WO2025031098A1 - Sortilin 1-specific nanoantibody, recombinant aav containing same, and use - Google Patents

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 nano antibody specific for sortilin 1, a recombinant AAV containing the nano antibody and its application Technical Field

The present invention relates to a nano antibody specific for sortilin 1, a recombinant AAV containing the nano antibody and application thereof, belonging to the field of biotechnology.

Background Art

Sortilin (Sort1), as a member of the vacuolar sorting factor 10 (VPS10P) domain receptor family, acts as a cellular receptor or co-receptor, mediating the targeted transport of different proteins into the cell. The N-terminus of sortilin 1 is located in the extracellular region, and the C-terminus is located in the intracellular region, connecting its extracellular and intracellular regions through a transmembrane structure. Sortilin 1 has a wide distribution on the cell membrane, but can also be found in the endoplasmic reticulum, Golgi apparatus, and early endocytic vesicles inside the cell. Sortilin 1 is widely expressed in a variety of tissues and organs, especially in the nervous system, where the expression level of sortilin 1 is high. It is expressed in multiple regions of the brain, including the cortex, hippocampus, striatum, and cerebellum. In addition, sortilin 1 is also expressed in multiple tissues and organs such as the heart, liver, lung, kidney, intestine, and immune system. Sortilin 1 plays an important role in intracellular protein transport, 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, 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. Secondly, Sortilin 1 plays an important role in the nervous system. It is involved in the growth and development regulation of neurons and is related to neurodegenerative diseases. Sortilin 1 interacts with multiple signaling pathways in neurons to regulate cell survival, apoptosis and differentiation processes. It is also closely related to neural functions such as synaptic transmission, synaptic plasticity and memory formation. In addition, Sortilin 1 also plays an important role in metabolic regulation. It is involved in cholesterol metabolism and insulin release regulation. It regulates cholesterol endocytosis and transport by binding to low-density lipoprotein receptor-related protein (LRP); in pancreatic islet cells, Sortilin 1 is involved in the maturation and secretion of insulin granules.

Tumor treatment is one aspect of the clinical application research of Sort1 antibodies. Studies have shown that sortin 1 plays an important role in tumorigenesis. For example, in the process of liver cancer, sortin 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 tumor cancers 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 expression level of Sort1, the worse the prognosis of the related disease. These findings suggest that by specifically targeting Sort1, it may be possible to achieve accurate identification of 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 expression of Sort1, and activate immune cells to specifically eliminate lesions through processes such as antibody-dependent cell-mediated cytotoxicity (ADCC); or conjugate antibody molecules with killing molecules such as chemotherapy drugs and radioactive isotopes, so that these drugs enter cancer cells with high expression of Sort1 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 molecule to a peptide molecule targeting Sort1. The peptide drug conjugate (PDC) named TH1902 has entered multiple clinical trials for solid tumors. Early clinical results show that the peptide drug conjugate can slow disease progression, increase immune cell infiltration, and inhibit angiogenesis, etc., showing good effectiveness.

On the other hand, in terms of clinical application, Sortilin 1 has been widely studied for regulating the level of Progranulin (PGRN), thereby affecting the function of the nervous system. Progranulin (PGRN) is an extracellular secretory protein composed of 588 amino acids. It is widely distributed in the human body, especially in tissues and cells such as the central nervous system, immune system, endocrine system and kidneys. It plays an important role in inflammation regulation, neuroprotection and tumor development. In the process of neuroprotection and neurodevelopment, progranulin can promote the growth, differentiation and survival of neurons, and participate in synapse formation and neurotransmission regulation. The lack of progranulin may lead to an increase in inflammatory response, induce toxic damage to neurons, interfere with neuronal growth and the formation of synaptic connections, and lead to neural network dysfunction. Progranulin deficiency may also lead to abnormal autophagy and protein metabolism disorders, further affecting cell function.

Sortilin 1 is one of the main degradation pathways of progranulin: the extracellular domain of Sortilin 1 binds to progranulin, and the endocytosis mediated by Sortilin 1 brings progranulin from the outside of the cell into the inside of the cell. By binding to vesicles and lysosomes, Sortilin 1 promotes progranulin to enter the degradation pathway, thereby reducing the concentration of progranulin outside the cell. The decrease in the level of progranulin can lead to an increase in inflammatory response, induce toxic damage to neurons, interfere with neuronal growth and the formation of synaptic connections, and lead to neural network dysfunction. Accordingly, inhibiting the function of Sortilin 1 can reduce the degradation of progranulin, thereby increasing the level of progranulin inside and outside the cell. When the function of Sortilin 1 is abnormal or the expression level of Sortilin 1 changes, it will lead to abnormal changes in the level of progranulin. By inhibiting the function of Sortilin 1, the abnormal degradation of progranulin can be prevented, thereby increasing the available amount of progranulin. Studies have shown that in neurodegenerative diseases such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD), the reduction of progranulin levels is associated with the occurrence and progression of the disease. Therefore, inhibiting the function of sortilin 1 to increase the level of progranulin may help restore the physiological function of progranulin, thereby having therapeutic potential for FTD, ALS, PD, AD and other related diseases.

