WO2023207574A1 - 一种穿越血脑屏障的重组hARSA - Google Patents
一种穿越血脑屏障的重组hARSA Download PDFInfo
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- fusion protein
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- arsa
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Classifications
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- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Definitions
- the present application relates to the field of biomedicine, specifically to a fusion protein containing transferrin-binding protein and arylsulfatase A, and its use.
- Arylsulfatase A (Arylsulfatase A or ARSA, also known as acyl sulfatase A, cerebroside-sulfatase/cerebroside-sulfatase or arylsulfatase A) is a type of lysosomal hydrolase that can decompose Sulfatide decomposes "cerebroside 3-sulfate" into cerebroside and sulfate.
- ARSA mainly plays a biological role in lysosomes and participates in the catabolism of polyphospholipids.
- 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.
- the present application provides a fusion protein, which includes: transferrin-binding protein, and arylsulfatase A (Arylsulfatase A, ARSA) or a functionally active fragment thereof.
- the fusion protein described in this application uses transferrin-targeted single-domain antibody technology to construct a protein or polypeptide fragment fused with a single-domain antibody, maintaining good binding activity with transferrin without affecting transferrin and transferrin.
- the binding of protein receptors has the function of extending half-life and crossing the blood-brain barrier.
- the present application provides a fusion protein, which includes: a transferrin-binding protein, and an arylsulfatase A (Arylsulfatase A, ARSA) or a functionally active fragment thereof, the transferrin-binding protein comprising a protein capable of binding Transferrin polypeptides, antibodies or antigen-binding fragments thereof.
- a transferrin-binding protein and an arylsulfatase A (Arylsulfatase A, ARSA) or a functionally active fragment thereof, the transferrin-binding protein comprising a protein capable of binding Transferrin polypeptides, antibodies or antigen-binding fragments thereof.
- 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) Ability to deliver the ARSA to cells expressing transferrin receptors.
- the transferrin is human transferrin.
- the antibody is selected from one or more of the group consisting of single domain antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
- the antigen-binding fragments include Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, VHH, di-scFv and/or dAb.
- the transferrin binding protein is VHH.
- the transferrin binding protein comprises CDR3, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3.
- the transferrin binding protein comprises CDR2, and the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2.
- the transferrin binding protein includes CDR1, and the CDR1 includes the amino acid sequence set forth in SEQ ID NO: 1.
- the transferrin binding protein comprises a VHH
- the VHH comprises the amino acid sequence set forth in SEQ ID NO:8.
- the ARSA is human ARSA.
- the ARSA or functionally active fragment thereof comprises a variant of ARSA or functionally active fragment thereof.
- the ARSA or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 10.
- the transferrin binding protein and the ARSA or functionally active fragment thereof are directly or indirectly linked.
- the N-terminus of the transferrin-binding protein and the C-terminus of the ARSA or functionally active fragment thereof are directly or indirectly connected.
- the C-terminus of the transferrin-binding protein and the N-terminus of the raw ARSA or functionally active fragment thereof are directly or indirectly connected.
- the transferrin-binding protein and the ARSA or functionally active fragment thereof are connected through a linker.
- the linker comprises a peptide linker.
- the linker comprises the amino acid sequence set forth in SEQ ID NO: 9.
- the fusion protein includes the amino acid sequence shown in SEQ ID NO: 10.
- the fusion protein is capable of binding transferrin and is capable of catalytically degrading cerebroside sulfate, a substrate of arylsulfatase A.
- the application also provides one or more isolated nucleic acid molecules encoding the fusion protein.
- the present application provides a vector comprising the nucleic acid molecule described in the present application.
- the present application provides a cell comprising the nucleic acid molecule, and/or the vector.
- the present application provides a pharmaceutical composition
- a pharmaceutical composition comprising the fusion protein, the nucleic acid molecule, the vector or the cell, and optionally a pharmaceutically acceptable carrier.
- this 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 medicines. For the prevention and/or treatment of diseases and/or conditions.
- the diseases and/or conditions include those caused by abnormalities in ARSA.
- the diseases and/or disorders include neurological disorders resulting from cerebroside sulfate substrate deposition.
- the disease and/or disorder includes metachromatic leukodystrophy (MLD).
- MLD metachromatic leukodystrophy
- the diseases and/or conditions include neurodegenerative diseases.
- the disease and/or disorder includes Parkinson's disease.
