Sortilin 1-specific nanoantibody, recombinant AAV containing same, and use

Abstract

A sortilin 1-specific nanoantibody, a recombinant AAV containing same, and a use, relating to the technical field of biology. Provided are a plurality of sortilin 1 nanoantibodies which are significantly different from existing sortilin 1 monoclonal antibodies AL001 and have better clinical application prospects. Experimental results show that the obtained anti-sortilin 1 nanoantibodies can effectively bind to sortilin 1 on a cell surface and be efficiently endocytosed into cells, and have the potential to be developed into antibody-drug conjugates targeting sortilin 1. More importantly, the plurality of obtained nanoantibodies can inhibit the function of sortilin 1 by means of AAV endogenous expression and significantly improve the level of progranulin in an experimental animal body, and have the ability to treat various neurodegenerative diseases and tumors.

Description

A nanobody specific for sortilin 1, a recombinant AAV containing the same, and its application Technical Field

[0001] The present invention relates to a sortilin 1-specific nanobody, a recombinant AAV containing the nanobody and applications thereof, and belongs to the field of biotechnology. Background Art

[0002] Sortilin (Sort1), a member of the vacuolar sorting factor 10 (VPS10P) domain receptor family, acts as a cellular receptor or co-receptor, mediating the targeted trafficking of various proteins into the cell. The N-terminus of Sortilin 1 is located in the extracellular domain, while the C-terminus is located in the intracellular domain, with a transmembrane structure connecting the extracellular and intracellular regions. Sortilin 1 is widely distributed on the cell membrane but can also be found within the cell in locations such as the endoplasmic reticulum, Golgi apparatus, and early endocytic vesicles. Sortilin 1 is widely expressed in various tissues and organs, with particularly high expression levels in the nervous system. It is expressed in multiple regions of the brain, including the cortex, hippocampus, striatum, and cerebellum. It is also expressed in various tissues and organs, including the heart, liver, lungs, kidneys, intestine, and immune system. Sortilin 1 plays an important role in intracellular protein trafficking, neural development, and metabolic regulation. First, Sortilin 1 is involved in the localization and transport of intracellular proteins. It can bind to a variety of proteins and ligands, such as nerve growth factor, neurotrophic factor, and glycoproteins, and bring them from the cell surface into the endoplasmic reticulum and transport vesicle system through endocytosis, where they participate in their localization and transport. Second, Sortilin 1 plays a crucial role in the nervous system, regulating neuronal growth and development and implicated in neurodegenerative diseases. Sortilin 1 interacts with multiple signaling pathways in neurons, regulating cell survival, apoptosis, and differentiation. It is also closely related to neural functions such as synaptic transmission, synaptic plasticity, and memory formation. Furthermore, Sortilin 1 plays a crucial role in metabolic regulation, participating in the regulation of cholesterol metabolism and insulin release. It regulates cholesterol endocytosis and transport by binding to low-density lipoprotein receptor-related protein (LRP). In pancreatic islet cells, Sortilin 1 participates in the maturation and secretion of insulin granules.

[0003] Tumor treatment is one aspect of the clinical application research of Sort1 antibodies. Studies have shown that sortilin 1 plays an important role in tumorigenesis. For example, in the development of liver cancer, sortilin 1 can affect the secretome of cancer cells, thereby affecting the fat generation process and the energy supply required for cancer cell proliferation. Studies have shown that overexpression of Sort1 protein has been found in refractory solid tumors including prostate cancer, ovarian cancer, triple-negative breast cancer, skin cancer, lung cancer, colorectal cancer, head and neck cancer, and pancreatic cancer; and the higher the Sort1 expression level, the worse the prognosis of the related disease. These findings suggest that by specifically targeting Sort1, it may be possible to accurately identify these tumor tissues and even develop new treatments for these solid tumors. For example, anti-Sort1 monoclonal antibodies with effector functions can efficiently target cancer cells with high Sort1 expression, activating immune cells to specifically eliminate lesions through processes such as antibody-dependent cell-mediated cytotoxicity (ADCC). Alternatively, antibody molecules can be conjugated to killing molecules such as chemotherapy drugs and radioisotopes, allowing these drugs to enter cancer cells with high Sort1 expression through antibody-mediated endocytosis, thereby targetedly killing these refractory cancer cells. Currently, the Canadian biotechnology company Thera Technologies Inc. has conjugated the chemotherapy drug docetaxel to a peptide molecule targeting Sort1. This peptide-drug conjugate (PDC), named TH1902, has entered clinical trials for multiple solid tumors. Early clinical results show that this peptide-drug conjugate can slow disease progression, increase immune cell infiltration, and inhibit angiogenesis, etc., demonstrating good effectiveness.

[0004] Another area of ​​clinical application currently under investigation is the role of Sortilin 1 in regulating progranulin (PGRN) levels, thereby influencing nervous system function. Progranulin (PGRN) is an extracellular secretory protein composed of 588 amino acids. It is widely distributed throughout the human body and is particularly abundant in tissues and cells such as the central nervous system, immune system, endocrine system, and kidneys. It plays an important role in regulating inflammation, neuroprotection, and tumor development. During neuroprotection and neural development, progranulin promotes neuronal growth, differentiation, and survival, and participates in synapse formation and neurotransmission regulation. Progranulin deficiency may lead to increased inflammatory responses, triggering toxic damage to neurons, interfering with neuronal growth and synaptic formation, and causing neural network dysfunction. Progranulin deficiency may also lead to abnormal autophagy and disrupted protein metabolism, further impairing cellular function.

[0005] Sortilin 1 is a major degradation pathway for progranulin. The extracellular domain of sortilin 1 binds to progranulin, mediated by endocytosis, bringing progranulin from the extracellular space into the cell interior. Through association with vesicles and lysosomes, sortilin 1 forces progranulin into the degradation pathway, thereby reducing its extracellular concentration. Decreased progranulin levels can lead to increased inflammation, toxic damage to neurons, and disruption of neuronal growth and synaptic formation, leading to neural network dysfunction. Accordingly, inhibiting sortilin 1 can reduce progranulin degradation, thereby increasing both intracellular and extracellular progranulin levels. Abnormal sortilin 1 function or altered sortilin 1 expression levels can lead to abnormal changes in progranulin levels. Inhibiting sortilin 1 can prevent abnormal progranulin degradation, thereby increasing the availability of progranulin. Studies have shown that reduced levels of progranulin are associated with the onset and progression of neurodegenerative diseases such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). Therefore, inhibiting Sortilin-1 to increase progranulin levels may help restore the physiological function of progranulin, potentially offering therapeutic potential for FTD, ALS, PD, AD, and other related diseases.

[0006] Using antibodies to inhibit Sortilin 1 function is a potential strategy for increasing progranulin levels. As specific molecular tools, antibodies can specifically bind to Sortilin 1 and affect its binding to Sortilin 1, thereby blocking its degradation pathway. This strategy is expected to increase the biological activity and functional expression of progranulin, potentially benefiting the treatment of related diseases. Several pharmaceutical and biotechnology companies worldwide, including Lundbeck, Sanofi, Takeda, and GSK, are investing in the development of Sortilin 1 antibodies. Latozinemab (AL001), a monoclonal antibody developed by the US biotechnology company Alector Therapeutics, has entered Phase III clinical trials for FTD, suggesting that Sortilin 1 antibodies have therapeutic potential for various neurodegenerative diseases. Despite this, no Sortilin 1 antibodies have been approved for marketing globally, necessitating the development of new, differentiated, and clinically suitable Sortilin 1 antibodies.

