Tissue targeting nano antibody and application thereof

Abstract

The invention provides a tissue targeting nano antibody and application thereof, and relates to the technical field of biological medicine and gene therapy. Membrane proteins (including an L-type calcium ion channel protein alpha2delta subunit CACNA2D1, a sodium ion channel protein alpha subunit SCN5A, a vascular epicardial substance BVES, a coxsackie virus adenovirus receptor CAR and the like) highly expressed in the heart and skeletal muscle are selected as targets, and a nano antibody synthesis library is used for screening. A series of nano antibodies with tissue targeting are successfully obtained by means of high-throughput sequencing, streaming and the like. The nano antibodies not only can be combined with target spots with high affinity, but also can effectively load and deliver proteins or nucleic acids, so that the enrichment amount and the treatment efficiency of the drugs in target tissues are remarkably improved, and a basis is provided for developing an efficient and safe DMD precise treatment scheme.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and gene therapy, and particularly relates to a tissue-targeted nanobody and application thereof. BACKGROUND

[0002] Duchenne Muscular Dystrophy (DMD) is an X-linked recessive lethal genetic disease caused by mutation of the dystrophin gene. The disease can affect skeletal muscle, myocardium and brain, and patients often die of heart failure. At present, antisense oligonucleotides of exon skipping and CTGF inhibitors for muscle fibrosis are often used to restore partial dystrophin activity or delay muscle fibrosis progression. Traditional non-targeted drug delivery methods often require a high dose to take effect, which not only increases the risk of liver and kidney toxicity, but also reduces the treatment effect on DMD due to the lack of efficient and specific muscle, heart and brain targeting. Therefore, developing a delivery tool that can specifically target skeletal muscle, heart and brain is the key to breaking through the current DMD treatment dilemma.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a tissue-targeted nanobody and application thereof. The present application selects membrane proteins (including CACNA2D1, SCN5A, BVES and CAR, etc.) that are highly expressed in the heart and skeletal muscle from the protein expression amount in the human protein atlas database as targets, uses a nanobody synthesis library for screening, and successfully obtains a series of tissue-targeted nanobodies by using high-throughput sequencing, flow cytometry, etc. These nanobodies can not only bind to the target with high affinity, but also can effectively load and deliver proteins or nucleic acids, significantly improving the enrichment amount and treatment efficiency of these drugs in the target tissue, and providing a basis for developing efficient and safe DMD precise treatment programs.

[0005] In order to achieve the purpose of the present application, the following technical solutions are adopted: The present application provides a tissue-targeted nanobody and application thereof. The nanobody comprises a complementarity determining region, and the complementarity determining region is composed of CDR1, CDR2 and CDR3 sequences. The amino acid sequence of CDR1 is shown as SEQ ID NO. 1-SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8-SEQ ID NO. 16, and CDR3 is shown as SEQ ID NO. 17-SEQ ID NO. 23. The complementarity determining region of the nanobody is selected from any one of the following combinations: (a) SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 17; (b) SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 18; (c) SEQ ID NO. 2, SEQ ID NO. 10, SEQ ID NO. 19; (d) SEQ ID NO. 3, SEQ ID NO. 11, SEQ ID NO. 20; (e) SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 21; (f) SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 22; (g) SEQ ID NO. 6, SEQ ID NO. 14, SEQ ID NO. 21; (h) SEQ ID NO. 6, SEQ ID NO. 15, SEQ ID NO. 23; (i) SEQ ID NO. 7, SEQ ID NO. 16, SEQ ID NO. 19.

[0006] SEQ ID NO. 1 RIFRRNT SEQ ID NO. 2 GSISANFTM SEQ ID NO. 3 GTIFVSQNM SEQ ID NO. 4 GNIFFAPYM SEQ ID NO. 5 GYISVHSEM SEQ ID NO. 6 GYIFQYVYM SEQ ID NO. 7 GNIFEPTWM SEQ ID NO. 8 TISRSGRINY SEQ ID NO. 9 AITTSGGSTT SEQ ID NO. 10 EFVAGIAYGTNTNY SEQ ID NO. 11 EFVASITPGANTNY SEQ ID NO. 12 EFVASIGSGTITNY SEQ ID NO. 13 ELVAGITRGGNTNY SEQ ID NO. 14 EFVAGINYGAITYY SEQ ID NO. 15 ELVAGIDYGSTTNY SEQ ID NO. 16 ELVAAISGGGNTYY SEQ ID NO. 17 ADIRRGGGRAAAG SEQ ID NO. 18 AASSRFGSSSARR SEQ ID NO. 19 AVAPVRQFNSKTYFDY SEQ ID NO. 20 AVTPTRDGAYLAPFYY SEQ ID NO. 21 AVSTWYGHAHLY SEQ ID NO. 22 AVYHYRSRWHGY SEQ ID NO. 23 AVIGYDGPPVFAPHLY Further, the tissue-targeting nanobody also comprises a framework region FR1, FR2, FR3 and FR4, wherein the amino acid sequence of FR1 is as shown in SEQ ID NO. 24, the amino acid sequence of FR2 is as shown in SEQ ID NO. 25, the amino acid sequence of FR3 is as shown in SEQ ID NO. 26, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 27.

