A recombinant hARSA that crosses the blood-brain barrier

Abstract

A fusion protein comprising: a transferrin binding protein and arylsulfatase A (ARSA) or a functionally active fragment thereof, wherein the transferrin binding protein comprises a polypeptide, an antibody or an antigen-binding fragment thereof capable of binding to transferrin. A preparation method and use of the fusion protein are also provided.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a fusion protein comprising transferrin binding protein and arylsulfatase A, and uses thereof. Background Art

[0002] Arylsulfatase A (ARSA, also known as acylsulfatase A, cerebroside-sulfatase or arylsulfatase A) is a type of lysosomal hydrolase that can decompose sulfatide, that is, decompose "cerebroside 3-sulfate" into cerebroside and sulfate. ARSA mainly plays a biological role in lysosomes and participates in the degradation of polyphospholipids. At the same time, in the cell cytoplasm, the direct binding of ARSA enzyme and α-synuclein effectively prevents the latter's protein aggregation and extracellular secretion. Therefore, the absence or reduced activity of ARSA enzyme is the direct cause of metachromatic leukodystrophy (MLD, a lysosomal storage disease that seriously affects life), and is also related to the occurrence and development of various degenerative neurological diseases such as Parkinson's disease. Therefore, ARSA enzyme replacement therapy, especially enzyme replacement therapy targeting the central nervous system, is expected to provide necessary solutions for the treatment of these diseases that seriously affect human life and health and for which there is currently no effective treatment. Summary of the invention

[0003] The present application provides a fusion protein, which comprises: transferrin binding protein, and arylsulfatase A (ARSA) or its functionally active fragment. The fusion protein described in the present application adopts the single-domain antibody technology targeting transferrin to construct a protein or polypeptide fragment fused with the single-domain antibody, maintains good binding activity with transferrin, does not affect the binding of transferrin to transferrin receptor, and has the function of extending half-life and crossing the blood-brain barrier.

[0004] On the one hand, the present application provides a fusion protein comprising: a transferrin binding protein and arylsulfatase A (ARSA) or a functionally active fragment thereof, wherein the transferrin binding protein comprises a polypeptide, an antibody or an antigen-binding fragment thereof capable of binding to transferrin.

[0005] In certain embodiments, the fusion protein has one or more of the following properties: 1) capable of extending the in vivo half-life of the ARSA; 2) capable of crossing the blood-brain barrier; 3) capable of oral delivery; and 4) capable of delivering the ARSA to cells expressing the transferrin receptor.

[0006] In certain embodiments, the transferrin is human transferrin.

[0007] In certain embodiments, the antibody is selected from one or more of the following groups: a single domain antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0008] In certain embodiments, the antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, VHH, di-scFv and / or dAb.

[0009] In certain embodiments, the transferrin binding protein is a VHH.

[0010] In certain embodiments, the transferrin binding protein comprises CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0011] In certain embodiments, the transferrin binding protein comprises CDR2, and the CDR2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0012] In certain embodiments, the transferrin binding protein comprises CDR1, and the CDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0013] In certain embodiments, the transferrin binding protein comprises VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0014] In certain embodiments, the ARSA is human ARSA.

[0015] In certain embodiments, the ARSA or a functionally active fragment thereof comprises a variant of ARSA or a functionally active fragment thereof.

[0016] In certain embodiments, the ARSA or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.

[0017] In certain embodiments, in the fusion protein, the transferrin binding protein and the ARSA or a functionally active fragment thereof are directly or indirectly linked.

[0018] In certain embodiments, in the fusion protein, the N-terminus of the transferrin binding protein and the C-terminus of the ARSA or a functionally active fragment thereof are directly or indirectly linked.

[0019] In certain embodiments, in the fusion protein, the C-terminus of the transferrin binding protein and the N-terminus of the raw ARSA or a functionally active fragment thereof are directly or indirectly linked.

[0020] In certain embodiments, in the fusion protein, the transferrin binding protein and the ARSA or a functionally active fragment thereof are connected via a linker.

[0021] In certain embodiments, the linker comprises a peptide linker.

[0022] In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:9.

[0023] In certain embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:11.

[0024] In certain embodiments, the fusion protein is capable of binding to transferrin and catalyzing the degradation of sulfatide, a substrate of arylsulfatase A.

[0025] In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the fusion protein.

[0026] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0027] On the other hand, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0028] On the other hand, the present application provides a pharmaceutical composition, which comprises the fusion protein, the nucleic acid molecule, the vector or the cell, and optionally a pharmaceutically acceptable carrier.

[0029] On the other hand, the present application provides the use of the fusion protein, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease and / or condition.

[0030] In certain embodiments, the disease and / or condition comprises a disease and / or condition caused by abnormalities in ARSA.

[0031] In certain embodiments, the disease and / or condition comprises a neurological disease caused by deposition of sulfatide substrates.

[0032] In certain embodiments, the disease and / or condition comprises Metachromatic Leukodystrophy (MLD).

[0033] In certain embodiments, the disease and / or disorder comprises a neurodegenerative disease.

[0034] In certain embodiments, the disease and / or condition comprises Parkinson's disease.

[0035] On the other hand, the present application provides a method for preventing and / or treating a disease and / or condition, comprising administering the fusion protein, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition to a subject in need thereof.

[0036] In certain embodiments, the disease and / or condition comprises a disease and / or condition caused by abnormalities in ARSA.

[0037] In certain embodiments, the disease and / or condition comprises a neurological disease caused by deposition of sulfatide substrates.

[0038] In certain embodiments, the disease and / or condition comprises Metachromatic Leukodystrophy (MLD).

[0039] In certain embodiments, the disease and / or disorder comprises a neurodegenerative disease.

[0040] In certain embodiments, the disease and / or condition comprises Parkinson's disease.

[0041] On the other hand, the present application also provides the fusion protein, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition, which are used for preventing and / or treating diseases and / or conditions.

[0042] In certain embodiments, the disease and / or condition comprises a disease and / or condition caused by abnormalities in ARSA.

[0043] In certain embodiments, the disease and / or condition comprises a neurological disease caused by deposition of sulfatide substrates.

[0044] In certain embodiments, the disease and / or condition comprises Metachromatic Leukodystrophy (MLD).

[0045] In certain embodiments, the disease and / or disorder comprises a neurodegenerative disease.

[0046] In certain embodiments, the disease and / or condition comprises Parkinson's disease.

[0047] On the other hand, the present application also provides a method for delivering a drug across the blood-brain barrier, which comprises administering the fusion protein.

[0048] On the other hand, the present application also provides a method for prolonging the half-life of ARSA in vivo, which comprises administering the fusion protein.

[0049] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The features and advantages of the inventions of the present application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0051] Figure 1 It was shown that the single-domain antibody VHH binding to transferrin significantly prolonged the half-life of recombinant ovalbumin in the blood of mice and enhanced its ability to transport across the blood-brain barrier.

[0052] Figure 2 Shown is the SDS-PAGE image of rhARSA related recombinant proteins.

[0053] Figure 3 Shown is the binding assay of rhARSA-related recombinant proteins to hTf.

[0054] Figure 4 Shown are the results of FACS detection of the binding of rhARSA-related recombinant proteins to hTf / hTfR1.

[0055] Figure 5 A shows that fusion of VHH with hARSA protein does not affect M6PR binding. Figure 5 B shows mARSA - / - The amount of 9056VHH-hARSA taken up by fibroblasts was 5.57 times that of hARSA (10 μM), which could be partially blocked by M6P. Figure 5C shows that the cellular uptake of 9056VHH-hARSA is time-dependent.

[0056] Figure 6 Shown is confocal laser scanning microscopy of mARSA - / - Intracellular localization of ARSA recombinant protein after uptake by fibroblasts.

[0057] Figure 7A-7B Shown is an in vitro recombinant ARSA enzyme specific activity assay (substrate pNCS).

[0058] Figure 8 Displayed is mARSA - / - Alcian blue / fast red staining results after fibroblasts were cultured with 10 μM, 3 μM, and 0 μM hARSA, 9056VHH-hARSA, and hARSA-9056VHH for 48 hours.

[0059] Figures 9A-9B Shown are the in vitro plasma stability results of recombinant ARSA enzyme.

[0060] Fig.10 Shown are the metabolic half-lives of hARSA, hARSA-9056VHH, and 9056VHH-hARSA in mouse blood.

[0061] Figures 11A-11B Shown is the efficiency of hARSA and 9056VHH-ARSA in crossing the blood-brain barrier in wild-type C57bl / 6 mice.

