SKP2 fragment, variant thereof, and use thereof

Abstract

The present invention relates to a SKP2 fragment, a variant thereof, and use thereof. Compared to a native SKP2 fragment, the SKP2 fragment variant according to the present invention has increased binding affinity to s-phase kinase associated protein 1 (SKP1). In addition, compared to a fusion protein comprising a native SKP2 fragment and a target binding site, a fusion protein comprising said SKP2 fragment variant and a target binding site can more effectively induce degradation of a target protein. Therefore, the SKP2 fragment variant of the present invention is expected to be usable in the development of PROTAC drug with improved therapeutic effect.

Description

SKP2 fragments and variants thereof, and uses thereof

[0001] The present invention relates to SKP2 fragments and variants thereof, and uses thereof.

[0002] To date, new drugs have been developed based on the principle of inhibiting the active site or ligand binding site of disease-causing proteins. Of the approximately 20,000 human proteins, only about 3% are known to be FDA-approved drug targets. Approximately 3,100 proteins (16%), representing the majority of disease-causing proteins, are considered inapplicable to current drug development technologies. Furthermore, existing active site-directed drugs often suffer from issues related to drug resistance.

[0003] Proteolysis-targeting chimeras (PROTACs) are dual-bonded molecules that bind to disease-causing proteins and induce proteolysis via the proteasome. PROTACs utilize the ubiquitin-proteasome system (UPS), the cell's endogenous protein degradation mechanism, enabling induced proteolysis of proteins previously inaccessible to conventional technologies. Therefore, they are attracting attention as a new paradigm for overcoming the limitations of traditional drug development.

[0004] Protacs are composed of two protein-binding molecules, each of which binds to an E3 ubiquitin ligase and a target protein. Through this binding, Protacs bring the target protein close to the E3 ligase, thereby inducing ubiquitination. Ubiquitination involves three steps: activation, conjugation, and ligation, carried out by a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). As a result of this cascade, ubiquitin is covalently bound to the target protein, and the ubiquitinated protein is degraded by the proteasome.

[0005] Protac technology was first introduced in 2001 and is currently receiving significant attention, with active development of related technologies (Sakamoto et al., PNAS, 98:8554 (2001), Huang et al., Cell Res, 26:484-498 (2016)). Most protac drugs have been developed as small-molecule compounds. While known protac drugs are very useful, improvements are needed to address the inherent low solubility of small-molecule compounds, reduced protein degradation efficiency due to reduced intracellular penetration, and safety concerns. Therefore, there is a growing need for protac drugs that address these issues.

[0006] Accordingly, the inventors of the present invention conducted research to develop a more effective protactic drug, discovered a mutant of a fragment of SKP2 (S-phase kinase-associated protein 2), and confirmed that the mutant forms a stronger E3 ubiquitin ligase complex compared to the natural type, thereby completing the present invention.

[0007] To achieve the above purpose, one aspect of the present invention provides a SKP2 fragment or a variant thereof, and a fusion protein comprising the SKP2 fragment or variant thereof; and a target binding site.

[0008] Another aspect of the present invention provides a polynucleotide encoding the SKP2 fragment or a variant thereof, or the fusion protein, a vector loaded with the polynucleotide, and a host cell transformed with the vector.

[0009] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a disease, comprising the fusion protein, a polynucleotide encoding the fusion protein, or a vector comprising the polynucleotide as an active ingredient.

[0010] Another aspect of the present invention provides a use of the fusion protein, a polynucleotide encoding the fusion protein, or a vector comprising the polynucleotide for the prevention or treatment of a disease.

[0011] Another aspect of the present invention provides a method for preventing or treating a disease, comprising administering to a subject the fusion protein, a polynucleotide encoding the fusion protein, or a vector comprising the polynucleotide.

[0012] The SKP2 fragment variant according to the present invention has increased binding affinity to SKP1 (S-phase kinase associated protein 1) compared to the native SKP2 fragment. Furthermore, the fusion protein comprising the SKP2 fragment variant and the target binding site was able to induce degradation of the target protein more effectively compared to the fusion protein comprising the native SKP2 fragment and the target binding site. Therefore, the SKP2 fragment variant of the present invention is expected to be useful in developing protease drugs with improved therapeutic effects.

[0013] Figures 1a to 1d are SKP2 2-176 This diagram shows the results of confirming the binding of a mutant protein library to SKP1.

[0014] Figures 2a to 2c are secondary SKP2 2-176This diagram shows the results of confirming the binding of a mutant protein library to SKP1.

[0015] Figure 3 is the first SKP2 2-176 Mutant protein library (clone 3, clone 15) and secondary SKP2 2-176 A diagram showing the amino acid sequences of mutants selected from a mutant protein library (clone E1, clone E2, clone E4 to clone E6).

[0016] Figures 4a to 4c are SKP2 2-176 This is a graph showing the results of confirming the binding affinity of SKP1 and its mutant proteins (clone E1, clone E2, clone E4 to clone E6).

[0017] Figure 5 is a specific example of SKP2 of the present invention. 2-176 Or a drawing showing the results of Western blot analysis of the degradation ability of STAT3 protein of a fusion protein containing a mutant thereof and an anti-ALFA tag nanobody (NbALFA) (top) and a graph showing the results of quantification of the results (bottom).

[0018] Figure 6 is a specific example of SKP2 of the present invention. 2-176 Or, a drawing (top) showing the results of Western blot analysis of the degradation ability of STAT3 protein of a fusion protein including a mutant thereof and an anti-STAT3 antibody (ETI01_A4 antibody mutant), and a graph (bottom) showing the results of quantifying the results.

[0019] SKP2 fragments and their variants

[0020] One aspect of the present invention provides a SKP2 fragment or a variant thereof.

[0021] As used herein, the term "SKP2 (S-phase kinase-associated protein 2)" refers to SCF Skp2It is an F-box protein that constitutes the ubiquitin ligase (E3 ligase) complex. The SKP2 protein contains a total of 424 amino acids, including an F-box domain (94 aa to 140 aa) consisting of approximately 40 amino acids from the N-terminus and 10 LRRs (leucin rich regions).