Using antibodies to inhibit the function of sortilin 1 is a potential strategy to achieve the goal of increasing the level of progranulin. As a specific molecular tool, antibodies can specifically bind to sortilin 1 and affect the binding of progranulin to sortilin 1, thereby blocking the degradation pathway of progranulin. This strategy is expected to increase the biological activity and functional expression of progranulin, which may have potential benefits for the treatment of related diseases. Many pharmaceutical companies and biotechnology companies around the world, including Lundbeck, Sanofi, Takeda and GSK, have invested in the research and development of sortilin 1 antibodies. Among them, the monoclonal antibody Latozinemab (AL001) developed by the American biotechnology company Alector Therapeutics has entered the third phase of clinical trials for FTD, suggesting that sortilin 1 antibodies have therapeutic potential for a variety of neurodegenerative diseases. Despite this, no sortilin 1 antibody has been approved for marketing worldwide, so there is still a need to develop new, significantly differentiated sortilin 1 antibodies that are more suitable for clinical applications.

There are many problems in the development and application of conventional IgG antibodies, such as long R&D cycle, high production cost, poor stability, high immunogenicity, etc., which limit its application scope in clinical development. In 1993, Hamers et al. reported that camels have this natural lack of light chain and heavy chain constant region 1 (CH1) heavy chain antibody, cloned its variable region to obtain a single domain antibody consisting of only one heavy chain variable region, called VHH (variable domain of heavy chain of heavy-chain antibody), now named "nanobody" (Nb). The molecular weight of nanobody is about 15kD, which is the smallest antigen binding fragment with complete binding function. Its structure is elliptical, with a diameter of 2.5nm and a length of 4nm. Compared with ordinary IgG monoclonal antibodies, nanobody can more easily pass through tissue barriers and increase the local concentration of therapeutic drugs due to its small size and good permeability. Due to the small size and the structure of a single variable domain, the risk of nanobody triggering an immune response in the human body is relatively low, which reduces the immune-related problems caused by patients using nanoantibodies and increases their acceptability in long-term treatment. Nanobodies have good environmental tolerance, stable conformation and are easy to synthesize. These unique properties make nanobodies have unique application value in disease diagnosis and targeted therapy. Unfortunately, because nanoantibody molecules are too small, nanoantibodies cannot be retained by the blood during glomerular filtration and are easily metabolized and cleared in the body, resulting in a very short half-life of nanoantibodies after administration, and they cannot maintain effective drug concentrations in the body, thus affecting the effect and feasibility of their widespread application. In order to extend the half-life of nanoantibodies in the body, antibody experts have adopted a variety of methods, such as polyethylene glycol modification, engineered Fc fragment fusion, antibody fragment splicing, and other fusion protein splicing. Although these methods have extended the half-life of nanoantibodies to a certain extent, they have also increased the volume and molecular weight of drug molecules, affecting the tissue permeability of nanoantibodies to a certain extent. In addition, these modifications increase the production cost of nanoantibody drugs and may lead to higher immunogenicity, thereby causing nanoantibodies to lose their key advantage as the smallest antigen-binding fragment. The field of drug research has been looking for methods that can effectively maintain the drug concentration of nanoantibodies in the body without changing their basic molecular characteristics.

In summary, despite the natural advantages of nano-antibodies such as high specificity and easy production, the performance of nano-antibodies against sort1 needs to be further improved, and so far, there have been no reports of using nano-antibodies to target Sort1 to treat diseases. There are two main reasons: First, the half-life of pure nano-antibodies is too short, and it is impossible to achieve the ideal drug concentration in the body through exogenous administration, making it difficult to achieve long-term therapeutic effects on neurodegenerative diseases; second, the study of Sort1 as a tumor target has just begun, and the functional verification of peptide-drug conjugates has only recently been completed, and subsequent antibody-based anti-tumor research has not yet been reported.

Summary of the invention

To solve the above problems, the present invention provides an anti-sortilin 1 nanobody and its application, aiming to provide a variety of sortilin 1 antibodies that are significantly differentiated from the current sortilin 1 monoclonal antibody AL001 and have better clinical application prospects. The antibodies of the present invention have broad biological and clinical application values, and their applications involve diagnosis and treatment of diseases related to sortilin 1, basic medical research, biological research and other fields.