[0007] SEQ ID NO. 24 EVQLQASGGGFVQPGGSLRLSCAASG SEQ ID NO. 25 MGWFRQAPGKEREFVSAIS SEQ ID NO. 26 YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA SEQ ID NO. 27 YWGQGTQVTVSS The application also provides a fusion protein comprising the above tissue-targeted nanobody and a heterologous molecule.

[0008] Further, the heterologous molecule is selected from any one or a combination of a reporter protein, a therapeutic protein or a nucleic acid binding protein.

[0009] Further, the reporter protein is mCherry, the therapeutic protein is anti-CTGF scFv, and the nucleic acid binding protein is monomeric streptavidin mSA.

[0010] The application also provides a drug delivery system comprising the above tissue-targeted nanobody and an active molecule.

[0011] Further, the active molecule is selected from a small molecule compound, a protein, a polypeptide, a nucleic acid, a viral vector, a lipid nanoparticle or an imaging agent.

[0012] The application also provides a pharmaceutical composition comprising the above tissue-targeted nanobody or the above fusion protein.

[0013] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0014] A disease diagnostic reagent comprising the above tissue-targeted nanobody or the above fusion protein.

[0015] Further, the disease is any one of a brain disease, a muscle disease or a heart disease.

[0016] The application also provides an enhanced drug delivery carrier or drug delivery system, wherein the enhanced drug delivery carrier is linked to or modified with the above tissue-targeted nanobody or the above fusion protein.

[0017] The application also provides use of the above tissue-targeted nanobody or fusion protein in the preparation of a medicament for treating Duchenne muscular dystrophy.

