Proteolysis targeted coupling nano antibody PROTNC as well as preparation method and application thereof

Abstract

The invention discloses a proteolysis targeted coupling nano antibody PROTNC as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention aims to provide a micromolecule coupled nano antibody type degradation agent which can degrade intracellular protein in a targeted manner. The invention provides a proteolysis targeted coupled nano antibody PROTNC. A degradation agent is prepared from the following components: a nano antibody, a connexon, an E3 ubiquitin ligase small molecule ligand and an intracellular delivery module. The PROTNC can be used for degrading intracellular proteins in a targeted manner, and particularly can be used for realizing targeted degradation on inherent disordered proteins.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a protein hydrolysis targeted coupled nano antibody PROTNC and a preparation method and application thereof. Background Art

[0002] Proteolysis targeting chimera (PROTAC) is a drug development technology that uses the ubiquitin-proteasome system (UPS) to degrade target proteins. At present, PROTAC is limited to classic drug targets, because small molecule inhibitors of these targets have clinically proven effects and can be used to prepare PROTAC molecules. However, there are still many pathogenic proteins in cells that are difficult to target with traditional small molecules, including (unligandable) target proteins that lack surface-fixed binding pockets, and a large number of inherently disordered proteins. For example, the binding pocket of unligandable proteins may be shallow and wide, making it difficult to form a stable binding with small molecules; while other unligandable proteins have a "smooth" surface and provide few binding sites for small molecules. For intrinsically disordered proteins, their structures are highly dynamic, and it is difficult to rationally design and screen specific small molecule ligands. Therefore, it has also become the main defect of the current small molecule PROTAC technology. Summary of the invention

[0003] The purpose of the present invention is to provide a small molecule coupled nano antibody type degradation agent that can target and degrade intracellular proteins.

[0004] The present invention provides a proteolysis-targeted coupled nano antibody PROTNC, wherein the degrader consists of the following components: a nano antibody, a linker, an E3 ubiquitin ligase small molecule ligand and an intracellular delivery module.

[0005] It is further defined that the connection method of the nanobody, linker, E3 ubiquitin ligase small molecule ligand and intracellular delivery module is: the E3 ubiquitin ligase small molecule ligand and the nanobody are connected through the linker to obtain a conjugate, and then the intracellular delivery module is connected to the conjugate.

[0006] It is further defined that the nanobody is used to target and recognize nanobodies or fluorescent protein nanobodies that recognize intracellular pathogenic proteins.

[0007] It is further defined that the nanobody is a nanobody targeting α-synuclein, a nanobody targeting phase separation protein, a nanobody targeting hsTPX2, a nanobody targeting P53, a nanobody targeting RhoA, a green fluorescent nanobody or a red fluorescent nanobody;

[0008] It is further defined that the linker is a PEG chain or a flexible protein linker composed of amino acids; the E3 ubiquitin ligase small molecule ligand is a small molecule compound targeting the E3 ubiquitin ligase in the cell, namely Pomalidomide and AHPC, Pomalidomide targets CRBN, and AHPC targets VHL.

[0009] It is further defined that the intracellular delivery module is a cyclic transmembrane peptide.

[0010] It is further defined that the cyclic transmembrane peptide is a cyclic deca-arginine, and the structural formula of the cyclic deca-arginine is as follows: n is zero or a natural number,

[0011]

[0012] The present invention provides a method for preparing the above-mentioned proteolysis-targeted coupled nanoantibody PROTNC, and the specific steps of the method are as follows:

[0013] Step 1: Chemical synthesis of cysteine-linker-E3 ubiquitin ligase small molecule ligand;

[0014] Step 2: chemically coupling the nanobody with the small molecule ligand obtained in step 1 to obtain a conjugate;

[0015] Step 3: Conjugate the intracellular delivery module with the conjugate obtained in step 2 to obtain a degrader.

[0016] The present invention provides an application of the above-mentioned proteolysis-targeted coupled nanoantibody PROTNC in degrading intracellular proteins for the purpose of non-disease diagnosis and treatment.

[0017] Further defined, the protein is an intrinsically disordered protein and a target protein lacking a surface-immobilized binding pocket.

[0018] Beneficial effects: The structural composition of the degrader designed by the present invention is divided into four parts: nano-antibodies are used to target and recognize intracellular pathogenic proteins, small molecule ligands are used to recognize and bind to intracellular E3 ubiquitin ligases, and cyclic transmembrane peptides and linkers are used for intracellular delivery, which can target and degrade intracellular proteins, especially intrinsically disordered proteins, and can achieve targeted degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the gel run result of GBP-intein-His6 expressed and purified by E. coli;

[0020] Figure 2Figure 1 is a diagram showing the preparation and characterization results of cRGA; A is the characterization result of the EPL reaction between GBP-intein (I) and Cys-AHPC, B is the characterization result of the reverse nickel column purification of GBP-AHPC, and C is the characterization result of the preparation of the cRGA molecule;

[0021] Figure 3 This is the result of laser confocal microscopy of the dose-dependency experiment of cRGA on EGFP degradation;

[0022] Figure 4 This is a graph showing the results of a dose-dependency experiment on the degradation of EGFP by cRGA;

[0023] Figure 5 This is a graph showing the time-dependent experimental results of cRGA degradation of EGFP;

[0024] Figure 6 This is the result diagram showing that cRGA can effectively induce the degradation of the intracellular protein EGFP-γtubulin;

[0025] Figure 7 The figures are microscopic and WB results showing that cRGA can induce EGFP degradation in serum; A is the WB result of cRGA inducing EGFP degradation in serum, and B is the microscopic result of cRGA inducing EGFP degradation in serum;

[0026] Figure 8 This is the WB result diagram showing that cRGA can induce the degradation of the membrane-localized protein EGFP-CAAX;

[0027] Fig. 9 This is the result of EGFP degradation by co-incubation of cRGA and MG132;

[0028] Fig.10 The graph shows the degradation of EGFP by co-incubation of cRGA with different inhibitors;

[0029] Fig.11 The preparation and characterization results of mCherry-RhoNb-AHPC are shown in Figure 1. A is the characterization result of the EPL reaction of mCherry-RhoNb-intein and Cys-AHPC, B is the characterization result of the reverse nickel column purification of mCherry-RhoNb-AHPC, and C is the characterization result of the molecular sieve purification of mCherry-RhoNb-AHPC.