- the present application provides a method for preventing and/or treating diseases and/or disorders, which includes administering the fusion protein, the nucleic acid molecule, and the vector to a subject in need, The cells, and/or the pharmaceutical composition.
- the diseases and/or conditions include those caused by abnormalities in ARSA.
- the diseases and/or disorders include neurological disorders resulting from cerebroside sulfate substrate deposition.
- the disease and/or disorder includes metachromatic leukodystrophy (MLD).
- MLD metachromatic leukodystrophy
- the diseases and/or conditions include neurodegenerative diseases.
- the disease and/or disorder includes Parkinson's disease.
- this application also provides the fusion protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition, which is used to prevent and/or treat diseases. and/or illness.
- the diseases and/or conditions include those caused by abnormalities in ARSA.
- the diseases and/or disorders include neurological disorders resulting from cerebroside sulfate substrate deposition.
- the disease and/or disorder includes metachromatic leukodystrophy (MLD).
- MLD metachromatic leukodystrophy
- the diseases and/or conditions include neurodegenerative diseases.
- the disease and/or disorder includes Parkinson's disease.
- the present application also provides a method for delivering drugs across the blood-brain barrier, which includes administering the fusion protein.
- the present application also provides a method for extending the half-life of ARSA in vivo, which includes administering the fusion protein.
- Figure 1 shows that the single-domain antibody VHH that binds to transferrin significantly extends the half-life of recombinant ovalbumin in mouse blood and enhances its transport ability across the blood-brain barrier.
- Figure 2 shows the SDS-PAGE image of rhARSA-related recombinant protein.
- Figure 3 shows the binding detection of rhARSA-related recombinant protein and hTf.
- Figure 4 shows the results of FACS detection of the binding of rhARSA-related recombinant proteins to hTf/hTfR1.
- Figure 5A shows that fusion of VHH and hARSA protein does not affect M6PR binding.
- Figure 5B shows that the amount of 9056VHH-hARSA taken up by mARSA ⁇ / ⁇ fibroblasts is 5.57 times that of hARSA (10 ⁇ M), and part of it can be blocked by M6P.
- Figure 5C shows the time-dependent cellular uptake of 9056VHH-hARSA.
- Figure 6 shows the intracellular localization of ARSA recombinant protein taken up by mARSA -/- fibroblasts observed under laser confocal microscopy.
- Figures 7A-7B show the in vitro recombinant ARSA enzyme specific activity assay (substrate pNCS).
- Figure 8 shows the alcian blue/fast red staining results of mARSA -/- fibroblasts cultured for 48 hours with 10 ⁇ M, 3 ⁇ M, and 0 ⁇ M hARSA, 9056VHH-hARSA, and hARSA-9056VHH respectively.
- Figures 9A-9B show the in vitro plasma stability results of recombinant ARSA enzyme.
- Figure 10 shows the metabolic half-lives of hARSA, hARSA-9056VHH and 9056VHH-hARSA in mouse blood.
- Figures 11A-11B show the blood-brain barrier crossing efficiency of hARSA and 9056VHH-ARSA in wild-type C57bl/6 mice.
- Figures 12A-12C show the blood-brain barrier crossing efficiency of 9056VHH-ARSA in wild-type C57bl/6 mice at increasing doses.
- Figure 13 shows the drug distribution and organelle localization 2 hours after intravenous injection of hARSA-9056VHH in MLD model mice and 30MPK administration.
- Figures 14A-14C show the results of PK studies of recombinant hARSA-9056VHH across the blood-brain barrier in cynomolgus monkeys.
- Figure 15 shows the assessment of exercise capacity of mARSA-deficient mice after hARSA-9056VHH treatment.
- Figures 16A-16F show TLC detection of cerebroside sulfate substrate levels in the brain tissue of mARSA-deficient mice after hARSA-9056VHH treatment.
- Figure 17 shows the detection of cerebroside sulfate substrate levels in the brain tissue of mARSA-deficient mice by ABS staining after hARSA-9056VHH treatment.
- Figure 18 shows the TLC/ABS staining to detect cerebroside sulfate substrate levels in the sciatic nerve of mARSA-deficient mice after hARSA-9056VHH treatment.
- Figure 19 shows the cerebroside sulfate substrate levels in the kidneys of mARSA-deficient mice after hARSA-9056VHH treatment.