[0018] The application has the following technical effects: Nanobodies can specifically target the heart, skeletal muscle and brain, solve the pain point of targeting difficulty in treating neuromuscular related diseases, achieve precise drug delivery to diseased tissues, successfully achieve efficient loading and delivery of different types of biological macromolecules, and show great potential as a universal technology platform. Nanobodies have small molecular weight and low immunogenicity, and have no risk of Fc fragment-mediated side effects. Targeted delivery mediated by nanobodies can significantly increase the accumulation of drugs in target tissues, thereby significantly increasing the therapeutic effect of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0020] Figure 1 Figure 1 is a graph of the enrichment amount of nanobodies with CACNA2D1 as the target, wherein a is the recovery amount of each round; b is the number of nanobodies obtained by high-throughput sequencing; Figure 2 Figure 2 is a graph of the recovery efficiency of nanobodies with BVES as the target, wherein a is the recovery efficiency of each round; b is a detection graph of the binding rate of candidate nanobodies and target proteins; Figure 3 Figure 3 is a graph of the recovery efficiency of nanobodies with CAR as the target, wherein a is the recovery amount of each round; b is a detection graph of the binding rate of candidate nanobodies and target proteins; Figure 4 Figure 4 is a graph of the enrichment amount of nanobodies with SCN5A as the target, wherein a is the recovery amount of each round; b is a detection graph of the binding rate of candidate nanobodies and target proteins; Figure 5 Figure 5 is a graph of the tissue distribution results of nanobodies with CACNA2D1 as the target and fused with mCherry protein after intravenous injection, wherein a is a graph of the fluorescence intensity of each tissue; b is the enrichment of fluorescence signal in muscle; c is the enrichment in the heart; Figure 6 Figure 6 is a graph of the tissue distribution results of nanobodies with BVES as the target and fused with mCherry protein after intravenous injection, wherein a is a graph of the fluorescence intensity of each tissue; b is the enrichment of fluorescence signal in muscle; c is the enrichment in the heart; Figure 7Fig. 6 is a diagram of the tissue distribution results of the nanobody fused with mCherry protein as a target of CAR after intravenous injection, wherein a is a fluorescence intensity diagram of each tissue; b is the enrichment of fluorescence signal in muscle; c is the enrichment in heart; Figure 8 Fig. 7 is a diagram of the tissue distribution results of the nanobody fused with mCherry protein as a target of SCN5A after intravenous injection, wherein a is a fluorescence intensity diagram of each tissue; b is the enrichment of fluorescence signal in muscle; c is the enrichment in heart; Figure 9 Fig. 8 is a diagram of the affinity of the nanobody NCA2 as a target protein of CACNA2D1 and the target protein; Figure 10 Fig. 9 is a diagram of the affinity of the nanobody as a target protein of BVES and the target protein, wherein a is the affinity diagram of NBVD1 and the target protein, b is the affinity diagram of NBVD2 and the target protein; Figure 11 Fig. 10 is a diagram of the affinity of the nanobody as a target protein of CAR and the target protein, wherein a is the affinity diagram of NCRC2 and the target protein, b is the affinity diagram of NCRF6 and the target protein; Figure 12 Fig. 11 is a diagram of the affinity of the nanobody as a target protein of SCN5A and the target protein, wherein a is the affinity diagram of NSCE9 and the target protein, b is the affinity diagram of NSCE10 and the target protein, c is the affinity diagram of NSCH6 and the target protein; Figure 13 Fig. 12 is a diagram of the delivery capability of NCA2 loaded with PMO, wherein a is the IHC result of the recovery of dystrophin protein in TA muscle of NCA2 loaded with PMO, b is the quantitative column chart of the recovery of dystrophin protein in IHC, c is the WB diagram of the recovery efficiency of dystrophin protein in TA muscle of NCA2 loaded with PMO, d is the quantitative result of the recovery of dystrophin protein in WB; Figure 14 Fig. 13 is a diagram of the delivery capability of the nanobody NBVD1 / NBVD2 loaded with PMO in TA muscle, wherein a is the IHC result of the recovery of dystrophin protein in TA muscle of the nanobody NBVD1 / NBVD2 loaded with PMO, b is the quantitative column chart of the recovery of dystrophin protein in IHC; Figure 15 Fig. 14 is a diagram of the delivery capability of the nanobody NCRC2 / NCRF6 loaded with PMO in TA muscle, wherein a is the IHC result of the recovery of dystrophin protein in TA muscle of the nanobody NCRC2 / NCRF6 loaded with PMO, b is the quantitative column chart of the recovery of dystrophin protein in IHC; Figure 16Figure 6: Delivery ability of nanobody NSCE10 / NSCH6 loaded PMO in TA muscle, a is IHC result of dystrophin restoration in TA muscle of NSCE10 / NSCH6 loaded PMO, b is quantitative column chart of dystrophin restoration in IHC; Figure 17 Figure 7: Delivery result of nanobody NCA2 loaded anti-CTGF scFv protein, a is echocardiography result of mouse heart, b is ejection fraction and fractional shortening, c is Masson staining and hematoxylin-eosin staining result of heart tissue, d is WB chart of collagen expression in heart; Figure 18 Figure 8: Delivery result of nanobody NBVD1 / NBVD2 loaded anti-CTGF scFv protein, a is echocardiography result of mouse heart, b is ejection fraction, c is fractional shortening, d is Masson staining and hematoxylin-eosin staining result of heart tissue, e is quantitative chart of fibrosis area of heart tissue; Figure 19 Figure 9: Delivery result of nanobody NCRC2 / NCRF2 loaded anti-CTGF scFv protein, a is echocardiography result of mouse heart, b is ejection fraction, c is fractional shortening, d is Masson staining and hematoxylin-eosin staining result of heart tissue, e is quantitative chart of fibrosis area of heart tissue; Figure 20 Figure 10: Delivery result of nanobody NSCE10 / NSCH6 loaded anti-CTGF scFv protein, a is echocardiography result of mouse heart, b is ejection fraction, c is fractional shortening, d is Masson staining and hematoxylin-eosin staining result of heart tissue, e is quantitative chart of fibrosis area of heart tissue. DETAILED DESCRIPTION

[0021] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present application.

[0022] In a first aspect, the present application provides a tissue-targeting nanobody, which comprises a complementarity determining region consisting of CDR1, CDR2 and CDR3 sequences, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1-SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8-SEQ ID NO. 16, and CDR3 is shown as SEQ ID NO. 17-SEQ ID NO. 23; The nanobody comprises complementarity determining regions selected from any one of the following combinations: (a) SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 17; (b) SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 18; (c) SEQ ID NO. 2, SEQ ID NO. 10, SEQ ID NO. 19; (d) SEQ ID NO. 3, SEQ ID NO. 11, SEQ ID NO. 20; (e) SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 21; (f) SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 22; (g) SEQ ID NO. 6, SEQ ID NO. 14, SEQ ID NO. 21; (h) SEQ ID NO. 6, SEQ ID NO. 15, SEQ ID NO. 23; (i) SEQ ID NO. 7, SEQ ID NO. 16, SEQ ID NO. 19.

[0023] In some embodiments, the nanobody further comprises framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequence of FR1 is shown in SEQ ID NO. 24, the amino acid sequence of FR2 is shown in SEQ ID NO. 25, the amino acid sequence of FR3 is shown in SEQ ID NO. 26, and the amino acid sequence of FR4 is shown in SEQ ID NO. 27.

[0024] In a second aspect, the present application further provides a fusion protein comprising the above-mentioned nanobody and a heterologous molecule.

[0025] In some embodiments, the heterologous molecule is selected from any one or a combination of a reporter protein, a therapeutic protein or a nucleic acid binding protein.