[0030] Fig.12 The preparation and characterization results of RBP-cR10* are shown in Figure 1. A is the EPL reaction characterization result of RBP-intein and cystein, B is the reverse nickel column purification result of RBP-Cys, and C is the characterization result of RBP-cR10*. * Preparation and characterization results of

[0031] Fig.13 This is the result of mCherry-RhoNb-AHPC co-incubated with RBP-cR10* into cells;

[0032] Fig.14 The figure shows that the delivery system can effectively enter the cell and effectively degrade the target protein RhoA; A is the WB result of different concentrations of the delivery system entering the cell, and B is the WB result of the delivery system degrading endogenous RhoA in a dose-dependent manner;

[0033] Fig.15 The preparation and characterization results of cRPA are shown in Figure 1. A is the EPL reaction characterization result of p53Nb-intein and Cys-intein, B is the reverse nickel column characterization result of p53Nb-AHPC, and C is the preparation characterization result of cRPA.

[0034] Fig.16 The WB result diagram shows that cRPA can induce the degradation of endogenous P53 in cells;

[0035] Fig.17 The preparation and characterization results of cRTA; A is the preparation result of EPL reaction of TBP-intein and Cys-AHPC, B is the reverse nickel column characterization result of TBP-AHPC, and C is the characterization result of cRTA preparation;

[0036] Fig.18 The figure shows the results of the dose-dependent experiment of cRTA inducing endogenous TPX2 degradation in cells;

[0037] Fig.19 This is a graph showing the experimental results of the time-dependent degradation of endogenous TPX2 induced by cRTA;

[0038] Fig. 20 The figure shows the results of the dose-dependent experiment of cRTP inducing endogenous TPX2 degradation in cells;

[0039] Fig.21 The preparation and characterization of cRSA and the results of inducing the dose-dependent degradation of Flag-α-synuclein; A is the preparation and characterization of cRSA, and B is the WB result of cRSA-induced Flag-α-synuclein degradation;

[0040] Fig. 22 The figure shows the results of the dose-dependency experiment of cRGA inducing endogenous and exogenous TPX2 degradation;

[0041] Fig.23 The figure is the result of the dose-dependency experiment of cRGA inducing endogenous and exogenous hEB1 degradation;

[0042] Fig.24 The figure shows the results of WB experiment that cRGA analogs with different linker lengths induce EGFP degradation. DETAILED DESCRIPTION

[0043] Experimental materials: target gene, Kumei Biotechnology Co., Ltd.; commercial plasmid, Miaoling plasmid platform; restriction endonuclease, T4 ligase, NEB; PCR high-fidelity enzyme, APE×BIO; DNTP, kanamycin, ampicillin, tetracycline, gentamicin, X-gal, Beijing Solebao Technology Co., Ltd.; single fragment seamless cloning kit, Mona Biotechnology Co., Ltd.; gel excision recovery kit, plasmid mini-preparation kit, Novozyme Biotechnology Co., Ltd.; nucleic acid dye, IPTG, BioShap; yeast extract, tryptone, OXOID; Rosseta, DH5α, Shanghai Vita Biotechnology Co., Ltd.; Cys-AHPC is a self-synthesized molecule, and other reagents used in the buffer were purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0044] Experimental instruments: PR-96E gradient PCR instrument, Hangzhou Miou Instrument Co., Ltd.; EPS 300 electrophoresis instrument power supply, Tanon; PWT-P7513 constant temperature incubator, Dascar Biotechnology Co., Ltd.; K5600 / C ultra-micro spectrophotometer, Kaio Technology Co., Ltd.; V-1200 visible spectrophotometer, Shanghai Meixi Instrument Co., Ltd.; AH-NANO high-pressure homogenizer, Zhengzhou Ketai Experimental Equipment Co., Ltd.; MVS-83 vertical pressure steam sterilizer, Panasonic; ZEISS LSM880, ZEISS; AKTA, Superdex200 gel filtration column, GE Healthcare; IS-RSDA constant temperature oscillator, IS-RSV1 vertical constant temperature oscillator, IS-RDA2 constant temperature oscillator, CI-191C CO2 cell constant temperature incubator, Crystal; BIR-250A / B low temperature biochemistry Shanghai Santeng Co., Ltd.; SORVALL LYNX 600 high-speed centrifuge, Thermo Scientific; Fc ImageStudio near-infrared and chemiluminescence imaging system, Odyssey.

[0045] Experimental plan and process:

[0046] Vector construction: pET28a(+), EGFP-C1, EGFP-N1, and pTXB1 are commercial plasmids, and other target genes are synthesized by Kumei Biotech Co., Ltd. EGFP-C1 and EGFP-N1 were double-digested at different restriction sites to obtain vector fragments, PCR amplified target genes of other proteins, and agarose gel electrophoresis was performed. The target fragments and empty vectors were recovered by gel cutting, and then inserted into the target gene by recombinase ligation to obtain EGFP-hsTPX2, EGFP-γtubulin, EGFP-hsEB1, and EGFP-CAAX. pET28a(+) was inserted into His8-EGFP-TEV and His8-EGFP-TEV-hsTPX2 by the same subcloning method, and Nanobody was cloned into pTXB1 plasmid as a universal plasmid for prokaryotic expression of proteins. Gene sequencing services are provided by Kumei Biotech Co., Ltd. or Ruibo Biotech Co., Ltd.