[0062] Figures 12A-12C Shown is the efficiency of 9056VHH-ARSA crossing the blood-brain barrier in wild-type C57bl / 6 mice at increasing doses.

[0063] Fig.13 Shown is the drug distribution and organelle localization of hARSA-9056VHH in MLD model mice 2 hours after intravenous injection of 30MPK.

[0064] Figures 14A-14C Shown are the results of a PK study of recombinant hARSA-9056 VHH crossing the blood-brain barrier in cynomolgus monkeys.

[0065] Fig.15 Shown is the evaluation of motor ability of mARSA-deficient mice after hARSA-9056VHH treatment.

[0066] FIG. 16 shows the level of sulfatide substrate in the brain tissue of mARSA-deficient mice detected by TLC after hARSA-9056VHH treatment.

[0067] Fig.17Shown is the level of sulfatide substrates detected by ABS staining in the brain tissue of mARSA-deficient mice after hARSA-9056VHH treatment.

[0068] Fig.18 Shown are TLC / ABS staining to detect sulfatide substrate levels in the sciatic nerve of mARSA-deficient mice after hARSA-9056VHH treatment.

[0069] Fig.19 Shown are sulfatide substrate levels in the kidneys of mARSA-deficient mice after hARSA-9056VHH treatment. DETAILED DESCRIPTION

[0070] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0071] Definition of terms

[0072] In the present application, the term "fusion protein" generally refers to a protein obtained by fusing two or more proteins or polypeptides. Fusion proteins can be artificially prepared by recombinant DNA technology. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be connected to each other to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion protein. Translation of the fusion gene can produce a single polypeptide, which can have the properties of at least one or even each of the two or more proteins or polypeptides before fusion.

[0073] In the present application, the term "transferrin" generally refers to a glycoprotein that can bind and transport multivalent ions. For example, transferrin can be a single-chain glycoprotein. For example, transferrin can have at least one ion binding site. For example, the ion binding site can have different affinities with iron ions. For example, multivalent iron ions can be iron ions, chromium ions, manganese ions, cadmium ions or nickel ions. For example, each molecule of transferrin can bind two trivalent iron atoms. For example, transferrin can be iron-containing Holo-transferrin, or iron-free apo-transferrin. For example, transferrin can be mouse transferrin. For example, the amino acid sequence of mouse transferrin can be that specified in GenBank. EDL21066.1, AAL34533.1, or AAL34533.1. For example, transferrin can be human transferrin. For example, the amino acid sequence of human transferrin can be that specified in GenBank. AAH59367.1, AAH59367.1, or AAB22049.1. In the present application, the term "transferrin" may include functionally active fragments, homologues, analogues and / or variants thereof.

[0074] In the present application, the term "transferrin binding protein" generally refers to a protein comprising a transferrin binding moiety, and optionally allows the antigen binding moiety to adopt a support or skeleton portion that promotes the conformation of the antigen binding protein to bind to the antigen. For example, the transferrin binding protein described in the present application may include but is not limited to an antibody, an antigen binding fragment (Fab, Fab', F(ab)2, Fv fragment, F(ab')2, VHH, scFv, two scFvs and / or dAb), an immunoconjugate, a multispecific antibody, an antibody fragment, an antibody derivative, an antibody analog or a fusion protein, as long as they can show the required antigen binding activity. For example, the antigen binding protein can specifically bind to transferrin. For example, the transferrin binding protein may not interfere with the interaction between transferrin and transferrin receptor 1. For example, the transferrin binding protein may not affect the combination of Tf / TfR1. For example, the transferrin binding protein can maintain the normal physiological function of iron transportation.

[0075] In this application, the term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. For example, immunoglobulin genes may include κ, λ, α, γ, δ, ε and μ constant region genes, as well as numerous immunoglobulin variable region genes. For example, light chains may be classified as κ or λ, which may define the immunoglobulin types: Igκ and Igλ, respectively. Heavy chains may be classified as γ, μ, α, δ or ε, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD and IgE, respectively. For example, an antibody may have a structural unit comprising a tetramer, each tetramer may be composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kD) and a "heavy chain" (about 50-70 kD), and the N-terminus of each member may define a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. For example, the terms "light chain variable region (VL)" and "heavy chain variable region (VH)" generally refer to the variable region regions of the light chain and heavy chain, respectively. Antibodies can exist as complete immunoglobulins or as many well-characterized fragments produced by digestion with various peptidases or de novo expression. In the present application, the antibody may also comprise only the heavy chain variable region. For example, the antibody may be a single domain antibody.

[0076] In the present invention, the term "antigen binding fragment" generally refers to one or more parts of a full-length antibody, which substantially retains the ability to bind to the same antigen (e.g., CD38) to which the antibody binds, and can compete with the full-length antibody for specific binding to the antigen. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), and the entire text is incorporated into this application by reference. Antigen binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen binding fragments include Fab, Fab', F(ab')2, (Fab)2, Fd, Fv, dAb and complementary determining region (CDR) fragments, VHH, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies. For example, pepsin can digest antibodies below the disulfide bonds in the hinge region to produce F(ab')2.

[0077] In the present application, the term "VHH" generally refers to an antibody comprising a variable antigen binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as a nanobody (Nanobody) (Nb) and / or a single domain antibody.

[0078] In this application, the term "functionally active fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to another molecule. For example, a functionally active fragment of arylsulfatase A (ARSA) can retain or partially retain the biological activity function of the full-length ARSA that causes cell proliferation.

[0079] In the present application, the term "arylsulfatase A" and "ARSA" can be used interchangeably, and the term can include wild-type ARSA and modified ARSA. For example, the ARSA can include its homologues, analogs, derivatives and / or functional variants. For example, the ARSA can include full-length ARSA, and the ARSA can include functionally active fragments of ARSA. In the present application, the ARSA can include human ARSA ("hARSA").

[0080] In this application, the term "Fab" generally refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains.

[0081] In this application, the term "Fab'" generally refers to an antibody fragment that has a few additional residues at the carboxyl terminus of the CH1 domain compared to the Fab fragment. For example, Fab' may include one or more cysteines from the antibody hinge region.

[0082] In the present application, the term "F(ab)2" generally refers to an antigen-binding fragment obtained by a pair of cysteine-linked Fab fragments.

[0083] In this application, the term "dAb fragment" generally refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341: 544-546 (1989)).

[0084] In the present application, the term "complementarity determining region CDR" generally refers to the three hypervariable regions (HVR) of the light chain variable region (VL) and the heavy chain variable region (VH). Because this part can form precise complementarity with the antigenic determinant cluster in terms of spatial structure, the hypervariable region is also called the complementarity determining region.

[0085] In the present application, the term "Fv fragment" generally refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0086] In the present application, the term "scFv" generally refers to a molecule formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide linker, also known as a single-chain antibody.

[0087] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the proteins or polypeptides.

[0088] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide. For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example 1-30, 1-20 or 1-10, for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0089] As used in this application, the term "sequence homology" generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program (e.g., Emboss Needle or BestFit) to determine the sequence identity, similarity or homology between two different amino acid sequences, the default settings can be used, or a suitable scoring matrix (such as blosum45 or blosum80) can be selected to optimize identity, similarity or homology scores. In some embodiments, homologous polynucleotides are those sequences that hybridize under stringent conditions, and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity compared to those sequences. When aligning sequences with considerable length, homologous polypeptides have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity, or have at least 99% sequence identity.

[0090] Generally, in a polypeptide chain, an amino group is connected to another carboxyl group in the polypeptide chain to form a chain, but at the two ends of the protein, there are amino acid residues that do not form a peptide bond, respectively, the end of the polypeptide chain carrying a free amino group and the end of the polypeptide chain carrying a carboxyl group. In this application, the term "N-terminal" generally refers to the end of the polypeptide chain where the amino acid residue carries a free amino group. In this application, the term "C-terminal" generally refers to the end of the polypeptide chain where the amino acid residue carries a free carboxyl group.

[0091] In this application, the term "nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.

[0092] In the present invention, the term "vector" generally refers to a nucleic acid carrier into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include: plasmids; bacteriophages; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. A vector may also include components that assist it in entering cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0093] In this application, the term "pharmaceutical composition" generally refers to a preparation in a form that permits the biological activity of the active ingredient to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is to be administered. For example, these preparations may be sterile.