[0007] The development and application of conventional IgG antibodies present numerous challenges, such as long R&D cycles, high production costs, poor stability, and high immunogenicity, which limit their clinical application. In 1993, Hamers et al. reported the presence of a naturally occurring heavy-chain antibody in camels that lacks both the light chain and the heavy chain constant region 1 (CH1). Cloning the variable region yielded a single-domain antibody consisting solely of a single heavy chain variable region, known as a VHH (variable domain of heavy chain antibody), now known as a "nanobody" (Nb). With a molecular weight of approximately 15 kDa, nanobodies are the smallest antigen-binding fragments with complete binding function. They are oval in shape, 2.5 nm in diameter and 4 nm in length. Compared to conventional IgG monoclonal antibodies, nanobodies, due to their small size and excellent permeability, can more easily cross tissue barriers, increasing the local concentration of therapeutic drugs. Due to their small size and single variable domain structure, nanobodies have a relatively low risk of triggering an immune response in humans, reducing the risk of immune-related issues associated with their use in patients and increasing their acceptability for long-term treatment. Nanobodies have excellent environmental tolerance, stable conformation, and are easy to synthesize. These unique properties make them particularly valuable for applications in disease diagnosis and targeted therapy. Unfortunately, due to their small size, nanobodies cannot be retained in the blood during glomerular filtration and are easily metabolized and cleared from the body. This results in a very short half-life after administration, making it difficult to maintain effective drug concentrations in the body, thus limiting their effectiveness and feasibility for widespread application. To extend the half-life of nanobodies in vivo, antibody researchers have employed various approaches, such as polyethylene glycol modification, engineered Fc fragment fusion, antibody fragment splicing, and other fusion protein splicing. While these approaches have somewhat extended the half-life of nanobodies, they also increase the size and molecular weight of the drug molecule, which in turn affects their tissue penetration. Furthermore, these modifications increase the production cost of nanobody drugs and may lead to higher immunogenicity, thus negating their key advantage as the smallest antigen-binding fragment. The pharmaceutical research community continues to search for methods that can effectively maintain drug concentrations in the body without altering their fundamental molecular characteristics.

[0008] In summary, despite the inherent advantages of nanobodies, such as high specificity and ease of production, the performance of nanobodies against sort1 needs to be further improved. To date, there have been no reports of using nanobodies to target sort1 for disease treatment. This is primarily due to two factors: first, the short half-life of nanobodies alone prevents exogenous administration from achieving ideal in vivo drug concentrations, making it difficult to achieve long-term therapeutic effects for neurodegenerative diseases. Second, research on sort1 as a tumor target has only recently begun, with functional validation of peptide-drug conjugates only recently completed, and subsequent antibody-based anti-tumor studies have yet to be reported.

[0009] Summary of the Invention

[0010] To address the above issues, the present invention provides anti-sortilin-1 Nanobodies and their applications. The goal is to provide multiple Sortilin-1 antibodies that are significantly differentiated from the current Sortilin-1 monoclonal antibody AL001 and have improved clinical application prospects. The antibodies of the present invention have broad biological and clinical applications, encompassing the diagnosis and treatment of Sortilin-1-related diseases, basic medical research, biological research, and other fields.

[0011] The first object of the present invention is to provide a Nanobody specific for Sortilin 1, wherein the heavy chain variable region of the Nanobody comprises complementarity determining regions CDR1, CDR2, and CDR3, and the complementarity determining regions comprise any combination of the following or a sequence with at least 90% homology thereto:

[0012] 1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;

[0013] 2) CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 9, and CDR3 shown in SEQ ID NO. 10;

[0014] 3) CDR1 shown in SEQ ID NO. 15, CDR2 shown in SEQ ID NO. 16, and CDR3 shown in SEQ ID NO. 17;

[0015] 4) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23, and CDR3 shown in SEQ ID NO. 24;

[0016] 5) CDR1 shown in SEQ ID NO. 29, CDR2 shown in SEQ ID NO. 30, and CDR3 shown in SEQ ID NO. 31;

[0017] 6) CDR1 shown in SEQ ID NO. 36, CDR2 shown in SEQ ID NO. 37, and CDR3 shown in SEQ ID NO. 38;

[0018] 7) CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44, and CDR3 shown in SEQ ID NO. 45;

[0019] 8) CDR1 shown in SEQ ID NO. 50, CDR2 shown in SEQ ID NO. 51, and CDR3 shown in SEQ ID NO. 52;

[0020] 9) CDR1 shown in SEQ ID NO. 57, CDR2 shown in SEQ ID NO. 58, and CDR3 shown in SEQ ID NO. 59;

[0021] 10) CDR1 shown in SEQ ID NO. 120, CDR2 shown in SEQ ID NO. 121, and CDR3 shown in SEQ ID NO. 122.

[0022] Furthermore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and optimally more than 98%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the above-mentioned CDR sequences.

[0023] Furthermore, the sequence with a homology of not less than 90% is obtained by amino acid substitution of CDR1, CDR2 and / or CDR3, and the amino acid substitution is selected from one or more of the following:

[0024] 1) Alanine is replaced by valine, leucine or isoleucine,

[0025] 2) Arginine is replaced by lysine, glutamine or asparagine,

[0026] 3) Asparagine is replaced by glutamine, histidine, lysine or arginine,

[0027] 4) Aspartic acid is replaced by glutamic acid,

[0028] 5) Cysteine ​​is replaced by serine,

[0029] 6) Glutamine is replaced by asparagine,

[0030] 7) Glutamic acid is replaced by aspartic acid,

[0031] 8) Glycine is replaced by proline or alanine,

[0032] 9) Histidine is replaced by asparagine, glutamine, lysine or arginine,

[0033] 10) Isoleucine is replaced by leucine, valine, methionine, alanine or phenylalanine,

[0034] 11) Leucine is replaced by isoleucine, valine, methionine, alanine or phenylalanine,

[0035] 12) Lysine is replaced by arginine, glutamine or asparagine,

[0036] 13) Methionine is replaced by leucine, phenylalanine or isoleucine,

[0037] 14) Phenylalanine is replaced by leucine, valine, isoleucine, alanine or tyrosine,

[0038] 15) Proline is replaced by alanine or glycine,

[0039] 16) Serine is replaced by threonine,

[0040] 17) threonine is replaced by serine,

[0041] 18) Tryptophan is replaced by tyrosine or phenylalanine,

[0042] 19) Tyrosine is replaced by tryptophan, phenylalanine, threonine or serine,

[0043] 20) Valine is replaced by isoleucine, leucine, methionine, phenylalanine or alanine.

[0044] Furthermore, the variable region of the Nanobody comprises framework regions FR1, FR2, FR3, and FR4, with complementarity determining regions (CDR1, CDR2, and CDR3) disposed between adjacent framework regions. Thus, the Nanobody is provided with FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in the order described.

[0045] Furthermore, the framework region includes any one of the following or a sequence having a homology of not less than 90% thereto:

[0046] 1) FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6, and FR4 shown in SEQ ID NO. 7;

[0047] 2) FR1 shown in SEQ ID NO.11, FR2 shown in SEQ ID NO.12, FR3 shown in SEQ ID NO.13, and FR4 shown in SEQ ID NO.14;

[0048] 3) FR1 shown in SEQ ID NO. 18, FR2 shown in SEQ ID NO. 19, FR3 shown in SEQ ID NO. 20, and FR4 shown in SEQ ID NO. 21;

[0049] 4) FR1 shown in SEQ ID NO. 25, FR2 shown in SEQ ID NO. 26, FR3 shown in SEQ ID NO. 27, and FR4 shown in SEQ ID NO. 28;

[0050] 5) FR1 shown in SEQ ID NO. 32, FR2 shown in SEQ ID NO. 33, FR3 shown in SEQ ID NO. 34, and FR4 shown in SEQ ID NO. 35;

[0051] 6) FR1 shown in SEQ ID NO.39, FR2 shown in SEQ ID NO.40, FR3 shown in SEQ ID NO.41, and FR4 shown in SEQ ID NO.42;

[0052] 7) FR1 shown in SEQ ID NO.46, FR2 shown in SEQ ID NO.47, FR3 shown in SEQ ID NO.48, and FR4 shown in SEQ ID NO.49;

[0053] 8) FR1 shown in SEQ ID NO.53, FR2 shown in SEQ ID NO.54, FR3 shown in SEQ ID NO.55, and FR4 shown in SEQ ID NO.56;

[0054] 9) FR1 shown in SEQ ID NO. 60, FR2 shown in SEQ ID NO. 61, FR3 shown in SEQ ID NO. 62, and FR4 shown in SEQ ID NO. 63;

[0055] 10) FR1 shown in SEQ ID NO. 123, FR2 shown in SEQ ID NO. 124, FR3 shown in SEQ ID NO. 125, and FR4 shown in SEQ ID NO. 126.

[0056] Furthermore, the Nanobody contains a sequence as shown in any one of SEQ ID NO.148-SEQ ID NO.156 and SEQ ID NO.165.