[0022] F-box proteins are classified into three groups: Fbxws, which contain a WD40 repeat domain; Fbxls, which contain a leucin-rich repeat domain; and Fbxos, which contain other protein-protein interaction sites or lack a recognition motif. F-box proteins are one of the proteins that constitute the SCF (SKP1-cullin-F-box) ubiquitin ligase complex and function as substrate recognition factors. Therefore, different substrate proteins can bind depending on the type of F-box. In addition, they bind to SKP1 through the F-box domain to form the SCF ubiquitin complex.

[0023] The above SKP2 protein is an F-box protein belonging to Fbxls, which binds to cell cycle regulatory proteins such as p27 and p21 and regulates their degradation.

[0024] The SKP2 protein may preferably be, but is not limited to, a human SKP2 protein. The amino acid sequence and polynucleotide sequence of the SKP2 protein may be obtained from known databases such as GenBank of the National Institutes of Health (NCBI) of the United States, and may preferably include the amino acid sequence of SEQ ID NO: 47.

[0025] In addition, it may be composed of a sequence in which one or more amino acids of the protein are added, deleted, or substituted, as long as it has the same activity as the protein or has the same gene location on the chromosome encoding the SKP2 protein. The SKP2 protein may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 47. Here, binding to SKP1 can be measured by a method known to those skilled in the art.

[0026] The term "SKP2 fragment" used herein refers to a fragment in which a portion of the N-terminus and / or C-terminus of the native SKP2 protein is deleted (truncated), and may exhibit the same binding affinity to SKP1 as the native SKP2 protein. The "wild type" includes all proteins found in nature or nucleic acids encoding the same, and may be described interchangeably with the wild type.

[0027] Specifically, the fragment may include a site that binds to SKP1. More specifically, it may include an F-box domain that binds to SKP1. In this case, the F-box domain is the same as described above.

[0028] More specifically, the SKP2 fragment is sequentially 139, 140, 141, 142, 143, 144, 145, 146, 147, 178, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, It may contain a sequence of 174 or 175 amino acids. In one specific example, the SKP2 fragment may contain or consist of the amino acid sequence of SEQ ID NO: 1.

[0029] Additionally, the SKP2 fragment may be composed of a sequence in which one or more amino acids of the fragment are added, deleted, or substituted, as long as it has the same binding affinity to the SKP2 fragment for the SKP1 protein or has the same gene location encoding the SKP2 protein on the chromosome. Specifically, the SKP2 protein may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity, or 100% identity, with the amino acid sequence of SEQ ID NO: 1.

[0030] The term "SKP2 fragment variant" used herein refers to a form in which some of the amino acids of the SKP2 fragment described above are substituted. That is, the SKP2 fragment variant refers to a peptide having a different sequence from the native SKP2 fragment and having the function of binding to SKP1. At this time, the SKP2 fragment variant can exhibit binding affinity for binding to SKP1 that is about 10 times to about 650 times improved compared to the native SKP2 fragment. At this time, "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule and its binding partner.

[0031] Here, the binding affinity to SKP1 can be measured by methods known to those skilled in the art.

[0032] Specifically, the SKP2 fragment variant may be one in which 7 to 14 amino acids are substituted in the native SKP2 fragment.

[0033] More specifically, the SKP2 fragment variant may be one in which any one amino acid selected from the group consisting of the 2nd, 4th, 11th, 12th, 19th, 24th, 29th, 33rd, 44th, 59th, 78th, 98th, 102nd, 107th, 111th, 112th, 113th, 124th, 128th, 130th, 133rd, 135th, 137th, 138th, 139th, 148th, 156th, 161st, 176th, and combinations thereof in the amino acid sequence of SEQ ID NO: 1 is substituted.

[0034] More specifically, the variant may be one in which any one amino acid selected from the group consisting of H2, K4, D11, L12, S19, W24, T29, L33, E44, L59, D78, D98, D102, G107, C111, L112, C113, C124, Y128, L130, D133, S135, W137, Q138, T139, H148, L156, I161, E176 and combinations thereof in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0035] At this time, the "amino acid" introduced by the above substitution may be any one selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, and proline.

[0036] More specifically, the variant may be substituted with an amino acid selected from the group consisting of H2R, K4R, D11G, L12P, S19G, W24R, T29A, L33P, E44K, L59P, D78G, D98V, D102N, G107R, C111R, L112P, L112R, C113R, C124S, Y128C, L130Q, D133E, S135P, W137R, Q138K, T139S, H148R, L156Q, L156R, I161A, I161V, E176G and combinations thereof in the amino acid sequence of SEQ ID NO: 1. In this case, when L112 is substituted, it may be substituted with L112P or L112R. When L156 is substituted, it can be substituted with L156Q or L156R. When I161 is substituted, it can be substituted with I161A or I161V.

[0037] As a specific example, the SKP2 fragment variant may comprise or consist of any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 8.

[0038] fusion protein

[0039] Another aspect of the present invention provides a fusion protein comprising an SKP2 fragment or a variant thereof and a target protein binding site. The SKP2 fragment and the variant thereof are the same as described above.

[0040] As used herein, the term "target protein" means a protein that is specifically expressed in a target (purpose) cell, tissue, or disease environment or that can cause a disease.