[0094] In the present application, the term "pharmaceutically acceptable" generally refers to an adjuvant that is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. For example, a pharmaceutically acceptable adjuvant may refer to those adjuvants for animals (more particularly for humans) that are approved by a regulatory agency (such as the U.S. Food and Drug Administration, the China Food and Drug Administration or the European Medicines Agency) or listed in a generally recognized pharmacopoeia (such as the U.S. Pharmacopoeia, the Chinese Pharmacopoeia or the European Pharmacopoeia).

[0095] In this application, the term "comprising" generally means including, encompassing, containing or encompassing. In some cases, it also means "for", "consisting of...".

[0096] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. DETAILED DESCRIPTION OF THE INVENTION

[0098] In one aspect, the present application provides a fusion protein comprising: a transferrin binding protein and arylsulfatase A (ARSA) or a functionally active fragment thereof. For example, the transferrin binding protein may comprise a polypeptide, an antibody or an antigen-binding fragment thereof that can bind to transferrin.

[0099] In the present application, the fusion protein may have one or more properties. For example, the fusion protein can extend the in vivo half-life of the ARSA. For example, the fusion protein can cross the blood-brain barrier to achieve drug delivery. For example, the fusion protein can be delivered orally. For example, the fusion protein can deliver the ARSA to cells expressing transferrin receptors.

[0100] In the present application, the transferrin may be human transferrin.

[0101] In the present application, the antibody capable of binding to transferrin may include one or more selected from the group consisting of a single domain antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

[0102] In the present application, the antigen-binding fragment capable of binding to transferrin may include Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, VHH, di-scFv and / or dAb.

[0103] In the present application, the transferrin binding protein may comprise a VHH capable of binding to transferrin.

[0104] For example, the transferrin binding protein may comprise at least one CDR in VHH, and the VHH may comprise the amino acid sequence shown in SEQ ID NO:8.

[0105] In the present application, the CDR of an antibody is also called the complementary determining region, which is a part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. The antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat / Chothia, etc. These coding systems are known in the art, and specific references can be made to, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In the present application, the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR being substituted, deleted and / or added with one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologues thereof are also contemplated, and the homologues may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR. In certain embodiments, the CDR of the transferrin binding protein described herein may be defined by the Kabat coding system.

[0106] For example, the transferrin binding protein may comprise CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0107] For example, the transferrin binding protein may comprise CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.

[0108] For example, the transferrin binding protein may comprise CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:1.

[0109] For example, the transferrin binding protein may comprise CDR1, CDR2 and CDR3, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0110] For example, the transferrin binding protein may include the framework region FR1 of VHH, and the FR1 may include the amino acid sequence shown in SEQ ID NO: 4. For example, the transferrin binding protein may include the framework region FR2 of VHH, and the FR2 may include the amino acid sequence shown in SEQ ID NO: 5. For example, the transferrin binding protein may include the framework region FR3 of VHH, and the FR3 may include the amino acid sequence shown in SEQ ID NO: 6. For example, the transferrin binding protein may include the framework region FR4 of VHH, and the FR4 may include the amino acid sequence shown in SEQ ID NO: 7.

[0111] In the present application, the transferrin binding protein may comprise a VHH capable of binding to transferrin, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0112] In the present application, the ARSA or its functionally active fragment in the fusion protein can be human ARSA or its functionally active fragment. In the present application, the ARSA or its functionally active fragment can be modified so that it still retains the function and / or activity of ARSA.

[0113] In the present application, ARSA or a functionally active fragment thereof in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO:10.

[0114] The "ARSA or its functionally active fragment" described in the present application may also include variants, truncations, homologs, and analogs thereof. For example, the ARSA or its functionally active fragment may also include an amino acid sequence having at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%% sequence homology with the amino acid sequence shown in SEQ ID NO: 10. For example, the ARSA or a functionally active fragment thereof may comprise an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acid mutations compared to SEQ ID NO: 10.

[0115] In the present application, in the fusion protein, the transferrin binding protein can be directly or indirectly linked to the ARSA or a functionally active fragment thereof.

[0116] In the present application, in the fusion protein, the N-terminus of the transferrin binding protein can be directly or indirectly connected to the C-terminus of the ARSA or its functionally active fragment.

[0117] In the present application, in the fusion protein, the C-terminus of the transferrin binding protein can be directly or indirectly connected to the N-terminus of the ARSA or its functionally active fragment.

[0118] In the present application, in the fusion protein, the transferrin-binding protein can be connected to the ARSA or its functionally active fragment via a linker. For example, the linker can include a flexible linker. For example, the linker can include a peptide linker.

[0119] In the present application, in the fusion protein, the linker may comprise the amino acid sequence shown in SEQ ID NO:9.

[0120] In the present application, the fusion protein may comprise the amino acid sequence shown in SEQ ID NO:11.

[0121] In the present application, the fusion protein can bind to transferrin and catalyze the degradation of sulfatide, the substrate of arylsulfatase A.

[0122] Nucleic acid molecule, vector, cell, pharmaceutical composition, and preparation method

[0123] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the fusion protein.

[0124] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis.

[0125] Recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by TJ Silhavy, ML Bennan and LV Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, FM et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, the nucleic acids can be prepared from genomic DNA fragments, cDNA and RNA, all of which can be extracted directly from cells or recombinantly produced by various amplification methods (including but not limited to PCR and RT-PCR).

[0126] Direct chemical synthesis of nucleic acids generally involves sequentially adding 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide polymer chain, wherein each addition is achieved by nucleophilic attack on the terminal 5'-hydroxyl group of the growing chain at the 3'-position of the added monomer, which is generally a phosphorus derivative, such as a phosphotriester, a phosphoramidite, etc. See, for example, Matteuci et al., Tet. Lett. 521: 719 (1980); U.S. Patent No. 4,500,707 to Caruthers et al.; and U.S. Patent Nos. 5,436,327 and 5,700,637 to Southern et al.; On the other hand, the present application provides a vector comprising the isolated polynucleotide of the present application. The vector may be any linear nucleic acid, plasmid, bacteriophage, cosmid, RNA vector, viral vector, etc. Non-limiting examples of viral vectors may include retroviruses, adenoviruses, and adeno-associated viruses.

[0127] On the other hand, the present application provides one or more vectors, which contain the nucleic acid molecules. For example, the vector may contain one or more nucleic acid molecules described in the present application. One or more nucleic acid molecules may be contained in each vector. In addition, the vector may also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functionally connected to a nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. The vector may include, for example, a plasmid, a cosmid, a virus, a bacteriophage or other vectors commonly used in, for example, genetic engineering.

[0128] On the other hand, the present application provides a cell, the cell comprising the fusion protein, the nucleic acid molecule, or the vector. The cell may be a host cell. For example, the cell may include many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0129] For example, the vector can be stably or transiently introduced into the host cell by a variety of established techniques. For example, one method involves calcium chloride treatment, wherein the vector is introduced by calcium precipitation. Other salts, such as calcium phosphate, can also be used in a similar manner. In addition, electroporation (i.e., applying an electric current to increase the permeability of the cell to nucleic acids) can be used. Other examples of transformation methods include microinjection, DEAE dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers can also be used to transfect host cells.

[0130] In another aspect, the present application provides a method for preparing the fusion protein, which may include culturing the cells under conditions that allow the fusion protein to be expressed, for example, by using an appropriate culture medium, an appropriate temperature and a culture time, etc., which methods are known to those of ordinary skill in the art.

[0131] On the other hand, the present application provides a composition comprising the fusion protein or the nucleic acid molecule, and optionally a pharmaceutically acceptable carrier.

[0132] For example, the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex and / or a nonionic surfactant, etc.

[0133] In the present application, the composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques in the art, for example, according to the techniques disclosed in Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0134] In the present application, the pharmaceutical composition can be formulated for oral administration in the form of tablets, capsules, pills, powders, sustained-release preparations, solutions, suspensions. The pharmaceutical composition can be in a unit dosage form suitable for single administration of a precise dose. The pharmaceutical composition can further include a conventional pharmaceutical carrier or excipient. In addition, the pharmaceutical composition can include other drugs or medicaments, carriers, adjuvants, etc.

[0135] The pharmaceutical composition described in the present application may include a therapeutically effective amount of the fusion protein. The therapeutically effective amount is a dosage required to prevent and / or treat (at least partially treat) a condition or condition (e.g., a neurodegenerative disease) and / or any complications thereof in a subject suffering from or having a risk of development. The specific amount / concentration of the dosage can vary according to the method of administration and the patient's needs, and can be determined based on, for example, the patient's volume, viscosity, and / or body weight. It should be understood that, based on the conditions of a particular patient, formulation, and / or disease, a person skilled in the art (e.g., a doctor or pharmacist) can conveniently adjust these specific doses.