- fusion protein generally refers to a protein resulting from the fusion of two or more proteins or polypeptides. Fusion proteins can be artificially prepared through recombinant DNA technology. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or 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.
- transferrin generally refers to a glycoprotein capable of binding and transporting multivalent ions.
- transferrin can be a single-chain glycoprotein.
- transferrin may have at least one ion binding site.
- ion binding sites can have different affinities for iron ions.
- the polyvalent iron ions may be iron ions, chromium ions, manganese ions, cadmium ions or nickel ions.
- each molecule of transferrin can bind two ferric iron atoms.
- transferrin can be iron-containing Holo-transferrin, or iron-free apo-transferrin.
- the transferrin can be mouse transferrin.
- the amino acid sequence of mouse transferrin can be specified in GenBank. EDL21066.1, AAL34533.1, or AAL34533.1.
- the transferrin can be human transferrin.
- the amino acid sequence of human transferrin may be specified in GenBank. AAH59367.1, AAH59367.1, or AAB22049.1.
- the term "transferrin" may include functionally active fragments, homologs, analogs and/or variants thereof.
- transferrin-binding protein generally refers to a protein that contains a transferrin-binding portion and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen.
- transferrin-binding proteins described herein may include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, VHH, scFv, di-scFv and/or dAb), immunoconjugates, multispecific antibodies, antibody fragments, antibody derivatives, antibody analogs or fusion proteins, as long as they exhibit the desired antigen-binding activity.
- antigen-binding proteins can specifically bind to transferrin.
- the transferrin binding protein may not interfere with the interaction between transferrin and transferrin receptor 1.
- the transferrin binding protein may not affect Tf/TfR1 binding.
- transferrin-binding proteins maintain normal physiological functions of iron transport.
- antibody generally refers to a protein comprising one or more polypeptides essentially encoded by an immunoglobulin gene or immunoglobulin gene fragment.
- immunoglobulin genes may include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes.
- light chains can be classified as kappa or lambda, which can define the immunoglobulin types: Ig ⁇ and Ig ⁇ , respectively.
- Heavy chains can be classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD and IgE respectively.
- an antibody can have structural units that comprise tetramers, each of which can be composed of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD ), the N-terminus of each member can define a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition.
- variable light chain (VL) and variable heavy chain (VH) generally refer to the variable domain regions of the light and heavy chains, respectively.
- Antibodies may exist as intact immunoglobulins or as a number of well-characterized fragments generated by digestion with various peptidases or de novo expression. In this application, the antibody may also comprise only the heavy chain variable region. For example, the antibody may be a single domain antibody.
- the term "antigen-binding fragment” generally refers to one or more portions of a full-length antibody that retains substantially the ability to bind the same antigen (e.g., CD38) to which the antibody binds and is capable of binding to the same antigen as the full-length antibody. Compete for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated by reference in its entirety. This can be achieved by recombinant DNA technology or by intact antibodies. Enzymatic or chemical cleavage of generates antigen-binding fragments.
- the antigen-binding fragments include Fab, Fab', F(ab')2, (Fab)2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments , VHH, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
- scFv single chain antibodies
- chimeric antibodies diabodies
- polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide are known to those skilled in the art to use conventional techniques (e.g., recombinant DNA techniques or enzymatic or chemical (Fragmentation method)
- Antigen-binding fragments of an antibody are obtained from a given antibody and screened for specificity in the same manner as for intact antibodies. For example, pepsin can digest the antibody below the disulfide bond in the hinge
- VHH generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be called Nanobody (Nb) and/or single domain antibody.
- the term "functionally active fragment” generally refers to a fragment that has a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid.
- a functionally active fragment may retain or partially retain the ability of the full-length protein to bind another molecule.
- functionally active fragments of arylsulfatase A (ARSA) may retain or partially retain the biologically active function of full-length ARSA that causes cell proliferation.
- arylsulfatase A and “ARSA” are used interchangeably, and the term may include wild-type ARSA or modified ARSA.
- the ARSA may include homologs, analogs, derivatives and/or functional variants thereof.
- the ARSA can comprise full-length ARSA, and the ARSA can comprise functionally active fragments of ARSA.
- the ARSA may comprise human ARSA.
- Fab generally refers to an antibody fragment consisting of VL, VH, CL and CH1 domains.
- Fab' generally refers to an antibody fragment that has several additional residues at the carboxy terminus of the CH1 domain compared to a Fab fragment.
