βtrcp fragment and variant thereof, and use thereof

Abstract

The present invention relates to a βTrCP fragment and a variant thereof, and a use thereof. The βTrCP fragment variant according to the present invention has increased binding ability to s-phase kinase associated protein 1 (SKP1) as compared to wild-type βTrCP fragments. Therefore, it is expected that the βTrCP fragment variant of the present invention can be used to develop PROTAC drugs having improved therapeutic effects.

Description

βTRCP fragments and variants thereof, and uses thereof

[0001] The present invention relates to βTrCP 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 the βTrCP fragment, 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 βTrCP fragment or a variant thereof, and a fusion protein comprising the βTrCP fragment or variant thereof; and a target binding site.

[0008] Another aspect of the present invention provides a polynucleotide encoding the βTrCP fragment or a variant thereof, 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 mutant βTrCP fragment according to the present invention has increased binding affinity to SKP1 (S-phase kinase associated protein 1) compared to the native βTrCP fragment. Therefore, the mutant βTrCP fragment of the present invention is expected to be useful in developing protactic drugs with improved therapeutic effects.

[0013] Figures 1a to 1d are 1st βTrCP 2-263 This diagram shows the results of confirming the binding of a mutant protein library to SKP1.

[0014] Figures 2a to 2c are secondary βTrCP 2-263 This diagram shows the results of confirming the binding of a mutant protein library to SKP1.

[0015] Figure 3 is the first βTrCP 2-263Mutant protein library (clone 7, clone 9) and secondary βTrCP 2-263 This is a diagram showing the amino acid sequences of mutants selected from the mutant protein library (clones E1 to E6).

[0016] Figures 4a and 4b show secondary βTrCP under SKP1 concentration-dependent conditions. 2-263 This graph shows the results of confirming the binding affinity of variants selected from the mutant protein library (clones E1 to E6) to SKP1.

[0017] Figure 4c is a second βTrCP 2-263 Binding affinity (EC) of variants selected from the mutant protein library (clones E1 to E6) to SKP1 50 ) is a drawing showing the same.

[0018] βTrCP fragment and its variants

[0019] One aspect of the present invention provides βTrCP fragments and variants thereof.

[0020] As used herein, the term "βTrCP" is also known as F-box / WD repeat-containing protein 1A (FBXW1A), SCF βTrCP It is an F-box protein that constitutes the ubiquitin ligase (E3 ligase) complex. The βTrCP protein contains an F-box domain at the N terminus and a WD40 repeat domain at the C terminus.

[0021] F-box proteins are classified into three groups: Fbxws, which contain a WD40 repeat domain; Fbxls, which contains 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.

[0022] The above βTrCP protein is an F-box protein belonging to Fbxws, and binds to CDC25A, eIF4A, DEPTOR, IκB, and β-catenin to regulate their degradation.

[0023] The above βTrCP protein may preferably be, but is not limited to, human βTrCP protein. The amino acid sequence and polynucleotide sequence of the βTrCP protein can be obtained from known databases such as GenBank of the National Institutes of Health (NCBI) of the United States, and preferably includes the amino acid sequence of SEQ ID NO: 19.

[0024] 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 βTrCP protein. The βTrCP 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: 19. Here, binding to SKP1 can be measured by a method known to those skilled in the art.

[0025] The term "βTrCP fragment" as used herein refers to a fragment in which a portion of the N-terminus and / or C-terminus of the native βTrCP protein is deleted (truncated), and may exhibit the same binding affinity to SKP1 as the native βTrCP 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.

[0026] 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.

[0027] More specifically, the βTrCP fragment comprises 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202 amino acids sequentially from the second amino acid from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 19. 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222. 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242. It may comprise an amino acid sequence of 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 or 262. In one specific example, the βTrCP fragment may comprise or consist of the amino acid sequence of SEQ ID NO: 1.

[0028] In addition, the βTrCP 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 as the βTrCP fragment for the SKP1 protein or the gene location encoding the βTrCP protein on the chromosome is the same. Specifically, the βTrCP 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.

[0029] The term "βTrCP fragment variant" used herein refers to a form in which some of the amino acids of the above-described βTrCP fragment are substituted. That is, the βTrCP fragment variant refers to a peptide having a different sequence from the native βTrCP fragment and having the function of binding to SKP1. At this time, the βTrCP fragment variant can exhibit binding affinity for binding to SKP1 that is about 10 to about 120 times improved compared to the native βTrCP 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.

[0030] Here, the binding affinity to SKP1 can be measured by methods known to those skilled in the art.