[0026] In some embodiments, the reporter protein is mCherry, the therapeutic protein is anti-CTGF scFv, and the nucleic acid binding protein is monomeric streptavidin mSA.

[0027] In a third aspect, the present application provides a drug delivery system comprising the above-mentioned tissue-targeting nanobody and an active molecule.

[0028] In some embodiments, the active molecule is selected from the group consisting of a small molecule compound, a protein, a polypeptide, a nucleic acid, a viral vector, a lipid nanoparticle, or an imaging agent.

[0029] In a fourth aspect, the present application further provides a pharmaceutical composition comprising the above-mentioned tissue-targeting nanobody or the above-mentioned fusion protein.

[0030] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0031] In a fifth aspect, the present application further provides the above-mentioned enhanced drug delivery carrier or drug delivery system, wherein the enhanced drug delivery carrier is linked to or modified with the above-mentioned tissue-targeting nanobody or the above-mentioned fusion protein.

[0032] In a sixth aspect, the present application further provides use of the above-mentioned tissue-targeting nanobody or fusion protein in the preparation of a medicament for treating Duchenne muscular dystrophy.

[0033] The following will be described in conjunction with specific embodiments: Embodiment 1: Screening of heart and muscle-targeting nanobodies Membrane proteins with relatively high expression in heart and skeletal muscle were used as targets for screening of candidate nanobodies. The screening stringency was improved by reducing the amount of target protein in each round. Sequencing was used to obtain enriched amino acid sequences of nanobodies, and the targeting delivery ability of the candidate nanobodies was subsequently verified.

[0034] The specific steps are as follows: ① The candidate target proteins were expressed and purified using prokaryotic or eukaryotic 293T cells, and the target proteins were quantified using BSA. ② The candidate target proteins were used to bind and screen the nanobody library. The enriched nanobody library was subjected to secondary amplification, and the amount of target protein was reduced for the next round of screening until the recovery efficiency was no longer significantly enriched. ③ The obtained nanobodies were sequenced to obtain the amino acid sequences of the target nanobodies, which were compared with the database to verify the uniqueness of the sequences.

[0035] The screening of CACNA2D1-targeting nanobodies was performed for a total of 5 rounds, and the enrichment amount of each round was calculated as shown in Figure 1 a and Figure 1 b. The phages enriched in the 2nd, 3rd, and 4th rounds were subjected to high-throughput sequencing to obtain the 2 nanobodies with the highest proportion, which were converted into amino acid sequences and named NCA1 and NCA2.

[0036] The CDR1-CDR3 region amino acid sequences of NCA1 are as follows: RIFRRNT, TISRSGRINY, ADIRRGGGRAAAG; the CDR1-CDR3 region amino acid sequences of NCA2 are as follows: RIFRRNT, AITTSGGSTT, AASSRFGSSSARR; the FR1-FR4 region amino acid sequences of the nanobody are as follows: EVQLQASGGGFVQPGGSLRLSCAASG, MGWFRQAPGKEREFVSAIS, YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA, YWGQGTQVTVSS.

[0037] The screening of nanobodies targeting BVES was performed for a total of 3 rounds, and the recovery enrichment efficiency of each round was calculated. The nanobodies with high binding efficiency to the target protein BVES in the third round were detected by flow cytometry, and the amino acid sequences of the candidate nanobodies NBVD1 and NBVD2 were obtained by sequencing, as shown in Figure 2 a and Figure 2 b. The CDR1-CDR3 region amino acid sequences of NBVD1 are as follows: GSISANFTM, EFVAGIAYGTNTNY, AVAPVRQFNSKTYFDY; The CDR1-CDR3 region amino acid sequences of NBVD2 are as follows: GTIFVSQNM, EFVASITPGANTNY, AVTPTRDGAYLAPFYY.

[0038] The screening of nanobodies targeting CAR was performed for a total of 3 rounds, and the recovery enrichment efficiency of each round was calculated. The nanobodies with high binding efficiency to the target protein CAR in the third round were detected by flow cytometry, and the amino acid sequences of the candidate nanobodies NBVD1 and NBVD2 were obtained by sequencing, as shown in Figure 3 a and Figure 3 b. The CDR1-CDR3 region amino acid sequences of NCRC2 are as follows: GNIFFAPYM, EFVASIGSGTITNY, AVSTWYGHAHLY; the CDR1-CDR3 region amino acid sequences of NCRF6 are as follows: GYISVHSEM, ELVAGITRGGNTNY, AVYHYRSRWHGY.

