome transport domain, and ubiquitination enzyme domain. In certain embodiments, the polypeptide of the present application comprises, in order from the N-terminus, a target-binding domain, an exosome transport domain, and a ubiquitination enzyme domain, or, in order from the N-terminus, an exosome transport domain, a target-binding domain, and a ubiquitination enzyme domain. For example, when the exosome transfer domain is a membrane protein such as a tetraspanin family protein or a portion thereof, particularly when the exosome transfer domain is a membrane protein or a portion thereof with an even number of transmembrane regions (e.g., two, four, or six), the polypeptide of the present application may comprise, from the N-terminus, a target-binding domain, an exosome transfer domain, and a ubiquitinase domain, or, alternatively, a ubiquitinase domain, an exosome transfer domain, and a target-binding domain. When the exosome transfer domain is a membrane protein or a portion thereof with an odd number of transmembrane regions (e.g., one), the polypeptide of the present application may comprise, from the N-terminus, an exosome transfer domain, a target-binding domain, and a ubiquitinase domain, or, alternatively, an exosome transfer domain, a ubiquitinase domain, and a target-binding domain. When the exosome transfer domain is an acylation tag, the polypeptide of the present application may comprise, from the N-terminus, an exosome transfer domain, a target-binding domain, and a ubiquitinase domain. The arrangement of the ubiquitination enzyme domain and the target binding domain can be determined based on the structure of the original ubiquitination enzyme.For example, when a portion of the SRS of the CRL complex is used as the ubiquitination enzyme domain, if the substrate-binding region of the scaffold protein-binding region and substrate-binding region of the SRS is located on the N-terminus, the target-binding domain of the ubiquitination enzyme domain and target-binding domain can be positioned on the N-terminus, and if the substrate-binding region is located on the C-terminus, the target-binding domain can be positioned on the C-terminus. Similarly, when other ubiquitination enzyme domains are used, the positions of the ubiquitination enzyme domain and target-binding domain can be determined based on the structure of the original ubiquitination enzyme.

[0048] The polypeptide of the present application may have a tag sequence added thereto, such as a FLAG tag, an HA tag, a His tag, a Myc tag, or a V5 tag.

[0049] The total length of the polypeptide of the present application can be, but is not limited to, for example, 100 to 2000 or 200 to 1000 amino acids.

[0050] In certain embodiments, the polypeptides of the present application comprise or consist of an amino acid sequence having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the amino acid sequence of a polypeptide of the present application set forth in Table 3; or an amino acid sequence of a polypeptide of the present application set forth in Table 3, or an amino acid sequence of a polypeptide of the present application set forth in Table 3, wherein one or more amino acid substitutions, deletions, additions, and / or insertions have been made.

[0051] The polypeptides of the present application not only induce target ubiquitination and degradation in cells in which they are present, but also migrate into exosomes and are secreted from those cells, thereby inducing target ubiquitination and degradation in surrounding cells. Thus, the polypeptides of the present application have advantages not available over existing targeted protein degradation technologies such as PROTACs and bioPROTACs.

[0052] Nucleic Acids The present application also provides nucleic acids encoding the polypeptides of the present application. The nucleic acids encoding the polypeptides of the present application may be in any form of nucleic acid, such as DNA or RNA. The nucleic acids can be easily produced based on the amino acid sequence of the polypeptides of the present application and the sequence information of the DNA encoding them. The base sequence of the nucleic acids of the present application may be codon-optimized.

[0053] In certain embodiments, a nucleic acid encoding a polypeptide of the present application comprises or consists of a nucleotide sequence having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a nucleotide sequence encoding a polypeptide of the present application listed in Table 3; or a nucleotide sequence encoding a polypeptide of the present application listed in Table 3, or a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, and / or inserted in the nucleotide sequence encoding a polypeptide of the present application listed in Table 3.

[0054] Vector The present application also provides a vector comprising a nucleic acid encoding a polypeptide of the present application. A vector refers to a molecule capable of delivering a nucleic acid of the present application to a cell. The vector of the present application may be a plasmid vector or a viral vector. The vector of the present application may be used in gene therapy. When administered to a subject, the vector of the present application expresses the polypeptide of the present application in cells within the subject's body. The expressed polypeptide of the present application induces degradation of a target within the cell and may also be transported into exosomes and secreted from the cell. The secreted exosomes may be taken up by surrounding cells and induce degradation of targets in the surrounding cells. Thus, the vector of the present application can be used for the treatment or prevention of disease. Alternatively, the vector of the present application may be a vector used to prepare cells expressing the polypeptide of the present application or exosomes containing the polypeptide of the present application, as described below.

[0055] When the vector of the present application is a plasmid vector, the plasmid vector to be used is not particularly limited, and any plasmid vector can be used, such as a cloning plasmid vector or an expression plasmid vector. The plasmid vector can be prepared by inserting the nucleic acid of the present application into the plasmid vector using a known method.

[0056] When the vector of the present application is a viral vector, examples of the viral vector used include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, retroviral vectors, and Sendai viral vectors. The vector of the present application is, for example, an adeno-associated viral vector.

[0057] Viral vectors can be prepared by known methods, for example, by preparing a viral expression plasmid vector into which the nucleic acid of the present application has been inserted, transfecting the vector into suitable host cells, transiently producing a viral vector containing the nucleic acid of the present application, and recovering the viral vector.

[0058] The vectors of the present application may have regulatory sequences that regulate the expression of the polypeptides and nucleic acids of the present application. Regulatory sequences include, for example, promoters, terminators, enhancers, polyadenylation signal sequences, and replication origin sequences. The regulatory sequences may be positioned so as to functionally regulate the expression of the polypeptides and nucleic acids of the present application, and may be positioned according to known methods.