[0136] use

[0137] On the other hand, the present application provides the use of the fusion protein, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease and / or condition.

[0138] On the other hand, the present application provides a method for preventing and / or treating a disease and / or condition, comprising administering the fusion protein, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition to a subject in need thereof.

[0139] On the other hand, the present application provides the fusion protein, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition, which are used for preventing and / or treating diseases and / or conditions.

[0140] In the present application, the diseases and / or disorders may include diseases and / or disorders caused by abnormalities in ARSA.

[0141] In the present application, the diseases and / or disorders may include neurological diseases caused by deposition of sulfatide substrates.

[0142] In the present application, the disease and / or disorder may include Metachromatic Leukodystrophy (MLD).

[0143] In the present application, the diseases and / or disorders may include neurodegenerative diseases.

[0144] In the present application, the disease and / or disorder may include Parkinson's disease.

[0145] On the other hand, the present application also provides a method for delivering a drug across the blood-brain barrier, which may include administering the fusion protein.

[0146] On the other hand, the present application also provides a method for prolonging the half-life of ARSA in vivo, which may include administering the fusion protein.

[0147] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0148] Example

[0149] Example 1 Discovery of transferrin-specific single domain antibody VHH

[0150] Two 2-3 year old Asian alpacas were immunized with human transferrin purified from human plasma until the specific serum titer reached more than 10,000 (ELISA method). 200 ml of peripheral blood was taken to separate and purify mononuclear cells (PBMC), extract mRNA, reverse transcribe into cDNA, and use VHH-specific primers to amplify the nucleotide sequence encoding VHH, clone into a phage display vector to establish a VHH phage display library. According to the results of DNA sequencing of nearly 100 randomly selected clones, the effective capacity of the constructed phage display library is about 100 million (10 9 ) independent VHH sequences.

[0151] The VHH library was panned using a liquid phase method: 1-10 μg of biotinylated human transferrin and 10 12 Phages with CFU (colony forming units) were incubated in phosphate buffered saline (PBST-1% BSA) containing 1% bovine serum albumin and 0.05% Tween 20 for 1 hour, and streptavidin-coupled magnetic beads (Invitrogen, Dynabeads M-280streptavidin) pre-washed and blocked with PBST-1% BSA were added. After incubation at room temperature for 1 hour, the magnetic beads were washed 10 times with PBST. Phages bound to magnetic beads were dissociated with 10 μg / ml trypsin, infected and amplified in Escherichia coli, and used for the next round of panning, or single clones were picked for ELISA method to select VHH sequences that specifically bind to human transferrin but do not affect the binding of transferrin to human transferrin receptor. Among the multiple specific VHH sequences obtained, SLN9056 (the amino acid sequence of its CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 8) showed better human transferrin binding activity (pH 7.4, pH 6.0) ( Figure 1 AB), which can significantly prolong the half-life of the recombinant ovalbumin (OVA) fused with it in the blood of mice ( Figure 1C). Mice were injected IV with purified recombinant protein (e.g., VHH-OVA-His) at a dose of 10 mg / kg and received daily IP injections of 10 mg / kg human transferrin starting one day before dosing. Samples were taken at different time points after dosing, and the brains were collected after perfusion with 20 mL of PBS and frozen in liquid nitrogen. The content of VHH-OVA-His in brain homogenates was quantified by ELISA. Rabbit anti-OVA polyclonal antibody (Sigma C6534-2 mL) was coated and biotinylated rabbit anti-VHH antibody (GeneScript, A02015-200) was used for detection. Compared with the negative control (SLN0066, VHH that does not bind to transferrin, amino acid sequence SEQ ID NO:12), the VHH that binds to transferrin significantly increased the level of test article in brain tissue (for the selected VHH, the average %ID (injection dose) percentage was ~1.03% to ~3.5% after 24 hours of dosing) ( Figure 1 D).

[0152] Example 2 Production of recombinant protein

[0153] 2.1 Protein expression, purification and identification

[0154] Recombinant plasmids were constructed to produce 9056VHH-hARSA-his or hARSA-9056VHH-his proteins (hARSA consists of the amino acid sequence shown in SEQ ID NO: 10; the amino acid sequence of CDR1 of 9056VHH is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 8; the linker consists of the amino acid sequence shown in SEQ ID NO: 9). After sequencing to confirm the plasmid sequence, the recombinant plasmid was used as a vector and lipofectamine or PEI was used as a transfection reagent to transiently transfect HEK293F cells to mass produce recombinant proteins. Cultured in 30ml–100ml shake flask medium for 5-7 days. Cells were removed by centrifugation and the supernatant was purified by Ni-NTA agarose. Purified proteins were analyzed by 4-12% gradient SDS-PAGE gel under non-reducing (NR) or reducing conditions (R) ( Figure 2 ).

[0155] Example 3 Biological properties of recombinant protein

[0156] 3.1 ELISA test of the binding ability of recombinant protein to transferrin

[0157] The 96-well plate was coated with streptavidin (0.1 μg / ml, 100 μl / well) and incubated overnight at 4°C; after washing 3 times with PBST, it was blocked with 200 μl of PBST containing 1% bovine serum albumin (BSA) at room temperature for 1 hour. Washed 3 times with PBST, biotinylated human transferrin (1 μg / ml, 100 μl / well) was added and incubated at room temperature for 1 hour. Washed the plate 3 times with PBST, added serially diluted hARSA or hARSA-9056VHH protein, and incubated at room temperature for 1 hour. After washing 3 times with PBST, 0.125 ug / ml goat anti-hARSA antibody (R&D, AF2485) (100 μl / well) was added to each well and incubated at room temperature for 1 hour. Washed 3 times with PBST, 100 μl anti-goat IgG-HRP was added to each well, incubated at room temperature for 0.5 hours, and then washed as before. After adding 100 μl / well TMB substrate solution, incubate at room temperature for 15 minutes. Add 100 μl / well stop solution to terminate the reaction, and read the absorbance at 450 nm with an ELISA reader. The results are as follows: Figure 3 As shown, the results showed that hARSA-9056VHH could bind to human transferrin, while hARSA could not bind to human transferrin.

[0158] 3.2 FACS detection of recombinant protein binding to transferrin without affecting the binding of transferrin / transferrin receptor complex on the cell membrane

[0159] The 293F cell line stably expressing human transferrin receptor 1 (hTfR1, UniProt P02786) was used to detect the binding of the recombinant protein to transferrin / transferrin receptor on the cell membrane. 6 Cell density: 293F / hTfR1 cells were plated into 96-well plates, centrifuged at 1000 rpm for 5 minutes at 4°C, washed with 200 μl 1×PBS (pH7.4), and resuspended and blocked with 200 ul PBS containing 2% BSA (blocking solution). After incubation at 4°C for 30 minutes, the cells were washed as above and resuspended in blocking solution containing serially diluted recombinant protein samples, with or without natural human transferrin (Sigma, Cat NO.T3309). After incubation at 4°C for 30 minutes, mouse anti-VHH (Genescript) and AF647-labeled goat anti-mouse antibody (Abcam, CAT#ab150115) were added in sequence. After incubation at 4°C for 30 minutes, the cells were centrifuged, washed, and resuspended in blocking solution. The binding of cells to recombinant proteins was analyzed by flow cytometry. The results are shown in Figure 4 As shown, the results showed that hARSA-9056VHH could bind to human transferrin (hTf) and did not affect the binding of transferrin to the human transferrin receptor on the cell membrane surface.

[0160] 3.3 ELISA test of the binding ability of recombinant 9056VHH-hARSA protein to M6P receptor

[0161] M6P receptor (extracellular segment M6PR-Fc recombinantly expressed by HEK293F cells, 0.5 μg / well) was coated on a 96-well plate and incubated overnight at 4°C. Washed 3 times with PBST (phosphate buffer, 0.1% Tween-20, pH 7.4) and blocked with 200 μl 1% BSA / PBST at room temperature for 1 hour. Washed 3 times with PBST, added serially diluted rhARSA or rhARSA-9056VHH protein, and incubated at room temperature for 1 hour. Washed the plate 3 times with PBST, added 0.125ug / ml biotin-labeled goat anti-hARSA antibody (R&D, BAF2485) (100 μl / well), and incubated at room temperature for 1 hour. Washed 3 times with PBST, added 100 μl streptavidin-HRP (Sigma, CAT#S5512-1MG) to each well, and incubated at room temperature for 1 hour. Wash 3 times with PBST, add 100 μl TMB substrate solution, and incubate at room temperature for 15 minutes. After termination, read the absorbance at 450 nm with an ELISA reader, and analyze the data with Graph Pad 8.0 software. Figure 5 As shown in A, rhARSA-9056VHH and rhARSA have similar M6P receptor binding abilities.