The first object of the present invention is to provide a sortilin 1-specific nanobody, wherein the heavy chain variable region of the nanobody comprises complementary determining regions CDR1, CDR2, and CDR3, and the complementary determining regions comprise any combination of the following or a sequence with a homology of not less than 90% thereto:

1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, CDR3 shown in SEQ ID NO.3;

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

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

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

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

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

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

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 by SEQ ID NO.120, CDR2 shown by SEQ ID NO.121, and CDR3 shown by SEQ ID NO.122.

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

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:

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 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.

Furthermore, the variable region of the nanobody includes framework regions FR1, FR2, FR3, and FR4, and a complementary determining region is arranged between two adjacent framework regions, that is, CDR1, CDR2, and CDR3 are separated by framework regions FR1, FR2, FR3, and FR4. Therefore, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged on the nanobody in sequence.

Furthermore, the framework region includes any one of the following or a sequence having 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, 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 by SEQ ID NO.123, FR2 shown by SEQ ID NO.124, FR3 shown by SEQ ID NO.125, and FR4 shown by SEQ ID NO.126.

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

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

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.

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

Furthermore, the recombinant adeno-associated virus vector contains an inverted terminal repeat sequence (ITR) for AAV virus packaging, and the inverted terminal repeat sequence is connected to the upstream of the CAG promoter and the downstream of the SV40 polyA tail, respectively, and the two ITRs are exactly the same but in opposite directions. The recombinant vector is subsequently packaged by the adeno-associated virus to produce a related gene therapy product.

Further, 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,

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.

The fourth object of the present invention is to provide a host cell containing the above recombinant expression vector.

Furthermore, the host cell may be a prokaryotic cell or a eukaryotic cell, such as a plant cell, an animal cell, a microorganism, and the like.

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.

Furthermore, the recombinant protein contains

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

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

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

A-L-B(Ia),

B-L-A(Ib),

Wherein A is the above-mentioned anti-sortilin 1 nanobody, B is the Fc fragment of IgG, and L is no or a flexible linker. Preferably, the flexible linker is a peptide linker, and more preferably, the peptide linker has 1-20 amino acids. The Fc fragment of IgG includes the Fc fragment of human IgG or an engineered mutant thereof, and the Fc fragment of IgG is selected from the Fc fragment of IgG1, IgG2, IgG3, IgG4 or a combination thereof.

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

Furthermore, the antibody drug conjugate contains:

(a) an anti-sortilin-1 nanobody, or the above-mentioned recombinant protein, bivalent or multivalent antibody, for binding the drug to a target cell with high expression of sortilin-1;

(b) Cytotoxic payloads. These payloads become highly active drugs after being hydrolyzed and cleaved, and can effectively kill target cells or inhibit the function of target cells. Commonly used cytotoxic payloads include a variety of DNA-damaging drugs, microtubule inhibitors, cytokines, radionuclides, or RNA payloads such as siRNA and miRNA.

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

The seventh object of the present invention is to provide the use of the above-mentioned 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 form of the product includes but is not limited to medicaments, reagents, test plates, kits, etc.

Further, the product is used to detect the molecular level of sortilin 1, 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.

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

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.

Furthermore, the drug can also be a drug for preventing or treating central nervous system diseases, such as neurodegenerative diseases. The 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.

Beneficial effects of the present invention:

(1) The inventors have successfully obtained multiple anti-human sortilin 1 nanobodies 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 phage display technology in this form of antigen, thereby obtaining a sortilin 1-specific nanoantibody gene. The antibody of the present invention can block the interaction between sortilin 1 and granulin precursor on the cell surface, with high affinity and strong specificity; it can effectively bind to sortilin 1 protein, and its 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 granulin precursor in the culture medium of U251 cells, and the ability to increase the level of extracellular granulin precursor is close to or better than the control antibody AL001, and has the potential to treat a variety of neurodegenerative diseases.

(2) The present invention screens and optimizes the elements (including promoters, introns, signal peptides, polyA, etc.) on the recombinant expression vector of nano antibodies to obtain a vector that can improve the expression level of nano antibodies. The construction process of the expression vector is simple, and it can effectively improve the expression level of nano antibodies in mammals, helping nano antibodies 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 nano antibody 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, more than 20 times higher than commonly used expression element combinations, and can maintain a high level of nano antibodies in serum for a long time.

(3) The present invention uses an optimized AAV vector to highly express anti-sortin 1 nanobody in vivo, successfully solving the problem of short half-life of nanobody drugs when administered exogenously, and achieving effective pharmacokinetic function. Animal in vivo experiments have shown that a single AAV injection can continuously increase the level of progranulin in the serum and cerebrospinal fluid of humanized disease models. Given that there are currently no reports on the use of AAV to express nanobodies to treat or prevent diseases, the present invention has the potential to be developed into a new gene therapy drug for the treatment of a variety of neurodegenerative diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the SDS-PAGE detection result of the recombinant protein in Example 1.