[0041] The target protein may be a protein present on the cell surface or within the cell. For example, the target protein may include a structural protein, a receptor, an enzyme, a cell surface protein, etc. The target protein can be, for example, any peptide or small molecule that binds to: FoxOl, HDAC, DP-1, E2F, ABL, AMPK, BRK, BRSK I, BRSK2, BTK, CAMKK1, CAMKK alpha, CAMKK beta, Rb, Suv39HI, SCF, p19INK4D, GSK-3, pi8 INK4, myc, cyclin E, CDK2, CDK9, CDG4 / 6, cyclin D, pl6 INK4A, cdc25A, BMI1, SCF, Akt, CHKl / 2, C 1 delta, CK1 gamma, C2, CLK2, CSK, DDR2, DYRK1A / 2 / 3, EF2K, EPH-A2 / A4 / B 1 / B2 / B3 / B4, EIF2A 3, Smad2, Smad3, Smad4, Smad7, p53, p21 Cipl, PAX, Fyn, CAS, C3G, SOS, Tal, Raptor, RACK-1, CRK, Rapl, Rac, KRas, NRas, HRas, GRB2, FAK, PI3K, spred, Spry, mTOR, MPK, LKBl, PAK 1 / 2 / 4 / 5 / 6, PDGFRA, PYK2, Src, SRPK1, PLC, PKC, PKA, PKB alpha / beta, PKC alpha / gamma / zeta, PKD, PLKl, PRAK, PRK2, WAVE-2, TSC2, DAPKI, BAD, IMP, C-TAK1, TAKl, TAOl, TBK1, TESK1, TGFBR1, TIE2, TLK1, TrkA, TSSK1, TTBK1 / 2, TTK, Tpl2 / cotl, MEK1, MEK2, PLDL Erk1, Erk2, Erk5, Erk8, p90RSK, PEA-15, SRF, p27 KIP1, TIF la, HMGN1, ER81, MKP-3, c-Fos, FGF-R1, GCK, GSK3 beta, HER4, HIPK1 / 2 / 3 / ,IGF-1R, cdc25, UBF, LAMTOR2, Statl, Stat3, StaO, CREB, JAK, Src, PTEN, NF-kappa B, HECTH9, Bax, HSP70, HSP90, Apaf-1, Cyto c, BCL-2, Bcl-xL, Smac, XIAP, Caspase-9, Caspase-3, Caspase-6, Caspase-7, CDC37, TAB, IKK, TRADD, TRAF2, R1P1, FLIP, TAKl, JNKl / 2 / 3, Lck, A-Raf, B-Raf, C-Raf, MOS, MLKl / 3, MN 1 / 2, MSKl, MST2 / 3 / 4, MPSK1, MEKKI, ME K4, MEL, ASK1, MINK1, MKK 1 / 2 / 3 / 4 / 6 / 7, NE 2a / 6 / 7, NUAK1, OSR1, SAP, STK33, Syk, Lyn, PDK1, PHK, PIM 1 / 2 / 3, Ataxin-1, mTORCl, MDM2, p21 Wafl, cyclin Dl, Lamln A, Tpl2, Myc, catenin, Wnt, IKK-beta, IKK-gamma, IKK-alpha, IKK-epsilon, ELK, p65RelA, IRAKI, IRA 2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK 1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNK l, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 K3, p38 alpha / beta / delta / gamma MAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK 1 / 2 / 3 / 4, Mucl, SHC, CXCR4, Gap-1, Myc, beta-catenin / TCF, Cbl, BRM, Mcl-1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, ERct, and variants thereof.

[0042] As used herein, the term "target protein binding site" refers to a site that binds to a target protein. Specifically, it may be a ligand (peptide), antibody, partner binding protein, etc. that binds to a target protein. The antibody may include an antibody fragment. The antibody fragment may include a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single-chain Fv protein (scFv), a bis-scFv (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb, nanobody), a heavy chain-only antibody (e.g., camelid VHH, camelid nanobody, shark Ig NAR), an affibody, an intrabody, etc. Specifically, it may be a single-domain antibody.

[0043] As used herein, the term "single domain antibody (sdAb)" refers to a nanobody, an antibody fragment composed of a single variable region fragment of an antibody. While sdAbs derived primarily from the heavy chain are used, single variable region fragments derived from the light chain have also been reported to specifically bind to antigens.

[0044] In one specific example, the target binding protein may be a nanobody that binds to Stat3. Specifically, the target binding protein may comprise the amino acid sequence of SEQ ID NO: 65.

[0045] The SKP2 fragment or variant thereof and the target protein binding site may be connected via a linker. In this case, the linker may be a peptide linker.

[0046] Specifically, the above fusion protein may be composed of the following structural formula (I) or (II).

[0047] N'-TB-(L)n-SR-C' (I)

[0048] N'-SR-(L)n-TB-C' (II)

[0049] At this time, in the structural formulas (I) and (II),

[0050] The above N' is the N-terminus of the fusion protein,

[0051] The above C' is the C-terminus of the fusion protein,

[0052] The above TB is a target protein binding site (target binder),

[0053] The above SR is a SKP2 fragment or a variant thereof,

[0054] The above L is a peptide linker,

[0055] The above n is 0 or 1.

[0056] At this time, the target protein binding site and SKP2 fragment or variant thereof are the same as described above.

[0057] The peptide linker may be composed of 1 to 30 consecutive amino acids, or 2 to 20 consecutive amino acids, or 2 to 10 amino acids. In one specific example, the peptide linker may be (GS)n (wherein n is an integer from 1 to 10). In this case, n in (GS)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The peptide linker may use, without particular limitation, a sequence linker known in the art, such as GS, GGGGS, (GGGGS)2, ASTKGP, or ASTKGPSVFPLAP, which provides structural flexibility without being cleaved by proteolytic enzymes. In one embodiment, the peptide linker may be composed of an amino acid sequence of SEQ ID NO: 48 (GS).

[0058] Polynucleotide encoding a SKP2 fragment or a variant thereof, or a fusion protein

[0059] Another aspect of the present invention provides a polynucleotide encoding an SKP2 fragment or a variant thereof, or the fusion protein. The SKP2 fragment or variant thereof, and the fusion protein are the same as described above.

[0060] Specifically, the polynucleotide encoding the SKP2 fragment may include the base sequence of SEQ ID NO: 23. In addition, the polynucleotide encoding the variant of the SKP2 fragment may include any one base sequence selected from the group consisting of SEQ ID NOs: 24 to 30.

[0061] The polynucleotide encoding the fusion protein may comprise a base sequence encoding a target protein binding site and an SKP2 fragment or a variant thereof. In one specific example, the target protein binding site may comprise the base sequence of SEQ ID NO: 66. Accordingly, in one specific example, the polynucleotide encoding the fusion protein may comprise any one base sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 64.

[0062] Additionally, if the polynucleotide encodes the same polypeptide, one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. When producing a polynucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on solid supports.

[0063] Specifically, the polynucleotide may include a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity with the base sequences of SEQ ID NOs: 23 to 30 and 57 to 64, respectively.