- Fab' may include one or more cysteines from the hinge region of the antibody.
- F(ab)2 generally refers to an antigen-binding fragment resulting from pairs of Fab fragments linked by cysteines.
- dAb fragment generally refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)).
- CDR complementarity determining region
- VL light chain variable region
- VH heavy chain variable region
- Fv fragment generally refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody.
- scFv generally refers to a molecule composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide linker, also known as a single-chain antibody.
- proteins, polypeptides and/or amino acid sequences involved should also be understood to include at least the following range: variants or homologues that have the same or similar functions as the protein or polypeptide.
- 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 polypeptide.
- the functional variant may comprise one that has been replaced by at least 1, such as 1-30, 1-20 or 1-10, such as 1, 2, 3, 4 or 5 amino acids. , deletion and/or insertion of proteins or polypeptides with amino acid changes.
- the functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (eg, substitution, deletion, or addition).
- the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding ability) of the protein or polypeptide prior to the alteration.
- the substitutions may be conservative substitutions.
- sequence homology generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences.
- a program such as Emboss Needle or BestFit
- the default settings can be used, or an appropriate scoring matrix can be selected (such as blosum45 or blosum80) to optimize identity, similarity, or homology scores.
- homologous polynucleotides are those sequences that hybridize under stringent conditions and are 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.
- 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. Identity.
- the amino group is connected to another carboxyl group in the polypeptide chain to make it a chain.
- the two ends of the protein there are remaining amino acid residues without peptide bonds, which carry free
- the end of the polypeptide chain containing the amino group and the end of the polypeptide chain carrying the carboxyl group.
- N-terminus generally refers to the end of a polypeptide chain in which the amino acid residue carries a free amino group.
- the term “C-terminus” generally refers to the end of a polypeptide chain where the amino acid residue carries a free carboxyl group.
- nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or artificially synthesized.
- vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed.
- the vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries are expressed in the host cell.
- vectors include: plasmids; phages; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animals Viruses etc.
- the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
- Viruses (such as SV40).
- a vector may contain a variety of elements that control expression, including promoters The kinematic sequence, transcription initiation sequence, enhancer sequence, selection element and reporter gene.
- the vector may also contain an origin of replication site. Vectors may also contain components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but they are not the only ones.
- the term "pharmaceutical composition” generally refers to a formulation in a form that is effective in allowing the biological activity of the active ingredient and which does not contain additional ingredients that would be unacceptable toxicity to the subject to whom the formulation is to be administered. .
- these preparations may be sterile.
- a pharmaceutically acceptable adjuvant generally means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic reaction, or other problems or complications, Adjuvants with reasonable benefit/risk ratios.
- a pharmaceutically acceptable adjuvant may mean one approved by a regulatory agency (such as the U.S. Food and Drug Administration, China Food and Drug Administration, or the European Medicines Agency) or listed in a generally recognized pharmacopeia (such as the United States Pharmacopeia, the Chinese Pharmacopeia, or the European Pharmacopoeia). Pharmacopoeia) for use in animals, more particularly in humans.
- the term “comprising” generally means including, encompassing, containing or encompassing. In some cases, it also means “for” or “composed of”.
- the term "about” generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
- the present application provides a fusion protein, which includes: transferrin binding protein, and arylsulfatase A (Arylsulfatase A, ARSA) or a functionally active fragment thereof.
- transferrin binding protein and arylsulfatase A (Arylsulfatase A, ARSA) or a functionally active fragment thereof.
- the transferrin-binding protein may comprise a polypeptide, an antibody, or an antigen-binding fragment thereof capable of binding transferrin.
- the fusion protein may have one or more properties.
- the fusion protein can extend the in vivo half-life of the ARSA.
- the fusion protein can cross the blood-brain barrier to achieve drug delivery.
- the fusion protein can be delivered orally.
- the fusion protein is capable of delivering the ARSA to cells expressing transferrin receptors.
- the transferrin may be human transferrin.
- the antibody capable of binding transferrin may include one or more selected from the group consisting of single domain antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
- the antigen-binding fragment capable of binding transferrin may include Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, VHH, di-scFv and/or dAb .
- the transferrin binding protein may comprise a VHH capable of binding transferrin.
- 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.
- the CDR of an antibody is also called the complementarity determining region, which is part of the variable region.
- the amino acid residues in this region may contact the antigen or antigenic epitope.