[0059] Cells. The present application also provides cells expressing the polypeptides of the present application. The cells may be mammalian cells, for example, human cells. Examples of the cells include established cell lines such as 293T cells and 293F cells, mesenchymal stem cells (e.g., derived from bone marrow, adipose tissue, umbilical cord, or dental pulp), and stem cells such as iPS cells and ES cells. In one embodiment, the cells are mesenchymal stem cells. When administered to a subject, the cells may be autologous or allogeneic. The cells may, for example, contain a vector of the present application. Introduction of the vector of the present application into cells can be carried out by known methods such as the calcium phosphate method, the DEAE-dextran method, electroporation, or lipofection. The cells of the present application may be used in cell therapy. When administered to a subject, the cells of the present application may secrete exosomes containing the polypeptides of the present application within the subject's body. The secreted exosomes may be taken up by surrounding cells and induce target degradation in the surrounding cells. Thus, the cells of the present application may be used for the treatment or prevention of disease. Alternatively, the cells of the present application can be used to prepare exosomes containing the polypeptides of the present application, as described below. Alternatively, the cells of the present application can be cells for preparing the vectors of the present application, as described above.

[0060] The present application also provides exosomes containing the polypeptide of the present application. Exosomes are extracellular vesicles with a lipid bilayer structure and a particle diameter of approximately 40 to 150 nm. They are formed during endocytosis and secreted from cells by exocytosis. The exosomes of the present application can be obtained using cells expressing the polypeptide of the present application. For example, the exosomes of the present application can be contained in the culture supernatant of cells expressing the polypeptide of the present application. Those skilled in the art can appropriately determine cell culture conditions suitable for obtaining exosomes. The culture supernatant may be processed as needed to purify or concentrate exosomes, for example, by chromatography such as size exclusion chromatography, ion exchange chromatography, or affinity chromatography, filtration, centrifugation, or other techniques. The exosomes of the present application can be used to treat or prevent diseases by administering them to a subject.

[0061] Pharmaceutical Compositions The present application also provides pharmaceutical compositions comprising the polypeptides, nucleic acids, vectors, cells, or exosomes of the present application. In some embodiments, the pharmaceutical compositions comprise the vectors of the present application. In some embodiments, the pharmaceutical compositions comprise the cells of the present application. In some embodiments, the pharmaceutical compositions comprise the exosomes of the present application.

[0062] The pharmaceutical composition of the present application can be used for the treatment or prevention of a disease that can be treated or prevented by degradation of the target of the polypeptide of the present application, and is not limited to a specific disease and can be appropriately selected depending on the target.

[0063] In certain embodiments, the disease is cancer. The cancer to be treated or prevented by the pharmaceutical composition of the present application is not particularly limited, and examples thereof include pancreatic cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, melanoma, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, liver cancer, head and neck squamous cell carcinoma, thyroid cancer, skin cancer, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, pleurima, male embryonal tumor, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic sarcoma, leiomyosarcoma, and Wilms' tumor. The cancer treated or prevented by the pharmaceutical composition of the present application is, for example, pancreatic cancer. When the disease is pancreatic cancer, the target of the polypeptide of the present application is, for example, KRAS or activated KRAS, and the target binding domain includes, for example, the RBD domain and / or the CRD domain.

[0064] In another embodiment, the disease is Parkinson's disease, multiple system atrophy, or dementia with Lewy bodies. When the disease is Parkinson's disease, multiple system atrophy, or dementia with Lewy bodies, the target of the polypeptide of the present application is, for example, α-synuclein, and the target binding domain is, for example, an antibody (e.g., a nanobody) that binds to α-synuclein.

[0065] As used herein, "treating" or "treatment" means reducing or eliminating the cause of a disease, slowing or halting its progression, and / or reducing, alleviating, ameliorating or eliminating its symptoms in a subject with a disease.

[0066] As used herein, "preventing" or "prevention" means preventing the onset of a disease or reducing the likelihood of developing a disease in a subject, particularly in a subject who is likely to develop the disease but has not yet done so, where onset of the disease includes recurrence.

[0067] Subjects for disease treatment or prevention include animals, typically mammals (for example, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), and particularly humans.

[0068] The compositions of the present application may be frozen. Freezing and thawing of compositions containing polypeptides, nucleic acids, vectors, cells, or exosomes can be performed using known methods. When the compositions of the present application contain nucleic acids or vectors, the vectors may be encapsulated in particles such as polymer particles or liposomes. When the compositions of the present application contain cells, the compositions of the present application may contain cryoprotectants such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, and sucrose. When the compositions of the present application contain exosomes, the compositions of the present application may contain components of the cell culture supernatant.

[0069] The compositions of the present application may contain pharmaceutically acceptable carriers or additives. Examples of such carriers or additives include isotonicity agents, thickeners, sugars, preservatives, pH adjusters, stabilizers, gel bases, humectants, suspending agents, excipients, buffers, and antioxidants. Specific carriers or additives include, but are not limited to, water for injection, physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, and amino acid solutions. One or more pharmaceutically acceptable carriers or additives may be used, or two or more may be mixed together.

[0070] The composition of the present application may be, for example, an injection, a liquid, an oral agent, a patch, an implantable preparation, a gel preparation, or the like.

[0071] The administration routes of the composition of the present application include, for example, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, lymphatic administration, intrathecal administration, intraperitoneal administration, rectal administration, vaginal administration, transdermal administration, implantation, direct administration to an organ, and local transplantation.