[0162] 3.4mARSA - / - Uptake of recombinant proteins by mouse skin fibroblasts

[0163] mARSA - / - Mouse skin fibroblasts were obtained from C57BL / 6J-Arsaem1cyagen (KOCMP-11883-Arsa-B6J) mice, which were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. Tissue block culture was performed as usual, and the culture medium used was high-glucose DMEM (containing 4.0 mM L-Glutamine, Sodium Pyruvate) (HyClone, SH30243.01), 10% fetal bovine serum (Gibco, 10099-141C). When the cells crawled out into sheets and grew well, they were passaged for later use.

[0164] 3.4.1 Dose-dependent cellular uptake experiment

[0165] 1) Take well-growing P1 mARSA - / - Mouse skin fibroblasts were seeded into 96-well plates at 2 × 10 4 Cells / well, culture overnight.

[0166] 2) Prepare 9056VHH-hARSA, hARSA (starting at 20μM, 10-fold gradient dilution) and 10mM M6P solution (D-mannose-6-phosphate disodium salt hydrate (J&K, 530168)) with 2-fold drug concentration respectively; the group without M6P was replaced with blank culture medium. Discard the culture medium in the 96-well plate, add 10mM M6P first, incubate for 15 minutes, then add equal volumes of drugs in the corresponding groups (final concentration starting at 10μM, 10-fold gradient dilution), and continue to culture for 24 hours.

[0167] 3) Collect cell lysis buffer: add 1× protease inhibitor to the cell lysis buffer, mix well and set aside; discard the supernatant of cells and wash with PBS three times; add 60 μl of cell lysis buffer to each well, collect into 1.5 ml EP tube after fully lysing cells; centrifuge at 12000 rpm, take the supernatant, determine the total protein concentration, and store at -80 degrees for detection.

[0168] ELISA detection of drug content in cell lysate: 96-well ELISA plate was coated with goat anti-hARSA antibody (R&D, AF2485) at 0.25 μg / ml, washed 3 times with PBST (Tween-20, 0.1%), and blocked with 200 μl PBST containing 1% BSA at room temperature for 1 hour. After washing 3 times with PBST, serial dilution test samples were added (with serial dilution standard rhARSA and 9056VHH-rhARSA as standard curve). After incubation at room temperature for 1 hour, the plate was washed 3 times with PBST, and 0.125 μg / ml biotin-labeled goat anti-hARSA antibody (R&D, BAF2485) was added and incubated at room temperature for 1 hour. After washing 3 times with PBST, streptavidin-labeled horseradish peroxidase (HRP) 1:5000 (Sigma, CAT#S5512-1MG) was added and incubated at room temperature for 1 hour. Wash 3 times with PBST, add 100 μl TMB substrate solution and incubate for 15 minutes. Read the absorbance at 450 nm after termination. Calculate the drug concentration in each sample according to the standard curve. Cell uptake = drug concentration in sample / total protein concentration. Figure 5 B shows mARSA - / - The amount of 9056VHH-hARSA taken up by fibroblasts was 5.57 times that of hARSA (10 μM), which could be partially blocked by M6P.

[0169] 3.4.2 Time-dependent cellular uptake experiments

[0170] 1) As in experiment 3.4.1, prepare 20 μM drug and dilute 10-fold to 2 μM (final concentration 1 μM); discard the culture medium in the 96-well plate, add 10 mM M6P first and incubate for 15 min, then add the corresponding groups of drugs, continue to culture for 48 hours, 24 hours, 6 hours, 2 hours and 0 hours, collect the cell lysate, centrifuge at 12000 rpm, take the supernatant, determine the total protein concentration, and store at -80 degrees for detection.

[0171] 2) As in 3.4.1, ELISA was used to detect the drug content in the cell lysate, and the cellular uptake of the drug at each time point was calculated = drug concentration in the sample / total protein concentration. Figure 5 C shows mARSA - / - The uptake of 9056VHH-rhARSA by fibroblasts was time-dependent.

[0172] 3.5 mARSA - / - Intracellular localization of recombinant proteins taken up by mouse skin fibroblasts

[0173] 3.5.1 Drug incubation

[0174] As in experiment 3.4, culture mARSA- / - mouse skin fibroblasts. Inoculate cells in good growth state into a 24-well plate with a coverslip and culture the cells until they are 70-80% confluent. Discard the old culture medium and add the culture medium containing 2μM 9056VHH-hARSA or hARSA, and continue to culture for 24 hours.

[0175] 3.5.2 Immunofluorescence staining and laser confocal microscopy observation

[0176] 1) Fixation: Discard the culture medium, wash the cells three times with pre-cooled PBS, fix with 4% PBS pH 7.4 paraformaldehyde at room temperature for 10 minutes, and wash the cells three times with pre-cooled PBS.

[0177] 2) Permeabilization: Incubate the sample in PBS containing 0.1% Triton X-100 at room temperature for 10 minutes. Wash the cells 3 times with pre-cooled PBS.

[0178] 3) Blocking: Block with PBST (PBS + 0.05% Tween 20) containing 1% BSA for 30 minutes. Wash the cells 3 times with pre-cooled PBS.

[0179] 4) Immunofluorescence staining: Dilute the primary goat anti-human ARSA antibody (R&D, AF2486) with 1% BSAPBST 1:100 and incubate at room temperature for 1 hour. Wash the cells 3 times with pre-cooled PBS. Dilute the PE-labeled secondary donkey anti-goat IgG antibody (R&D, F0107) and lysosomal marker antibody CD107a (LAMP-1) (eBio1D4B (1D4B)) (AlexaFluor 488, eBioscience) with 1% BSAPBST 1:100 TM ,53-1071-82) at room temperature in the dark for 1 hour. Wash the cells three times with pre-cooled PBS.

[0180] 5) Mounting medium The slides were sealed with Gold Antifade Reagent with DAPI (CST, #8961), stored at 4°C in the dark, and observed and photographed under a laser confocal microscope.

[0181] The results are as follows Figure 6 As shown: A large number of 9056VHH-hARSA (A) and hARSA (B) recombinant proteins can be cleaved by mARS - / - It is internalized by mouse skin fibroblasts and transported to lysosomes (arrows).

[0182] Example 4 Biological Activity Detection of Recombinant Protein

[0183] 4.1 In vitro enzyme activity assay (pNCs artificial substrate) 6

[0184]

[0185] Prepare the solution:

[0186] 1) p-nitrocatechol (p-NC, TCI, Lot. N5V4L-RT) standard solution: pNC, 1 mM dissolved in the stop solution (MW = 155.11, 5 mg in 32.23 mL).

[0187] 2) Stop solution: 0.2M NaOH (MW=40, 0.8g in 100mL).

[0188] 3) Assay Buffer: 50 mM NaOAc, 0.5 M NaCl, pH 4.5 (NaOAc, NaCl, HOAc were used to adjust the pH to 4.5).

[0189] 4) p-nitrocatechol sulfate (p-NCs, sigma, Lot#MKCN3971) stock solution: pNCS, 2 mM dissolved in reaction buffer (MW=311.35, 10 mg in 16 mL).

[0190] Experimental steps:

[0191] 1) p-NC standard curve: Add 0, 20, 40, 60, 80, 100 μL of pNC standard to a 96-well plate; adjust the total volume to 100 μL / well with stop solution. The corresponding pNC contents are 0, 20, 40, 60, 80, 100 nmol / well.

[0192] 2) dilute rhARSA or 9056VHH-hARSA to 200 nM (final concentration 100 nM) with reaction buffer; add or not add hTf to the reaction system.

[0193] 3) Dilute pNCS to 2 mM with reaction buffer (final concentration 1 mM).

[0194] 4) Take 75 μL of 200 nM rhARSA or 9056VHH-hARSA and 75 μL of 2 mM pNCS and mix thoroughly; 75 μL of reaction buffer and 75 μL of substrate mixture were used as blank controls; react at 37°C for 0, 5, 10, 15 and 30 minutes.

[0195] 5) Add 150 μL 0.2 M NaOH to terminate the reaction.