- Antibody CDRs can be determined through a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, taking Kabat/Chothia into consideration, etc. These encoding systems are known in the art and can be found, for example, at http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different encoding systems, there may be differences in the CDR area.
- the CDR covers CDR sequences classified according to any CDR classification method; it also covers its variants, which include substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR. .
- the homologues may be at least about 85% identical to the amino acid sequence of the CDR (for example, having at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences that have about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater) sequence homology.
- the CDRs of the transferrin-binding proteins described herein can be defined by the Kabat coding system.
- the transferrin binding protein may comprise CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.
- the transferrin-binding protein may comprise CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2.
- the transferrin binding protein may comprise CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1.
- the transferrin-binding protein may include CDR1, CDR2 and CDR3, and the CDR1 may include the amino acid sequence shown in SEQ ID NO:1, and the CDR2 may include the amino acid sequence shown in SEQ ID NO:2, And the CDR3 may include the amino acid sequence shown in SEQ ID NO:3.
- the transferrin-binding protein may comprise the framework region FR1 of VHH, and the FR1 may comprise the amino acid sequence shown in SEQ ID NO:4.
- the transferrin-binding protein may comprise the framework region FR2 of VHH, and the FR2 may comprise the amino acid sequence shown in SEQ ID NO:5.
- the transferrin-binding protein may comprise the framework region FR3 of VHH, and the FR3 may comprise the amino acid sequence shown in SEQ ID NO: 6.
- the transferrin-binding protein may comprise the framework region FR4 of VHH, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7.
- the transferrin-binding protein may comprise a VHH capable of binding transferrin, and the VHH comprises the amino acid sequence shown in SEQ ID NO: 8.
- ARSA or functionally active fragments thereof in the fusion protein may be human ARSA or functionally active fragments thereof.
- the ARSA or functionally active fragment thereof can be modified so that it still retains the function and/or activity of ARSA.
- ARSA or its functionally active fragment in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 10.
- ARSA or functionally active fragments thereof described in this application may also include variants, truncations, homologs, and analogs thereof.
- the ARSA or functionally active fragment thereof may also comprise at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74% with the amino acid sequence shown in SEQ ID NO:10.
- the ARSA or functionally active fragment thereof may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 compared to SEQ ID NO: 10. Amino acid sequences with 1, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more amino acid mutations.
- the transferrin-binding protein in the fusion protein, can be directly or indirectly connected to the ARSA or its functionally active fragment.
- the N-terminus of the transferrin-binding protein in the fusion protein, can be directly or indirectly connected to the C-terminus of the ARSA or its functionally active fragment.
- the C-terminus of the transferrin-binding protein in the fusion protein, can be directly or indirectly connected to the N-terminus of the ARSA or its functionally active fragment.
- the transferrin-binding protein in the fusion protein, can be connected to the ARSA or its functionally active fragment through a linker.
- the linker may comprise a flexible linker.
- the linker may comprise a peptide linker.
- the linker in the fusion protein, may comprise the amino acid sequence shown in SEQ ID NO: 9.
- the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 11.
- the fusion protein is capable of binding to transferrin and capable of catalytically degrading cerebroside sulfate, a substrate of arylsulfatase A.
- the application provides one or more isolated nucleic acid molecules encoding said fusion protein.
- the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification , for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis.
- amplification in vitro such as by polymerase chain reaction (PCR) amplification
- PCR polymerase chain reaction
- production by clonal recombination e.g., clonal recombination
- purification for example by enzymatic digestion and gel electrophoresis fractionation
- synthetic for example by chemical synthesis.
- 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.
- Direct chemical synthesis of nucleic acids typically involves the sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide polymer chain, where each addition passes through a nucleophilic This is achieved by attacking the terminal 5'-hydroxyl group of the growing chain at the 3'-position of the added monomer, which is usually a phosphorus derivative such as a phosphotriester, 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.
- Vectors comprising the isolated polynucleotides of the present application are provided.
- the vector can be any linear nucleic acid, plasmid, phage, cosmid, RNA vector, viral vector, etc.
- Non-limiting examples of viral vectors may include retroviruses, adenoviruses, and adeno-associated viruses.
- the application provides one or more vectors comprising said nucleic acid molecules.
- the vector may comprise one or more nucleic acid molecules described herein.
- One or more such nucleic acid molecules may be included in each vector.
- other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
- the vector may contain expression control elements that allow correct expression of the coding region in an appropriate host.