Figure 2 shows the situation of human Sortilin 1 binding nanoantibodies in bacterial periplasmic space extracts identified by ELISA after amplification and induction of expression of 576 monoclonal clones randomly selected from the Sortilin 1 positive library after three rounds of screening.

FIG. 3 shows the ELISA identification results of 26 anti-human sortilin 1 nanobodies competitively inhibiting the binding of human sortilin 1 to its ligand granule protein precursor.

Figures 4 to 9 are the results of flow cytometry detection of the ability of different antibodies to bind to cell surface sortilin 1.

FIG10 shows the results of Nb426 cross-recognition of sort1 from different species.

Figures 11-16 are the results of the Gator affinity assay experiment for detecting the binding of nanobodies to human sortilin 1 recombinant protein.

Figure 17 shows the results of the Gator Epitope Binning experiment to detect multiple nanoantibodies binding to human Sortilin 1 recombinant protein.

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

Figure 19 shows the ELISA test results of the level of progranulin in the cell culture medium after U251 cells were treated with nanoantibodies.

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).

FIG. 21 is a schematic diagram of the construction of humanized mice expressing sort1 protein.

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

Figure 23 is a pscAAV-RNY20-VHH plasmid map.

FIG. 24 shows the results of Progranulin detection in serum after injection of AAV encoding Sort1 nanobody and AL001.

FIG. 25 shows the results of the detection of Progranulin levels in the cerebrospinal fluid of mice 45 days after administration.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Explanation of terms involved in the following embodiments:

In the present invention, the terms "antibody of the present invention", "antibodies 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 can be used interchangeably, all referring to Nanobodies that specifically recognize and bind to sortilin 1 (including human sortilin 1).

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

In the present invention, the terms "single domain antibody", "VHH", "nanobody", "single domain antibody" (sdAb, or nanobody) have the same meaning and can be used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

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

In the present invention, the term "variable" means that some parts of the variable region in the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light chain and heavy chain. 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 together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Pub1. No. 91-3242, Volume I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

In the present invention, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules and antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the anti-Sort1 nanobody or fragments thereof.

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

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

In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to Sortilin 1, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

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.

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. The polypeptide fragments, derivatives or analogs of the present invention may be: (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol) or a polypeptide (such as a polypeptide that prolongs the half-life, such as a nanobody against serum albumin, or an engineered antibody Fc domain), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6xHis tag). According to the teachings herein, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art.

The antibody of the present invention refers to a polypeptide having sortilin 1 binding activity and comprising the above CDR region. The term also includes variant forms of polypeptides comprising the above CDR region and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

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.

The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids 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.

The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

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

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

The present invention also relates to polynucleotides that hybridize with 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 can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The 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 the protein sequence of the present invention by chemical synthesis.

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.

Host cells can be prokaryotic cells, such as bacterial cells; or 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 of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture 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 by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using 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, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

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.

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 a variety of DNA damaging drugs, or microtubule inhibitors; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

Sortilin 1 is a cell membrane receptor protein belonging 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 participates in the growth and development regulation of neurons 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. 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.

Pharmaceutical composition

The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, 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 with 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.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or its conjugate) of the present invention 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, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 milligrams/kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

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 usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 100 milligrams/kg body weight, preferably the dosage is about 10 micrograms/kg body weight to about 50 milligrams/kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

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

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.

Labeled Antibodies

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

Colloidal gold labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the antibody of sortilin 1 is labeled with colloidal gold to obtain a colloidal gold-labeled antibody.

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

Detection Methods

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.

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 solution.

Reagent test kit

The present invention also provides a kit containing the antibody (or fragment thereof) or the 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, and the like.

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 universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

Another key content of the present invention is to use AAV vectors to endogenously express nanobodies against sorting protein 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 (ITR) 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 sorting protein 1 in patients or experimental animals. As a result, nanobodies mediated by AAV endogenous expression can effectively inhibit the function of sorting protein 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 administration.

The experimental methods in the following examples where specific conditions are not specified are generally carried out under 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 stated, percentages and parts are calculated by weight.

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

Table 1 List of Nanobody Sequences

Table 4 List of Nanobody Sequences

Example 1 Expression of human sortilin 1 extracellular segment fusion protein

Expression and purification of human Sortilin-Fc fusion protein: Transient expression of human sortilin 1 extracellular segment protein using mammalian cells Expi-293F: Mix the pcDNA3.1-H-SORT1-hFc recombinant plasmid cloned with the human sortilin 1 extracellular segment gene with the transfection reagent polyethyleneimine (PEI), and transfect it into Expi-293F cells; culture in a _shaker incubator at 37°C, 5% CO2 for 5 days; then collect the cell supernatant and bind it to Protein A beads at room temperature for 1 hour; then wash the beads with phosphate buffer pH 7.0, and elute the protein with 0.1M pH3.0 glycine solution; then ultrafilter the eluted protein into phosphate buffer solution, determine the yield and take a sample 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 can be used for camel immunization and antibody screening (where M represents Marker, and lane 1 is human sortilin 1 protein with Fc domain).