[0064] vector loaded with polynucleotides

[0065] Another aspect of the present invention provides a vector loaded with a polynucleotide encoding an SKP2 fragment or a variant thereof, or the fusion protein. In this case, the polynucleotide encoding the SKP2 fragment may include the base sequence of SEQ ID NO: 23. In addition, the polynucleotide encoding the variant of the SKP2 fragment may include any one base sequence selected from the group consisting of SEQ ID NOs: 24 to 30. As a specific example of the polynucleotide encoding the fusion protein, the polynucleotide may include any one base sequence selected from the group consisting of SEQ ID NOs: 57 to 66. The SKP2 fragment or variant thereof and the fusion protein are the same as described above.

[0066] As used herein, the term "vector" refers to a nucleic acid vector that can be introduced into a host cell and recombined and integrated into the host cell genome. Alternatively, the vector is understood to be a nucleic acid vehicle containing a nucleotide sequence capable of autonomously replicating as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, mini-chromosomes, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0067] Specifically, the vector may be a plasmid DNA, phage DNA, etc., and may be a commercially developed plasmid (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (e.g., pUB110, pTP5, etc.), a yeast-derived plasmid (e.g., YEp13, YEp24, YCp50, etc.), a phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.), etc. There is. Since the protein expression amount and formula of the above vector differ depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0068] The vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Examples of sequences to be fused include FLAG (IBI, USA), glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), and 6Х His (hexahistidine; Quiagen, USA).

[0069] In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.

[0070] Transformed cells

[0071] Another aspect of the present invention provides a transformed host cell into which an expression vector comprising a polynucleotide encoding a SKP2 fragment or a variant thereof, or the fusion protein, is introduced.

[0072] As used herein, the term "transformed host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. The transformed cell can be produced by introducing the vector into a host cell and transforming it. In addition, the polynucleotide contained in the vector can be expressed to produce the SKP2 fragment of the present invention, a variant thereof, or the fusion protein.

[0073] The above transformation can be performed by various methods. It is not particularly limited thereto, as long as it can produce the SKP2 fragment of the present invention or a variant thereof, or the fusion protein. Specifically, the transformation method may be a CaCl2 precipitation method, a Hanahan method that increases efficiency by using a reducing substance called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, a calcium phosphate precipitation method, a protoplast fusion method, a stirring method using silicon carbide fibers, an Agrobacterium-mediated transformation method, a PEG-based transformation method, a dextran sulfate, lipofectamine, and a drying / inhibition-mediated transformation method. In addition, a target object can be delivered into a cell using a virus particle by infection. In addition, a vector can be introduced into a host cell by gene bombardment, etc.

[0074] In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the antibody of the present invention. Specifically, the host cell may include, but is not limited to, prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cellular origin. An example of the prokaryotic cell may be Escherichia coli. In addition, an example of the eukaryotic cell may be yeast. In addition, the mammalian cell may be CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, or HEK293T cells, but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art may be used.

[0075] Additionally, to optimize the therapeutic properties of the antibody or for other purposes, the glycosylation-related genes of the host cell can be manipulated using methods known to those skilled in the art to adjust the sugar chain pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation).

[0076] A pharmaceutical composition comprising a fusion protein, a polynucleotide encoding the fusion protein, or a vector comprising the polynucleotide

[0077] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a disease, comprising as an active ingredient a fusion protein comprising a SKP2 fragment or a variant thereof; and a target protein binding site, a polynucleotide encoding the fusion protein, or a vector comprising the polynucleotide.

[0078] The above terms, “fusion protein”, “polynucleotide encoding fusion protein” and “vector comprising polynucleotide” are the same as described above.

[0079] The polynucleotide may be DNA, mRNA, plasmid DNA, etc., and the polynucleotide or a vector containing the same may be delivered into a cell by a cell-penetrating functional nano carrier to exhibit the same target protein degradation efficacy as the fusion protein.

[0080] The above disease may be selected from the group consisting of cancer, stroke, ischemic disease, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson's disease, Alzheimer's disease, fibrosis, diabetes, ALS, pathogenic disease, inflammatory disease, arthritis, anemia, genetic disorder, hyperglycemia, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, and obesity.

[0081] The term "treatment" as used herein may be used to encompass both therapeutic and preventative treatments, and includes any application or form of medication for treating a disease in mammals, including humans. Furthermore, the term encompasses inhibiting or slowing the progression of a disease; restoring or repairing damaged or defective functions, thereby partially or completely alleviating a disease; or stimulating an ineffective process; or alleviating a serious disease. The term "prevention" may be used to encompass alleviating or reducing a pathological condition or disease in a subject.

[0082] In the present invention, the nucleic acid may be used together with various carriers such as lipid nanoparticles (LNPs), liposomes, or vesicles, which are known to effectively deliver polynucleotides into cells, but are not limited thereto.

[0083] In the present invention, the pharmaceutical composition may be administered systemically via parenteral administration. Parenteral administration may include, but is not limited to, intranasal, intranasal, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intradermal, intraperitoneal, enteral, topical, sublingual, or rectal administration.

[0084] The pharmaceutical composition may be prepared in the form of, for example, a powder, a tablet, a capsule, a liquid, an ointment, a cream, a gel, a hydrogel, an aerosol, a spray, a micellar solution, a transdermal patch, a liposomal suspension, a polyplex, an emulsion, a lipid nanoparticle (LNP) (having RNA on its surface or encapsulated therein) or any other suitable form that can be administered to a human or mammal in need of treatment.

[0085] Another aspect of the present invention provides a use of the fusion protein, a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, or a pharmaceutical composition comprising the same for the prevention or treatment of a disease.

[0086] Another aspect of the present invention provides a method for preventing or treating a disease, comprising administering to a subject the fusion protein, a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, or a pharmaceutical composition comprising the same.

[0087] The above fusion protein, the polynucleotide encoding the same, the vector containing the polynucleotide, and the pharmaceutical composition are the same as described above.