[0072] The dosage and frequency of administration of the composition of the present application can be appropriately determined by those skilled in the art depending on the species of animal to be administered, the health condition, age, weight, route of administration, and dosage form of the subject, so that an effective amount of the active ingredient is administered to the subject. For example, the composition of the present application can be administered once to several times a day, or once to several times a day or several days, or once a week or several weeks, for example, once every 1 to 4 weeks, but is not limited thereto. The effective amount in a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. For example, when the composition of the present application contains a vector, the amount can be, but is not limited to, 10 9 ~10 15 For example, when the composition of the present application contains cells, the vector can be administered in an amount of, but not limited to, 10 ３ pieces / kg weight ~10 ７ Cells / kg body weight may be administered.

[0073] The present composition can be used alone or in combination with one or more additional active ingredients, particularly active ingredients for the treatment or prevention of diseases. "Combined use" of ingredients refers not only to the use of a dosage form containing all ingredients or the use of a combination of dosage forms containing each ingredient separately, but also to the simultaneous, sequential, or delayed administration of each ingredient, as long as they are used for the treatment or prevention of diseases. Two or more additional active ingredients can also be used in combination. Active ingredients suitable for combination use include, for example, anti-inflammatory agents, antibacterial agents, antifungal agents, antiviral agents, immunosuppressants, molecular target drugs, etc.

[0074] Treatment Methods The present application also provides a method for treating a disease, comprising administering a pharmaceutical composition of the present application to a subject in need thereof. The present application also provides use of a polypeptide, nucleic acid, vector, cell, or exosome of the present application for the manufacture of a pharmaceutical composition for treating a disease. The present application further provides a polypeptide, nucleic acid, vector, cell, or exosome of the present application for use in treating a disease. Examples of diseases are as described above.

[0075] Prevention Methods The present application also provides a method for preventing a disease, comprising administering the pharmaceutical composition of the present application to a subject in need thereof. The present application also provides use of the polypeptide, nucleic acid, vector, cell, or exosome of the present application for the manufacture of a pharmaceutical composition for preventing a disease. The present application further provides the polypeptide, nucleic acid, vector, cell, or exosome of the present application for use in preventing a disease. Examples of diseases are as described above.

[0076]

[0077] The present disclosure includes, for example, the following: [1] A polypeptide comprising a ubiquitination enzyme domain, an exosome transport domain, and a target binding domain. [2] The polypeptide according to [1], wherein the ubiquitination enzyme domain comprises Skp2, FBXO6, FBXO21, FBXO27, FBXO41, FBW1B, FBXW5, FBXW9, FBXW12, WSB1, VHL, CRBN, SOCS1, SPOP, or FBXL6, or a portion thereof, or wherein the ubiquitination enzyme domain comprises Skp2, FBXO6, FBXO21, FBXO27, FBXO41, FBW1B, FBXW5, FBXW9, FBXW12, WSB1, VHL, CRBN, SOCS1, or SPOP, or a portion thereof. [3] The polypeptide according to [2] above, wherein the ubiquitination enzyme domain comprises or consists of an amino acid sequence having about 70% or more sequence identity with the amino acid sequence of a SEQ ID NO shown in Table 1-1 or Table 1-2, or a portion thereof; or an amino acid sequence of a SEQ ID NO shown in Table 1-1 or Table 1-2, or a portion thereof, or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of a SEQ ID NO shown in Table 1-1 or Table 1-2, or a portion thereof. [4] The polypeptide according to [3] above, wherein the ubiquitination enzyme domain comprises or consists of the amino acid sequence of a SEQ ID NO shown in Table 1-1 or Table 1-2, or a portion thereof. [5] The polypeptide according to [2] above, wherein the ubiquitination enzyme domain comprises WSB1 or a portion thereof. [6] The polypeptide according to [5] above, wherein the ubiquitination enzyme domain comprises or consists of an amino acid sequence having about 70% or more sequence identity with the amino acid sequence of positions X1 to Y1 of the SEQ ID NO: shown in Table 1-1 or Table 1-2; or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of positions X1 to Y1 of the SEQ ID NO: shown in Table 1-1 or Table 1-2.[7] The polypeptide according to [6] above, wherein the ubiquitination enzyme domain comprises or consists of the amino acid sequence of positions 372 to 421 of SEQ ID NO: 1, or the amino acid sequence of positions 372 to 421 of the amino acid sequence of SEQ ID NO: 1 in which T at position 372 is replaced with D. [8] The polypeptide according to any of [1] to [7] above, wherein the exosome transport domain comprises a tetraspanin family protein or a portion thereof. [9] The polypeptide according to [8] above, wherein the portion of the tetraspanin family protein comprises part or all of the N-terminal intracellular region, the first and fourth transmembrane regions from the N-terminus, and part or all of the C-terminal intracellular region of the tetraspanin family protein, but does not include the second and third transmembrane regions from the N-terminus or the intracellular region between these transmembrane regions.

[10] The polypeptide according to [8] or [9] above, wherein the tetraspanin family protein is CD9, CD63, CD81, CD82, or CD151.

[11] The polypeptide according to

[10] above, wherein the exosome transfer domain comprises or consists of an amino acid sequence having about 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

[12] The polypeptide according to

[11] above, wherein the exosome transfer domain comprises or consists of the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

[13] The polypeptide according to

[10] above, wherein the tetraspanin family protein is CD9.

[14] The polypeptide according to

[13] above, wherein the exosome transport domain comprises or consists of an amino acid sequence having about 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 31 or 33; or the amino acid sequence of SEQ ID NO: 31 or 33, or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 31 or 33.