Expression and purification of human Sortilin-HisTag fusion protein: transient expression of human Sortilin 1 extracellular segment protein using mammalian cells Expi-293F: pcDNA3.1-H-SORT1-Hisx6 recombinant plasmid cloned with human Sortilin 1 extracellular segment gene was mixed with transfection reagent PEI and transfected into Expi-293F cells; cultured in a shaking incubator at 37°C and 5% CO ₂ for 5 days; then the cell supernatant was collected, bound to Ni affinity chromatography column beads, and the protein was eluted with 300mM imidazole and 500mM imidazole solution; the eluted protein was then ultrafiltered into PBS solution, and the yield was measured and sampled for SDS-PAGE detection. The detection results are shown in B in Figure 1. The purity of the expressed and purified H-SORT1-Hisx6 protein is greater than 90%, which can be used for camel immunization and antibody screening (where M represents Marker, and lane 2 is human Sortilin protein with Hisx6 tag).

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

Purified human Sortilin-Hisx6 fusion protein was mixed with Freund's adjuvant in a 1:1 ratio, and a healthy alpaca was immunized four times by multi-point subcutaneous injection. During the period, a small amount of blood was drawn to separate serum for serum titer detection. 100 mL of peripheral blood was collected 7-10 days after the last immunization, and Ficoll lymphocyte separation medium (GE Healthcare 17-1440-03 FICOLL PAQUE PLUS) was used to separate mononuclear cells (PBMC).

Total mRNA of PBMC was extracted with phenol chloroform, and RNA was converted into cDNA using SuperScript ^TM IV First-Strand cDNA Synthesis Reaction Kit (Thermo, 18091200). Using cDNA as a template, nested PCR was used to amplify the heavy chain variable region fragment (Q5 High-Fidelity 2X Master NEB, M0492L). The primers used in the two rounds of PCR are shown in Table 3 below:

Table 3 Primers used for nested PCR amplification of VHH genes

The antibody fragments recovered from the gel of the second round of PCR and the phage vector pADL were digested separately with Not1 and Bstx1 restriction endonucleases, and the digested products were subjected to agarose electrophoresis and purified, followed by enzyme ligation reaction. The enzyme ligation products were electrotransformed into TG1 competent cells (Weidi Biotechnology, DE1055M) to construct an Escherichia coli library containing nanobody fragments. The clone count showed that the library capacity was 5E8; 40 single clones were randomly selected for sequencing, and the sequencing results showed that the insertion rate of the nanobody gene was about 95%.

After the obtained sorted protein 1 immune library was amplified, VCSM13 helper phage was added in the logarithmic growth phase, and the bacteria were infected at 37°C for 1 hour; after centrifuging the bacterial culture, the supernatant was discarded and the bacteria were resuspended in 2xYT medium with antibiotics and 0.5% glucose, and the phage was packaged overnight in a constant temperature shaker at 30°C and 225rpm. The next day, the phage particles were precipitated with polyethylene glycol/sodium chloride, and the library was screened using phage display technology. After three rounds of "adsorption-washing-enrichment" screening, the nano-antibody phage group that binds to the H-Sort1-Hisx6 fusion protein was enriched. 576 monoclones randomly selected from the above enriched phage group were amplified and IPTG-induced expression was performed. The periplasmic protein extract (PPE) was obtained by bursting the bacterial outer wall with a hypotonic solution PPB (phosphate peptone buffer), and the binding of PPE to human sorted protein 1 (ECD) protein was identified by ELISA. The results showed that 117 out of 576 random clones bound to Sortlilin ( FIG. 2 , clones with ELISA readings more than 5 times the background were defined as positive clones).

In order 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.

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 with the human Fc domain encoding gene, and Expi-293F cells were transfected 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.

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

ELISA plates were coated with 50 μL 4 μg/mL H-Sort1-His antigen at 4°C overnight; washed 3 times with 0.1% PBST (phosphate-buffered saline-Tween buffer), and 150 μL 5% BSA (bovine serum albumin) was added for 1 hour at room temperature; washed 3 times with 0.1% PBST, 6 ug/mL Bio-PGRN (biotin-modified human progranulin) and 4 ug/mL VHH-Sort1 were mixed and added, and 6 ug/mL Bio-PGRN alone was used as a control 1, and 6 ug/mL B Bio-PGRN and 4ug/mL Hyhel-VHH (non-specific control nanobody) as control 2, 1 hour at room temperature; wash 3 times with 0.1% PBST, add 50μL horseradish peroxidase-labeled streptavidin (1:5000) (diluted with 5% BSA), 1 hour at room temperature; wash 3 times with 0.1% PBST, add 50μLTMB (3,3',5,5'-tetramethylbenzidine) for color development for 8-10 minutes, and terminate the reaction with 50μL stop solution; read the absorbance of each well at a wavelength of 450nm. If the nanobody competes for the binding of sortilin 1 to the granulin precursor, it will lead to a decrease in Bio-PGRN bound to sortilin 1, and the reading of the corresponding sample will be lower than that of the sample without the nanobody.