[0088] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0089] Manufacturing Example 1. Manufacturing of anti-STAT3 nanobody

[0090] The gene encoding the anti-STAT3 nanobody VHH13 (referred to as SBT-100 in US9,695,234) was synthesized (IDT Technology) and inserted into pYD5, a yeast surface expression vector constructed with reference to the paper (Wang Z et al. "A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities". Protein Eng Des Sel. (2005) 18:337-343, doi:10.1093 / protein / gzi036). Afterwards, the CDR of the plasmid was divided into five regions (H1, H2A, H2B, H3A, H3B), and primers were synthesized (IDT Technology) to induce random mutations in the regions, and PCR was performed using these primers. The final 5 ug of randomly mutated PCR product was inserted into 1 ug of pYD5 vector and transformed into EBY100 (ATCC, Cat no. MYA-4941) yeast to produce approximately 0.5 × 10 7 ~1.0×10 7 We built a library with a variety of features.

[0091] Using the above library, the ETI01_A4 nanobody with high binding affinity to STAT3 was prepared. The specific sequence of ETI01_A4 is as follows.

[0092] SEQ ID NO NAME TYPE LENGTH Sequence 65ETI01_A4PRT127aaHVQLVESGGGSVQAGGSLRLSCAASGFNSGDSCIGWFRQVPGKEREGVSVISSGGEIRYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSVFLCYPRTWFNRY MYNSWGQGTQVTVSS66ETI01_A4polynucleotideDNA381bpCATGTTCAGCTTGTAGAAAGTGGGGGAGGCTCCGTTCAAGCTGGTGGGAGCCTTCGGCTGTCTTGTGCAGCGAGCGGTTTTTAACTCGGGTGATAGT TGCATTGGTTGGTTCCGACAGGTTCCGGGTAAAGAGCGAGAGGGCGTAAGTGTGATTTCTTCCGGCGGGGAGATTAGGTATTATGCTGATAGTGTTAAGGGACGCTTTACAATCTCTCAGGATAATACCAAAAACACCCTGT ATTTGCAAATGAACTCTCTTAAGCCTGAAGATACGGCAATGTACTACTGCGCCACCTCCGTTTTTCTGTGTTACCCGAGGACGTGGTTCAATAGGTATGTATAACTCATGGGGTCAGGGGAACCCAGGTAACGGTCAGTAGC

[0093] Example 1. Construction of a primary library for SKP2 2-176 mutant proteins.

[0094] Amino acids 2 to 176 of S-phase Kinase Associated Protein-2 (Uniprot ID: Q13309) (SKP2) 2-176,The gene encoding sequence number 1) was synthesized (IDT Technology) and inserted into the yeast surface expression vector pYD5. PCR was performed using the Diversify PCR Random mutagenesis kit (Clonetech, Cat no. 630703) to induce 8.1 random mutations per 1,000 bp using the plasmid as a template. The final 2.5 ug of randomly mutated PCR products were each inserted into 0.5 ug of the pYD5 vector. EBY100 (ATCC, Cat no. MYA-4941) yeast was transformed with each of the vectors, and 6.0 × 10 6 We built a library with a variety of dog breeds.

[0095] Example 2. Binding affinity and single-clone analysis of the SKP2 2-176 mutant protein library to SKP1.

[0096] The constructed primary library was cultured at 30°C for approximately 16 h using SD-CAA medium (containing 20 g glucose (SIGMA, Cat no. G7528), 14.7 g sodium citrate (SIGMA, Cat no. C8532), 4.3 g citric acid monohydrate (SIGMA, Cat no. C0706), 6.7 g yeast nitrogen base (BD Difco, Cat no. 291940), 5 g bacto casamino acid (BD Difco, Cat no. 223050), and 100 ug / mL kanamycin (Biosesang, Cat no. KC1001-025-02) per 1 L of distilled water). After culture, the culture was transferred to SG-CAA medium at OD 600 The value Dilute to 1 and re-culture at 30℃ for 18 hours to add SKP2 to the yeast surface. 2-176 The mutant protein was induced to be expressed. 0.5×10 cultured yeast 7Primary staining was performed by mixing 500 nM of biotinylated SKP1 protein (Sino biological, Cat no. 14161-H40E-B) and mouse anti-V5 antibody (Invitrogen, Cat no. R960-25) with 0.1% BSA (BOVOGEN, Cat no. BSAS0.1) (1:500 dilution in wash buffer containing PBS pH7.4 (Biosesang, Cat no. PR2004-100-74)) and reacting at room temperature for 30 minutes. Secondary staining was performed with Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Invitrogen, Cat no. SA10044) and anti-mouse IgG(H+L)-FITC antibody (Invitrogen, Cat no. 11-4011-85) diluted in wash buffer at a ratio of 1:100 and incubated at 4°C for 20 minutes. The stained reaction product was analyzed using a flow cytometer (SONY, SH800S) for SKP2 2-176 Among the yeast populations with enhanced binding affinity for SKP1, individuals within the top 0.1% were isolated. The above process was repeated three times, and SKP2 was isolated from Round 0 to the final Round 3. 2-176 The binding affinity of the mutant protein library to SKP1 was confirmed (Fig. 1a).

[0097] After the final round, single clones from the isolated yeast population were analyzed using the same method as above under conditions of a biotinylated SKP1 protein concentration of 250 nM (Figs. 1b to 1d). Among the single clones, clones '3' (SEQ ID NO: 2) and '15' (SEQ ID NO: 3) with high binding affinity to SKP1 were selected, and secondary random mutations were generated using the sequences of these two types of clones.

[0098] Example 3. Construction of a secondary library for SKP2 2-176 mutant proteins.

[0099] PCR was performed using the plasmids in which the sequences of clone '3' and clone '15' selected through the method of Example 2 were inserted as templates, respectively, inducing 3.5 and 5.8 mutations per 1,000 bp in the same manner as Example 1. The final 2.5 ug of randomly mutated PCR products were inserted into 0.5 ug of pYD5 vector, and EBY100 yeast was transformed with each vector to obtain 7.0×10 6 9.0×10 6 We built four libraries with a variety of features.

[0100] Example 4. Binding affinity and single-clone analysis of SKP2 2-176_3 and SKP22-176_15 mutant libraries to SKP1.