- control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
- the expression control sequences are tunable elements.
- the specific structure of the expression control sequence can vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, GA cap sequence, CAAT sequence, etc.
- the 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid.
- the expression control sequences may also include enhancer sequences or upstream activator sequences.
- the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
- the present application provides a cell comprising the fusion protein, the nucleic acid molecule, or the vector.
- the cell may be a host cell.
- the cells may include many cell types such as prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or fibroblasts, CHO animal cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
- the vector can be introduced into a host cell stably or transiently by a variety of established techniques.
- one method involves calcium chloride treatment, in which the carrier is introduced by calcium precipitation.
- Other salts such as calcium phosphate, may be used in a similar manner.
- electroporation ie, the application of electrical current to increase the permeability of cells to nucleic acids
- transformation methods include microinjection, DEAE dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipoplexes, liposomes, and dendrimers can also be used to transfect host cells.
- the present application provides a method for preparing the fusion protein, which may include culturing the cells under conditions enabling the expression of the fusion protein. For example, by using appropriate culture medium, appropriate temperature and culture time, etc., these methods are understood by those of ordinary skill in the art.
- the present application provides a composition comprising the fusion protein, or the nucleic acid molecule, and optionally a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and/or non-ions. Surfactants, etc.
- composition may be formulated with a pharmaceutically acceptable carrier or diluent and any other known auxiliaries and excipients according to conventional techniques in the art, for example, according to Remington: The Science and Practice of Pharmacy, Nineteenth Edition, Gennaro Editor, Mack Publishing Co., Easton, PA, 1995.
- the pharmaceutical composition may be formulated for oral administration, such as tablets, capsules, pills, powders, sustained-release preparations, solutions, and suspensions.
- the pharmaceutical compositions may be in unit dosage form suitable for single administration of precise doses.
- the pharmaceutical composition may further comprise conventional pharmaceutical carriers or excipients.
- the pharmaceutical composition may include other drugs or agents, carriers, adjuvants, and the like.
- the pharmaceutical compositions described herein may comprise a therapeutically effective amount of the fusion protein.
- the therapeutically effective amount is that required to prevent and/or treat (at least in part) the disorder or condition (e.g., neurodegenerative disease) and/or any complications thereof in a subject suffering from or at risk of developing it. dose.
- the specific amount/concentration of the dosage may vary depending on the method of administration and patient needs, and may be determined based on, for example, patient volume, viscosity, and/or body weight. It should be understood that based on the specific patient, formulation and/or disease conditions, one skilled in the art (e.g., a physician or pharmacist) can readily adjust these specific doses.
- this 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 medicines. For the prevention and/or treatment of diseases and/or conditions.
- the present application provides a method for preventing and/or treating diseases and/or disorders, which includes administering the fusion protein, the nucleic acid molecule, and the vector to a subject in need, The cells, and/or the pharmaceutical composition.
- the present application provides the fusion protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition, which is used to prevent and/or treat diseases and /or illness.
- the diseases and/or conditions may include diseases and/or conditions caused by abnormalities in ARSA.
- the diseases and/or disorders may include neurological diseases resulting from deposition of cerebroside sulfate substrates.
- the disease and/or disorder may include metachromatic leukodystrophy (MLD).
- MLD metachromatic leukodystrophy
- the diseases and/or conditions may include neurodegenerative diseases.
- the disease and/or disorder may include Parkinson's disease.
- the present application also provides a method of delivering a drug across the blood-brain barrier, which may include administering the fusion protein.
- the present application also provides a method for extending the half-life of ARSA in vivo, which may include administering the fusion protein.
- Purified human transferrin from human plasma was used to immunize two Asian alpacas aged 2-3 years until the specific serum titer reached over 10,000 (ELISA method).
- 200 ml of peripheral blood were taken to isolate and purify mononuclear cells (PBMC), and the mRNA was extracted and reverse transcribed into cDNA.
- VHH-specific primers were used to amplify the nucleotide sequence encoding VHH, and cloned into a phage display vector to establish a VHH phage display library. Based on the DNA sequence determination results of nearly 100 randomly selected clones, it is estimated that the effective capacity of the constructed phage display library is approximately 100 million (10 9 ) independent VHH sequences.