[0057] A second object of the present invention is to provide a polynucleotide encoding the above-mentioned Nanobody.

[0058] The third object of the present invention is to provide a recombinant adeno-associated virus (AAV) expression vector containing the above-mentioned polynucleotide and used for endogenously expressing anti-sortilin 1 nanobody.

[0059] Furthermore, the recombinant expression vector is an optimized recombinant adeno-associated virus vector, which is used to increase the expression level of nanobodies endogenously expressed by the recombinant adenovirus. The expression elements contained therein include but are not limited to: a promoter, a polyA tail, etc. required for the gene expression unit, and a signal peptide may also be set before the polynucleotide. Preferably, it contains a CMV promoter or a CAG promoter (including a CMV enhancer and a chicken β-actin promoter), a BM40 signal peptide, a polynucleotide and an SV40 polyA tail, and does not contain introns, the CAG promoter and the BM40 signal peptide are located upstream of the polynucleotide, and the SV40 polyA tail is located downstream of the polynucleotide.

[0060] Furthermore, the recombinant adeno-associated virus (AAV) vector contains inverted terminal repeats (ITRs) for AAV packaging. These ITRs are linked upstream of the CAG promoter and downstream of the SV40 polyA tail, with the two ITRs being identical but oriented in opposite directions. The recombinant vector can then be packaged with AAV to produce a gene therapy product.

[0061] Furthermore, the sequence of the CAG promoter is shown in SEQ ID NO.169, the sequence of the BM40 signal peptide is shown in SEQ ID NO.170, the amino acid sequence encoded by it is shown in SEQ ID NO.171, the sequence of the SV40 polyA tail is shown in SEQ ID NO.172, and the sequence of the inverted terminal repeat sequence is shown in SEQ ID NO.173. Specifically,

[0062] Furthermore, adeno-associated viruses used to express anti-sortilin-1 nanobodies in vivo can be selected from a variety of viral serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAVrh10, AAVrh20, AAVrh32, AAVrh33, AAVrh34, AAVrh39, AAVrh43, AAVrh51, AAVrh52, AAVrh53, AAVrh58, AAVrh61, AAVrh64 and other commonly used serotypes, as well as other engineered adeno-associated virus serotypes.

[0063] The fourth object of the present invention is to provide a host cell containing the above-mentioned recombinant expression vector.

[0064] Furthermore, the host cell may be a prokaryotic cell or a eukaryotic cell, such as a plant cell, an animal cell, a microorganism, etc.

[0065] A fifth object of the present invention is to provide a monovalent antibody, a bivalent antibody, a multivalent antibody or a recombinant protein containing the above-mentioned Nanobody.

[0066] Furthermore, the recombinant protein contains

[0067] (a) an anti-sortilin-1 Nanobody, or a bivalent or multivalent antibody as described above;

[0068] (b) Tag sequences that facilitate expression and / or purification.

[0069] Preferably, the tag sequence includes an Fc tag, an HA tag, or a 6xHis tag. For example, if the fusion protein is formed with an Fc fragment, the structure of the fusion protein from the N-terminus to the C-terminus is as shown in Formula Ia or Ib:

[0070] ALB(Ia),

[0071] BLA(Ib),

[0072] Wherein A is the anti-sortilin-1 nanobody described above, B is an IgG Fc fragment, and L is either null or a flexible linker. Preferably, the flexible linker is a peptide linker, more preferably, the peptide linker has 1-20 amino acids. The IgG Fc fragment includes a human IgG Fc fragment or an engineered mutant thereof, and the IgG Fc fragment is selected from the Fc fragments of IgG1, IgG2, IgG3, IgG4, or a combination thereof.

[0073] The sixth object of the present invention is to provide an antibody-drug conjugate (ADC) containing the above-mentioned nanobody.

[0074] Furthermore, the antibody drug conjugate contains:

[0075] (a) Anti-sortilin-1 nanobody, or the above-mentioned recombinant protein, bivalent or multivalent antibody, for binding the drug to target cells with high expression of sortilin-1;

[0076] (b) Cytotoxic payloads. These payloads become highly active drugs after hydrolysis and cleavage, which can effectively kill target cells or inhibit the function of target cells. Commonly used cytotoxic payloads include various DNA-damaging drugs, microtubule inhibitors, cytokines, radionuclides, or RNA payloads such as siRNA and miRNA.

[0077] (c) Linkers used to link antibodies and toxic payloads. When the antibody-drug conjugate reaches the cell, the ADC enters the cell through endocytosis, and the connection between the linker and the cytotoxic payload is cut by hydrolysis or other means, thereby releasing the cytotoxic substance so that it can perform its function. There are two types of linkers: cleavable linkers and non-cleavable linkers.

[0078] A seventh object of the present invention is to provide the use of the aforementioned Nanobodies, polynucleotides, recombinant expression vectors, host cells, monovalent antibodies, bivalent antibodies, multivalent antibodies, recombinant proteins, or antibody-drug conjugates in the preparation of diagnostic, preventive, or therapeutic products. The forms of such products include, but are not limited to, pharmaceutical agents, reagents, test panels, and kits.

[0079] Furthermore, the product is used to detect the level of sortilin 1 molecules, target cells with high expression of sortilin 1, inhibit the function of sortilin 1 or increase the level of progranulin (in vitro or in vivo), specifically, to block the interaction between progranulin and sortilin 1. Of course, those skilled in the art will know that various pharmaceutical carriers can be used to deliver nanobodies in vivo, such as the strategy of expressing sortilin 1 nanobodies using AAV viral vectors or LNPs, for endogenous expression of sortilin 1 nanobodies in vivo.

[0080] Furthermore, the detection includes flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay (ELISA) detection, etc.

[0081] Furthermore, the drug is a drug for preventing or treating tumors, and can be used to prevent or treat various solid tumors and non-solid tumors, such as colon cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, kidney cancer, malignant melanoma, gallbladder cancer, brain glioma, pancreatic cancer, etc.

[0082] Furthermore, the drug can also be a drug for preventing or treating central nervous system diseases, such as neurodegenerative diseases. Diseases that can be prevented or treated by the drug include, but are not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD), depression, neuropsyciatric disorder, vascular dementia, seizures, retinal dystrophy, age-related macular degeneration, glaucoma, traumatic brain injury, aging, wound healing, stroke, arthritis, atherosclerosis, etc.

[0083] Beneficial effects of the present invention:

[0084] (1) The inventors have successfully obtained multiple anti-human sortilin 1 nanoantibodies through extensive and in-depth research and a large number of screenings. Specifically, the present invention uses human sortilin 1 extracellular segment antigen protein to immunize camels to obtain a high-quality immune nanoantibody gene library. Then, the sortilin 1 protein molecule is coupled to an ELISA plate, and the immune nanoantibody gene library (camel heavy chain antibody phage display gene library) is screened using this form of antigen using phage display technology, thereby obtaining a sortilin 1-specific nanoantibody gene. The antibodies of the present invention can block the interaction between sortilin 1 and granule protein precursor on the cell surface, with high affinity and strong specificity; they can effectively bind to sortilin 1 protein, and their binding activity is similar to that of the control antibody AL001. In addition, relevant experimental results show that the multiple nanoantibodies obtained in the present invention can significantly increase the level of granule protein precursor in the U251 cell culture medium, and the ability to increase the level of extracellular granule protein precursor is close to or better than the control antibody AL001, and have the potential to treat a variety of neurodegenerative diseases.

[0085] (2) The present invention screens and optimizes the elements (including promoters, introns, signal peptides, polyA, etc.) on the nanobody recombinant expression vector to obtain a vector that can enhance the expression level of nanobodies. The construction process of this expression vector is simple, and it can effectively enhance the expression level of nanobodies in mammals, helping nanobodies to be expressed continuously and efficiently in vivo, thereby solving the problem of low in vivo concentration due to short half-life currently faced by nanobody drugs. The AAV delivery gene based on this expression element combination has an expression level that is more than 10 times higher than other preferred combinations and more than 20 times higher than commonly used expression element combinations, and can maintain a high level of nanobodies in serum for a long time.