[0101] The constructed library was cultured in the same manner as in Example 2 above, and SKP2 was added to the yeast surface. 2-176 The mutant protein was induced to be expressed. Cultured yeast (0.5 × 10 7 The primary staining was performed by mixing 5 nM biotinylated SKP1 and mouse anti-V5 antibody diluted in wash buffer at a ratio of 1:500 and reacting at room temperature for 30 minutes. The secondary staining was performed by reacting Streptavidin, R-Phycoerythrin Conjugate (SAPE) and anti-mouse IgG(H+L)-FITC antibody diluted in wash buffer at a ratio of 1:100 and reacting in the dark at 4°C for 20 minutes. The reaction product was analyzed by flow cytometry to determine the binding affinity for SKP1 compared to SKP2. 2-176 _3 clones and SKP2 2-176 _15 Individuals within the top 0.1% of the improved yeast population compared to clones were isolated. The above process was repeated three times, and SKP2 was used from Round 0 to the final Round 3. 2-176 We confirmed changes in binding affinity of the mutant protein library to SKP1 (Fig. 2a and Fig. 2b).

[0102] After the last round, the yeast individual clones separated were analyzed using the above method, and among the clones with high binding affinity, clones E1 (SEQ ID NO: 4), E2 (SEQ ID NO: 5), E4 (SEQ ID NO: 6), E5 (SEQ ID NO: 7), and E6 (SEQ ID NO: 8) were finally selected (Fig. 2c), and the sequences of each of the clones are shown in Fig. 3.

[0103] Example 5. Analysis of binding affinity of selected SKP2 2-176 mutant proteins to SKP1.

[0104] SKP2 2-176 And to compare the binding affinity of each mutant, EC was measured using a flow cytometer. 50 was measured. Six final mutants (E1, E2, E4, E5, and E6) were induced to be expressed on the yeast surface using the same method as in Example 2. 0.5×10 cultured yeast 7 Primary staining was performed by mixing the mouse anti-V5 antibody at a ratio of 1:500 and 16 concentrations of biotinylated SKP1 serially diluted by 1 / 2 from 0 nM to 1000 nM and reacting at room temperature for 30 minutes. Secondary staining was performed by diluting Streptavidin, R-Phycoerythrin Conjugate (SAPE) and anti-mouse IgG(H+L)-FITC antibody in wash buffer at a ratio of 1:100 and reacting in the dark at 4°C for 20 minutes. The reaction products were measured for the binding patterns of the final clones to biotinylated SKP1 at different concentrations using a flow cytometer (SONY, SH800S) and the results were analyzed with the flow-jo program to determine the EC 50 was derived (Fig. 4a to Fig. 4c).

[0105] Manufacturing Example 2. Production of an expression vector to confirm target protein degradation ability.

[0106] SKP2 2-176To evaluate the target protein degradation ability of SKP2 and its variants, we referred to the previous paper ("The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications" Nat. Commun. 10:4403 (2019)). Specifically, SKP2 2-176 Or, a mutant thereof, and an expression vector of a fusion protein (SKP2.GS.NbALFA) in which an anti-ALFA tag nanobody (NbALFA) is linked via a GS linker (GS, SEQ ID NO: 48) and a plasmid in which an ALFA tag is conjugated to STAT3 (RefSeq. NM_139276.2, 2358 bp) were constructed.

[0107] More specifically, the pCMV6 (Origene, Cat no. PS100001) vector for expression in mammalian cells was treated with a restriction enzyme, and then 'Flag tag-SKP2' containing a Flag tag in the direction from the N-terminus to the C-terminus of the cleaved site was constructed. 2-176 .GS.NbALFA' (SEQ ID NO: 9) and 'Flag tag-SKP2 2-176 Polynucleotides encoding mutants (E1, E2, E4, E5, E6).GS.NbALFA' (SEQ ID NO: 10 to SEQ ID NO: 14) were each inserted. The STAT3 expression vector (ALFA-Myc tag.STAT3, SEQ ID NO: 15) was constructed by adding ALFA tag and Myc tag to the N terminus using pCMV3_STAT3 (Sinobiological., Cat no. HG10034-NM, SEQ ID NO: 16).

[0108] 이이번호이름아미노산 이이9Flag tag-SKP2(2-176) WQTLDLTGKNLHPDVTGRLLSQGVIAFRCPRSFMDQPLAEGSEVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYCHVLEDRVDSFHDYWGQGTQVTVSS10Flag tag-E1.GS.NbALFADYKDDDDKTRRRRHLQEIPGLSSNVATSFTWGRDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNPGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLRIFSRLPELLKVSGVSKRWYRLASDESLWKSL DLTGKNLHPDVTGRLLSQGVVAFRCPRSFMDQPLAGGSEVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYCHVLEDRVDSFHDYWGQGTQVTVSS11Flag tag-E2.GS.NbALFADYKDDDDKTTRRRRHLQEIPGLSSNVATSFTWGRDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNPGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLRIFSRLRLPELLKVSGVSKRWCRLASDESLWKTLDLTGK NLHPDVTGRLLSQGVAAFRCPRSFMDQPLAGGSEVQLQESGGGLVQPGGSLRLSCTASGVTISANLAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYCHVLEDRVDSFHDYWGQGTQVTVSS12Flag tag-E4.GS.NbALFADYKDDDDKTRHRKHLQEIPDPSSNVATSFTWGWDSSKTSELPSGMGVSALEKEEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKGFVIVRRPKLNRENFPGVSWVSLPDELLLGIFSCPCLPELLKVSGVCKRWCRLASDEPLWQTLDLTGKNLRPDVTGRLRRSQGVIAFRCPRSFMDQPLAEGSEVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS13Flag tag-E5.GS.NbALFADYKDDDDKTRHRKHLQEIPDPSSNVATSFTWGWDSSKTSELPSGMGVSALEKKEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKGFVIVRRPKLNRENFPGVSWVSLPDELLLLGIFSCPPLELLKVSGVCKRWCRLASDEPLRQTL DLTGKNLRPDVTGRLLSQGVIAFRCPRSFMDQPLAEGSEVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAvyYCHVLEDRVDSFHDYWGQGTQVTVSS14Flag tag-E6.GS.NbALFADYKDDDDKTRHRKHLQEIPDPSSNVATGFTWGWDSSKASELPSGMGVSALEKEEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKGFVIVRRPKLNRENFPGVSWVSLPNELLLGIFSCRCLPELLKVSGVCKRWCRQASEEPLWQTLDLT GKNLRPDVTGRLQSQGVIAFRCPRSFMDQPLAEGSEVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS.