[0039] The screening of nanobodies targeting SCN5A was performed for a total of 3 rounds, and the recovery enrichment efficiency of each round was calculated. The nanobodies with high binding efficiency to the target protein SCN5A in the third round were detected by flow cytometry, and the amino acid sequences of the candidate nanobodies NSCE9, NSCE10 and NSCH6 were obtained by sequencing, as shown in Figure 4 a and Figure 4b. The CDR1-CDR3 region amino acid sequences of NSCE9 are as follows: GYIFQYVYM, EFVAGINYGAITYY, AVSTWYGHAHLY; The CDR1-CDR3 region amino acid sequences of NSCE10 are as follows: GYIFQYVYM, ELVAGIDYGSTTNY, AVIGYDGPPVFAPHLY. The CDR1-CDR3 region amino acid sequences of NSCH6 are as follows: GNIFEPTWM, ELVAAISGGGNTYY, AVAPVRQFNSKTYFDY.

[0040] Example 2: Identification of tissue distribution of heart and muscle targeting nanobodies To confirm whether the candidate nanobodies can mediate the targeted delivery of mCherry protein to skeletal muscle, heart, the candidate nanobodies were expressed in fusion with mCherry protein, with NGFP as a negative control, and C57BL / 6J mice were injected with 20 μg / g dose of tail vein. Compared with Nb-GFP-mCherry, the candidate nanobodies that can target the delivery of proteins to the heart, skeletal muscle and brain were successfully obtained.

[0041] The specific steps are as follows: ① The candidate and nanobodies or NGFP and mCherry fusion proteins were expressed and purified by E. coli, and the protein concentration was quantified; ② The above proteins were injected into C57BL / 6J mice at a dose of 20 μg / g of tail vein, and circulated in vivo for 1 h; ③ The mouse heart H, gastrocnemius muscle G, quadriceps muscle Q, tibialis anterior muscle TA, triceps brachii muscle T, liver Li, kidney K tissues were taken, and the content of mCherry protein in different tissues of mice was detected by small animal imaging.

[0042] The CACNA2D1 target nanobodies were expressed in fusion with mCherry protein, and the fluorescence intensity in muscle and heart was detected by tail vein injection of 20 μg / g fusion protein. The small animal imaging detection showed that, compared with the control group, the NCA2 loaded mCherry protein was significantly enriched in the fluorescence intensity in the heart and gastrocnemius muscle and quadriceps muscle, and the experimental results were as shown in Figure 5 a- Figure 5 c.

[0043] The BVES target nanobodies were expressed in fusion with mCherry protein, and the mice were injected by tail vein. The small animal imaging detection showed that, compared with the control group, the NBVD1 / NBVD2 loaded mCherry protein was significantly enriched in the fluorescence intensity in the heart and skeletal muscle, and the experimental results were as shown in Figure 6 a-6c.

[0044] The mice were injected with the CAR-targeted nanobodies fused with mCherry protein via the tail vein, and small animal imaging detection showed that, compared with the control group, the fluorescence intensity of NCRC2 / NCRF6 loaded with mCherry protein was significantly enriched in the heart and skeletal muscle, and the experimental results were as shown in Figure 7 a-7c.

[0045] The mice were injected with the SCN5A-targeted nanobodies fused with mCherry protein via the tail vein, and small animal imaging detection showed that, compared with the control group, the fluorescence intensity of NSCE9 / NSCE10 / NSCH6 loaded with mCherry protein was significantly enriched in the heart and skeletal muscle, and the experimental results were as shown in Figure 8 a-8c.

[0046] Example 3: Affinity detection of nanobodies and target proteins To verify the binding of the candidate nanobodies to the corresponding target proteins, BLI was used for detection, and the results showed that the candidate nanobodies and the target proteins both had high affinity levels. The above results showed that the present patent successfully screened nanobodies that can bind to target proteins with high efficiency and can mediate the targeted delivery of fluorescent proteins to the heart and skeletal muscle.

[0047] The specific steps are as follows: ① Use E. coli or eukaryotic 293T cells to express and purify the target protein and perform protein quantification; ② Biotinylate the target protein or use His tag to fix it to the chip and perform blocking and solidification. Block for 180 s and solidify for 100 s. At the same time, use an empty chip without target protein as a negative control to detect whether there is non-specific binding between the empty chip and the nanobody; ③ Dilute the candidate nanobody by a factor of 15.63-1000 nM, and then perform binding and dissociation with the corresponding chip containing the target protein, with a binding time of 480 s and a dissociation time of 480 s. Finally, calculate the final binding force according to the KD values measured at different concentrations.

[0048] Nanobody targeting CACNA2D1: The target protein CACNA2D1 and the nanobody NCA2 protein were expressed and purified, respectively. The CACNA2D1 protein was biotinylated and fixed on the chip. BLI was used to detect the affinity of NCA2 to the target protein, and the results showed that the KD value was 86 pM, indicating that NCA2 had extremely high affinity to the target protein CACNA2D1, and the experimental results were as shown in Figure 9 .