- VHH libraries were panned using a liquid phase method: 1-10 ⁇ g of biotinylated human transferrin was mixed with 10 12 CFU (clonogenic monomers). The phages at position) were incubated for 1 hour in phosphate buffered saline (PBST-1% BSA) containing 1% bovine serum albumin, 0.05% Tween 20, and Streptomyces pre-washed and blocked with PBST-1% BSA was added. Avidin streptavidin-coupled magnetic beads (Invitrogen, Dynabeads M-280 streptavidin). After incubation at room temperature for 1 hour, the magnetic beads were washed 10 times with PBST.
- PBST-1% BSA phosphate buffered saline
- Tween 20 1% bovine serum albumin
- Streptomyces pre-washed and blocked with PBST-1% BSA was added.
- Avidin streptavidin-coupled magnetic beads Invitrogen, Dynabead
- the phage bound to the magnetic beads are dissociated with 10 ⁇ g/ml trypsin, infected and amplified in E. coli, and used for the next round of panning, or single clones are selected for ELISA method to specifically bind to human transferrin. , but does not affect the VHH sequence that binds transferrin to the human transferrin receptor.
- SLN9056 shows better binding activity to human transferrin (pH7.4, pH6.0) ( Figure 1A-B), and can significantly extend the recombinant egg white fused with it.
- Half-life of protein (OVA) in mouse blood Figure 1C).
- mice were intravenously injected with purified recombinant protein (such as VHH-OVA-His) at a dose of 10 mg/kg, and intraperitoneally injected 10 mg/kg human transferrin daily starting the day before administration. Samples were taken at different time points after administration, and the brains were collected after perfusion with 20 mL of PBS and frozen in liquid nitrogen. ELISA quantitatively determined the content of VHH-OVA-His in brain tissue homogenate. Rabbit anti-OVA polyclonal antibody (Sigma C6534-2mL) was used for coating, and biotinylated rabbit anti-VHH antibody (GeneScript, A02015-200) was used for detection.
- VHH-OVA-His purified recombinant protein
- transferrin-binding VHHs significantly increased the levels of test article in brain tissue (average % ID of injected dose for selected VHHs) dose) percentage, ranging from ⁇ 1.03% to ⁇ 3.5% 4 hours after dosing (Fig. 1D).
- 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, and the amino acid sequence of CDR2 The sequence is as shown in SEQ ID NO:2, the amino acid sequence of CDR3 is as shown in SEQ ID NO:3, the amino acid sequence of VHH is as shown in SEQ ID NO:8; the linker is composed of the amino acid sequence as shown in SEQ ID NO:9).
- the recombinant plasmid is used as the vector, lipofectamine or PEI is used as the transfection reagent, and HEK293F cells are transiently transfected to produce the recombinant protein in batches.
- the 96-well plate was coated with streptavidin (0.1 ⁇ g/100 ⁇ l/well) and incubated at 4°C overnight; after washing three times with PBST, the plate was incubated at room temperature.
- the cells were blocked with 200 ⁇ l of PBST containing 1% bovine serum albumin (BSA) for 1 hour. Wash three times with PBST, add biotinylated human transferrin (1 ⁇ g/ml, 100 ⁇ l/well) and incubate at room temperature for 1 hour. Wash the plate three times with PBST, add serially diluted hARSA or hARSA-9056VHH protein, and incubate at room temperature for 1 hour.
- BSA bovine serum albumin
- 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 surface.
- M6P receptor extracellular segment M6PR-Fc recombinantly expressed in HEK293F cells, 0.5 ⁇ g/well
- PBST phosphate buffer saline, 0.1% Tween-20, pH7.4
- mARSA -/- mouse skin fibroblasts were obtained from C57BL/6J-Arsaem1cyagen (KOCMP-11883-Arsa-B6J) Mice, the model mice were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.
- the tissue blocks were cultured as usual, and the culture medium used high-glucose DMEM (containing 4.0mM L-Glutamine, Sodium Pyruvate) (HyClone, SH30243.01) and 10% fetal bovine serum (Gibco, 10099-141C). When the cells crawl out into sheets and grow well, they are ready for passage.
- Collect cell lysate Add 1 ⁇ protease inhibitor to the cell lysate, mix well and set aside; discard the supernatant and wash 3 times with PBS; add 60 ⁇ l of cell lysate to each well, fully lyse the cells and collect into a 1.5ml EP tube medium; centrifuge at 12,000 rpm, take the supernatant, determine the total protein concentration, and store at -80 degrees for detection.