[0047] Plasmid construction: The vector used for cloning was pTXB1 (NEB, N6707S), PEGFP-C1, PmCherry-C1 and PmCherry-N1 were all purchased from commercial sources.

[0048] The cloning method is Gibson cloning or T4 ligase ligation. The desired gene is amplified from a plasmid containing the desired gene by PCR using hyPerFUsion high-fidelity polymerase (APExBIO, Cat#1032).

[0049] The GFP nanobody encoding gene (GBP), RhoA nanobody (RhoNb), p53 nanobody (p53Nb), α-Synuclein nanobody (αSynNb), and TPX2 nanobody (TBP) were synthesized by Jilin Kumei.

[0050] Restriction enzymes and T4 ligase were purchased from New England BioLabs (NEB).

[0051] RBP, i.e. the gene sequence of mCherry red fluorescent protein nanobody: (SEQ ID NO.1)

[0052] ATGGCGCAGGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGGCGGGTGGTTCTCT

[0053] GCGTCTGTCTTGCGCGACCTCTGGTTTCACCTTCTCTGACTACGCGATGGGTTGGTTCC

[0054] GTCAGGCGCCGGGTAAAGAACGTGAATTCGTTGCGGCGATCTCTTGGTCTGGTCACGTT

[0055] ACCGACTACGCGGACTCTGTTAAAGGTCGTTTCACCATCTCTCGTGACAACGTTAAAAA

[0056] CACCGTTTACCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCGGTTTACTCTTGC

[0057] GCGGCGGCGAAATCTGGTACCTGGTGGTACCAGCGTTCTGAAAACGACTTCGGTTCTT

[0058] GGGGTCAGGGTACCCAGGTTACCGTTTCTAAAGAAGCGATC;

[0059] GBP, that is, the gene sequence of the GFP green fluorescent protein nanobody: (SEQ ID NO.2)

[0060] GTTCAACTGGTTGAATCTGGCGGCGCACTGGTTCAACCGGGCGGTAGTCTGCGTCTGA

[0061] GTTGCGCAGCATCTGGTTTTCCGGTTAATCGTTATAGCATGCGTTGGTATCGTCAAGCAC

[0062] CGGGTAAAGAACGCGAGTGGGTTGCAGGTATGAGTAGCGCAGGCGATCGTAGTAGTTA

[0063] CGAAGACAGCGTCAAAGGCCGTTTTACCATTAGCCGTGACGACGCACGTAATACCGTTT

[0064] ATCTGCAGATGAACAGCCTGAAACCGGAAGACACCGCGGTGTATTATTGCAACGTCAA

[0065] CGTCGGTTTCGAATATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGT;

[0066] RhoNb, which is the gene sequence of RhoA nanobody: (SEQ ID NO.3)

[0067] GAGGTGCAGCTGCAGGCCAGCGGCGGAGGATTTGTGCAGCCTGGCGGCAGCCTGAGA

[0068] CTGTCCTGTGCCGCCAGCGGCGACACCTGGTGGAGCTCTGCCATGGGCTGGTTTAGAC

[0069] AGGCCCCTGGCAAGGAGAGAGAGTTTGTGAGCGCCATCTCCTTCTACCCCACCGAGTA

[0070] CACCTACTACGCCGATTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAATTCCAAGA

[0071] ATACCGTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACAGCCACATACTACTGT

[0072] GCCTGGATCGCCTGGGGCCCTTGGATGAGAACATCCTGGTACTGGGGCCAGGGCACCC

[0073] AGGTGACCGTGAGCAGC;

[0074] p53Nb, which is the gene sequence of p53 nanobody: (SEQ ID NO.4)

[0075] GCGCAGGTTCAGCTGCAGGAATCTGGTGGTGGTCTGGTTCAGGCGGGTGGTTCTCTGC

[0076] GTCTGTCTTGCGCGGCGTCTGAACGTACCTTCTCTACCTACGCGATGGGTTGGTTCCGT

[0077] CAGGCGCCGGGTCGTGAACGTGAATTCCTGGCGCAGATCAACTGGTCTGGTACCACCA

[0078] CCTACTACGCGGAATCTGTTAAAGACCGTACCACCATCTCTCGTGACAACGCGAAAAA

[0079] CACCGTTTACCTGGAAATGAACAACCTGAACGCGGACGACACCGGTATCTACTTCTGC

[0080] GCGGCGCACCCGCAGCGTGGTTGGGGTTCTACCCTGGGTTGGACCTACTGGGGTCAGG

[0081] GTACCCAGGTTACCGTTTCT;

[0082] αSynNb, namely the gene sequence of α-Synuclein nanobody: (SEQ ID NO.5)

[0083] CAAGTGCAGCTGCAGGAATCTGGTGGTGGTAGCGTACAGGCTGGCGGCAGCCTGCGTC

[0084] TGTCTTGCACCATCTCTGACGCGATCTTTTCTCGTTACGCAGTAGGTTGGTTTCGCCAGG

[0085] CTCCTGGCAAAGAATGTGAACTGGTTTCTACCATCACTCCAGACTCCACGACTACCCAC

[0086] TCCGACTTCGTAAAAGGTCGTTTCACGCTGTCTCGCGACAACGCGAAAAACACCGTAT

[0087] ACCTGCAGATGCACTCTCTGAAACCTGATGACACTGCCGTCTACTACTGTGCCTCCCGC

[0088] TGGCGTTCTGTCTCCGAAGGTTGTGGTGGTCAGGGTACCCAGGTTACTGTAAGCTCTGCTGCAGCTTACCCTTACGATGTGCCGGATTATGGTTCT;