[0086] (3) The present invention uses an optimized AAV vector to highly express anti-sortilin 1 nanobodies in vivo, successfully resolving the problem of short half-life of nanoantibody drugs when administered exogenously, and achieving effective pharmacokinetic function. In vivo animal experiments have shown that a single AAV injection can consistently increase the level of progranulin in the serum and cerebrospinal fluid of humanized disease models. Given that there are currently no reports of using AAV to express nanoantibodies to treat or prevent diseases, the present invention has the potential to be developed into a new gene therapy drug for the treatment of various neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG1 is the SDS-PAGE detection result of the recombinant protein in Example 1.

[0088] Figure 2 shows the situation of enzyme-linked immunosorbent assay to identify human sortilin 1 binding nanoantibodies in bacterial periplasmic space extracts after amplification and induced expression of 576 monoclonal clones randomly selected from the sortilin 1 positive library after three rounds of screening.

[0089] Figure 3 shows the ELISA identification results of 26 anti-human sortilin 1 nanobodies that competitively inhibit the binding of human sortilin 1 to its ligand granule protein precursor.

[0090] Figures 4 to 9 are the results of flow cytometry detection of the binding ability of different antibodies to cell surface sortilin 1.

[0091] Figure 10 shows the results of sort1 cross-recognition of different species sources by Nb426.

[0092] Figures 11-16 are the results of Gator affinity determination experiments detecting the binding of nanobodies to human sortilin 1 recombinant protein.

[0093] FIG17 shows the results of an epitope binning experiment using Gator to detect the binding of multiple nanobodies to human sortilin-1 recombinant protein.

[0094] FIG18 shows the results of immunofluorescence observation of the internalization of nanoantibodies after binding to cell surface Sort1 at different temperatures.

[0095] Figure 19 shows the ELISA results of detecting the level of progranulin in the cell culture medium after U251 cells were treated with nanobodies.

[0096] Figure 20 shows the results of detecting the level of progranulin in the cell culture medium after U251 cells were treated with different concentrations of sortilin 1 antibodies (including nanobodies Nb171, Nb184 and AL001).

[0097] FIG21 is a schematic diagram of the construction of humanized mice expressing sort1 protein.

[0098] Figure 22 shows the changes in the levels of progranulin in mouse serum after intravenous injection of different anti-sortilin-1 nanobodies.

[0099] Figure 23 is a pscAAV-RNY20-VHH plasmid map.

[0100] FIG24 shows the results of Progranulin detection in serum after injection of AAV encoding Sort1 nanobody and AL001.

[0101] FIG25 shows the results of the detection of Progranulin levels in the cerebrospinal fluid of mice 45 days after administration. DETAILED DESCRIPTION

[0102] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0103] Explanation of terms involved in the following embodiments:

[0104] In the present invention, the terms "antibody of the present invention", "antibody of the present invention", "anti-sortilin 1 Nanobody of the present invention", "sortilin 1 Nanobody of the present invention", "anti-sortilin 1 Nanobody", and "sortilin 1 Nanobody" have the same meaning and are used interchangeably, all referring to Nanobodies that specifically recognize and bind to sortilin 1 (including human sortilin 1).

[0105] For the purposes of the present invention, the term "antibody" or "immunoglobulin" refers to an IgG heterotetrameric glycoprotein of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0106] In the present invention, the terms "single domain antibody," "VHH," "nanobody," and "single domain antibody" (sdAb, or nanobody) have the same meaning and are used interchangeably. They refer to the construction of a single domain antibody (VHH) consisting solely of a single heavy chain variable region by cloning the variable region of an antibody heavy chain. This is the smallest antigen-binding fragment with complete function. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0107] In the present invention, "multivalent" refers to a protein comprising multiple anti-sortilin-1 Nanobody VHH chains, anti-sortilin-1 Nanobody, or a fusion protein comprising an anti-sortilin-1 Nanobody.

[0108] In the present invention, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the entire antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a beta-folded configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial beta-folded structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pp. 647-669 (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0109] In the present invention, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments thereof of the present invention to form conjugates. The present invention also includes cell surface markers or antigens that bind to the anti-Sort1 nanobody or fragment thereof.

[0110] In the present invention, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0111] In the present invention, the terms "variable region" and "complementarity determining region" are used interchangeably.

[0112] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to Sortilin 1, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0113] Generally, an antibody's antigen-binding properties are described by three specific regions within the variable region of the heavy chain, known as the variable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0114] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0115] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be: (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol) or a polypeptide (e.g., a polypeptide that extends the half-life of the polypeptide, such as a Nanobody against serum albumin, or an engineered antibody Fc domain); or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence for purifying the polypeptide, or a fusion protein formed with a 6xHis tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0116] The antibodies of the present invention refer to polypeptides that have sortilin-1 binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides comprising the aforementioned CDR regions that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0117] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0118] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the antibodies of the invention. Typically, the fragments comprise at least about 50 contiguous amino acids of the antibodies of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0119] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated according to the amino acid substitutions described above.

[0120] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0121] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0122] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0123] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 90%, preferably at least 95%, and more preferably at least 98% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0124] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0125] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0126] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0127] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0128] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0129] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0130] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0131] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0132] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0133] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0134] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins, including various DNA damaging drugs, or microtubule inhibitors; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0135] Sortilin 1 is a cell membrane receptor protein that belongs to the VPS10P superfamily. It plays an important role in intracellular protein transport, neural development, and metabolic regulation. Sortilin 1 can bind to a variety of proteins and ligands, such as nerve growth factor, neurotrophic factor, glycoprotein, etc., and bring them from the cell surface into the endoplasmic reticulum and transport vesicle system through endocytosis, participating in their localization and transport process. Sortilin 1 interacts with multiple signaling pathways in neurons. It is involved in the regulation of neuronal growth and development, and is also closely related to neural functions such as synaptic transmission, synaptic plasticity, and memory formation. Sortilin 1 plays an important role in the nervous system, and studies have shown that the function of Sortilin 1 is related to neurodegenerative diseases. In addition, Sortilin 1 is overexpressed in cancers such as prostate cancer, ovarian cancer, triple-negative breast cancer, skin cancer, lung cancer, endometrial cancer, colorectal cancer, and pancreatic cancer, and helps the adsorption and invasion of cancer cells during breast cancer metastasis.

[0136] Pharmaceutical composition

[0137] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody, active fragment thereof, or fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0138] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0139] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 100 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 50 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0140] Typically, the anti-sortilin-1 nanobody may include at least two VHH chains, and the VHH chains are connected by a linker.

[0141] In the present invention, the linker is selected from the following sequence: (GaSb)x—(GmSn)y, wherein a, b, m, n, x, y = 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a = 4 and b = 1, m = 3 and n = 1), that is, the linker is selected from the following group: GGGGSGGGS.

[0142] Labeled antibodies

[0143] In the present invention, the antibody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label, or a fluorescent label.

[0144] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, antibodies against sortilin 1 are labeled with colloidal gold to obtain colloidal gold-labeled antibodies.

[0145] The anti-sortilin 1 nanobody of the present invention can effectively bind to the sortilin 1 protein on the cell surface.

[0146] Detection method

[0147] The present invention also relates to a method for detecting Sortilin 1 protein. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of Sortilin 1 protein in the dissolved sample.

[0148] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage medium.

[0149] Reagent test kit

[0150] The present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0151] The present invention also provides a detection kit for detecting the level of sortilin 1, which includes an antibody that recognizes sortilin 1 protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. The detection kit can be an in vitro diagnostic device.

[0152] Another key content of the present invention is the use of AAV vectors to endogenously express nanobodies against sortilin 1. We use our optimized expression elements, including specific promoters, signal peptides, terminators, etc., to improve the efficiency of nanobodies expressed in cells and secreted out of cells, and add inverted repeat sequences (ITRs) upstream and downstream of the expression frame to obtain gene vectors for AAV packaging. The AAV virus carrying the target gene is packaged and purified by conventional methods, which can be used to express nanobodies that recognize sortilin 1 in patients or experimental animals. As a result, nanobodies mediated by AAV endogenous expression can effectively inhibit the function of sortilin 1, reduce the degradation of granule protein precursor (PGRN), and indirectly increase the level of functional PGRN, thereby achieving the treatment of various neurodegenerative diseases. This scheme of using AAV to endogenously express nanobodies at a high level solves the problem of too short half-life of nanobody drugs, and can achieve the therapeutic purpose of long-term benefits from a single dose.