[0031] Specifically, the βTrCP fragment variant may be one in which 5 to 18 amino acids are substituted in the native βTrCP fragment.

[0032] More specifically, the βTrCP fragment variant has the 8th, 10th, 11th, 16th, 18th, 28th, 33rd, 35th, 38th, 47th, 91st, 95th, 98th, 102nd, 105th, 110th, 114th, 115th, 120th, 122nd, 124th, 125th, 127th, 132nd, 144th, 146th, 148th, 159th, 170th, 172nd, 173rd, 183rd, 185th, 192nd, 197th, 198th, 202nd, 203rd, 204th, in the amino acid sequence of SEQ ID NO: 1. Any one amino acid selected from the group consisting of the 225th, 234th, 236th, 240th, 241st, 242nd, 250th, 251st, 260th, and combinations thereof may be substituted.

[0033] More specifically, the variants are L8, E10, K11, M16, S18, E28, I33, P35, N38, C47, S91, E95, K98, V102, F105, E110, V114, E115, L120, S122, M124, C125, Y127, I132, F144, T146, L148, N159, C170, A172, E173, T183, D185, L192, V197, R198, L202, W203, R204, N225, A234, Y236, I240, Q241, D242, W250, R251, I260 and their amino acid sequences in SEQ ID NO: 1. Any one amino acid selected from the group consisting of combinations may be substituted for another amino acid.

[0034] 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.

[0035] More specifically, the variants are L8P, E10V, K11E, M16L, S18T, E28G, I33T, P35A, N38D, C47S, S91T, E95G, K98R, V102A, F105L, E110G, V114A, E115G, L120P, S122P, M124V, C125R, Y127C, I132T, F144S, T146P, L148P, N159K, C170R, A172V, E173V, T183S, D185G, D185E, L192P, V197A, R198G, L202Q, L202R, W203R in the amino acid sequence of SEQ ID NO: 1. It can be substituted with any one amino acid selected from the group consisting of R204G, N225Y, A234V, Y236C, I240M, Q241R, D242E, W250R, R251G, I260S, and combinations thereof. In this case, when D185 is substituted, it can be substituted with D185G or D185E. In addition, L202 can be substituted with L202Q or L202R.

[0036] As a specific example, the βTrCP 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: 9.

[0037] fusion protein

[0038] Another aspect of the present invention provides a fusion protein comprising a βTrCP fragment or a variant thereof and a target protein binding site. The βTrCP fragment and the variant thereof are the same as described above.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The above βTrCP 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.

[0044] Specifically, the above fusion protein may be composed of the following structural formula (I) or (II).

[0045] N'-TB-(L)n-SR-C' (I)

[0046] N'-SR-(L)n-TB-C' (II)

[0047] At this time, in the structural formulas (I) and (II),

[0048] The above N' is the N-terminus of the fusion protein,

[0049] The above C' is the C-terminus of the fusion protein,

[0050] The above TB is a target protein binding site (target binder),

[0051] The above SR is a βTrCP fragment or a variant thereof,

[0052] The above L is a peptide linker,

[0053] The above n is 0 or 1.

[0054] At this time, the target protein binding site and the βTrCP fragment or variant thereof are the same as described above.

[0055] 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 specific example, the peptide linker may include an amino acid sequence of SEQ ID NO: 20 (amino acid sequence: GS).

[0056] Polynucleotide encoding a βTrCP fragment or a variant thereof, or a fusion protein

[0057] Another aspect of the present invention provides a polynucleotide encoding a βTrCP fragment or a variant thereof, or the fusion protein. The βTrCP fragment or variant thereof, and the fusion protein are the same as described above.

[0058] Specifically, the polynucleotide encoding the βTrCP fragment may include the base sequence of SEQ ID NO: 10. In addition, the polynucleotide encoding the variant of the βTrCP fragment may include any one base sequence selected from the group consisting of SEQ ID NOs: 11 to 18.

[0059] The polynucleotide encoding the above fusion protein may include a base sequence encoding a target protein binding site and a βTrCP fragment or a variant thereof.

[0060] 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.

[0061] 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 to each of SEQ ID NOs. 11 to 18 and the base sequence encoding the fusion protein.

[0062] vector loaded with polynucleotides

[0063] Another aspect of the present invention provides a vector loaded with a polynucleotide encoding a βTrCP fragment or a variant thereof, or the fusion protein. In one specific example, the polynucleotide encoding the βTrCP fragment may comprise the base sequence of SEQ ID NO: 10. In one specific example, the polynucleotide encoding the variant of the βTrCP fragment may comprise any one base sequence selected from the group consisting of SEQ ID NOs: 11 to 18. The βTrCP fragment or variant thereof and the fusion protein are the same as described above.