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, and we selected these 25 antibodies for further characterization analysis.

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

The pcDNA3.1-HSort1 recombinant plasmid cloned with the full-length human sortilin 1 protein gene was mixed with the transfection reagent PEI and transfected into 293T cells. After 48 hours of transfection, the cells were collected by blowing and suspension, the culture medium was removed by centrifugation, and the cells were washed twice with pre-cooled PBS, and the cells were suspended in a pre-cooled PBS solution containing 1% BSA. After counting the cells, 100,000 cells/well were transferred to a U-shaped bottom 96-well plate, and purified anti-sortilin 1 nano antibody VHH-Fc was added to an antibody concentration of 50ng/ml, and incubated on ice for 30 minutes; the cells were washed twice with pre-cooled PBS, and the secondary antibody with anti-human Fc domain labeled with APC fluorescence was added, and incubated on ice for 30 minutes, and the cells were washed twice with cold PBS, and suspended in a pre-cooled PBS solution containing 1% BSA, and the binding of the antibody to the cell surface antigen was analyzed by flow cytometry.

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.

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, as 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 the untransfected parental 293T cells, indicating that these nanoantibodies have high sortilin 1 specificity.

Table 4 Flow cytometry results

Example 5 Cross-recognition of Nb426

Plasmids pcDNA3.1-HSort1, pcDNA3.1-MSort1, and pcDNA3.1-CSort1 containing the protein coding genes of human, mouse, and monkey Sort1 were constructed, and the sequences were confirmed to be correct after sequencing and alignment. 293T cells were transfected respectively to obtain 293T cells with high expression of Sort1 of different species on the cell surface, namely hSort1-293T, mSor1-293T, and cSort-T. 48 hours after transfection, cells were collected and washed, and suspended in pre-cooled PBS solution containing 1% BSA, and purified anti-Sort1 nanoantibody Nb426-Fc or control antibody AL001 with human Fc tag was added to the antibody concentration of 50ng/ml, and incubated on ice for 30 minutes; cells were washed twice with pre-cooled PBS, and secondary antibodies with APC fluorescent labeling against human Fc domain were added, incubated on ice for 30 minutes, and cells were washed twice with cold PBS, suspended in pre-cooled PBS solution containing 1% BSA, and the binding effect of antibodies on Sort1 proteins of different species on the cell surface was analyzed by flow cytometry. The same antibody and process were used for untransfected wild-type 293T cells as a control for non-specific binding.

As shown in Figure 10, compared with the binding to wild-type 293T cells, the binding of anti-Sort1 nanobody Nb426 to 293T cells transfected with human, mouse, and monkey Sort1 proteins was significantly increased, indicating that the nanobody clone can recognize Sort1 proteins from three species; in comparison, the control antibody AL001 can bind to Sort1 proteins from humans and monkeys, but has a weak binding to mouse Sort1. This result not only shows that Nb426 has better Sort1 species recognition characteristics, but also that the antigen epitope it binds is different from the control antibody AL001.

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

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

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 300-1.2 nM of human Sortilin-Hisx6 fusion protein, including a no sortin 1 protein control (0 nM), for about 180 seconds, followed by a 180-second dissociation step. The association and dissociation curves were plotted and calculated by GatorPrime (Gator Bio) software to obtain the 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).

The same method was used to determine the binding kinetics of the control antibody Latozinemab to the human sortilin 1 fusion protein. The binding kinetics 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.

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

Example 7 Using the double epitope distribution assay to determine the grouping of antigen epitopes recognized by nanobodies on sortilin 1

Biolayer interferometry was performed using the Gator Prime system (Gator Bio) v2.7.3.0728 (https://www.gatorbio.com/) to evaluate the antigenic epitopes to which each nanobody binds to the sortilin-1 fusion protein.

The glass His-tag probe (Gator Bio) was first immersed in Q buffer (Gator Bio) for about 30 seconds to obtain a baseline signal. Then, the probe was loaded with 100nM human Sortilin-Hisx6 fusion protein diluted in Q buffer for 180 seconds; followed by a 30-second Q buffer wash step. Next, the probe bound to Sortilin-Hisx6 was bound to 300nM control antibody AL001 diluted in Q buffer for about 180 seconds; then the probe bound to Sortilin-AL001 was immersed in a 200nM nanobody-Fc fusion protein (VHH-Fc) solution diluted in Q buffer for a 180-second double-binding step. The double-binding curve was drawn and calculated by GatorPrime (Gator Bio) software to obtain the grouping of different antibodies against the original binding epitope.