[0153] The experimental methods in the following examples, for which specific conditions are not specified, were generally performed according to conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are calculated by weight.

[0154] The sequence information involved in the present invention is shown in the following table.

[0155] Table 1 List of Nanobody Sequences

[0156] Table 4 Nanobody sequence list

[0157] Example 1 Expression of Human Sortilin-1 Extracellular Segment Fusion Protein

[0158] Expression and purification of human Sortilin-Fc fusion protein: Transient expression of human Sortilin 1 extracellular segment protein using mammalian cells Expi-293F: The pcDNA3.1-H-SORT1-hFc recombinant plasmid containing the cloned human Sortilin 1 extracellular segment gene was mixed with the transfection reagent polyethyleneimine (PEI) and transfected into Expi-293F cells; cultured in a shaker incubator at 37°C, 5% CO2 for 5 days; then the cell supernatant was collected and bound to Protein A beads at room temperature for 1 hour; the beads were then washed with phosphate buffer pH 7.0 and the protein was eluted with 0.1M pH 3.0 glycine solution; the eluted protein was then ultrafiltered into phosphate buffer solution, and after determining the yield, a sample was taken for SDS-PAGE detection. The test results are shown in Figure 1A. The purity of the expressed and purified H-SORT1-hFc protein is greater than 90%, and it can be used for camel immunization and antibody screening (where M represents Marker, and lane 1 represents human sortilin 1 protein with an Fc domain).

[0159] Expression and purification of the human Sortilin-HisTag fusion protein: Transient expression of the human Sortilin 1 extracellular domain protein in Expi-293F mammalian cells: The pcDNA3.1-H-SORT1-Hisx6 recombinant plasmid, containing the cloned human Sortilin 1 extracellular domain gene, was mixed with the transfection reagent PEI and transfected into Expi-293F cells. The cells were cultured in a shaking incubator at 37°C with 5% CO2 for 5 days. The cell supernatant was then collected and bound to Ni affinity chromatography beads. The protein was then eluted with 300mM imidazole and 500mM imidazole solutions. The eluted protein was then ultrafiltered into PBS, the yield was determined, and a sample was analyzed by SDS-PAGE. The results are shown in Figure 1B. The expressed and purified H-SORT1-Hisx6 protein is greater than 90% pure and suitable for camel immunization and antibody screening (where M represents marker and lane 2 represents Hisx6-tagged human Sortilin protein).

[0160] Example 2 Construction of camel immune library and screening, expression and purification of anti-human sortilin 1 nanobody

[0161] Purified human Sortilin-Hisx6 fusion protein was mixed with Freund's adjuvant in a 1:1 ratio and administered subcutaneously to a healthy alpaca for four rounds of immunization. During each round, a small amount of blood was drawn for serum separation and titer testing. Seven to ten days after the final immunization, 100 mL of peripheral blood was collected and mononuclear cells (PBMCs) were isolated using Ficoll lymphocyte separation medium (GE Healthcare 17-1440-03 FICOLL PAQUE PLUS).

[0162] Total mRNA of PBMC was extracted with phenol-chloroform and analyzed using SuperScript TM The RNA was converted into cDNA using IV First-Strand cDNA Synthesis Reaction Kit (Thermo, 18091200). The heavy chain variable region fragment was amplified using nested PCR using cDNA as a template (Q5 High-Fidelity 2X Master The primers used in the two rounds of PCR are shown in Table 3 below:

[0163] Table 3 Primers used for nested PCR amplification of VHH genes

[0164] The antibody fragments recovered from the second round of PCR and the phage vector pADL were digested separately using Not1 and Bstx1 restriction endonucleases. The digestion products were subjected to agarose gel electrophoresis and purified, followed by enzyme ligation. The ligation products were then electroporated into TG1 competent cells (Weidi Biotech, DE1055M) to construct an E. coli library containing the nanobody fragments. Colony counts indicated a library capacity of 5E8. Sequencing of 40 randomly selected single clones revealed an insertion rate of the nanobody gene of approximately 95%.

[0165] After amplification of the Sortilin 1 immune library, VCSM13 helper phage was added during logarithmic growth phase and the bacteria were infected at 37°C for 1 hour. After centrifugation, the supernatant was discarded and the bacteria were resuspended in 2xYT medium supplemented with antibiotics and 0.5% glucose. Phage were packaged overnight in a shaker at 30°C, 225 rpm. The next day, phage particles were precipitated with polyethylene glycol / sodium chloride and the library was screened using phage display technology. After three rounds of "adsorption-wash-enrichment" screening, a population of phage expressing nanobodies that bind to the H-Sort1-Hisx6 fusion protein was enriched. From this enriched population, 576 single clones were randomly selected, amplified, and after IPTG-induced expression, the bacterial outer wall was disrupted with a hypotonic solution, PPB (phosphate peptone buffer), to obtain the periplasmic protein extract (PPE). Binding of the PPE to the human Sortilin 1 (ECD) protein was analyzed by ELISA. The results showed that 117 out of 576 random clones bound to Sortlilin ( FIG2 , clones with ELISA readings more than 5 times the background were defined as positive clones).

[0166] To identify whether these clones have specific binding to Sortilin-1, the selected 117 clones were used to interact with insulin-coated ELISA plates. The results showed that 108 of the 117 clones did not interact with insulin, suggesting that the binding of these clones to Sortilin-1 is specific.

[0167] All 108 clones that specifically bound to sortilin 1 were sequenced and identified. The 25 non-repetitive VHH coding sequences obtained by sequencing were cloned into the pcDNA3.1-VHH-hFc plasmid containing the human Fc domain encoding gene and transfected into Expi-293F cells to express VHH-hFc fusion protein. After affinity binding with Protein-A magnetic beads and elution with glycine solution, the purified nanobody-Fc fusion protein was obtained.

[0168] Example 3 ELISA experiment of anti-Sort1 nanobody competing for the binding of progranulin to sortilin 1

[0169] ELISA plates were coated with 50 μL of 4 μg / mL H-Sort1-His antigen at 4°C overnight; washed three times with 0.1% PBST (phosphate-buffered saline, Tween buffer), and 150 μL of 5% BSA (bovine serum albumin) was added for 1 hour at room temperature; washed three times with 0.1% PBST, and 6 μg / mL Bio-PGRN (biotin-modified human progranulin) and 4 μg / mL VHH-Sort1 were mixed and added, with 6 μg / mL Bio-PGRN alone as a control, and 6 μg / mL Bio-PGRN and 4 μg / mL VHH-Sort1 were added as a control. Hyhel-VHH (a nonspecific control Nanobody) was used as control 2 and incubated at room temperature for 1 hour. The cells were washed three times with 0.1% PBST, and 50 μL of horseradish peroxidase-conjugated streptavidin (1:5000) (diluted in 5% BSA) was added and incubated at room temperature for 1 hour. The cells were washed three times with 0.1% PBST, and 50 μL of TMB (3,3',5,5'-tetramethylbenzidine) was added for color development for 8-10 minutes. The reaction was terminated with 50 μL of stop solution. The absorbance of each well was read at 450 nm. If the Nanobody competes for the binding of Sortilin 1 to progranulin, this will result in a decrease in Bio-PGRN bound to Sortilin 1, and the corresponding sample reading will be lower than that of the sample without the Nanobody.

[0170] The effects of the 25 candidate antibodies on the binding of Sortilin-1 to its ligand, progranulin, are shown in Figure 3. The results showed that 24 of the 25 candidate antibodies were able to inhibit the interaction between Sortilin-His fusion protein and progranulin in ELISA experiments. We selected these 25 antibodies for further characterization.

[0171] Example 4 Detection of Binding Activity of Anti-Sortilin-1 Nanobody to Cell Surface Antigens

[0172] The pcDNA3.1-HSort1 recombinant plasmid, cloned with the full-length human Sortilin 1 gene, was mixed with the transfection reagent PEI and transfected into 293T cells. Forty-eight hours after transfection, cells were harvested by pipetting and suspended, the culture medium removed by centrifugation, and washed twice with ice-cold PBS. The cells were suspended in ice-cold PBS containing 1% BSA, counted, and transferred to a 96-well U-bottom plate at a density of 100,000 cells / well. Purified anti-Sortilin 1 nanobody VHH-Fc was added to a concentration of 50 ng / ml and incubated on ice for 30 minutes. The cells were washed twice with ice-cold PBS, and a secondary antibody against the human Fc domain conjugated to APC fluorescence was added and incubated on ice for 30 minutes. The cells were washed twice with ice-cold PBS and suspended in ice-cold PBS containing 1% BSA. Antibody binding to cell surface antigens was analyzed by flow cytometry.