[0049] Nanoantibody with BVES as target protein: the target protein BVES and nanoantibody NBVD1 and NBVD2 proteins were expressed and purified respectively, BVES was fixed on the chip by His tag, and the affinity of NBVD1 / NBVD2 and target protein was detected by BLI. The results showed that the KD value was 3.06 nM, 3.01 nM, and the experimental results of NBVD1 are shown in Figure 10 a, and the experimental results of NBVD2 are shown in Figure 10 b.

[0050] Nanoantibody with CAR as target protein: the target protein CAR and nanoantibody NCRC2 and NCRF6 proteins were expressed and purified respectively, CAR was fixed on the chip by His tag, and the affinity of NCRC2 and NCRF6 and target protein was detected by BLI. The results showed that the KD value was 33.4 nM, 44.8 nM, and the experimental results of NCRC2 are shown in Figure 11 a, and the experimental results of NCRF6 are shown in Figure 11 b.

[0051] Nanoantibody with SCN5A as target protein: the target protein SCN5A and nanoantibody NSCE9, NSCE10 and NSCH6 proteins were expressed and purified respectively, CAR was fixed on the chip by His tag, and the affinity of NSCE9, NSCE10 and NSCH6 and target protein was detected by BLI. The results showed that the KD value was 30.37 nM, 3.315 nM, 10.22 nM, and the experimental results of NSCE9 are shown in Figure 12 a, the experimental results of NSCE10 are shown in Figure 12 b, and the experimental results of NSCH6 are shown in Figure 12 c.

[0052] Example 4: Detection of the delivery ability of candidate nanoantibody loaded PMO To prove the delivery ability of candidate nanoantibody to mediate nucleic acid drugs, candidate nanoantibody was expressed and fused with monomeric streptavidin mSA respectively, and then incubated with biotinylated PMO to achieve the purpose of nanoantibody loading PMO. TA muscle injection containing 1 μg PMO complex on each side. IHC and WB results showed that candidate nanoantibody can promote the delivery ability of PMO, and significantly increase the recovery of dystrophin protein in TA muscle.

[0053] The specific steps are as follows: ① Purify Nb-mSA fusion protein by using E. coli, and quantify the protein concentration; ② Incubate Nb-mSA protein with biotinylated PMO at a molar ratio of 1:1 at 4°C for 2 h; ③ Inject the experimental group (Nb-mSA / PMO) and the control group (mSA / PMO) products into the left and right leg TA muscles of mdx mice respectively; ④ Take the material after 2 weeks, and detect the recovery of dystrophin protein in TA muscle by IHC or WB.

[0054] Immunohistochemistry (IHC) detection, steps as follows: ① Take the tissue frozen section out of -80℃, place it at room temperature, and immerse it in dPBS solution for 10 min to remove OCT; ② Blocking: use immunohistochemical pen to enclose the tissue, and block it with blocking solution at room temperature for 1 h; ③ Antibody incubation: dilute dystrophin antibody (1:200) with blocking solution according to the instructions, incubate at 4℃ overnight; after overnight, transfer the section to room temperature for 30 min, wash with dPBS for 3 times; dilute monkey anti-rabbit 594 fluorescent secondary antibody with blocking solution 1:500, incubate at room temperature for 1 h; after secondary antibody incubation, wash with dPBS for 3 times; ④ Mounting: mount with mounting agent containing DAPI, avoid air bubbles, store the section in the dark, and take photos under a microscope.

[0055] WB detection, steps as follows: ① Cut the TA tissue stored in frozen state with a frozen section machine and store it in a nuclease-free centrifuge tube; ② Take 100 μl of 5% β-mercaptoethanol protein lysate and place it in the centrifuge tube, and crush the tissue thoroughly; ③ Incubate on ice for 30 min, heat with a metal bath at 100℃ for 10 min, centrifuge at 12000 rpm / min to take the supernatant, and determine the protein concentration in the supernatant; ④ Take 100 μg of tissue and add loading to prepare the sample and heat it with a metal bath for 10 min; ⑤ Use 6% separation gel electrophoresis, voltage is 80 V, 30 min, then 120 V, 120 min, stop when the loading reaches the bottom of the gel plate; ⑥ After electrophoresis, place the PVDF membrane and the separation gel in the "black gel white membrane" mode, transfer the membrane at 110 mA constant current at 4℃ overnight; block with 5% skim milk for about 2 h ⑦ Cut the PVDF membrane according to different protein bands, and incubate different antibodies in plastic bags, 4℃ shaking bed overnight (antibody dilution ratio: dystrophin: 1:200 / α-actinin: 1:3000; 5% skim milk wash primary antibody, 10 min / time, 3 times; secondary antibody incubation 2 h (goat anti mouse: 1:5000); PBST wash 3 times, 10 min each time; ⑧ Add the light-emitting liquid drop by drop in the dark room using film exposure, and develop and fix.