[15] The polypeptide according to

[14] above, wherein the exosome transport domain comprises or consists of the amino acid sequence of SEQ ID NO: 31 or 33.

[16] The polypeptide according to any of [1] to

[15] above, wherein the polypeptide comprises, in order from the N-terminus, a target-binding domain, an exosome transport domain, and a ubiquitination enzyme domain, or comprises, in order from the N-terminus, a ubiquitination enzyme domain, an exosome transport domain, and a target-binding domain.

[17] The polypeptide according to any one of [1] to

[16] , wherein the target-binding domain binds to KRAS, NRAS, HRAS, RHoA, α-synuclein, or TDP43.

[18] The polypeptide according to

[17] , wherein the target-binding domain binds to KRAS.

[19] The polypeptide according to

[18] , wherein KRAS is activated KRAS.

[20] The polypeptide according to

[17] , wherein the target-binding domain binds to α-synuclein.

[21] The polypeptide according to any one of [1] to

[20] , wherein the target-binding domain is a protein or peptide, or a portion thereof, capable of binding to a target.

[22] The polypeptide according to any one of [1] to

[21] , wherein the target-binding domain is an antibody.

[23] The polypeptide according to

[22] , wherein the antibody is a nanobody.

[24] The polypeptide according to any one of [1] to

[21] , wherein the target-binding domain comprises a RAS-binding domain and / or a cysteine-rich domain.

[0078]

[25] A nucleic acid encoding the polypeptide according to any one of [1] to

[24] .

[26] A vector comprising the nucleic acid according to

[25] .

[27] A cell expressing the polypeptide according to any one of [1] to

[24] .

[28] An exosome comprising the polypeptide according to any one of [1] to

[24] .

[0079]

[29] A pharmaceutical composition comprising the polypeptide according to any one of [1] to

[24] , the nucleic acid according to

[25] , the vector according to

[26] , the cell according to

[27] , or the exosome according to

[28] .

[30] The pharmaceutical composition according to

[29] , comprising the vector according to

[26] .

[31] The pharmaceutical composition according to

[30] , wherein the vector is an adeno-associated virus vector.

[32] The pharmaceutical composition according to

[29] , comprising the cell according to

[27] .

[33] The pharmaceutical composition according to

[29] , comprising the exosome according to

[28] .

[34] The pharmaceutical composition according to any one of

[29] to

[33] , for treating or preventing cancer.

[35] The pharmaceutical composition according to

[34] , wherein the cancer is pancreatic cancer.

[36] The pharmaceutical composition according to any one of

[29] to

[33] , for treating or preventing a neurodegenerative disease.

[37] The pharmaceutical composition according to the above

[36] , wherein the neurodegenerative disease is Parkinson's disease, multiple system atrophy, or dementia with Lewy bodies.

[0080]

[38] A polypeptide comprising an exosome transfer domain, wherein the exosome transfer domain comprises a portion of a tetraspanin family protein.

[39] The polypeptide according to

[38] , wherein the portion of the tetraspanin family protein comprises part or all of the N-terminal intracellular region of the tetraspanin family protein, the first and fourth transmembrane regions from the N-terminus, and part or all of the C-terminal intracellular region, but does not comprise the second and third transmembrane regions from the N-terminus or the intracellular region between these transmembrane regions.

[40] The polypeptide according to

[38] or

[39] , wherein the portion of the tetraspanin family protein comprises an extracellular region of 10 to 50, 10 to 40, 10 to 30, or 10 to 20 amino acids between the first and fourth transmembrane regions from the N-terminus of the tetraspanin family protein.

[41] The polypeptide according to

[40] , wherein the extracellular region comprises part or all of SEL and / or part or all of LEL.

[42] The polypeptide according to any of

[38] to

[41] above, wherein the tetraspanin family protein is CD9, CD63, CD81, CD82, or CD151.

[43] The polypeptide according to any of

[38] to

[42] above, wherein the exosome transport domain comprises or consists of an amino acid sequence having about 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 33, 37, 41, 45, or 49; or the amino acid sequence of SEQ ID NO: 33, 37, 41, 45, or 49, or an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 33, 37, 41, 45, or 49.

[44] The polypeptide according to any of

[43] above, wherein the exosome transport domain comprises or consists of the amino acid sequence of SEQ ID NO: 33, 37, 41, 45, or 49.

[0081] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples in any way.

[0082] Example 1 Identification of a ubiquitinating enzyme that transports into exosomes We aimed to achieve the goal of constructing a chimeric protein consisting of an exosome transport domain, a target-binding domain, and a ubiquitinating enzyme domain, which would degrade a target within the expressing cell while spreading the effect to surrounding cells.

[0083] An acylation tag was selected as the exosome-transfer tag (the acylation tag was found to be identical to the Xpack tag (SBI) and will hereafter be referred to as XP). We first used a GFP nanobody (VHH antibody) as the target-binding domain and performed screening based on GFP resolution. Because a compact molecular size is desirable for exosome transport, we chose the substrate recognition subunit (SRS), part of the cullin ubiquitin ligase complex (CRL complex), as the ubiquitination enzyme. SRSs are classified into several categories containing F-boxes, such as FBXL, FBXO, and FBXW. We individually estimated the localization of each of these proteins using Uniprot (https: / / www.uniprot.org) and the Human Protein Atlas (https: / / v14.proteinatlas.org / subcellular), and excluded those showing clear nuclear targeting or with unclear SRS domains. From the selected SRSs, we created plasmids encoding chimeric proteins (Figure 1A) containing the adapter region (CRL binding: F-box domain, etc.) required for CRL complex formation. A FLAG tag (DYKDDDDK tag; SEQ ID NO: 86) was added to this chimeric protein for biochemical analysis. The nucleic acid sequence encoding the FLAG tag is shown in SEQ ID NO: 87. The order of the CRL-binding site and GFP nanobody was created to mimic the sequence order in the original SRS protein. The amino acid sequences of each artificial protein and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 88 to 115.