[0173] In a parallel experiment of the control group, the same nanoantibody was used to treat untransfected 293T cells, and the background binding of the antibody to nonspecific proteins on the cell surface was detected by flow cytometry.

[0174] The results showed that among the 25 candidate nanoantibodies, 10 nanoantibodies could efficiently bind to the human sortilin 1 protein highly expressed on the surface of 293T cells, and the binding activity was similar to that of the control antibody AL001. The results are shown in Figures 4-9 and Table 4; the other 15 nanoantibodies had lower efficiency in binding to the human sortilin 1 on the cell surface, indicating that their affinity was weaker than that of the previous 10 antibodies; importantly, all 25 nanoantibodies did not bind to untransfected parental 293T cells, indicating that these nanoantibodies have high sortilin 1 specificity.

[0175] Table 4 Flow cytometry test results

[0176] Example 5 Cross-recognition of Nb426

[0177] Plasmids pcDNA3.1-HSort1, pcDNA3.1-MSort1, and pcDNA3.1-CSort1, carrying the genes encoding human, mouse, and monkey Sort1 proteins, respectively, were constructed and sequenced and confirmed correct. These plasmids were then transfected into 293T cells to generate cells expressing high expression of Sort1 from different species on their cell surfaces, namely hSort1-293T, mSor1-293T, and cSort-T. 48 hours after transfection, cells were harvested and washed, then suspended in pre-chilled PBS containing 1% BSA. Purified human Fc-tagged anti-Sort1 nanobody Nb426-Fc or control antibody AL001 were added to an antibody concentration of 50 ng / ml and incubated on ice for 30 minutes. Cells were washed twice with pre-chilled PBS, and a secondary antibody with an APC fluorescent-labeled anti-human Fc domain was added and incubated on ice for 30 minutes. Cells were washed twice with cold PBS and suspended in pre-chilled PBS containing 1% BSA. Antibody binding to cell surface Sort1 proteins from different species was analyzed by flow cytometry. The same antibody and protocol were used on untransfected wild-type 293T cells as a control for nonspecific binding.

[0178] As shown in Figure 10, the anti-Sort1 nanobody Nb426 showed significantly increased binding to 293T cells transfected with human, mouse, and monkey Sort1 proteins compared to wild-type 293T cells, demonstrating that this nanobody clone can recognize Sort1 proteins from all three species. In comparison, the control antibody AL001 binds to human and monkey Sort1 proteins but binds only weakly to mouse Sort1. This result demonstrates not only that Nb426 has superior species recognition for Sort1 but also that it binds to a different epitope than the control antibody AL001.

[0179] Example 6 Determination of the affinity of nanobodies for sortilin 1 using biomembrane interferometry

[0180] Biolayer interferometry assays were performed to evaluate the binding kinetics of the nanobody to the sortilin-1 fusion protein using the Gator Prime system (Gator Bio) v2.7.3.0728 (https: / / www.gatorbio.com / ).

[0181] The glass hFC probe (Gator Bio) was first immersed in Q buffer (Gator Bio) for about 30 seconds to obtain a baseline signal. The probe was then loaded with 3-5 μg / ml of nanobody-Fc fusion protein (VHH-Fc) diluted in Q buffer for 180 seconds, followed by a 30-second Q buffer wash step. Next, the VHH-Fc-bound probe was bound to 300-1.2nM of human Sortilin-Hisx6 fusion protein, including a no sortilin 1 protein control (0nM), for about 180 seconds, followed by a 180-second dissociation step. Association and dissociation curves were drawn and calculated using GatorPrime (Gator Bio) software to obtain binding kinetic values ​​(dissociation rate constant koff, association rate constant Kon, and dissociation constant KD). The results are shown in Figures 11-16 (clones with too low koff are not shown).

[0182] The binding kinetics of the control antibody Latozinemab to the human Sortilin-1 fusion protein were determined using the same method. The binding kinetic values ​​(koff, Kon, and KD) obtained under the same conditions can be used for parallel comparison with the VHH-Fc affinity. The results are shown in Table 5.

[0183] Table 5 Gator determination of affinity data of various nanobodies (VHH-Fc) for human Sortilin-Hisx6 fusion protein

[0184] Example 7: Determination of the epitope grouping of sortilin 1 by nanobodies using a dual epitope partitioning assay

[0185] Biolayer interferometry was performed using the Gator Prime system (Gator Bio) v2.7.3.0728 ( https: / / www.gatorbio.com / ) to evaluate the epitope to which each nanobody bound to the sortilin-1 fusion protein.

[0186] A glass His-tag probe (Gator Bio) was first immersed in Q buffer (Gator Bio) for approximately 30 seconds to obtain a baseline signal. The probe was then loaded with 100 nM human Sortilin-Hisx6 fusion protein diluted in Q buffer for 180 seconds, followed by a 30-second Q buffer wash step. Next, the Sortilin-Hisx6-bound probe was bound to 300 nM control antibody AL001 diluted in Q buffer for approximately 180 seconds. The Sortilin-AL001-bound probe was then immersed in a 200 nM nanobody-Fc fusion protein (VHH-Fc) solution diluted in Q buffer for a 180-second doublet binding step. Doublet binding curves were plotted and calculated using Gator Prime (Gator Bio) software to determine the grouping of different antibodies against the original epitope.

[0187] The results are shown in Figure 17. It shows that the antigen epitopes bound by the selected nanobodies are different from the antigen epitopes bound by the control antibody AL001, indicating that the nanobodies we developed have obvious differences from the control antibody AL001.

[0188] Example 8 Immunofluorescence observation of the process of nanoantibodies binding to cell surface Sort1 and then endocytosed into the cell

[0189] After the protein receptors on the cell membrane surface are bound by antibodies, they may undergo endocytosis due to conformational changes. This is also the key reason why most antibody-drug conjugates (ADCs) kill cancer cells. The human glioma cell line U251 has a high level of Sort1 receptor protein on its surface, which can be used as a functional detection tool for anti-Sort1 antibodies. When the antibody binds to the cell surface receptor, endocytosis is a relatively fast process. In order to better observe this process, we took advantage of the principle that low temperature can inhibit endocytosis and conducted experiments at two different temperatures, 4°C and 37°C, to observe the localization changes of nanoantibodies in cell fluorescence staining experiments after binding to U251 cells, thereby observing the occurrence of endocytosis. The following is the specific experimental plan:

[0190] Take U251 cells and divide them into 1*10 6Cells were plated at a density of 100 cells / mL on a culture dish mounted on a slide and cultured for 24 hours to allow attachment. The culture medium was removed, and a PBS solution containing 1 μg / mL anti-Sort1 nanobody-Fc fusion protein was added. The culture dishes were incubated at 37°C and 4°C for 15 minutes respectively. The cell slides in the culture dishes were washed with PBS for a total of 3 washes for 3 minutes each. The cell slides were fixed with 4% paraformaldehyde for 15 minutes and then washed with PBS for 3 minutes each to remove excess paraformaldehyde. The cells were permeabilized with 0.5% Triton X-100 at room temperature for 20 minutes and then washed with PBS for 3 minutes each to remove excess Triton X-100. The cells were blocked with PBS containing 5% BSA at room temperature for 30 minutes. A diluted anti-human IgG Fc-FITC antibody (1:500) was added and incubated in the dark for 1 hour at room temperature to bind to the previously used anti-Sort1 nanobody-Fc fusion protein. The cells were then washed with PBS three times to remove excess fluorescent secondary antibody. Finally, add a drop of anti-fluorescence quencher containing DAPI, seal the slide, and observe under a microscope.

[0191] The results are shown in Figure 18 (Nb07 is Nb171, Nb08 is Nb184). The results of the immunofluorescence experiment showed that under the conditions of a warm water bath at 37°C, the anti-Sort1 nanobody can quickly enter the cell within 15 minutes, and its fluorescent signal will appear in the cytoplasm in the form of endocytic bodies and gradually accumulate in the area near the cell nucleus. On the contrary, under low-temperature treatment at 4°C, the anti-Sort1 nanobody will only bind to the Sort1 protein on the cell membrane and will not be internalized. The fluorescent signal can reflect the complete shape of the entire cell. These results show that after our nanobody binds to Sort1, it can be efficiently internalized into the cell and has the potential to be developed into an ADC-type anti-tumor drug.