[0064] 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.

[0065] Specifically, the vector may be a plasmid DNA, a phage DNA, etc., and may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.), etc. Since the above vector exhibits different protein expression levels and expression patterns depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0066] 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).

[0067] 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.

[0068] Transformed cells

[0069] Another aspect of the present invention provides a transformed host cell into which an expression vector comprising a βTrCP fragment or a variant thereof, or a polynucleotide encoding the fusion protein is introduced.

[0070] 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 βTrCP fragment of the present invention, a variant thereof, or the fusion protein.

[0071] The above transformation can be performed by various methods. It is not particularly limited thereto, as long as it can produce the βTrCP 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 transformation method using PEG, 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 means of infection. In addition, a vector can be introduced into a host cell by gene bombardment, etc.

[0072] 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.

[0073] 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).

[0074] A pharmaceutical composition comprising a fusion protein, a polynucleotide encoding the fusion protein, or a vector comprising the polynucleotide

[0075] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a disease, comprising as an active ingredient a βTrCP fragment or a variant thereof; and a fusion protein comprising a target protein binding site, a polynucleotide encoding the same, or a vector comprising the polynucleotide.

[0076] The above “fusion protein”, “polynucleotide encoding the fusion protein” and “vector comprising the polynucleotide” are the same as described above.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] Another aspect of the present invention provides a use of the fusion protein, a polynucleotide encoding the same, a vector comprising the polynucleotide, or a pharmaceutical composition comprising the same for the prevention or treatment of a disease.

[0084] Another aspect of the present invention provides a use 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.

[0085] The above fusion protein, the polynucleotide encoding the same, the vector containing the polynucleotide, and the pharmaceutical composition are the same as described above.

[0086] 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.

[0087] Example 1. Construction of a primary library for βTrCP2-263 mutant proteins.

[0088] Amino acids 2-263 of βTrCP (Uniprot ID: Q9Y297) 2-263, The gene encoding sequence number 1) 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). At this time, βTrCP 2-263 The c-Myc tag (SEQ ID NO: 21) was linked to the C-terminus via a linker (SEQ ID NO: 20, GS). PCR was performed using the Diversify PCR Random mutagenesis kit (Clontech, Cat no. 630703) to induce 8.1 random mutations per 1,000 bp using the plasmid as a template.

[0089] 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 inoculate 2.74 × 10 7 We built a library with a variety of features.

[0090] Example 2. Binding affinity and monoclonal analysis of a βTrCP2-263 mutant protein library to SKP1.

[0091] The primary library constructed by the method of Example 1 was cultured at 30°C for about 16 hours using SD-CAA medium [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), 100 ug / mL kanamycin (Biosesang, Cat no. KC1001-025-02) in 1 L distilled water]. The culture was added to SG-CAA medium at OD 600 The yeast was diluted to a value of 1 and re-cultured at 30°C for 16 hours, and then induced to express βTrCP mutant protein on the yeast surface. 0.5×10 of the cultured yeast 7 Primary 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 for βTrCP using a flow cytometer (SONY, SH800S).2-263 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 from Round 0 to the final Round 3, βTrCP was isolated. 2-263 We confirmed changes in the binding affinity of the mutant protein library to SKP1 (Fig. 1a).

[0092] After the final round, single clones were isolated from the yeast population and analyzed using the same method as above under conditions of a biotinylated SKP1 protein concentration of 200 nM (Figs. 1b to 1d). Among the single clones, clones '7' (SEQ ID NO: 2) and '9' (SEQ ID NO: 3) with high binding affinity to SKP1 were selected, and the sequences of these two clones were used as templates to generate secondary random mutations.

[0093] Example 3. Construction of a secondary library for βTrCP2-263 mutant proteins.

[0094] PCR was performed using plasmids each having the sequences of clone '7' (SEQ ID NO: 2) and clone '9' (SEQ ID NO: 3) selected through the method of Example 2 as templates, inducing 3.5 and 5.8 mutations per 1000 bp in the same manner as Example 1. The final 5 ug of randomly mutated PCR products were inserted into 1 ug of pYD5 vector, and EBY100 yeast was transformed with each vector, and 6 × 10 6 7×10 6 We built four libraries with a variety of features.

[0095] Example 4. Enhanced binding affinity and single-clone analysis of the βTrCP2-263_7 and βTrCP2-263_9 mutant libraries for SKP1.