[0089] The gene sequence of CAAX: (SEQ ID NO.6)

[0090] AAGATGAGCAAAGATGGTAAAAAGAAGAAAAAGAAGTCAAAGACAAAGTGTGTAATTATG。

[0091] Amino acid sequence of EGFP target protein: (SEQ ID NO.7)

[0092] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL

[0093] VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

[0094] VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK. Amino acid sequence of mCherry protein: (SEQ ID NO.8)

[0095] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLP

[0096] FAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQD

[0097] GEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0098] Preparation of PROTAC molecules based on nanoantibodies:

[0099] (1) The expressed and purified Nanobody-Intein protein was concentrated by ultrafiltration and the buffer was exchanged to a buffer containing 0.75 mM β-mercaptoethanol. The final concentration was controlled at 15 mg / ml.

[0100] (2) Add 1 / 2 volume of MENSNa and 1 / 2 volume of MPAA as catalysts, cut the protein with 1.5 mM cys-AHPC, and place in a cold room for reaction for two days.

[0101] (3) SDS-PAGE was run to identify the progress of the reaction. After the reaction was completed, the Nanobody-AHPC was purified by reverse nickel column.

[0102] (4) The purified Nanobody-AHPC was ultrafiltered into Buffer A, 2 eq of TCEP was added, centrifuged, sonicated, argon was added, and the reaction was carried out on ice for 45 min.

[0103] (5) Add 10 eq of DTNB and react on ice for 1 h. Ultrafiltration exchange buffer to DB buffer to remove excess DTNB, add 3 eq of cR10 and react on ice overnight.

[0104] (6) Gel run identification. After successful ligation, exchange into PBS buffer and aliquot and freeze.

[0105] AHPC is a small molecule ligand targeting E3 ubiquitin ligase. AHPC is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide;

[0106] Pomalidomide.

[0107] cR10* is a cyclic transmembrane peptide which is a cyclic deca-arginine. The structural formula of the cyclic deca-arginine is as follows: n is zero or a natural number,

[0108]

[0109] Example 1. Method for preparing small molecule coupled nano antibody degradation agent

[0110] The structure of the degrader designed by the present invention is divided into four parts: nano-antibodies for targeting and recognizing intracellular pathogenic proteins, small molecule ligands for recognizing and binding intracellular E3 ubiquitin ligases, and cyclic transmembrane peptides and linkers for intracellular delivery. The structure of the degrader is as follows:

[0111] Nanobody-linker-E3 ubiquitin ligase small molecule ligand-intracellular delivery module

[0112] (1) Select appropriate nanoantibodies for binding to intracellular proteins and obtain the gene fragments of the nanoantibodies through gene synthesis.

[0113] (2) Chemical synthesis of cysteine-linker-E3 ubiquitin ligase ligand (the main function of the linker is to connect small molecules and nanoantibodies, so that the two molecules have different distances between them. There is only a difference in length and no restriction on type).

[0114]

[0115] (3) Through vector construction, the gene fragment of the nanobody was inserted into the plasmid pTXB1_intein-His6 for subsequent protein expression.

[0116] (4) The constructed pTXB1_nanobody-intein-His6 plasmid was transfected into the Rosetta competent cell, and a single colony was selected for protein expression and purification.

[0117] (5) Select small molecule ligands that target intracellular E3 ubiquitin ligases.

[0118] (6) Chemically coupling the expressed nanobody with a small molecule ligand.

[0119] (7) The obtained small molecule nanobody conjugate is coupled with a cyclic transmembrane peptide through a disulfide bond method to obtain a small molecule coupled nanobody degrader.

[0120] (8) Adding the obtained small molecule-coupled nanoantibody degrader to cells and incubating them for a certain period of time to degrade intracellular proteins.

[0121] 1. Synthesis of Cys-AHPC (cysteine-linker-APC ligand):

[0122]

[0123] Reaction Scheme 1 | Synthesis route of Cys-AHPC (1). i) HBTU, DIEA, DMF, RT, ON, 62%.

[0124] ii) TFA, 2.5% v TIPS, RT, Ar, 6h, 99%.;

[0125] Abbreviations: Boc, tert-butoxycarbamate protection group; Trt, tritylprotection group; Cys, L-cysteine; PEG, polyethylene glycol;

[0126] HBTU,2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate;

[0127] DIEA, N, N-diisopropylethylamine; RT, room temperature; ON, overnight; TFA, trifluoroacetic acid; TIPS, triisopropylsilane; Ar, argon gas.

[0128] Synthesis of Cys-AHPC (1) and related intermediates

[0129]

[0130] Boc-Cys(Trt)-PEG-COOH (3) was coupled with H2N-AHPC (4) using the coupling reagent HBTU. Briefly, the carboxylate intermediate (3, 39 mg, 0.05 mmol) was first dissolved in anhydrous DMF (0.52 ml), and HBTU (21.6 mg, 0.057 mmol) and DIEA (15.9 mg, 0.123 mmol) were added to activate the carboxylate. After 10 minutes, H2N-AHPC (4, 20.3 mg, 0.047 mmol) was added. The reaction solution was stirred at room temperature overnight until TLC analysis (EtOAc:MeOH 6:1, Rf ~ 0.4) showed that the starting material was completely converted to the product. The reaction mixture was extracted with Na2CO3 and the organic phase was separated. After the aqueous phase was repeatedly extracted several times, all organic phases were combined, dried over saturated brine and anhydrous Na2SO4, filtered, concentrated and purified by silica gel chromatography to produce 37.3 mg of white solid as the product with a yield of 62%.