[0056] The nanobody NCA2 was fused with monomeric streptavidin mSA and co-incubated with biotinylated PMO, and TA muscle of mdx mice was injected with mSA alone as negative control. WB and IHC results showed that NCA2 could significantly promote the recovery efficiency of PMO in dystrophin protein of TA muscle, and the experimental results were as shown in Figure 13 a- Figure 13 d.

[0057] The nanobody NBVD1 / NBVD2 was fused with monomeric streptavidin mSA and co-incubated with biotinylated PMO and injected into TA muscle. IHC results showed that compared with the mdx control group, NBVD1 / NBVD2 could significantly promote the recovery of dystrophin protein in TA muscle, and the experimental results were as shown in Figure 14 a- Figure 14 b.

[0058] The nanobody NCRC2 / NCRF6 was fused with monomeric streptavidin mSA and co-incubated with biotinylated PMO and injected into TA muscle. IHC results showed that compared with the mdx control group, NCRC2 / NCRF6 could significantly promote the recovery of dystrophin protein in TA muscle, and the experimental results were as shown in Figure 15 a- Figure 15 b.

[0059] The nanobody NSCE10 / NSCH6 was fused with monomeric streptavidin mSA and co-incubated with biotinylated PMO and injected into TA muscle. IHC results showed that compared with the mdx control group, NSCE10 / NSCH6 could significantly promote the recovery of dystrophin protein in TA muscle, and the experimental results were as shown in Figure 16 a- Figure 16 b.

[0060] Example 5: Detection of the delivery ability of candidate nanobody loaded with anti-CTGF scFv protein To test the therapeutic effect of candidate nanobody-mediated anti-CTGF scFv protein in a disease model, candidate nanobody was fused with anti-CTGF scFv for expression, and doxorubicin-induced mdx mouse cardiac fibrosis model was treated. Compared with the NGFP control group, candidate nanobody delivery of anti-CTGF scFv protein can significantly maintain the cardiac function of mice and inhibit the progression of cardiac fibrosis in mice The specific operation is as follows: ① DOX was injected into 12-week-old (25 g) mdx male mice at a dose of 5 mg / kg once a week for 4 consecutive weeks; ② Treatment started 2 days after DOX injection, and each group was injected with fusion-expressed therapeutic protein 3 times a week via tail vein at a dose of 4 mg / kg for 4 consecutive weeks; ③ After 4 weeks, the cardiac function of mice was determined using a mouse ultrahigh-resolution small animal ultrasound imaging system, and the degree of cardiac fibrosis of mice was detected using masson and WB.

[0061] Cardiac ultrasound detection, the steps are as follows: ① Anesthetize the mouse: the mouse is depilated in the thoracic cavity one day in advance, anesthetized with isoflurane, then fixed on a flat plate with four feet, and the coupling agent is applied between the feet and the plate. Connect the ECG line on the plate, adjust the anesthetic and oxygen flow to adjust the heart rate of the mouse to be in the range of 400-450; ② Measure the long axis of the heart: place the flat plate horizontally, the ultrasound probe is parallel to the heart, at an angle of 45 degrees, fine-tune the x-axis to ensure that the apex of the heart is visible, the outflow tract is unobstructed, and there is no artifact. Click Save to save; ③ Measure the short axis of the heart: rotate the probe 90 degrees (perpendicular to the heart) and measure. Click Save Clip to save.

[0062] Masson staining detection, the steps are as follows: ① De-waxing: immerse the paraffin section in xylene for 30 min, then sequentially immerse in 100%, 95%, 80% and 70% ethanol for 5 min each time; ② A1 and A2 reagents are configured as Weigert iron hematoxylin staining solution at a ratio of 1:1, dropped onto the section for 10 min, and washed with distilled water until no obvious fading; ③ Lichunhong staining solution was used for staining for 10 min, and distilled water was used for washing until no obvious fading; ④ Add phosphomolybdic acid solution for staining for 2 min, discard the solution, and directly add aniline blue solution for staining for 1.5 min, and wash with distilled water until no obvious fading; ⑤ 95% ethanol, anhydrous ethanol, xylene transparency for 5 min, then neutral resin mounting, and microscopic photography to calculate the fibrosis area.

[0063] WB detection, the steps are as follows: ① The frozen heart tissue is sliced using a frozen section machine and stored in a nuclease-free centrifuge tube; ② 100 μL of 5% β-mercaptoethanol protein lysate is added to the centrifuge tube and the tissue is thoroughly crushed; ③ Incubate on ice for 30 min, add 10 min using a metal bath at 100°C, centrifuge at 12000 rpm / min to obtain the supernatant, and determine the protein concentration in the supernatant; ④ Add 100-200 μg of tissue to the loading preparation sample and heat using a metal bath for 10 min; ⑤ Use 8% separation gel electrophoresis, voltage 80 V, 30 min, then 120 V, 120 min, stop when the loading reaches the bottom of the gel plate; 250 mA constant current, 4°C, transfer membrane for 2 h; 5% skim milk blocking for 1 h; ⑥ Cut the PVDF membrane according to different protein bands, and incubate different antibodies in plastic bags, 4°C shaking overnight (antibody dilution ratio: collagen I: 1:2000 / collagen III: 1:2000; 5% skim milk wash the primary antibody, 10 min / time, 3 times; ⑧ Put the PVDF membrane into a plastic bag, incubate the secondary antibody for 2 h (goat anti rabbit: 1:5000); wash with PBST for 3 times, 10 min each time; ⑨ After adding the luminescence solution, expose the film in a dark room, and develop and fix.