[0196] 6) Take 200 μL of each reaction into a new 96-well plate and read the absorbance at 515 nm using a microplate reader.

[0197] 7) Based on the standard curve, draw the enzyme activity reaction time relationship curve and calculate the enzyme specific activity.

[0198] The results are shown in FIG7 : the recombinant enzyme proteins produced by the CHO K1 (7A) expression system all maintained good enzyme activity; (7B) the binding of the recombinant enzyme to human transferrin did not affect the enzyme activity.

[0199] 4.2 Recombinant protein degradation of mARSA in vitro - / - Deposition of sulfatide substrates in mouse skin fibroblasts

[0200] Same as experiment 3.4, in vitro culture of mARSA - / - Mouse skin fibroblasts, when the cells are growing well, mARS - / -Fibroblasts were seeded into 24-well plates and cultured until the cells were 80-90% confluent; 9056VHH-hARSA 10μM, 3μM, and 0μM were added for 48 hours respectively; after discarding the culture medium, Alcian blue dye solution was added according to the instructions of the kit (Alcian blue-nuclear fast red staining kit (pH 1.0), Bio-Sky, C0153S), and the mixture was incubated at room temperature for 1 hour; after washing with distilled water for 3 times, fast red was added for counterstaining for 5 minutes, and the mixture was washed with tap water for 3 times; the results were observed under a microscope. Figure 8 As shown: After culturing mARSA- / - fibroblasts with 10μM, 3μM, and 0μM 9056VHH-hARSA for 48 hours, Alcian blue / fast red staining showed that the three groups of cells had different degrees of lake blue dots and block staining in the cytoplasm. The blue dye was a sulfate metabolite containing sulfate radicals; the blue dye in the cytoplasm of the 10μM group was diffuse and small dots, with few blocks; the size of the blue dye dots and blocks in the cytoplasm of the three groups of cells was: 10μM group < 3μM group < 0μM group; the amount of blue dye in the cytoplasm of the three groups of cells was: 10μM group < 3μM group = 0μM group. The 9056VHH-hARSA 10μM group played a role in degrading substrates to a certain extent in the cells.

[0201] Example 5 In vitro plasma stability test

[0202] 5.1 Mouse plasma stability test

[0203] 1) Sample preparation: The standard protein was prepared in mouse plasma at a certain concentration of 6 μg / ml, and incubated at 37°C for 1 hour, 4 hours, 8 hours, 24 hours and 48 hours, and then the samples were quickly frozen in liquid nitrogen and stored at -80°C until use. Protein samples without plasma were used as positive controls.

[0204] 2) ELISA detection: SLN7134 (anti-9056VHH, Linuo homemade) 1μg / ml, 100μL / well coated 96-well plate, 4℃ overnight. 1% BSA TBST, 200μL per well, blocked at 37℃ for 1 hour. 1% BSA TBST diluted protein, made a standard curve, starting concentration 200ng / ml, 2-fold gradient dilution, 100μL per well, incubated at room temperature for 1 hour. 1% plasma diluted samples to the appropriate concentration range, 100-fold, 200-fold, 300-fold dilution, 100ul per well, incubated at room temperature for 1 hour. 1% BSA TBST diluted SLN7165-Biotin (biotinylated anti-human ARSA antibody, Linuo homemade), the final concentration is 0.5μg / ml, add 100μL to each well and incubate at room temperature for 1 hour. 1% BSA TBST 1:5000 dilution of SA-HRP (Sigma, CAT#S5512-1MG), add 100 μL to each well, room temperature for 0.5 hours. Add 100 μL TMB to each well, color at room temperature in the dark for 15 minutes, then add 100 μL sulfuric acid to each well to terminate the reaction. Set the wavelength of the microplate reader to 450nm, read the plate, and analyze the data with GraphPAD 8.0. The results are shown in Figure 9 (A): rhARSA-9056VHH-his has good stability in mouse plasma.

[0205] 5.2 Human plasma stability test

[0206] 1) Prepare the recombinant protein in the same manner as in 5.1, incubate the sample in human plasma solution at 37 degrees, and detect the protein concentration in the sample using the 5.1 ELISA method. The results are shown in Figure 9 (B): rhARSA-9056VHH-his has good stability in human plasma.

[0207] Example 6 Pharmacokinetic (PK) study of recombinant protein in mice

[0208] 6.1 Establishment of PK detection method: Goat anti-hARSA antibody (R&D, AF2485) was used to coat 96-well enzyme-linked plates, washed 3 times with PBST (Tween-20, 0.1%) and blocked with 200 μl 1% BSAPBST at room temperature for 1 hour. After washing 3 times with PBST, 100 μl / well of samples containing 1% mouse serum (as sample matrix) and serial dilution standards (rhARSA or rhARSA-9056VHH) were added. After incubation at room temperature for 1 hour, PBST was washed 3 times, and biotin-labeled goat anti-hARSA antibody (R&D, BAF2485) was added and incubated at room temperature for 1 hour. After washing 3 times with PBST, streptavidin-labeled horseradish peroxidase (HRP) (Sigma, CAT#S5512-1MG) was added and incubated at room temperature for 30 minutes. After washing three times with PBST, 100 μl of TMB substrate solution was added and incubated for 15 min. After adding the stop solution, the absorbance at 450 nm was read. Fig.10 The validation results of the PK method are shown. The results of the validation using three standard concentrations (high, medium, and low) indicate that the precision (CV% <20%) and accuracy (RE% + / - 25%) of this method meet the sample testing requirements.

[0209] 6.2 PK study of single dose administration: PK study was conducted in 6-8 week old male C57BL / 6 mice. Animals were divided into groups according to their weight and human transferrin was injected intraperitoneally at 10 mg / kg on the day before administration and every day during the study. The single tail vein administration dose of rhARSA, rhARSA-9056VHH and 9056VHH-rhARSA was 3MPK. Blood samples were collected 2, 4, 8, 24 and 48 hours after administration to separate serum. The 6.1PK method was used for quantitative analysis of drugs in serum, and PK sovler software was used to calculate PK parameters. Fig.10 The results showed that at a dose of 3MPK, the blood half-life of rhARSA-9056VHH (5.29 hours) and 9056VHH-rhARSA (1.50 hours) were significantly higher than that of rhARSA (blood half-life less than 0.76 hours). The blood half-life of rhARSA was significantly prolonged by binding transferrin with 9056VHH.

[0210] 6.3 PK study of single-dose administration across the blood-brain barrier: The PK study was conducted in 6-8 week old male C57BL / 6 mice. The animals were grouped according to their weight and intraperitoneally injected with 10 mg / Kg of human transferrin one day before administration and every day during the study. The tail vein administration dose of hARSA or 9056VHH-hARSA was 10 MPK. Blood samples were collected to separate serum and cerebrospinal fluid at 4, 24, and 48 hours after administration; brain tissue was collected after perfusion with 20 ml PBS. The drug content in serum, cerebrospinal fluid and brain tissue homogenate was detected using 6.1 PK method. Graph Pad 8.0 software was used to analyze the data and calculate the efficiency of crossing the blood-brain barrier (injection dose percentage %ID = brain tissue drug concentration (ng / mg tissue) / 10 mg / kg×100%). The results are as follows Figures 11A-11B As shown, Fig.11A The results showed that the mean blood concentration of hARSA was about 45 ng / ml (4 h), the blood concentration of 9056VHH-hARSA was about 5000 ng / ml (4 h), ~400 ng / ml (24 h), and ~18 ng / ml (48 h), which were significantly higher than those of the hARSA group. Fig. 11B The results showed that hARSA-his could not be detected in brain tissue homogenate. The concentration of 9056VHH-hARSA in brain tissue homogenate was 4.6-10.8 ng / mg brain tissue (4 h) and 0.7-4.2 ng / mg brain tissue (24 h). Its efficiency of crossing the blood-brain barrier was much higher than that of the hARSA group.