[0131]

[0132] The product was prepared by deprotecting Boc-Cys(Trt)-PEG-AHPC (5) prepared in the previous step. In brief, Boc Cys(Trt)-PEG-AHPC (5, 23.5 mg, 0.020 mmol) was dissolved in 0.5 ml of trifluoroacetic acid (TFA) and 12.5 μl of triisopropylsilane (2.5% v) was added. The reaction solution was stirred at RT under Ar for 6 h to completely deprotect the Boc- and Trt-protecting groups. The reaction mixture was then vacuum dried and distributed in ddH 2O / EtOAc. The aqueous phase was carefully separated and washed twice more with EtOAc to remove impurities. The aqueous phase was concentrated under reduced pressure by rotary evaporation, and the residue was carefully dried in a vacuum to obtain 18.9 mg of a crystalline white solid as a pure product with a yield of 99%.

[0133] 2. Synthesis of Cys-Pom (Cysteine-Linker-Pomalidomide Ligand):

[0134]

[0135] Reaction Scheme 2 | Synthesis route of Cys-Pom (1). i) HBTU, DIEA, DMF, RT, ON, 44%.

[0136] ii) TFA, 2.5% v TIPS, RT, Ar, 6h, 87%.;

[0137] Abbreviations: Boc, tert-butoxycarbamate protection group; Trt, tritylprotection group; Cys, L-cysteine; PEG, polyethylene glycol;

[0138] HBTU,2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate;

[0139] DIEA, N, N-diisopropylethylamine; RT, room temperature; ON, overnight; TFA, trifluoroacetic acid; TIPS, triisopropylsilane; Ar, argon gas.

[0140] Synthesis of Cys-Pom(1) and related intermediates

[0141]

[0142] Boc-Cys(Trt)-PEG-COOH(3) was coupled with Pomalidomide-NH2(6) using the coupling reagent HBTU. In short, the carboxylate intermediate (3, 39 mg, 0.05 mmol) was first dissolved in anhydrous DMF (0.52 ml), and HBTU (21.6 mg, 0.057 mmol) and DIEA (15.9 mg, 0.123 mmol) were added to activate the carboxylate. After 10 minutes, Pomalidomide-NH2 (6, 14.9 mg, 0.047 mmol) was added. The reaction solution was stirred at room temperature overnight until thin layer chromatography analysis showed that the raw material was completely converted into the product. The reaction mixture was extracted with Na2CO3 and the organic phase was separated. After the aqueous phase was repeatedly extracted several times, all organic phases were combined, dried with saturated brine and anhydrous Na2SO4, filtered, concentrated and purified by silica gel chromatography to produce 23.8 mg of green solid as the product with a yield of 44%.

[0143]

[0144] The product was prepared by deprotecting BocCys(Trt)-Pom (7) prepared in the previous step. Briefly, BocCys(Trt)-Pom (7, 17.2 mg, 0.016 mmol) was dissolved in 0.5 ml of trifluoroacetic acid (TFA) and 12.5 μl of triisopropylsilane (2.5% v) was added. The reaction solution was stirred at RT under Ar for 6 h to completely deprotect the Boc- and Trt-protecting groups. The reaction mixture was then vacuum dried and distributed in ddH 2O / EtOAc. The aqueous phase was carefully separated and washed twice more with EtOAc to remove impurities. The aqueous phase was concentrated under reduced pressure by rotary evaporation, and the residue was carefully dried in a vacuum to obtain 12.2 mg of a green solid as the pure product with a yield of 87%.

[0145] Example 2. Preparation of cRGA

[0146] 1. Expression and purification of GBP-Intein fusion protein in E. coli

[0147] Based on the sequence of the known EGFP nanoantibody GBP, its gene fragment was obtained by gene synthesis, and then cloned into the commercial pTXB1_Intein empty vector by PCR amplification to obtain pTXB1_GBP-intein-His6. After successful plasmid sequencing and identification, it was transformed into Rosetta competent cells, and induced overnight by shaking and IPTG. The next day, the bacteria were collected by centrifugation, crushed by high-pressure homogenization, and the supernatant was collected by centrifugation and purified by Ni column to collect the purified GBP-intein-His6 (I). The purification results are shown in Figure 2. Figure 1shown.

[0148] The purified GBP-Intein-His6 was ultrafiltered into buffer A, and 1 / 2 volume of MENSNa and 1 / 2 volume of MPAA were added. At the same time, 1.5mM Cys-AHPC was used for cutting. The reaction was allowed to react in a cold room for three days, and the gel was run for identification. GBP-AHPC was purified by reverse nickel column and the impurity intein-His6 was removed to obtain GBP-AHPC. The obtained GBP-AHPC (III) was ultrafiltered to exchange the buffer and then connected to cR10* through a disulfide bond to obtain cRGA. The preparation results are shown in Figure 2 shown.

[0149] Example 3. Dose-dependency test of cRGA degradation of EGFP

[0150] 1. Transfect EGFP into Hela cells to overexpress it. Add different doses of cRGA 24 hours after transfection and observe under confocal microscope after 48 hours of incubation. Figure 3 As shown, EGFP has a clear concentration dependence.

[0151] The cell lysates transfected into 24-well plates and added with different doses of cRGA drugs were collected and the degradation effects were verified by WB. Figure 4 It can be seen that EGFP is significantly degraded, proving the effect of cRGA on the targeted induction of EGFP degradation. And the degradation is obvious at a concentration of 5 μM.