[0192] Example 9 Determination of progranulin levels in cell culture medium after nanobody treatment of U251 cells

[0193] To test whether anti-Sort1 nanobodies could increase progranulin levels, we treated U251 cells cultured in vitro with the antibodies. Specifically, U251 cells in logarithmic growth phase were plated in 96-well cell culture plates at 1E4 cells per well and incubated at 37°C in a 5% CO2 incubator. Twenty-four hours after plating, either a VHH-Fc nanobody at a working concentration of 40 nM or the control antibody AL001 at the same molar concentration was added. Seventy-two hours later, cell culture supernatants were collected and progranulin levels were measured using the Progranulin ELISA Kit (EAGLE BIOSCIENCES Cat#: E103). The assay method is as follows: dilute the cell supernatant 15-fold with the dilution buffer provided in the kit; add 50 μL of the antibody conjugate provided in the kit and 50 μL of diluted sample (including antibody-treated cell culture supernatant, progranulin standard solution, and positive control) to each ELISA well of the kit and incubate on a shaker at room temperature (≥350 rpm) for 1 hour; discard the supernatant and wash five times with wash buffer; add 100 μL of the enzyme conjugate provided in the kit and incubate on a shaker at room temperature (≥350 rpm) for 30 minutes; discard the supernatant and wash five times with wash buffer; add 100 μL of the substrate solution provided in the kit and incubate at room temperature in the dark for 30 minutes; add 100 μL of stop solution to each well and measure absorbance at 450 nm within 30 minutes. For data analysis, calculate the progranulin concentration in the cell culture supernatant based on the absorbance curve of the standard sample.

[0194] The results showed that the selected nanobodies were able to increase the level of progranulin in the culture medium of U251 cells, among which antibodies such as Nb128, Nb171, Nb184, and Nb252 had an effect of increasing progranulin that reached or exceeded that of the control antibody AL001 ( FIG. 19 ).

[0195] To further determine the relationship between antibody concentration and the level of progranulin secretion in U251 cells, U251 cells were treated with different concentration gradients of the Sortilin-1 nanobody VHH-Fc. After 72 hours of treatment, the progranulin concentration in the cell culture medium was measured by ELISA, and the antibody effect was calculated by comparing the progranulin concentration in the culture medium of U251 cells without antibody. The results showed that within the test range of 0.004ug / mL to 4.0ug / mL, the effect of Nb171 and Nb184 on inducing increased progranulin secretion was roughly positively correlated with the antibody concentration, with higher concentrations of the antibody inducing more pronounced increases in progranulin secretion. It is worth noting that at the same antibody concentration, Nb171 and Nb184 were both more effective than AL001 in inducing increased progranulin secretion (Figure 20).

[0196] Example 10 In vivo experiment

[0197] To further verify the in vivo effects of anti-sortilin 1 nanobodies, we constructed humanized mice targeting the Sort1 protein. Specifically, we replaced exon 2 in the Sort1 gene of wild-type C57B6 mice with the gene encoding the human Sort1 protein through gene cloning and homologous recombination, thereby achieving in situ expression of the human Sort1 protein in the mouse Sort1 gene. The offspring mice were genotyped and homozygous individuals expressing only the human Sort1 gene were selected for in vivo experiments (Figure 21).

[0198] The purified nanobody-Fc fusion protein was intravenously injected into Sort1 humanized mice at a dose of 100 mg / kg on day 1 and day 7, respectively. Peripheral blood was collected from the mice at multiple time points before and after administration, and the serum was separated and the concentration of progranulin in the serum was measured using a mouse progranulin ELISA KIT (R&D systems Cat#MPGRN0).

[0199] The results showed that intravenous injection of the anti-sortilin-1 nanobody significantly increased the level of progranulin in mouse serum. Even 11 days after antibody injection, serum progranulin levels were still more than three times the pre-dose level. This suggests that our nanobody can significantly increase the level of functional progranulin in vivo by preventing sortilin-1-mediated endocytosis and degradation of progranulin. The results are shown in Figure 22.

[0200] Example 11 Using AAV to Endogenously Highly Express Anti-Sort1 Nanobodies

[0201] In our patent “A recombinant adeno-associated virus vector expressing nanobodies and its application” (China Patent No. 2023114805791, CN117210503A), we disclosed a plasmid vector pssMMAAV-RNY-020 for recombinant adeno-associated virus expression of nanobodies. This plasmid vector carries a combination of expression elements such as CAG promoter, BM40 signal peptide, nanobody sequence, and SV40 poly (A) terminator between the two inverted terminal repeat sequences (ITR) of the AAV expression vector, which can achieve the highest expression level of secreted nanobodies. Based on this, we constructed an AAV plasmid vector that highly expresses anti-Sort1 nanobodies, which is shown in Figure 23.

[0202] AAV virus packaging uses the classic three-plasmid transfection method. These include: 1) the expression plasmid pAAV9-Cap-WT encoding the wild-type AAV9 REP-CAP gene; 2) the helper plasmid pADdeltaF6 encoding E4 / E2A / VA / L4 / L5; and 3) the optimized expression vector plasmid pscAAV-RNV20-VHH encoding the anti-Sort1 nanobody gene. The three plasmids were mixed in equal proportions and transfected into adherent HEK-293 cells cultured in DMEM medium with 10% FBS. After 72 hours, the cell culture supernatant was collected and centrifuged at 3000g for 10 seconds at room temperature to remove cell debris. The supernatant was then sterilized by filtration through a 0.22μm filter. The purified AAV virus carrying the nanobody gene was obtained through classic steps such as ultrafiltration concentration, buffer exchange, affinity chromatography to remove host cell proteins, and anion exchange chromatography to remove other trace impurities and viral shells. The viral titer in the sample was determined by qPCR. The upstream primer used for detection was fwd ITR primer: 5'-GGAACCCCTAGTGATGGAGTT, and the downstream primer used for detection was rev ITR primer: 5'-CGGCCTCAGTGAGCGA.

[0203] To evaluate the physiological function of endogenously expressed anti-Sort1 nanobodies in AAV, we selected 6-week-old humanized Sort1 mice for drug administration and analysis. On day 0 of the experiment, we administered a single dose of 1E12 vg of AAV9 virus encoding the nanobody via the tail vein. Blood samples were collected before and after drug administration at various time points to measure serum progranulin protein levels. The experiment was terminated on day 45 after drug administration. Mice were sacrificed and cerebrospinal fluid collected for analysis.

[0204] As a control, we selected the same mice for the experiment and injected them with 100 mg / kg of the anti-Sort1 monoclonal antibody AL001 twice, on day 0 and day 7. We collected blood samples and cerebrospinal fluid at the same time as the AAV-administered group to measure the levels of progranulin in the serum and cerebrospinal fluid of the control mice.

[0205] The results of serum progranulin detection are shown in Figure 24. A single tail vein injection of AAV9 encoding the Sort1 nanobody significantly increased mouse serum progranulin levels by more than double. This increase began to appear on the second day after administration and was maintained throughout the experiment, demonstrating the long-term effectiveness of AAV-expressed nanobodies. As a control, injection of the anti-Sort1 monoclonal antibody AL001 only temporarily increased mouse serum progranulin levels. This increase reached a peak two days after administration and then declined. The effect could only be maintained with further administration. 14 days after the last administration, serum progranulin levels had returned to pre-dose levels.

[0206] Progranulin levels in the cerebrospinal fluid (CSF) are important markers of efficacy in treating central nervous system diseases such as FTD. After 45 days of drug administration, mice were sacrificed and progranulin levels in their cerebrospinal fluid (CSF) were measured. The results are shown in Figure 25. As can be seen, 45 days after drug administration, the levels of progranulin in the cerebrospinal fluid (CSF) of mice injected with AAV endogenously expressing the anti-Sort1 nanobody were 2.5-5 times higher than those in the control group injected with the AL001 monoclonal antibody. This indicates that the AAV9-expressed nanobody has, to a certain extent, penetrated the brain tissue and exerted a therapeutic effect. This increase in cerebrospinal fluid progranulin levels exceeds the effects of long-term, repeated administration of AL001 published by companies such as Alector, indicating that AAV endogenously expressing the anti-Sort1 nanobody has greater therapeutic potential for neurodegenerative diseases such as FTD.