[0084] The artificial proteins were introduced into 293T cells, which constitutively express HiBiT-EGFP, using a PiggyBac vector to induce doxycycline-dependent expression. After two passages of selection with an antibiotic (puromycin), EGFP degradation due to the expression of the artificial proteins was evaluated using flow cytometry and the HibiT assay (Promega). The results are shown in Figure 1B. Relatively strong reductions were observed in Skp2, FBXO6, FBXW9, WSB1, VHL, SOCS1, and SPOP. Of these, we focused on WSB1, which showed the strongest degradation activity. DEGs, which are GFP-degrading proteins including WSB1, were also identified. GFP The amino acid sequence of (XP, WSB1) and the nucleic acid sequence encoding it are shown in SEQ ID NOs: 106 and 107. X (Y, Z) means an artificial protein that includes a target-binding domain that binds to target X, Y as an exosome transport domain, and Z as a ubiquitination enzyme domain.

[0085] Next, DEGs GFP Western blotting was performed to verify the GFP degradation ability of , and doxycycline-dependent degradation was confirmed as shown in Figure 1C.

[0086] Next, we examined the extent to which these proteins have the ability to diffuse into the surrounding area. GFP The C-terminus of (XP, WSB1) was modified to add a HiBiT tag (Promega) to enable tracking by luminescence, and a 293T cell line expressing this tag in a doxycycline-dependent manner was created. After culturing for 48 hours with doxycycline, the signal in exosomes simply purified from the culture supernatant was compared with the signal in the cell lysate, and the exosome transfer rate was calculated. GFP (XP, WSB1) was estimated to be approximately 5% (Figure 1D).

[0087] Next, to verify whether they have the ability to diffuse to the surrounding area, we compared cells that constitutively express EGFP-KRAS with cells that express DEGs in a doxycycline-dependent manner. GFPWe co-cultured cells expressing XP and WSB1 to examine whether EGFP-KRAS was degraded. Flow cytometry analysis confirmed that diffusion-mediated degradation was achieved (Figure 1E).

[0088] Example 2. Selection of exosome transport domain To develop a tag that transports into exosomes, we decided to base our work on exosome transport proteins. The four-transmembrane proteins tetraspanin (CD9, CD63, CD81) and LAMP2 were selected as proteins that exhibit good transport to the exosome membrane surface. First, we constructed plasmids expressing EGFP tagged with HiBiT and exosome transport tags, transiently expressed them in 293T cells, and examined the transport rate into exosomes. The tetraspanin group exhibited a higher transport rate than the acylation tag (XP), with CD9 showing a particularly high transport rate (Figure 2A).

[0089] Although tetraspanins exhibit good exosome translocation rates, they are relatively large (approximately 25 kDa) and possess diverse functions, potentially resulting in unpredictable effects when used as tags. Therefore, we attempted to avoid these unpredictable effects by removing most of their extracellular domains. From the CD9, CD63, and CD81 proteins used in the above validation, we selected CD9 and CD81. Both CD9 and CD81 contain a small extracellular loop (SEL) and a large extracellular loop (LEL) (Figure 2B), and the three-dimensional structure formed by the SEL and LEL is believed to be functionally important. Therefore, we created truncated CD9 (sCD9) and CD81 (sCD81) by ligating the SEL and LEL to remove the intermediate structure and trimming the unnecessary intracellular domain (Figure 2B). Given that CD9 exhibited higher exosome translocation in 293T cells and the significant amino acid sequence homology between CD9 and CD81, we narrowed our focus to sCD9 and added a HiBiT tag to track exosome translocation. The results showed that sCD9 was expressed at a higher level than CD9 (Fig. 2C) and exhibited high exosome transferability (Fig. 2D).

[0090] Next, we investigated whether this protein could degrade GFP when combined with the ubiquitin degradation machinery. GFP Three candidate designs are considered: sCD9-GFP nanobody-WSB1, GFP nanobody-sCD9-WSB1, and GFP nanobody-WSB1-sCD9. The amino acid sequences of each artificial protein and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 116-121. When assessed for their degrading activity against HiBiT-EGFP, only GFP nanobody-sCD9-WSB1 exhibited degrading activity (Figure 2E).

[0091] After exosome uptake, degradation in endosomes becomes a problem. To efficiently deliver target proteins via exosomes, it is necessary to promote their release from endosomes after uptake into target cells. Therefore, we attempted to increase delivery efficiency by inserting a peptide sequence that promotes membrane fusion at the SEL-LEL junction of sCD9. Candidate sequences (R9, FR, HL6, ZF5.3, INF, GALA, TAT, and SN21) were inserted into sCD9 (Table 4), and transient expression in HeLa cells was evaluated for exosome translocation, uptake, and cytosolic translocation (endosomal escape rate) using HiBiT (Figure 2F). Insertion of a sequence linking INF and TAT (INF-TAT sequence) significantly increased delivery.

[0092] The list of antibodies used in this example is shown in Table 5.

[0093] Example 3. Application to KRAS Although KRAS mutations are observed in over 90% of pancreatic cancers, making it a very promising target, there are currently no effective inhibitors, and pancreatic cancer remains a representative cancer type with a poor prognosis. We considered using the above-mentioned method to target activated KRAS.