[0152] 2. Time-dependent test of GBP-AHPC-cR10* degradation of EGFP: EGFP was transfected into Hela cells. 24 hours after transfection, 5 μM cRGA was added, and cell lysates were collected at different time points, and WB was performed to verify the degradation effect. Figure 5 visible.

[0153] 3. Study on the universality and substrate range of cRGA targeted degradation: In order to verify the universality of cRGA, EGFP-γtubulin was transfected into Hela cells. 24 hours after transfection, different concentrations of cRGA were added to the cells. After incubation for 48 hours, the cell lysate was collected and verified by WB. The results were visualized by WB Figure 6 It can be seen that cRGA can induce the effective degradation of EGFP-γ-tubulin in cells.

[0154] The PROTACs developed so far are mainly targeted at proteins in cells, and the applicable substrate range is relatively limited. In order to verify whether PROTACs of small molecules coupled to nanoantibodies can target proteins in serum or membrane-anchored proteins. First, exogenous 5μM EGFP and cRGA were added to the culture medium, and microscopic photos were taken after incubation for 48 hours. As shown in the figure, EGFP in the experimental group was significantly degraded. Cell lysates were collected for WB verification. Figure 7 It can also be seen that EGFP has been significantly degraded.

[0155] 4. Microscopy and WB results of cRGA inducing EGFP degradation in serum

[0156] After verifying that cRGA can effectively induce targeted degradation of extracellular proteins, the next step is to verify whether cRGA can have a targeted degradation effect on membrane-anchored proteins. EGFP-CAAX was transfected into Hela cells, and the cells were incubated with 5 μM cRGA 24 hours after transfection. Cell lysates were collected 48 hours later for WB verification. Figure 8 As shown, GBP-AHPC-cR10* can effectively induce the degradation of membrane-localized proteins.

[0157] Example 4: Study on the mechanism of cRGA-induced EGFP degradation

[0158] Hela cells were transfected with EGFP. 24 hours after transfection, 5 μM cRGA and 1 μM proteasome inhibitor MG132 were added. After incubation for 48 hours, proteins were collected and WB was performed to verify the degradation effect. Fig. 9 As shown in the figure, EGFP was not significantly degraded, while the cRGA positive control group without MG132 was degraded, indicating that the degradation of EGFP induced by cRGA is dependent on the proteasome.

[0159] EGFP was transfected into Hela cells. 24 hours after transfection, 5 μM cRGA and 10 μM cRGA competitive inhibitor AHPC and NEDD8 activating enzyme inhibitor MLN4924 were added. After incubation for 48 hours, the protein was collected and WB was used to verify the degradation effect. Fig.10 As shown, there was no obvious degradation of EGFP in the inhibitor group, while degradation occurred in the cRGA positive control group without inhibitor, indicating that cRGA-induced EGFP degradation is based on VHL ubiquitin ligase and ubiquitination-dependent degradation.

[0160] Example 5: Universal delivery system evaluation of Tag-PROTAC

[0161] In order to establish a rapid in vitro screening method for PROTAC, the RBP-cR10* delivery molecule can be prepared by fusing the mCherry tag to the PROTAC molecule (using the method of Example 1 to replace the GBP nanobody with the RBP nanobody). In this way, the PROTAC molecule can be quickly delivered to the cell to play a targeted degradation role. First, a PROTAC molecule (mCherry-RhoNb AHPC) that can target endogenous Rho A in the cell and a delivery molecule RBP-cR10* are prepared. The preparation results are shown in Figure 1. Fig.11 and Fig.12 .

[0162] Since PROTAC molecules carry mCherry, their entry into cells can be observed using confocal microscopy. * The cells were added in a 1:1 ratio and observed by confocal microscopy 12 hours later. Fig.13 As shown, RBP-cR10 was added * The experimental group can effectively enter the cells.

[0163] Then mCherry-Rh57-AHPC and RBP-cR10* were added to the cells at a ratio of 1:1. After incubation for 48 hours, the cell lysate was collected for WB verification. The degradation of endogenous RhoA by the delivery system was verified by WB. As shown in the figure, it can be seen that the delivery system has a significant degradation of endogenous cell RhoA.

[0164] Since Tag-PROTAC molecules carry mCherry tags, WB can be used to quantify the situation of molecules in cells. Fig.14 As shown,

[0165] Example 6: Targeted degradation of endogenous P53 by PROTAC based on nanobodies

[0166] 1. Preparation method of cRPA: Based on the known sequence of p53 nanoantibody Nbp53, its gene fragment was obtained by gene synthesis, and PCR amplification was used to clone it into the commercial pTXB1_Intein empty vector to obtain pTXB1-NbP53-intein-His6. After successful plasmid sequencing and identification, it was transformed into Rosetta competent medium, shaken and induced by IPTG overnight. The next day, the bacteria were collected by centrifugation, crushed by high pressure homogenization, and the supernatant was collected by centrifugation, purified by Ni column, and the purified Nbp53-intein-His6 was collected.

[0167] The purified NbP53-Intein-His6 was ultrafiltered into buffer A, and 1 / 2 volume of MENSNa and 1 / 2 volume of MPAA were added. At the same time, 1.5mM Cys-AHPC was used for cutting. The reaction was allowed to react in a cold room for three days, and the gel was run for identification. The NbP53-AHPC was purified by reverse nickel column and the impurity intein-His6 was removed to obtain NbP53-AHPC. The obtained NbP53-AHPC was ultrafiltered to exchange the buffer and then connected to cR10* through a disulfide bond to obtain cRPA.