[0211] 6.4 PK study of blood-brain barrier crossing with increasing doses: The experimental method was the same as 6.3. The doses of 9056VHH-hARSA administered via tail vein were 45 nmol / kg, 150 nmol / kg, and 450 nmol / kg, respectively. The drug content in serum, cerebrospinal fluid, and brain tissue homogenate was detected using the 6.1 PK method. Graph Pad 8.0 software was used to analyze the data and calculate the efficiency of crossing the blood-brain barrier (injection dose percentage %ID = brain tissue drug concentration (nmol / mg tissue) / injection dose nmol / kg × 100%). The results are shown in Figures 12A-12C Shown: Among them Fig. 12A The blood concentration of 9056VHH-hARSA at three doses of 45nmol / kg, 150nmol / kg, and 450nmol / kg; Fig. 12B The content of 9056VHH-hARSA in brain tissue homogenate, 4 hours after administration, the average percentage of injected dose (%ID) of 45nmol / kg-4h (3MPK); 450nmol / kg-4h (10MPK); 450nmol / kg-4h (30MPK) in each group were: 0.503%, 0.25%, 0.15% respectively; Fig. 12CIt showed that the content of 9056VHH-hARSA in CSF was low.

[0212] 6.5 Study on drug distribution in brain tissue after increasing dose administration in MLD mice: The experimental method was the same as 6.3, and the doses of rhARSA-9056VHH administered by tail vein were 150nmol / kg and 450nmol / kg respectively. Two hours after administration, the mice were euthanized, and the brain tissue was collected and fixed with 4% paraformaldehyde. Paraffin sections of brain tissue were prepared as usual, with 5μm sagittal sections (commissioned to Shanghai Ruibaohe Biological Co., Ltd.). hARSA / CD31 / Lamp1 immunofluorescence staining: Dewax, hydrate, and wash 3 times with PBS as usual. Blocking: Block for 30 minutes with 1% BSAPBST (PBS+0.05% Tween 20). Wash 3 times with PBS. Immunofluorescence staining: Dilute the primary goat anti-human ARSA antibody (R&D, AF2486) or rabbit anti-mouse CD31 antibody (Abcam, ab281583) with 1% BSAPBST1:100, and incubate at room temperature for 1 hour. Wash 3 times with pre-cooled PBS. PE-labeled secondary antibody donkey anti-goat IgG (R&D, F0107) and lysosomal marker antibody CD107a (LAMP-1) (eBio1D4B (1D4B)) (Alexa Fluor 488, eBioscience) were diluted 1:100 in 1% BSA PBST. TM ,53-1071-82) at room temperature in the dark for 1 hour. Wash 3 times with pre-cooled PBS. Mounting medium Gold Antifade Reagent with DAPI (CST, #8961) was used to seal the slides, and the slides were stored at 4°C in the dark. The slides were observed and photographed using a laser confocal microscope. Fig.13 As shown: Fig.13 A, drugs can cross the blood-brain barrier and enter brain tissue (anti-hARSA), vascular endothelial cells (anti-CD31,); there are also drugs in vascular endothelial cells; Fig.13 B, Drug (anti-hARSA) entering brain cells co-localizes with lysosomal membrane protein (anti-Lamp1).

[0213] 6.6 PET / CT in cynomolgus monkeys 124 I-marked single-dose PK across the blood-brain barrier:

[0214] 6.6.1 Animal in vivo experiments and sample collection

[0215] 1 per group Cynomolgus monkeys (male), intravenous infusion of 10MPK or 30MPK 124I-rhARSA-9056VHH, dynamic brain PET / CT scanning was started immediately for 2h (0-2h), and then PET / CT static scanning was performed at 7 time points: 6h, 10h, 24h, 48h, 72h, 96h, and 144h after administration.

[0216] 6.6.2 Data Analysis: Calculation 124 I-rhARSA-9056VHH brain penetration efficiency (Kp = brain (radiation dose) / blood (radiation dose) ratio. The results are as follows Figures 14A-14C As shown: Fig.14A , drug concentration-time curve of brain tissue; Fig. 14B , left ventricular drug concentration-time curve; Fig. 14C , brain tissue drug concentration / left ventricular drug concentration percentage: 20min to 30min after administration, the brain / plasma ratio of drug concentration increased significantly, and hARSA-9056VHH can accumulate and retain in brain tissue; the 10MPK group reached its peak at 96 hours, with a brain / plasma ratio of 53.3%; the 30MPK group reached its peak at 72 hours, with a brain / plasma ratio of 23.95%.

[0217] Example 7 Pharmacodynamic study of recombinant protein in mARSA-deficient mouse MLD model

[0218] 7.1 mARSA-deficient mice: The genotype of the mARSA gene (GenBank: Gene ID 11883)-deficient mice is C57BL / 6J-Arsa em1cyagen (KOCMP-11883-Arsa-B6J), verified by PCR and sequencing.

[0219] 7.2 In vivo pharmacodynamic study of rhARSA-9056VHH:

[0220] 7.2.1 Animal grouping: Select mARSA animals that are about 10-12 months old and in good health. - / - Homozygous mice; matched by sex / age; weigh and record the weight of mice; number mice and fill in cage card information. hTf 10mg / kg intraperitoneal injection, once a day, to maintain a certain level of hTf in mice;

[0221] 7.2.2 Administration: hARSA-9056VHH was administered, with a dosing frequency of BIW×4 or BIW×8 times; the dosage was 10MPK or 30MPK. The drug was premixed with hTf at a molar ratio of 1:1 at 4°C overnight the day before administration; the mice were weighed on the morning of administration and the administration volume was calculated; the mice were injected into the tail vein and the administration time was recorded.

[0222] Daily records: weigh and record the weight of mice; observe and record the status and activity level of mice.

[0223] 7.2.3 Behavioral assessment of mice: balance beam method

[0224] 1) Training period: On the first day, a balance beam was used to allow mice to pass through the square balance beam into the small dark box. Each mouse was trained 3 times. On the second day, the same steps as on the first day were repeated.

[0225] 2) Testing period: The third day was the testing phase, in which mice were made to pass through a square balance beam into a small dark box. The entire process was recorded, and the time it took to pass the balance beam was recorded.

[0226] 3) Compare the time taken by each group of mice to pass the balance beam. Fig.15 As shown: After 4 weeks of administration, the time taken by mice to pass the balance beam in the 30MPK and BIW×8 groups was significantly reduced, which was 83.53% of that before administration, P<0.001 (Dunnett's multiple comparisons).

[0227] 7.2.4 Sample collection: On the 32nd day (8 days after the last administration), the mice were euthanized and 0.2 ml of mouse urine was collected (4 h before euthanasia); stored at -20°C for testing. Whole blood was collected by puncturing the mouse orbital sinus venous plexus and placed in a serum separation tube. After standing for 10 min, it was centrifuged at 3000 g for 5 min, and the supernatant was collected into a 1.5 ml centrifuge tube and stored at -80°C. Tissues and organs were collected at the same time: kidneys were stored at -80°C; sagittal sections of the brain (including olfactory bulb, cerebellum, cerebrum, brainstem and part of the spinal cord) were cut, and the left brain and left kidney were quickly frozen in liquid nitrogen for TLC (cerebellum, cerebrum, brainstem and part of the spinal cord) and ELISA detection; the right brain and right kidney were fixed with 4% PFA for Alcian blue staining and immunofluorescence staining; and peripheral nerve tissue-sciatic nerve was fixed with 4% PFA for Alcian blue staining.

[0228] 7.2.5 Sample testing:

[0229] 7.2.5.1 Detection of sulfatide substrate in brain tissue:

[0230] 1) TLC analysis of sulfatide / GalCer ratio: Weigh 100 mg of mouse brain, 20 mg of cerebellum, and 20 mg of brainstem medulla oblongata from each group of mice, add 1 mL or 200 μl TBS pH 7.4 and steel beads according to the mass-volume ratio, and homogenize at 60 Hz for 60 seconds; add liquid to 2 mL of homogenate and centrifuge at 13000 rpm for 2 hours; discard the supernatant, and use the tissue fragments for lipid extraction. Add 5 mL of CH3Cl / CH3OH (2:1) to the precipitate, extract at 60°C for 4 hours; centrifuge, transfer the solvent supernatant, and extract the precipitate again with 5 mL of C / M (1:1) at 60°C for 4 hours; combine the two extracts (crude extraction), mix well, take half for subsequent operations, and keep half as a control; take about 5 mL of the extract, spin dry, and white adherent material can be seen, which is fully dissolved with 5 mL of MeOH. Add 250 μL 4N NaOH, incubate at 37°C for 2 hr, then stop with 40 μL 100% acetic acid. Evaporate the solvent and add 1 mL MeOH to dissolve. Add 1 mL 0.3 M ammonium acetate solution and load all the mixture. Desalting: Fill the column (Japan YMC, AAG12S50) with about 1 mL ODS filler (pre-activated with MeOH), and balance the column with 6 mL mixed solvent: C / M / 0.1M KCl 6:96:94. Add the mixed sample to the column, first wash and desalt with 6 mL H2O, then elute with 1 mL MeOH and 6 mL C / M (1:1), white turbidity appears during the elution process, and then gradually disappears. Concentration: Evaporate the eluent containing lipid molecules with a rotary evaporator (Shanghai Bochu Instrument Equipment Co., Ltd., LOOYE), and finally fully dissolve with 500 μL C / M 2:1 for TLC analysis. Load a certain volume of sample onto the silica gel plate, sulfatide (Matreya LLC; Cat.NO.: 1049-50mg; Lot No.: 24534), GalCer (Matreya LLC; Cat.NO.: 1066-10mg; Lot No.: 23332) as standard, the development time is about 10 minutes. Developing agent: chloroform: methanol: water 65:25:4 (v / v / v) (need to be prepared in advance to saturate the steam inside the development tank). Color development: Soak the developed silica gel plate in the color developing solution (10% CuSO4 in 10% phosphoric acid solution) for about 3 minutes, take it out, blow dry with a hair dryer, and place it in an oven at 130°C for 10 minutes, and black spots or stripes will appear. Gel imager scanning, image grayscale analysis, calculation of the sulfatide / GalCer ratio of brain tissue of each group of mice, mean+SD, statistical analysis of the difference in sulfatide / GalCer ratio in the cerebrum, cerebellum and brainstem tissues of the treated and untreated groups of mice. As shown in Figure 16: mARSA - / -Intravenous administration (BIW) of the drug for 4 weeks significantly reduced the accumulation of sulfatide substrate in the cerebrum (A, B), cerebellum (C, D), and brainstem (E, F), and showed a dose-dependent manner (p values ​​were 0.027, 0.026, and 0.059, respectively, One-way ANOVA).

[0231] 2) Alcian blue staining: Prepare frozen sections of brain tissue as usual, 10μm sagittal section (commissioned by Shanghai Ruibaohe Biological Co., Ltd.); prepare Alcian blue staining buffer (0.025M sodium acetate, pH 5.7, containing 0.3MMgCl2, 1% PFA), 0.05% Alcian blue staining solution (Alcian blue 8GX, sigma, A9186-10G) and dissolve in the staining buffer. After the frozen sections of brain tissue are dissolved at room temperature, they are fixed with 4% paraformaldehyde (PFA) for 1 minute; after being thoroughly washed with staining buffer, they are incubated with 0.05% Alcian blue staining solution at room temperature for 1 hour; after being thoroughly washed with staining buffer, they are counterstained with fast red for 2 minutes; after being thoroughly washed with tap water, the sections are sealed with glycerol; and they are observed and photographed under a microscope. The results are as follows Fig.17 :mARSA - / - Mice were treated with intravenous injection (BIW) for 4 weeks. There was no significant difference in the accumulation of substrate sulfatide in the cerebrum (B1, C1, D), cerebellum (B3, C3, E) and brainstem (B2, C2, F) of the 10MPK (B) and 30MPK (C) groups, but the average grayscale density was reduced, showing a dose-related trend.

[0232] 7.2.5.2 Detection of sulfatide substrate in sciatic nerve: The same method as above was used to detect sulfatide substrate in sciatic nerve. The sulfatide / cholesterol ratio was analyzed by Alcian blue staining and thin layer chromatography, and the deposition of sulfatide substrate in mice in the treatment group and the untreated group was analyzed. Fig.18 Shown: A, TLC results show mARSA - / - After 4 weeks of intravenous administration (BIW) to mice, the accumulation of substrate sulfatide in the sciatic nerve of the 10MPK and 30MPK groups was significantly reduced, and showed a dose-related relationship, with P values ​​less than 0.0001 (One-way ANOVA); B, The ABS staining results were consistent with the TLC results. The accumulation of substrate sulfatide in the 10MPK and 30MPK groups was significantly reduced, and showed a dose-related relationship, with P values ​​of 0.033 and 0.032, respectively (One-way ANOVA).

[0233] 7.2.5.3 Detection of sulfatide substrate in kidney: The same method as above was used to detect sulfatide substrate in brain tissue. The ratio of sulfatide / galcer was analyzed by thin layer chromatography and the deposition of sulfatide substrate in kidney of mice in the treatment group and the untreated group was analyzed. Fig.19 As shown: After 2 weeks of administration, the level of sulfatide substrate in the 30MPK, BIW×4 group was lower than that in the non-administered group, but there was no statistical difference; after 4 weeks of administration, the level of sulfatide substrate in the 30MPK, BIW×8 group was significantly reduced, P=0.0169 (One-way ANOVA).

Claims

1. A fusion protein comprising: a transferrin-binding protein, and arylsulfatase A (ARSA) or a functionally active fragment thereof, wherein the transferrin-binding protein comprises a VHH capable of binding to transferrin, wherein the VHH comprises CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of the CDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is as shown in SEQ ID NO:

3.

2. The fusion protein according to claim 1, having one or more of the following properties: 1) being able to prolong the in vivo half-life of the ARSA; 2) Ability to cross the blood-brain barrier; 3) capable of oral delivery; and 4) The ARSA can be delivered to cells expressing the transferrin receptor. The fusion protein according to claim 1 , wherein the transferrin is human transferrin. The fusion protein according to claim 1 , wherein the amino acid sequence of the VHH is as shown in SEQ ID NO:

8.

5. The fusion protein of claim 1, wherein the ARSA is human ARSA.

6. The fusion protein according to claim 1, wherein the ARSA or a functionally active fragment thereof comprises a variant of ARSA or a functionally active fragment thereof.

7. The fusion protein according to claim 1, wherein the amino acid sequence of ARSA or its functionally active fragment is as shown in SEQ ID NO:

10.

8. The fusion protein of claim 1, wherein the transferrin-binding protein and the ARSA or a functionally active fragment thereof are directly or indirectly connected.

9. The fusion protein according to claim 1, wherein the N-terminus of the transferrin-binding protein and the C-terminus of the ARSA or its functionally active fragment are directly or indirectly connected.

10. The fusion protein according to claim 1, wherein the C-terminus of the transferrin binding protein and the N-terminus of the ARSA or a functionally active fragment thereof are directly or indirectly connected.

11. The fusion protein according to claim 1, wherein the transferrin binding protein and the ARSA or a functionally active fragment thereof are connected via a linker. The fusion protein of claim 11 , wherein the linker comprises a peptide linker. The fusion protein according to claim 12 , wherein the amino acid sequence of the linker is shown in SEQ ID NO:

9. The fusion protein according to claim 1 , whose amino acid sequence is shown in SEQ ID NO:

11. The fusion protein according to claim 1 , which is capable of binding to transferrin and catalyzing the degradation of sulfatide, a substrate of arylsulfatase A.

16. One or more isolated nucleic acid molecules encoding the fusion protein of any one of claims 1-15. A vector comprising the nucleic acid molecule according to claim 16 .

18. A cell comprising the nucleic acid molecule of claim 16 and / or the vector of claim 17.

19. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17 or the cell of claim 18, and optionally a pharmaceutically acceptable carrier.

20. A method for preparing the fusion protein according to any one of claims 1 to 15, comprising culturing the cell according to claim 18 under conditions where the fusion protein is expressed.

21. Use of the fusion protein of any one of claims 1-15, the nucleic acid molecule of claim 16, the vector of claim 17, the cell of claim 18, and / or the pharmaceutical composition of claim 19 in the preparation of a medicament for preventing and / or treating a disease and / or condition, wherein the disease and / or condition is a nervous system disease caused by deposition of a sulfatide substrate.

22. Use of the fusion protein according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16, the vector according to claim 17, the cell according to claim 18, and / or the pharmaceutical composition according to claim 19 in the preparation of a medicament for preventing and / or treating metachromatic leukoencephalopathy Leukodystrophy, MLD).

23. Use of the fusion protein of any one of claims 1-15, the nucleic acid molecule of claim 16, the vector of claim 17, the cell of claim 18, and / or the pharmaceutical composition of claim 19 in the preparation of a drug for preventing and / or treating a neurodegenerative disease.

24. Use of the fusion protein of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17, the cell of claim 18, and / or the pharmaceutical composition of claim 19 in the preparation of a drug for preventing and / or treating Parkinson's syndrome.

25. Use of the fusion protein described in any one of claims 1-15 in the preparation of a medicament capable of prolonging the half-life of ARSA in vivo.

26. Use of the fusion protein according to any one of claims 1 to 15 in the preparation of a drug capable of delivering ARSA into the brain across the blood-brain barrier.

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