[0094] KRAS is a G protein whose activity is controlled by cycling between an activated form bound to GTP and an inactive form bound to GDP. However, specific mutations found in cancer sites result in a constant activated state, which has been experimentally shown to be a prerequisite for carcinogenesis in pancreatic cancer and necessary for tumor maintenance. While targeted degradation of specific amino acid mutations in pancreatic cancer is theoretically possible, this only covers those with specific mutations, and there is backup from normal KRAS alleles. Therefore, we considered it appropriate to target activated KRAS. One downstream effector protein that recognizes activated KRAS is RAF, and previous reports have shown that the Ras-binding domain (RBD) and cysteine-rich domain (CRD) of this protein are required for identifying activated KRAS. Therefore, we focused on the above-mentioned DEGs. GFP DEGs in which the target binding domain, GFP nanobody, of (XP, WSB1) was substituted for the RBD / CRD KRAS (XP, WSB1) were created (Figure 3A). KRAS The amino acid sequences of (XP, WSB1) and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 122 and 123, respectively.

[0095] DEG KRAS When DEGs (XP, WSB1) were expressed in HeLa cells in a doxycycline-dependent manner, potent KRAS degradation was observed, and this was almost completely suppressed by the proteasome inhibitor MG132 (Fig. 3B), suggesting that the mechanism of degradation was via the proteasome. KRAS We investigated whether (XP, WSB1) targets activated KRAS. Various activating mutations of KRAS have been reported, including G12C, G12D, and G13D, but it has been reported that activation is lost with the K104Q mutation. Therefore, we constitutively expressed wild-type (WT) and various mutants of KRAS tagged with HiBiT or HA in 293T cells, and investigated DEGs. KRASThe quantitative reduction of XP and WSB1 by the enzymes was measured by HiBiT assay or Western blot (Fig. 3C and 3D). A significant difference was observed between the active (G13D) and inactive (G13D / K104Q) mutants, demonstrating the active-form-specific degradation.

[0096] Next, we changed the exosome transfer domain from XP to sCD9 and identified DEGs. KRAS We created (sCD9, WSB1) and examined their degradation ability using the HIBiT assay. As a result, we observed similar degradation of KRAS (Fig. 3E). KRAS (sCD9, WSB1). KRAS The amino acid sequences of (sCD9, WSB1) and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 124 and 125, respectively.

[0097] First, we investigated the doxycycline-dependent DEG expression in pancreatic cancer cell lines PK-1, PK-45H, PK-59, T3M-4, and PANC-1 (all of which have been confirmed to have KRAS mutations). KRAS We established cell lines expressing sCD9 and WSB1, and assessed their doxycycline-dependent tumor growth. All cell lines, except PANC-1, showed a strong growth-suppressing effect, with some even dying (Figure 3F). To verify that this was due to KRAS degradation, we harvested cells 24 hours after doxycycline administration and analyzed them by Western blotting. We found that the cell lines that showed growth suppression exhibited a reduction in KRAS and almost complete suppression of pERK (Figure 3G).

[0098] Next, we examined whether KRAS degradation was possible through co-culture. KRAS (XP, WSB1) or DEG KRAS 293T cells expressing (sCD9, WSB1) (labeled with mCherry) were cocultured with 293T cells expressing HiBiT-KRAS G12D, and the HiBiT signal per cell in the mCherry-negative fraction was detected, demonstrating a bystander effect (Fig. 3H).

[0099] Next, DEGs KRAS (XP, WSB1) or DEG KRAS We investigated the growth inhibitory effect of co-culture of 293T cells expressing sCD9 and WSB1 with pancreatic cancer cells. PK-1 and T3M-4 were labeled with nanoluciferase, and DEGs were identified. KRAS (XP, WSB1) or DEG KRAS Co-culture with 293T cells expressing (sCD9, WSB1) revealed a significant growth-suppressing effect (Fig. 3I).

[0100] Next, UniWells TM We investigated the growth inhibitory effect of co-culture using a membrane that allows only exosomes and other particles of 0.6 μm or less to pass through. KRAS When co-cultured with 293T cells expressing sCD9 and WSB1 for 120 hours, the proliferation of PK-1 and T3M-4 cell lines was significantly suppressed (Fig. 3J). KRAS Exosomes containing (sCD9, WSB1) have been shown to have an inhibitory effect on pancreatic cancer growth.

[0101] Although 293T cells are easy to manipulate, they are not commonly used as cell preparations. Therefore, we focused on mesenchymal stem cells, which are already used clinically and are known to produce a large amount of exosomes. KRAS We generated mesenchymal stem cells expressing sCD9 and WSB1 in a doxycycline-dependent manner and co-cultured them with iRFP-labeled pancreatic cancer cell lines. After 5 days of culture, the cells were harvested and the iRFP signal in the pellet was measured, revealing a significant decrease (Figure 3K). This demonstrated the potential for suppressing pancreatic cancer through the bystander effect using mesenchymal stem cells.

[0102] Example 4. Application to α-synuclein α-synuclein (aS) forms Lewy bodies, which are aggregates in Parkinson's disease and multiple system atrophy, and is thought to be central to the pathogenesis. In Parkinson's disease, aS accumulates in neurons, while in multiple system atrophy, it accumulates inside the supporting cells, oligodendroglia. Multiple system atrophy is an intractable disease that progresses very rapidly and has no cure. We began development based on the belief that this technology could be used to remove aS.

[0103] aS exists within cells in various sizes, from monomers to polymers, but most are monomers, and some are thought to exhibit cytotoxicity by forming fibrils and precursors. A nanobody, NbSyn87, which recognizes the N-terminus of aS, has been developed and reported along with its sequence. Therefore, we investigated whether it is possible to use this to degrade aS.