[0168] 2.p53 is an important tumor suppressor gene and the most common mutated gene in human cancer. The protein it encodes is an intrinsic disordered protein in cells. Based on the known p53 nanoantibody sequence, Nbp53-AHPC-cR10*(cRPA) was prepared to target and degrade endogenous P53 protein in cells. The preparation results are shown in Figure 2. Fig.15

[0169] Different concentrations of Nbp53-AHPC-cR10* were added to Hela cells, and cell lysates were collected after 24 h of incubation. WB verification was performed. Fig.16 Endogenous p53 protein in the cells shown was degraded at a concentration of 5 μM.

[0170] Example 7: Targeted degradation of endogenous hsTPX2 by PROTAC based on nanobodies

[0171] 1. Preparation method of TBP-AHPC-cR10*(cRTA): Based on the sequence of the known TPX2 nanoantibody TBP, its gene fragment was obtained by gene synthesis, and PCR amplification was used to clone it into the commercial pTXB1_Intein empty vector to obtain pTXB1-TBP-intein-His6. After successful plasmid sequencing and identification, it was transformed into Rosetta competent cells, shaken and induced by IPTG overnight. The next day, the bacteria were collected by centrifugation, crushed by high-pressure homogenization, and the supernatant was collected by centrifugation, purified by Ni column, and the purified TBP-intein-His6 was collected.

[0172] The purified TBP-Intein-His6 was ultrafiltered into buffer A, and 1 / 2 volume of MENSNa and 1 / 2 volume of MPAA were added. At the same time, 1.5mM Cys-AHPC was used for cutting. The reaction was allowed to react in a cold room for three days, and the gel was run for identification. TBP-AHPC was purified by reverse nickel column and the impurity intein-His6 was removed to obtain TBP-AHPC. The obtained TBP-AHPC was ultrafiltered and exchanged with buffer, and then connected to cR10 by disulfide bond. * Get TBP-AHPC-cR10 *(cRTA).

[0173] 2.TPX2 is an important microtubule-associated protein. TPX2 is associated with the development and progression of various cancers. Its abnormal expression or overexpression has been observed in many tumor types, including breast cancer, lung cancer, gastric cancer, etc. TPX2 has become a potential therapeutic target in cancer research. Targeted degradation of TPX2 plays an important role in inhibiting the progression of cancer cells. Fig.17 .

[0174] Different concentrations of TBP-AHPC-cR10*(cRTA) were added to Hela cells. After 24 h of incubation, the cell lysate was collected for WB analysis to verify the degradation of endogenous hsTPX2 by cRTA. Fig.18 As shown, with the gradual increase of concentration, the degradation effect of hsTPX2 gradually increased.

[0175] 5uM cRTA was added to Hela cells, and cell lysates were collected at different time points for WB to verify the time dependence of degradation. The results of WB are shown in Fig.19 shown.

[0176] Example 8.

[0177] 1. Preparation of TBP-Pom-cR10* (cRTP) method: Based on the known sequence of TPX2 nanoantibody TBP, its gene fragment was obtained by gene synthesis, and PCR amplification was cloned into the commercial pTXB1_Intein empty vector to obtain pTXB1-TBP-intein-His6. After successful plasmid sequencing and identification, it was transformed into Rosetta competent cells, shaken and induced overnight by IPTG. The next day, the bacteria were collected by centrifugation, crushed by high-pressure homogenization, and the supernatant was collected by centrifugation, purified by Ni column, and the purified TBP-intein-His6 was collected.

[0178] The purified TBP-Intein was ultrafiltered into buffer A, and 1 / 2 volume of MENSNa and 1 / 2 volume of MPAA were added. At the same time, 1.5mM Cys-Pom was used for cutting. The reaction was allowed to react in a cold room for three days, and the gel was run for identification. TBP-AHPC was purified by reverse nickel column and the impurity protein intein-His6 was removed to obtain TBP-AHPC. The obtained TBP-AHPC was ultrafiltered and exchanged with buffer, and then connected to cR10* through a disulfide bond to obtain TBP-Pom-cR10* (cRTP).

[0179] 2. In order to verify the modular characteristics of small molecule-coupled nanoantibody degraders. Then, the small molecule ligand Pomalidomide targeting the E3 ubiquitin ligase CRBN was selected to replace AHPC, and TBP-Pomalidomide-cR10* (cRTP) was prepared by coupling TBP with cys-Pomalidomide. After adding different concentrations to Hela cells and incubating for 24 hours, the cell lysate was collected for WB analysis to verify the degradation of endogenous hsTPX2 by cRTP. The degradation effect is as follows Fig. 20 As shown, with the gradual increase of concentration, the degradation effect of hsTPX2 gradually increased.

[0180] Example 9: cRSA degrades intracellular α-synuclein

[0181] 1. cRSA preparation method: Based on the known sequence of α-synuclein nanoantibody αSynNb, its gene fragment was obtained by gene synthesis, and PCR amplification was used to clone it into the commercial pTXB1_Intein empty vector to obtain pTXB1-αSynNb-intein-His6. After successful plasmid sequencing and identification, it was transformed into Rosetta competent medium, and induced overnight by shaking and IPTG. The next day, the bacteria were collected by centrifugation, crushed by high-pressure homogenization, and the supernatant was collected by centrifugation, purified by Ni column, and the purified αSynNb-intein-His6 was collected.

[0182] The purified αSynNb-Intein-His6 was ultrafiltered into buffer A, and 1 / 2 volume of MENSNa and 1 / 2 volume of MPAA were added. At the same time, 1.5mM Cys-AHPC was used for cutting. The reaction was carried out in a cold room for three days, and the gel was run for identification. The flag nanoantibody-AHPC was purified by reverse nickel column and the impurity protein intein-His6 was removed to obtain αSynNb-AHPC. The obtained TBP-AHPC was ultrafiltered and exchanged with buffer, and then connected to cR10* through a disulfide bond to obtain αSynNb-AHPC-cR10* (cRSA).