[0109] In addition, the anti-STAT3 nanobody 'ETI01_A4' was conjugated to SKP2 via the 'GS' linker in the same manner as above. 2-176 and its mutants, respectively, were constructed. Specifically, after treating the pCMV6 vector with a restriction enzyme, 'SKP2' containing a Flag tag in the direction from the N-terminus to the C-terminus of the cleaved site was constructed. 2-176 .GS.ETI01_A4' (SEQ ID NO: 17) and 'SKP2 2-176 Each polynucleotide encoding mutants (E1, E2, E4, E5).GS.ETI01_A4' (SEQ ID NO: 18 to SEQ ID NO: 21) was inserted and produced.

[0110] 서열번호이름아미노산 서열17Flag tag-SKP2(2-176).GS.ETI01_A4DYKDDDDKTRHRKHLQEIPDLSSNVATSFTWGWDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLGIFSCLCLPELLKVSGVCKRWYRLASDESLWQTLDLTGKNLHPDVTGRLLSQGVIAFRCPRSFMDQPLAEGSHVQLVESGGGSVQAGGSLRLSCAASGFNSGDSCIGWFRQVPGKEREGVSVISSGGEIRYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSVFLCYPRTWFNRYMYNSWGQGTQVTVSS18Flag tag-E1.GS.ETI01_A4DYKDDDDKTRRRRHLQEIPGLSSNVATSFTWGRDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNPGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLRIFSRLRLPELLKVSGVSKRWYRLASDESLWKSLDLTGKNLHPDVTGRLLSQGVVAFRCPRSFMDQPLAGGSHVQLVESGGGSVQAGGSLRLSCAASGFNSGDSCIGWFRQVPGKEREGVSVISSGGEIRYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSVFLCYPRTWFNRYMYNSWGQGTQVTVSS19Flag tag-E2.GS.ETI01_A4DYKDDDDKTRRRRHLQEIPGLSSNVATSFTWGRDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNPGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLRIFSRLRLPELLKVSGVSKRWCRLASDESLWKTLDLTGKNLHPDVTGRLLSQGVAAFRCPRSFMDQPLAGGSHVQLVESGGGSVQAGGSLRLSCAASGFNSGDSCIGWFRQVPGKEREGVSVISSGGEIRYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSVFLCYPRTWFNRYMYNSWGQGTQVTVSS20Flag tag-E4.GS.ETI01_A4DYKDDDDKTRHRKHLQEIPDPSSNVATSFTWGWDSSKTSELPSGMGVSALEKEEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKGFVIVRRPKLNRENFPGVSWVSLPDELLLGIFSCPCLPELLKVSGVCKRWCRLASDEPLWQTLDLTGKNLRPDVTGRLRSQGVIAFRCPRSFMDQPLAEGSHVQLVESGGGSVQAGGSLRLSCAASGFNSGDSCIGWFRQVPGKEREGVSVISSGGEIRYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSVFLCYPRTWFNRYMYNSWGQGTQVTVSS21Flag tag-E5.GS.ETI01_A4DYKDDDDKTRHRKHLQEIPDPSSNVATSFTWGWDSSKTSELPSGMGVSALEKKEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKGFVIVRRPKLNRENFPGVSWVSLPDELLLGIFSCPCLPELLKVSGVCKRWCRLASDEPLRQTLDLTGKNLRPDVTGRLLSQGVIAFRCPRSFMDQPLAEGSHVQLVESGGGSVQAGGSLRLSCAASGFNSGDSCIGWFRQVPGKEREGVSVISSGGEIRYYADSVKGRFTISQDNTKNTLYLQMNSLKPEDTAMYYCATSVFLCYPRTWFNRYMYNSWGQGTQVTVSS.

[0111] Example 6. Confirmation of the target protein degradation ability of a fusion protein comprising an anti-ALFA tag nanobody and the SKP2 2-176 variant.

[0112] Anti-ALFA tag nanobody (NbALFA) and SKP2 produced by the method of Manufacturing Example 2 above 2-176 The target protein degradation ability of fusion proteins containing wild-type (WT) fragments or mutant proteins was confirmed.

[0113] Specifically, the expression vector was transfected into human kidney cell line HEK293 cells using Lipofectamine 3000 (Invitrogen). 24 hours after transfection, the cells were washed twice with cold PBS buffer (Gibco, 10010-023), and then treated with cell lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS) containing a protease inhibitor cocktail (Thermofisher, 78440) to obtain cell extracts. The cell extracts were homogenously lysed using a vortex and heated at 95°C for 10 minutes. The protein amount of the cell extracts was quantified using a BCA kit (ThermoFisher, 23227), and 20 μg of protein was electrophoresed on a 4-12% SDS PAGE gel (Invitrogen, NW04125BOX). The separated proteins were transferred to a PVDF membrane (Invitrogen, IB24002) and blocked for 30 minutes in a blocking buffer [1×PBS-T buffer solution (1×PBS, 0.1% Tween 20) containing 5% Skim milk (BD, 232100). Then, the membrane was treated with primary antibodies and reacted at 4°C for 16 hours. At this time, the primary antibodies were anti-ALFA antibody (Nanotag, N1583, 1:5000), anti-α-tubulin (GeneTex, GTX628802, 1:10000), and anti-FLAG antibody (Sigma, F1804, 1:2500), each diluted in the blocking buffer. After the primary antibody reaction, the membrane was washed three times for 5 minutes each with 1×PBS-T. Then, After treating the membrane with secondary antibodies, the membrane was incubated at room temperature for 1 hour. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (CST, 7074) or anti-mouse antibodies (CST, 7076), diluted in blocking buffer. After secondary antibody reaction, the membrane was washed three times with 1× PBS-T for 5 minutes each.The expression level of each protein was confirmed using an Imager (Invitrogen, iBright, CL1500) after treatment with ECL (Cytiva, RPN2232 or Thermofisher, 34096) solution.