[0207] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A nanobody specific for sortilin 1, characterized in that The heavy chain variable region of the Nanobody includes complementary determining regions CDR1, CDR2, and CDR3, and the complementary determining regions include any combination of the following or a sequence with a homology of not less than 90% thereto: 1) CDR1 shown in SEQ ID NO.43, CDR2 shown in SEQ ID NO.44, and CDR3 shown in SEQ ID NO.45; 2) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; 3) CDR1 shown in SEQ ID NO.8, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.10; 4) CDR1 shown in SEQ ID NO.15, CDR2 shown in SEQ ID NO.16, and CDR3 shown in SEQ ID NO.17; 5) CDR1 shown in SEQ ID NO.22, CDR2 shown in SEQ ID NO.23, and CDR3 shown in SEQ ID NO.24; 6) CDR1 shown in SEQ ID NO.29, CDR2 shown in SEQ ID NO.30, and CDR3 shown in SEQ ID NO.31; 7) CDR1 shown in SEQ ID NO.36, CDR2 shown in SEQ ID NO.37, and CDR3 shown in SEQ ID NO.38; 8) CDR1 shown in SEQ ID NO.50, CDR2 shown in SEQ ID NO.51, and CDR3 shown in SEQ ID NO.52; 9) CDR1 shown in SEQ ID NO.57, CDR2 shown in SEQ ID NO.58, and CDR3 shown in SEQ ID NO.59; 10) CDR1 shown in SEQ ID NO.120, CDR2 shown in SEQ ID NO.121, and CDR3 shown in SEQ ID NO.

122.

2. The Nanobody according to claim 1, characterized in that The sequence with a homology of not less than 90% is obtained by amino acid substitution of the CDR1, CDR2 and / or CDR3, wherein the amino acid substitution is selected from one or more of the following: 1) Alanine is replaced by valine, leucine or isoleucine, 2) Arginine is replaced by lysine, glutamine or asparagine, 3) Asparagine is replaced by glutamine, histidine, lysine or arginine, 4) Aspartic acid is replaced by glutamic acid, 5) Cysteine ​​is replaced by serine, 6) Glutamine is replaced by asparagine, 7) Glutamic acid is replaced by aspartic acid, 8) Glycine is replaced by proline or alanine, 9) Histidine is replaced by asparagine, glutamine, lysine or arginine, 10) isoleucine is replaced by leucine, valine, methionine, alanine or phenylalanine, 11) Leucine is replaced by isoleucine, valine, methionine, alanine or phenylalanine, 12) Lysine is replaced by arginine, glutamine or asparagine, 13) Methionine is replaced by leucine, phenylalanine or isoleucine, 14) Phenylalanine is replaced by leucine, valine, isoleucine, alanine or tyrosine, 15) Proline is replaced by alanine or glycine, 16) Serine is replaced by threonine, 17) threonine is replaced by serine, 18) Tryptophan is replaced by tyrosine or phenylalanine, 19) Tyrosine is replaced by tryptophan, phenylalanine, threonine or serine, 20) Valine is replaced by isoleucine, leucine, methionine, phenylalanine or alanine.

3. The Nanobody according to claim 1, characterized in that: The variable region of the Nanobody includes framework regions FR1, FR2, FR3, and FR4; the framework region includes any one of the following or a sequence with a homology of not less than 90% thereto: 1) FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.5, FR3 shown in SEQ ID NO.6, and FR4 shown in SEQ ID NO.7; 2) FR1 shown in SEQ ID NO.11, FR2 shown in SEQ ID NO.12, FR3 shown in SEQ ID NO.13, and FR4 shown in SEQ ID NO.14; 3) FR1 shown in SEQ ID NO.18, FR2 shown in SEQ ID NO.19, FR3 shown in SEQ ID NO.20, and FR4 shown in SEQ ID NO.21; 4) FR1 shown in SEQ ID NO.25, FR2 shown in SEQ ID NO.26, FR3 shown in SEQ ID NO.27, and FR4 shown in SEQ ID NO.28; 5) FR1 shown in SEQ ID NO.32, FR2 shown in SEQ ID NO.33, FR3 shown in SEQ ID NO.34, and FR4 shown in SEQ ID NO.35; 6) FR1 shown in SEQ ID NO.39, FR2 shown in SEQ ID NO.40, FR3 shown in SEQ ID NO.41, and FR4 shown in SEQ ID NO.42; 7) FR1 shown in SEQ ID NO.46, FR2 shown in SEQ ID NO.47, FR3 shown in SEQ ID NO.48, and FR4 shown in SEQ ID NO.49; 8) FR1 shown in SEQ ID NO.53, FR2 shown in SEQ ID NO.54, FR3 shown in SEQ ID NO.55, and FR4 shown in SEQ ID NO.56; 9) FR1 shown in SEQ ID NO.60, FR2 shown in SEQ ID NO.61, FR3 shown in SEQ ID NO.62, and FR4 shown in SEQ ID NO.63; 10) FR1 shown in SEQ ID NO.123, FR2 shown in SEQ ID NO.124, FR3 shown in SEQ ID NO.125, and FR4 shown in SEQ ID NO.

126.

4. The Nanobody according to claim 1, characterized in that: The nanobody contains a sequence as shown in any one of SEQ ID NO.148-SEQ ID NO.156 and SEQ ID NO.

165.

5. A polynucleotide encoding the Nanobody according to any one of claims 1 to 4. A recombinant expression vector comprising the polynucleotide according to claim 5.

7. The recombinant expression vector according to claim 6, characterized in that: The expression vector is selected from DNA, RNA, virus, plasmid, transposon, gene transfer system, liposome nanoparticle, or a combination thereof.

8. The recombinant expression vector according to claim 7, characterized in that: The recombinant expression vector is a recombinant adeno-associated virus vector; the recombinant adeno-associated virus vector contains: a promoter, a polynucleotide and a polyA tail.

9. The recombinant expression vector according to claim 8, characterized in that: The recombinant adeno-associated virus vector contains: a promoter, a signal peptide, a polynucleotide and a polyA tail.

10. The recombinant expression vector according to claim 9, characterized in that: The recombinant adeno-associated virus vector contains: CAG promoter or CMV promoter, BM40 signal peptide, polynucleotide and SV40 polyA tail, wherein the CAG promoter and BM40 signal peptide are located upstream of the nanobody coding sequence, and the SV40 polyA tail is located downstream of the nanobody coding sequence.

11. The recombinant expression vector according to claim 8, characterized in that: The recombinant adeno-associated virus vector contains an inverted terminal repeat sequence.

12. A recombinant cell containing the recombinant expression vector according to any one of claims 6 to 11.

13. A monovalent antibody, a bivalent antibody, a multivalent antibody or a recombinant protein containing the Nanobody according to any one of claims 1 to 4.

14. A drug, characterized in that: The drug contains the nanobody according to any one of claims 1 to 4 or the monovalent antibody, bivalent antibody, multivalent antibody or recombinant protein according to claim 13.

15. The drug according to claim 14, characterized in that: The drug includes an antibody-drug conjugate; the antibody-drug conjugate contains: (1) a nanobody, a monovalent antibody, a bivalent antibody, a multivalent antibody or a recombinant protein, (2) a cytotoxic load, and (3) a linker for linking the two.

16. Use of the Nanobody according to any one of claims 1 to 4, the polynucleotide according to claim 5, the recombinant expression vector according to any one of claims 6 to 11, the recombinant cell according to claim 12, or the monovalent antibody, bivalent antibody, multivalent antibody or recombinant protein according to claim 13 in the preparation of a diagnostic, preventive or therapeutic product.

17. The use according to claim 16, characterized in that: The product is used to inhibit the function of sortilin 1 molecule, identify cells with high expression of sortilin 1, or increase the level of granulin precursor.

18. The use according to claim 16, characterized in that: The product is a drug for preventing or treating tumors with high expression of sortilin 1.

19. The use according to claim 16, characterized in that: The product is a drug for preventing or treating central nervous system diseases related to granule protein levels.

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