[0183] 2. Later, the degradation of another intrinsically disordered protein, α-synuclein, was also demonstrated, which plays a vital role in Parkinson's disease. As an intrinsically disordered protein, its structure changes highly dynamically, and it is difficult to design a small molecule ligand that specifically binds to it. Therefore, it is impossible to degrade it by means of small molecule degraders. Therefore, targeting α-synuclein with nanoantibodies to design small molecule-coupled nanoantibody degraders has become a solution. Flag-α-synuclein was transfected in Hela cells, and different concentrations of cRSA were added to the cells 24 hours after transfection. After incubation for 48 hours, the cell lysate was collected for WB verification. The results were developed by WB Fig.21 It can be seen that cRSA can induce the effective degradation of Flag-α-synuclein in cells.

[0184] Example 10: Degradation of endogenous phase separation proteins using cRGA

[0185] Phase separation proteins, as a special type of intrinsically disordered proteins, are usually involved in the formation of liquid-liquid phase separation cell structures. Therefore, this type of protein can phase separate from exogenous EGFP-tagged proteins. By adding cRGA, endogenous phase separation proteins and exogenous proteins can be degraded simultaneously.

[0186] EGFP-hsTPX2 was transfected into Hela cells. 24 hours after transfection, different concentrations of cRGA were added to the cells. After incubation for 48 hours, the cell lysate was collected and verified by WB. Fig. 22 As shown, it can be seen that while GBP-AHPC-cR10 targets and degrades EGFP-hsTPX2, it also causes the degradation of endogenous TPX2 in the cell.

[0187] GBP-AHPC-cR10* was used to degrade endogenous phase separation proteins in cells. Hela cells were transfected with EGFP-hsEB1 and GBP-AHPC-cR10* was added 24 hours after transfection. Cell lysates were collected after 48 hours of incubation and the degradation effect was verified by WB. Fig.23 As shown, endogenous hsEB1 was degraded simultaneously with the degradation of EGFP-hsEB1.

[0188] Structurally, PROTAC consists of three parts: an E3 ubiquitin ligase ligand and a target protein ligand. The two active ligands are connected together by a specially designed "Linker", ultimately forming an active form of a heterologous bifunctional molecule. Among them, the linker is an important component, and the length of the linker is also an important factor affecting the degradation efficiency. Therefore, it is necessary to compare the degradation by designing linkers of different lengths. EGFP was transfected in Hela cells. 24 hours after transfection, 5uM of cRGA analogs with different linker lengths were added to the cells, such as Fig.24 As shown in Table 1, cRGA analogs with different linker lengths all had a degradation effect on EGFP.

[0189] The first type has no linker, the second type has GGSlinker, the third type has PEGlinker, and the fourth type has GGS+PEGlinker.

[0190] Table 1

[0191]

Claims

1. A proteolysis-targeted conjugated nanoantibody PROTNC, characterized in that: The degrader consists of the following components: nanoantibodies, linkers, E3 ubiquitin ligase small molecule ligands and intracellular delivery modules.

2. The proteolysis-targeted coupled nanoantibody PROTNC according to claim 1, characterized in that: The connection method of the nanobody, linker, E3 ubiquitin ligase small molecule ligand and intracellular delivery module is: the E3 ubiquitin ligase small molecule ligand and the nanobody are connected through the linker to obtain a conjugate, and then the intracellular delivery module is connected to the conjugate.

3. The proteolysis-targeted coupled nanoantibody PROTNC according to claim 1, characterized in that: The nanobody is used for targeting and recognizing the nanobody of the intracellular pathogenic protein or the fluorescent protein nanobody.

4. The proteolysis-targeted coupled nanoantibody PROTNC according to claim 3, characterized in that: The nanobody is a nanobody targeting α-synuclein, a nanobody targeting phase separation protein, a nanobody targeting hsTPX2, a nanobody targeting P53, a nanobody targeting Rho A, a green fluorescent nanobody or a red fluorescent nanobody.

5. The proteolysis-targeted coupled nanoantibody PROTNC according to claim 1, characterized in that: The linker is a PEG chain or a flexible protein linker composed of amino acids; the E3 ubiquitin ligase small molecule ligand is a small molecule compound targeting the E3 ubiquitin ligase in the cell, which is Pomalidomide and AHPC, Pomalidomide targets CRBN, and AHPC targets VHL.

6. The proteolysis-targeted coupled nanobody PROTNC according to claim 1, characterized in that: The intracellular delivery module is a cyclic transmembrane peptide.

7. The proteolysis-targeted coupled nanobody PROTNC according to claim 6, characterized in that: The cyclic transmembrane peptide is a cyclic deca-arginine, and the structural formula of the cyclic deca-arginine is as follows: n is zero or a natural number, 8. A method for preparing the proteolysis-targeted coupled nanoantibody PROTNC according to any one of claims 1 to 7, characterized in that: The specific steps of the method are as follows: Step 1: Chemical synthesis of cysteine-linker-E3 ubiquitin ligase small molecule ligand; Step 2: chemically coupling the nanobody with the small molecule ligand obtained in step 1 to obtain a conjugate; Step 3: Conjugate the intracellular delivery module with the conjugate obtained in step 2 to obtain a degrader.

9. Use of the proteolysis-targeted coupled nanoantibody PROTNC according to any one of claims 1 to 7 in degrading intracellular proteins for the purpose of non-disease diagnosis and treatment.

10. The use according to claim 9, characterized in that: The proteins are intrinsically disordered proteins and target proteins that lack surface-fixed binding pockets.

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