[0104] First, DEGs GFP (XP, WSB1) and DEGs GFP (sCD9, WSB1) DEGs in which the target binding domains were replaced with NbSyn87 aS (XP, WSB1) and DEGs aS (sCD9, WSB1) were created (Figure 4A). aS The amino acid sequences of (XP, WSB1) and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 110 and 111, respectively. aS The amino acid sequences of (sCD9, WSB1) and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 126 and 127, respectively. 293T cells constitutively expressing HiBiT-aS were constructed, and DEGs were isolated in a doxycycline-dependent manner. aS (XP, WSB1) or DEG aS When 293T cells constitutively expressed HiBiT-aS, the DEGs (sCD9, WSB1) were expressed, resulting in a decrease in the HiBiT signal (Fig. 4B). aS (XP, WSB1) or DEG aS When 293T cells expressing (sCD9, WSB1) were co-cultured and evaluated by HiBiT assay, a bystander effect was confirmed (Figure 4C).

[0105] Next, to verify the potential of viral vectors, we investigated the DEGs packaged with adeno-associated viral vector serotype 2 (AAV2) in SHSY-5Y cells (a human neuroblastoma cell line) that constitutively express aS driven by the CMV promoter. aS (sCD9, WSB1) was administered at 10,000 vg / cell, and the reduction of aS was confirmed by ELISA (Fig. 4D).

[0106] Next, to verify the bystander effect, we used the human oligodendroglial cell line MO3.13. MO3.13 cells were created that constitutively express aS and iRFP using the CMV promoter, and then co-cultured with SHSY-5Y cells, where they were then mediated by AAV2. First, we expressed EGFP using an improved neuron-specific promoter (Synapsin tet off), and found that EGFP expression was almost exclusively limited to SHSY-5Y. Next, we used the same culture system to express DEGs using the above promoter. aS Western blot analysis of iRFP-positive MO3.13 cells expressing sCD9 and WSB1 revealed a reduction in aS, demonstrating in vitro the bystander effect of aS reduction in neurons and neural supporting cells (Fig. 4E).

[0107] Example 5. Comparison of Various Exosome Transfer Domains To verify the exosome transfer rate, various tetraspanins carrying HiBiT-EGFP at their N-terminus were transiently expressed in 293T cells (1 ml of each in 12 wells). After 36 hours, the culture supernatant and cell pellet were collected, and the HiBiT signals were compared to calculate the exosome transfer rate (Figure 5A). The amino acid sequences of GFP nanobody-tetraspanin-WSB1 and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 118, 119, and 130-147.

[0108] Next, we generated a cell line expressing HiBiT-EGFP driven by the UBC promoter using 293T cells. We then transfected these cells with the GFP nanobody-tetraspanin WSB1 in a doxycycline-dependent manner and assessed the reduction in HiBiT-EGFP expression levels using the HiBiT assay (Fig. 5B). No significant differences were observed between the tetraspanins.

[0109] Next, we created a cell line based on 293T cells expressing HiBiT-EGFP-KRAS under the control of the EF1 promoter. 1) These cells were cocultured with 2) cells expressing the GFP nanobody-tetraspanin-WSB1 in a doxycycline-dependent manner. Because 2) cells were labeled with mCherry, we were able to separate the 1) population by measuring the mCherry-negative fraction after trypsin harvest. We then estimated the HiBiT-EGFP-KRAS signal per cell by calculating (the total HiBiT signal (KRAS molecule count) in the lysate) / (the total cell titer * mCherry-negative fraction in the lysate). The percentages (%) compared to the negative control values ​​are shown in Figure 5C. A relatively strong decrease in sCD9 and sCD63 was observed.

[0110] Example 6. Comparison of various ubiquitination enzymes DEGs that exhibit excellent resolution within the same cells GFPWe investigated whether the bystander effect of SRS (XP, WSB1) was superior to that of other SRSs. We established a cell line based on 293T cells expressing HiBiT-EGFP-KRAS under the EF1 promoter. 1) These cells were cocultured with 2) cells expressing Xpack-GFP nanobody-ubiquitinase in a doxycycline-dependent manner. Because 2) cells were labeled with mCherry, we were able to separate the 1) population by measuring the mCherry-negative fraction after trypsin harvest. We estimated the HiBiT-EGFP-KRAS signal per cell by calculating (the total HiBiT signal (KRAS molecule count) in the lysate) / (the total cell titer * mCherry-negative fraction in the lysate). The percentages (%) compared to the negative control values ​​are shown in Figure 6A. The strongest reductions were observed for WSB1 and VHL.

[0111] Next, we evaluated the degradation potential of exosomes by replacing the exosome trafficking tag with sCD9. Using HiBiT-EGFP-expressing 293T cells as the parent cell line, we engineered the ubiquitinase-sCD9-GFP nanobody (Skp2) or GFP nanobody-sCD9-ubiquitinase (WSB1, VHL, SPOP) to be inducible by doxycycline, and assessed the relative HiBiT / cell titer before and after (Figure 6B). The amino acid sequences of the GFP nanobody-sCD9-ubiquitinase constructs and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 118, 119, and 148-151. Degradation was confirmed for all constructs tested.

[0112] Next, 293T cells expressing GFP nanobody-sCD9-ubiquitinase were cocultured with 293T cells expressing HiBiT-EGFP-KRAS, and the quantitative change in HiBiT-EGFP-KRAS per cell was calculated using the same normalization method as above (Fig. 6C). Significant degradation of HiBiT-EGFP-KRAS was confirmed for both constructs.