[0096] The library constructed by the method of Example 3 above was cultured in the same manner as Example 2 above, and βTrCP was added to the yeast surface. 2-263The mutant protein was induced to be expressed. Cultured yeast (0.5 × 10 7 The primary staining was performed by mixing 10 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 using a flow cytometer to determine the binding affinity for SKP1. 2-263 _7 clone and βTrCP 2-263 _9 Individuals within the upper 0.1% of the yeast population were isolated compared to the clones. After repeating the above process three times, βTrCP was isolated from Round 0 to the final Round 3. 2-263 We confirmed changes in binding affinity of the mutant protein library to SKP1 (Fig. 2a and Fig. 2b).

[0097] 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), E3 (SEQ ID NO: 6), E4 (SEQ ID NO: 7), E5 (SEQ ID NO: 8), and E6 (SEQ ID NO: 9) were finally selected (Fig. 2c), and the sequences of each of the clones are shown in Fig. 3.

[0098] Example 5. Binding Ability Analysis of Selected βTrCP2-263 Mutant Proteins to SKP1

[0099] βTrCP 2-263 And to compare the binding affinity of each mutant, EC was measured using a flow cytometer. 50 was measured. Six final mutants (E1, E2, E3, 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 7Primary staining was performed by mixing the mouse anti-V5 antibody at a ratio of 1:500 and 18 concentrations of biotinylated SKP1 serially diluted by 1 / 2 from 0 nM to 400 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).

Claims

1. βTrCP fragment or a mutant thereof.

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

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

4. In paragraph 1, The above βTrCP fragment variant is a βTrCP fragment or a variant thereof, wherein 5 to 18 amino acids are substituted in the βTrCP fragment.

5. In paragraph 1, The above variant is the 8th, 10th, 11th, 16th, 18th, 28th, 33rd, 35th, 38th, 47th, 91st, 95th, 98th, 102nd, 105th, 110th, 114th, 115th, 120th, 122nd, 124th, 125th, 127th, 132nd, 144th, 146th, 148th, 159th, 170th, 172nd, 173rd, 183rd, 185th, 192nd, 197th, 198th, 202nd, 203rd, 204th, 225th, in the amino acid sequence of SEQ ID NO:

1. A βTrCP fragment or a variant thereof, wherein any one amino acid selected from the group consisting of the 234th, 236th, 240th, 241st, 242nd, 250th, 251st, 260th, and combinations thereof is substituted.

6. In paragraph 5, The above variants are composed of L8, E10, K11, M16, S18, E28, I33, P35, N38, C47, S91, E95, K98, V102, F105, E110, V114, E115, L120, S122, M124, C125, Y127, I132, F144, T146, L148, N159, C170, A172, E173, T183, D185, L192, V197, R198, L202, W203, R204, N225, A234, Y236, I240, Q241, D242, W250, R251, I260 and combinations thereof in the amino acid sequence of SEQ ID NO:

1. A βTrCP fragment or a variant thereof, wherein any one amino acid selected from the group is substituted.

7. In paragraph 6, The above variants are L8P, E10V, K11E, M16L, S18T, E28G, I33T, P35A, N38D, C47S, S91T, E95G, K98R, V102A, F105L, E110G, V114A, E115G, L120P, S122P, M124V, C125R, Y127C, I132T, F144S, T146P, L148P, N159K, C170R, A172V, E173V, T183S, D185G, D185E, L192P, V197A, R198G, L202Q, L202R, W203R, A βTrCP fragment or a variant thereof, wherein the βTrCP fragment is substituted with any one amino acid selected from the group consisting of R204G, N225Y, A234V, Y236C, I240M, Q241R, D242E, W250R, R251G, I260S, and combinations thereof.

8. In paragraph 7, A βTrCP 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:

9.

9. In paragraph 1, A βTrCP fragment or a variant thereof, wherein the mutant has a 10- to 120-fold improved binding ability to SKP1 compared to the βTrCP fragment.

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

11. In paragraph 10, A fusion protein, wherein the βTrCP 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 βTrCP fragment or a variant thereof, wherein L is a peptide linker, The above n is 0 or 1.

13. A polynucleotide encoding the βTrCP 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. A pharmaceutical composition for preventing or treating a disease, comprising as an active ingredient a fusion protein comprising a 18.βTrCP fragment or a variant thereof; and a target protein binding site.

19. A pharmaceutical composition for the prevention or treatment of a disease, comprising the polynucleotide of claim 14 or the vector of claim 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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