[0064] Nanobody NCA2 is fused with anti-CTGF scFv, NGFP is used as a negative control, and mdx mice induced by doxorubicin are treated for 4 weeks. After small animal ultrasound detection, the results show that NCA2-anti-CTGF scFv improves the decline of cardiac function of mdx mice caused by DOX and inhibits the fibrosis process of the heart. The experimental results are shown in Figure 17 a- Figure 17 d.

[0065] Nanobody NBVD1 / NBVD2 is fused with anti-CTGF scFv, and the mice after treatment are detected by small animal ultrasound. The results show that NBVD1 / NBVD2-anti-CTGF scFv improves the decline of cardiac function of mdx mice and inhibits the fibrosis process of the heart. The experimental results are shown in Figure 18 a- Figure 18 e.

[0066] The nanobody NCRC2 / NCRF2 is fused with anti CTGF scFv for expression, and the mice after treatment are detected by small animal ultrasound, the results show that NCRC2 / NCRF2-anti CTGF scFv improves the decline of cardiac function of mdx mice, and inhibits the fibrosis process of the heart, and the experimental results are as shown in Figure 19 a-19e.

[0067] The nanobody NSCE10 / NSCH6 is fused with anti CTGF scFv for expression, and the mice after treatment are detected by small animal ultrasound, the results show that NSCE10 / NSCH6-anti CTGF scFv improves the decline of cardiac function of mdx mice, and inhibits the fibrosis process of the heart, and the experimental results are as shown in Figure 20 a-20e.

[0068] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present application.

Claims

1. A tissue-targeted nanobody, characterized in that, The nanobody comprises complementarity determining regions consisting of CDR1, CDR2 and CDR3 sequences, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1-SEQ ID NO. 7, the amino acid sequence of CDR2 is shown as SEQ ID NO. 8-SEQ ID NO. 16, and CDR3 is shown as SEQ ID NO. 17-SEQ ID NO. 23; The nanobody comprises complementarity determining regions selected from any one of the following combinations: (a) SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 17; (b) SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 18; (c) SEQ ID NO. 2, SEQ ID NO. 10, SEQ ID NO. 19; (d) SEQ ID NO. 3, SEQ ID NO. 11, SEQ ID NO. 20; (e) SEQ ID NO. 4, SEQ ID NO. 12, SEQ ID NO. 21; (f) SEQ ID NO. 5, SEQ ID NO. 13, SEQ ID NO. 22; (g) SEQ ID NO. 6, SEQ ID NO. 14, SEQ ID NO. 21; (h) SEQ ID NO. 6, SEQ ID NO. 15, SEQ ID NO. 23; (i) SEQ ID NO. 7, SEQ ID NO. 16, SEQ ID NO.

19.

2. The tissue-targeted nanobody of claim 1, wherein, The nanobody further comprises framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequence of FR1 is shown as SEQ ID NO. 24, the amino acid sequence of FR2 is shown as SEQ ID NO. 25, the amino acid sequence of FR3 is shown as SEQ ID NO. 26, and the amino acid sequence of FR4 is shown as SEQ ID NO.

27.

3. A fusion protein, characterized in that, The fusion protein comprises the tissue-targeting nanobody of any one of claims 1-2 and a heterologous molecule.

4. A drug delivery system characterized by, The fusion protein comprises the tissue-targeting nanobody of any one of claims 1-2 and an active molecule.

5. The drug delivery system of claim 4, wherein, The active molecule is selected from the group consisting of a small molecule compound, a protein, a polypeptide, a nucleic acid, a viral vector, a lipid nanoparticle, or an imaging agent.

6. A pharmaceutical composition, characterized by, The fusion protein of claim 3.

7. A disease diagnostic reagent, characterized by comprising the polypeptide of claim 1 or 2. The fusion protein of claim 3.

8. The disease diagnostic reagent according to claim 7, characterized by, The disease is any one of a brain disease, a muscle disease, or a heart disease.

9. An enhanced drug delivery vehicle or drug delivery system, characterized in that, The enhanced drug delivery vehicle is linked or modified with the tissue-targeting nanobody of any one of claims 1-2, or the fusion protein of claim 3.

10. Use of a tissue-targeting nanobody according to any one of claims 1-2, or a fusion protein according to claim 3, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for the treatment of Duchenne Muscular Dystrophy.

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