[0113] Example 7. Degradation of target proteins using the ALFA tag The ALFA tag (PSRLLEEELRRRLTE) has been previously published along with its corresponding nanobody (https: / / www.nature.com / articles / s41467-019-12301-7). The amino acid sequence of the ALFA tag and the sequence of the nucleic acid encoding it are shown in SEQ ID NOs: 152 and 153. DEG GFP DEGs in which the target binding domains of (XP, WSB1) were replaced with nanobodies against the ALFA tag alfa (XP, WSB1) were created. alfa The amino acid sequence of (XP, WSB1) and the nucleic acid sequence encoding it are shown in SEQ ID NOs: 154 and 155. alfa Target proteins with HiBiT-ALFA tags attached to the N-terminus (EGF-NES, EGF-NLS, α-synuclein, TDP43, KRAS, and RhoA; "NLS" indicates the nuclear localization signal, and "NES" indicates the nuclear export signal) were constitutively expressed in 293T cells expressing XP and WSB1, and the increase or decrease in HiBiT / cell titer before and after the addition of doxycycline was plotted (Figure 7). Degradation of almost all proteins tested was possible.

[0114] Example 8. Degradation of Target Proteins Using the ALFA Tag Artificial proteins were created using sCD9 as the exosome transport domain, a nanobody targeting the ALFA tag as the target-binding domain, and Skp2 or WSB1 as the ubiquitination enzyme domain. The amino acid sequences of each artificial protein and the nucleic acid sequences encoding them are shown in SEQ ID NOs: 156-171. 293T cells expressing HiBiT-EGFP-ALFA tag under the UBC promoter were established, and each artificial protein was transiently introduced. 24 hours later, the HiBiT signal, corrected for cell mass (cell titer), was quantified. For both ubiquitination enzyme domains, placing sCD9 in the center resulted in better degradation. Furthermore, it was suggested that the positioning of the ubiquitination enzyme domain and target-binding domain should preferably mimic the structure of the original ubiquitination enzyme.

[0115] Example 9. Examination of the Positioning of Each Domain (1) 293T cells expressing artificial proteins in a doxycycline-dependent manner were generated using WSB1 as the ubiquitination enzyme domain, a nanobody targeting the ALFA tag as the target-binding domain, and exosome transport domains consisting of sCD81 (four), CD36 (two), CD81 (four), STEAP1 (six), or CD19 (one) (the number of transmembrane domains is indicated in parentheses), or a nanobody targeting the ALFA tag. These artificial proteins were generated using 293T cells expressing HiBiT-EGFP as the parent cell line. Cells were harvested 24 hours after the addition of doxycycline, and the HiBiT / cell titer change rate was plotted (Figure 9). Target protein degradation was observed to some extent in all cases. Furthermore, more efficient degradation was observed when the target-binding domain was closer to the N-terminus of WSB1.

[0116] Example 10. Examination of the Arrangement of Each Domain (2) 293T cells expressing artificial proteins in a doxycycline-dependent manner were prepared as in Example 9, with the exosome transport domain being sCD9, the target-binding domain being a nanobody for the ALFA tag, and the ubiquitination enzyme domain (FBXL6, SPOP) located at the N- or C-terminus, and then evaluated (Figure 10). The ubiquitination enzyme domain is located at the N-terminus for FBXL6 and at the C-terminus for SPOP. Similar to the results of Example 8, these results suggest that for degradation activity, it is desirable to mimic the arrangement of the ubiquitination enzyme domain and target-binding domain to resemble the structure of the original ubiquitination enzyme.

Claims

1. A polypeptide comprising a ubiquitination enzyme domain, an exosome transport domain, and a target binding domain.

2. The polypeptide of claim 1, wherein the ubiquitination enzyme domain comprises Skp2, FBXO6, FBXO21, FBXO27, FBXO41, FBW1B, FBXW5, FBXW9, FBXW12, WSB1, VHL, CRBN, SOCS1, or SPOP or a portion thereof.

3. The polypeptide of claim 2, wherein the ubiquitination enzyme domain comprises WSB1 or a portion thereof.

4. The polypeptide according to any one of claims 1 to 3, wherein the exosome transport domain comprises a tetraspanin family protein or a part thereof.

5. The polypeptide of claim 4, wherein the tetraspanin family protein is CD9, CD63, CD81, CD82, or CD151.

6. The polypeptide according to claim 5, wherein the tetraspanin family protein is CD9.

7. A polypeptide according to any one of claims 1 to 6, wherein the target binding domain binds to KRAS.

8. The polypeptide according to claim 7, wherein the KRAS is an activated KRAS.

9. A polypeptide according to any one of claims 1 to 6, wherein the target binding domain binds to alpha-synuclein.

10. A polypeptide according to any one of claims 1 to 9, wherein the target binding domain is an antibody.

11. The polypeptide of claim 10, wherein the antibody is a nanobody.

12. A polypeptide according to any one of claims 1 to 8, wherein the target-binding domain comprises a RAS-binding domain and / or a cysteine-rich domain.

13. A nucleic acid encoding a polypeptide according to any one of claims 1 to 12.

14. A vector comprising the nucleic acid of claim 13.

15. A cell expressing a polypeptide according to any one of claims 1 to 12.

16. An exosome comprising a polypeptide according to any one of claims 1 to 12.

17. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 12, the nucleic acid according to claim 13, the vector according to claim 14, the cell according to claim 15, or the exosome according to claim 16.

18. The pharmaceutical composition according to claim 17 for treating or preventing cancer.

19. The pharmaceutical composition according to claim 17 for treating or preventing a neurodegenerative disease.

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