[0114] As a result, as shown in Fig. 5, SKP2 compared to the control group (mock) 2-176 We confirmed that the expression of ALFA-Myc tag.STAT3 protein was reduced in cell groups expressing fusion proteins including WT or its mutants. In particular, SKP2 2-176 Among the mutants, fusion proteins containing E4, E5, or E6 exhibited superior target resolution compared to the WT. The above experiment was repeated three times, and the results were statistically analyzed using a one-way ANOVA method. As a result, it was confirmed that the fusion proteins containing the E4 mutant showed a statistically significant increase in STAT3 resolution by approximately 30% compared to the WT.

[0115] Example 7. Confirmation of STAT3 protein degradation ability of fusion proteins including anti-STAT3 nanobody and SKP2 2-176 or its variants

[0116] Anti-STAT3 nanobody (ETI01_A4) and SKP2 produced by the method of Manufacturing Example 2 above 2-176 The target protein degradation ability of a fusion protein comprising a wild-type (WT) fragment or a mutant thereof was confirmed. The target protein degradation was performed using the same method as in Example 6. At this time, an anti-c-Myc (tag) antibody was additionally used as the primary antibody.

[0117] As a result, as shown in Fig. 6, SKP2 compared to the control group (mock) 2-176 We confirmed that the expression of STAT3 protein was reduced in cell groups expressing fusion proteins including WT or its mutants. In particular, compared to WT, SKP2 2-176In cell groups expressing fusion proteins containing mutants, STAT3 protein degradation was further increased. The above experiment was repeated three times, and the results were statistically analyzed using one-way ANOVA. As a result, it was confirmed that fusion proteins containing E2 or E4 mutants showed a statistically significant increase in STAT3 degradation by approximately 50-70% compared to WT.

[0118] Through the above results, it was confirmed that STAT3 was degraded more efficiently by the SKP2 mutant than by the native SKP2 fragment, thereby confirming that the SKP2 mutant binds to SKP1 more strongly than the native SKP2 fragment.

Claims

1. SKP2 fragment or variant thereof.

2. In paragraph 1, An SKP2 fragment or variant thereof, wherein the SKP2 fragment or variant thereof comprises a binding site for SKP1.

3. In paragraph 1, The SKP2 fragment or a variant thereof, wherein the SKP2 fragment comprises the amino acid sequence of sequence number 1.

4. In paragraph 1, The above SKP2 fragment mutant is an SKP2 fragment or a mutant thereof, wherein 7 to 14 amino acids are substituted in the SKP2 fragment.

5. In paragraph 1, The above variant is an SKP2 fragment or a variant thereof, wherein any one amino acid selected from the group consisting of the 2nd, 4th, 11th, 12th, 19th, 24th, 29th, 33rd, 44th, 59th, 78th, 98th, 102nd, 107th, 111th, 112th, 113th, 124th, 128th, 130th, 133rd, 135th, 137th, 138th, 139th, 148th, 156th, 161st, 176th, and combinations thereof in the amino acid sequence of SEQ ID NO: 1 is substituted.

6. In paragraph 5, The above variant is an SKP2 fragment or a variant thereof, wherein any one amino acid selected from the group consisting of H2, K4, D11, L12, S19, W24, T29, L33, E44, L59, D78, D98, D102, G107, C111, L112, C113, C124, Y128, L130, D133, S135, W137, Q138, T139, H148, L156, I161, E176 and combinations thereof is substituted in the amino acid sequence of SEQ ID NO:

1.

7. In paragraph 6, The above variant is an SKP2 fragment or a variant thereof, wherein the variant is substituted with any one amino acid selected from the group consisting of H2R, K4R, D11G, L12P, S19G, W24R, T29A, L33P, E44K, L59P, D78G, D98V, D102N, G107R, C111R, L112P, L112R, C113R, C124S, Y128C, L130Q, D133E, S135P, W137R, Q138K, T139S, H148R, L156Q, L156R, I161A, I161V, E176G and combinations thereof in the amino acid sequence of SEQ ID NO:

1.

8. In paragraph 7, An SKP2 fragment or a variant thereof, wherein the variant comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

8.

9. In paragraph 1, The above variant is an SKP2 fragment or a variant thereof, wherein the binding ability to SKP1 is improved by 10 to 650 times compared to the SKP2 fragment.

10. A fusion protein comprising a fragment of SKP2 or a variant thereof; and a target protein binding site.

11. In paragraph 10, A fusion protein, wherein the SKP2 fragment or a variant thereof; and a target protein binding site are connected via a linker.

12. In paragraph 11, The above fusion protein is a fusion protein consisting of the following structural formula (I) or (II); N'-TB-(L)n-SR-C' (I) N'-SR-(L)n-TB-C' (II) At this time, in the structural formulas (I) and (II), The above N' is the N-terminus of the fusion protein, The above C' is the C-terminus of the fusion protein, The above TB is a target protein binding site (target binder), The above SR is a SKP2 fragment or a variant thereof, wherein L is a peptide linker, The above n is 0 or 1.

13. A polynucleotide encoding the SKP2 fragment of paragraph 1 or a variant thereof.

14. A polynucleotide encoding the fusion protein of clause 10.

15. A vector loaded with the polynucleotide of clause 13.

16. A vector loaded with the polynucleotide of clause 14.

17. A host cell transformed with the vector of claim 15 or 16.

18. A pharmaceutical composition for preventing or treating a disease, comprising a fragment of SKP2 or a variant thereof; and a fusion protein comprising a target protein binding site as an active ingredient.

19. A pharmaceutical composition for the prevention or treatment of a disease, comprising the polynucleotide of clause 14 or the vector of clause 16 as an active ingredient.

20. In paragraph 18 or 19, A pharmaceutical composition for preventing or treating a disease, wherein the disease is selected from the group consisting of cancer, stroke, ischemic disease, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson's disease, Alzheimer's disease, fibrosis, diabetes, ALS, pathogenic disease, inflammatory disease, arthritis, anemia, genetic disorder, hyperglycemia, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, and obesity.

21. Use of the fusion protein of clause 10, the polynucleotide of clause 14, or the vector of clause 16 for the prevention or treatment of a disease.

22. A method for preventing or treating a disease, comprising a step of administering to a subject the fusion protein of clause 10, the polynucleotide of clause 14, or the vector of clause 16.

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