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.

Example 8 Immunofluorescence observation of the process of nanoantibodies binding to cell surface Sort1 and then internalizing into the cell

After the protein receptors on the cell membrane surface are bound by antibodies, they may undergo endocytosis due to conformational changes, which is also the key reason why most antibody-drug conjugates (ADCs) kill cancer cells. The human glioma cell line U251 has a higher level of Sort1 receptor protein on its surface, which can be used as a functional detection tool for anti-Sort1 antibodies. When antibodies bind to cell surface receptors, endocytosis is a relatively fast process. In order to better observe this process, we used 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 a specific experimental plan:

U251 cells were taken and plated in a culture dish equipped with a glass slide at a density of 1*10 ⁶ cells and cultured for 24 hours to allow them to attach. Remove the culture medium, add PBS solution containing 1 μg/mL anti-Sort1 nanobody-Fc fusion protein, and incubate the culture dish at 37℃ and 4℃ for 15 minutes respectively; wash the cell slides in the culture dish with PBS for 3 times, 3 minutes each time; fix the cell slides with 4% paraformaldehyde for 15 minutes, then wash with PBS 3 times, 3 minutes each time, to remove excess paraformaldehyde; use 0.5% Triton X-100 for cell permeabilization at room temperature for 20 minutes, then wash with PBS 3 times, 3 minutes each time, to remove excess Triton X-100; use PBS solution containing 5% BSA for blocking treatment at room temperature for 30 minutes; add diluted anti-human IgG Fc-FITC antibody (ratio of 1:500), incubate at room temperature in the dark for 1 hour to bind the previous anti-Sort1 nanobody-Fc fusion protein; then wash with PBS 3 times to remove excess fluorescent secondary antibody. Finally, add anti-fluorescence quencher containing DAPI, seal the slides, and observe under a microscope.

The results are shown in Figure 18 (Nb07 is Nb171, Nb08 is Nb184). The results of the immunofluorescence experiment showed that under the condition of a warm water bath at 37°C, the anti-Sort1 nanobody can quickly enter the cell within 15 minutes, and its fluorescence signal will appear in the cytoplasm in the form of endosomes and gradually accumulate in the area near the 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 nanoantibodies bind to Sort1, they can be efficiently internalized into cells and have the potential to be developed into ADC anti-tumor drugs.

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

To test whether anti-Sort1 nanobodies can increase the level of progranulin, we treated U251 cells cultured in vitro with antibodies. Specifically, U251 cells in the logarithmic growth phase were plated in a 96-well cell culture plate, with 1E4 cells per well, and placed in a 37°C cell culture incubator containing 5% CO _2. 24 hours after plating, VHH-Fc nanobodies at a working concentration of 40nM or control antibody AL001 at the same molar concentration were added. After 72 hours, the cell culture supernatant was taken and the concentration of progranulin was detected using progranulin ELISA KIT (EAGLE BIOSCIENCES Cat#: E103). The detection method is as follows: take the cell supernatant and dilute it 15 times with the dilution buffer provided by the kit; add 50uL of the antibody conjugate provided in the kit and 50ul of the diluted sample (including the cell culture supernatant treated with the antibody, the granulin precursor standard solution and the positive control) to each ELISA well of the kit, and incubate at room temperature for 1 hour (≥350rpm); discard the supernatant and wash 5 times with the washing solution; add 100uL of the enzyme conjugate provided by the kit and incubate at room temperature for 30 minutes (≥350rpm); discard the supernatant and wash 5 times with the washing solution; add 100uL of the substrate solution provided by the kit and incubate at room temperature for 30 minutes in the dark; add 100uL of the stop solution to each well and detect the absorption light at 450nm within 30 minutes. When analyzing the data, the concentration of granulin precursor in the cell culture supernatant is calculated according to the absorbance curve of the standard sample.

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 achieved or exceeded the effect of the control antibody AL001 in increasing progranulin ( FIG. 19 ).

In order to further determine the relationship between antibody concentration and the secretion level of progranulin in U251 cells, U251 cells were treated with different concentration gradients of sortilin 1 nanoantibody VHH-Fc, and the concentration level of progranulin in the cell culture medium was determined by ELISA after 72 hours of treatment, and the effect of the antibody was calculated by comparing the concentration of progranulin in the culture medium of U251 cells without antibody. The results showed that within the test range of 0.004ug/mL-4.0ug/mL, the effect of Nb171 and Nb184 in inducing the increase of progranulin was roughly positively correlated with the antibody concentration, and the increase of progranulin induced by high concentration antibodies was more obvious. It is worth mentioning that at the same antibody concentration, the effects of Nb171 and Nb184 in inducing the increase of progranulin were better than AL001 (Figure 20).

Example 10 In vivo experiment

To further verify the in vivo effects of anti-sortilin 1 nanoantibodies, 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 the 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).

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

The results showed that after intravenous injection of anti-sortilin 1 nanobody, the level of progranulin in mouse serum increased significantly. Even 11 days after antibody injection, the level of progranulin in serum was still more than 3 times the level before administration. This shows 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.

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

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

AAV virus packaging adopts the classic three-plasmid transfection method. It includes 1) expression plasmid pAAV9-Cap-WT encoding wild-type AAV9REP-CAP gene; 2) helper plasmid pADdeltaF6 (Helper plasmid) encoding E4/E2A/VA/L4/L5, 3) optimized expression vector plasmid pscAAV-RNV20-VHH (transfer plasmid) encoding anti-Sort1 nanobody gene. The three plasmids were mixed in equal proportions and transfected into 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. After that, it was filtered and sterilized with a 0.22um filter, and then ultrafiltration concentration, buffer replacement, affinity chromatography to remove host cell proteins, anion exchange chromatography to remove other trace impurities and virus empty shells and other classic steps were performed to obtain purified AAV virus with nanobody gene. The virus titer in the samples was determined by qPCR method. 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.

To evaluate the physiological function of anti-Sort1 nanobodies endogenously expressed by AAV, we selected 6-week-old Sort1 humanized mice for administration and analysis. On day 0 of the experiment, we injected AAV9 virus carrying the encoded nanobodies by a single tail vein administration, and each mouse received 1E12 vg of virus. We collected blood samples at different time points before and after administration to measure the level of Progranulin protein in mouse serum. The experiment ended on the 45th day after administration. At the end of the experiment on the same day, the mice were killed and cerebrospinal fluid was collected for testing.

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

The results of the Progranulin detection in serum are shown in Figure 24. A single injection of AAV9 encoding the Sort1 nanobody into the tail vein can significantly increase the Progranulin level in the mouse serum by more than one fold. This increase began to appear on the second day after administration and was maintained until the end of the experiment, indicating that the AAV-expressed nanobody has long-term effectiveness. As a control, the injection of the anti-Sort1 monoclonal antibody AL001 can only temporarily increase the Progranulin level in the mouse serum. This increase reached the highest level 2 days after administration, and then decreased. The effect can only be maintained by re-administration. 14 days after the last administration, the Progranulin in the serum will drop to the level before administration.

The level of Progranulin in the cerebrospinal fluid is an important pharmacodynamic marker for the treatment of central nervous system diseases such as FTD. After 45 days of administration, we killed the mice and measured the level of Progranulin in the cerebrospinal fluid of the mice. The test results are shown in Figure 25. It can be seen that after 45 days of administration, the level of Progranulin in the cerebrospinal fluid of mice injected with AAV endogenously expressing anti-Sort1 nanoantibodies was 2.5-5 times higher than that of the control group injected with AL001 monoclonal antibodies, indicating that the AAV9-expressed nanoantibodies entered the brain tissue to a certain extent and played a therapeutic role. The degree of increase in cerebrospinal fluid Progranulin exceeded the effect of long-term repeated administration of AL001 published by Alector and other companies, indicating that AAV endogenously expressing anti-Sort1 nanoantibodies has better therapeutic potential for neurodegenerative diseases such as FTD.

Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims (19)

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. 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, and 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. 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, 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. The nanobody according to claim 1 is 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. A polynucleotide encoding the Nanobody according to any one of claims 1 to 4. A recombinant expression vector containing the polynucleotide according to claim 5. 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. The recombinant expression vector according to claim 7 is 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. The recombinant expression vector according to claim 8 is characterized in that the recombinant adeno-associated virus vector contains: a promoter, a signal peptide, a polynucleotide and a polyA tail. The recombinant expression vector according to claim 9 is characterized in that: the recombinant adeno-associated virus vector contains: CAG promoter or CMV promoter, BM40 signal peptide, polynucleotide and SV40 polyA tail, the CAG promoter and BM40 signal peptide are located upstream of the nanoantibody coding sequence, and the SV40 polyA tail is located downstream of the nanoantibody coding sequence. The recombinant expression vector according to claim 8 is characterized in that the recombinant adeno-associated virus vector contains an inverted terminal repeat sequence. A recombinant cell containing the recombinant expression vector according to any one of claims 6 to 11. 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. A drug, characterized in that it 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. The drug according to claim 14 is characterized in that: the drug comprises 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. 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. The use according to claim 16 is characterized in that the product is used to inhibit the function of sortilin 1 molecules, identify cells with high expression of sortilin 1, or increase the level of progranulin. The use according to claim 16 is characterized in that the product is a drug for preventing or treating tumors with high expression of sortilin 1. The use according to claim 16 is characterized in that the product is a drug for preventing or treating central nervous system diseases related to granule protein levels.