- ELISA detection of drug content in cell lysate Goat anti-human Anti-hARSA antibody (R&D, AF2485) 0.25 ⁇ g/ml coated 96-well enzyme-linked plate, washed 3 times with PBST (Tween-20, 0.1%), and then washed with 200 ⁇ l containing 1 Block in % BSA in PBST for 1 hour at room temperature. After washing three times with PBST, add serially diluted detection samples (using serially diluted standard rhARSA and 9056VHH-rhARSA as the standard curve).
- cellular uptake drug concentration in the sample/total protein concentration.
- the amount of 9056VHH-hARSA taken up by mARSA ⁇ / ⁇ fibroblasts was 5.57 times that of hARSA (10 ⁇ M), and part of it could be blocked by M6P.
- mARSA-/- mouse skin fibroblasts were cultured. Inoculate well-growing cells into a 24-well plate with a coverslip and culture the cells until they are 70-80% confluent. Discard the old culture medium, add culture medium containing 2 ⁇ M 9056VHH-hARSA or hARSA, and continue culturing for 24 hours.
- Fixation discard the culture medium, wash the cells 3 times with pre-cooled PBS; fix with 4% PBS pH 7.4 paraformaldehyde at room temperature for 10 minutes. Wash cells 3 times with pre-chilled PBS.
- Blocking Block with 1% BSA PBST (PBS+0.05% Tween 20) for 30 minutes. Wash cells 3 times with pre-chilled PBS.
- a large amount of 9056VHH-hARSA (A) and hARSA (B) recombinant proteins can be endocytosed by mARSA ⁇ / ⁇ mouse skin fibroblasts and transported to lysosomes (arrows).
- Reaction buffer Assay Buffer 50mM NaOAc, 0.5M NaCl, pH 4.5 (NaOAc, NaCl, HOAc adjusted Adjust pH to 4.5).
- p-nitrocatechol sulfate (p-NCs, sigma, Lot#MKCN3971) storage solution: pNCS, 2mM dissolved in reaction buffer (MW 311.35, 10mg in 16mL).
- p-NC standard curve Add pNC standards 0, 20, 40, 60, 80, and 100 ⁇ L into a 96-well plate respectively; use stop solution to adjust the total volume to 100 ⁇ L/well.
- the corresponding pNC contents are 0, 20, 40, 60, 80, and 100 nmol/well respectively.
- mARSA-/- fibroblasts were cultured for 48 hours with 10 ⁇ M, 3 ⁇ M, and 0 ⁇ M 9056VHH-hARSA. After Alcian Blue/Fast Red staining, varying degrees of lake-blue dots and lumpy staining could be seen in the cytoplasm of the cells in the three groups.
- the blue dye material is a sulfate metabolite containing sulfate radicals; in the cytoplasm of the cells in the 10 ⁇ M group
- the blue-dyed material was diffuse and fine dot-like, with few lumps; the size of the blue-dyed material spots and lumps in the cytoplasm of the cells in the three groups was: 10 ⁇ M group ⁇ 3 ⁇ M group ⁇ 0 ⁇ M group; the amount of blue dye material in the cytoplasm of the three groups
- 9056VHH-hARSA 10 ⁇ M group plays a role in degrading substrates in cells to a certain extent.
- Sample preparation Configure the standard protein in mouse plasma to a certain concentration: 6 ⁇ g/ml, and incubate it under constant temperature and moisturizing conditions at 37°C for 1 hour, 4 hours, 8 hours, 24 hours and 48 hours, and then Samples were snap frozen in liquid nitrogen and stored at ⁇ 80°C until use. Protein samples without plasma were used as positive controls.
- ELISA detection SLN7134 (anti-9056VHH, made by Lingnuo) 1 ⁇ g/ml, 100 ⁇ L/well coated 96-well plate, overnight at 4°C. 1% BSA TBST, 200 ⁇ L per well, blocked at 37°C for 1 hour. Dilute the protein with 1% BSA TBST and prepare a standard curve. The starting concentration is 200ng/ml, 2-fold gradient dilution, 100 ⁇ L per well, and incubate at room temperature for 1 hour. Dilute the sample to the appropriate concentration range with 1% plasma, dilute 100 times, 200 times, and 300 times, 100ul per well, and incubate at room temperature for 1 hour.
- PK studies were conducted in 6-8 week old male C57BL/6 mice. The animals were divided into groups according to their body weight, and 10 mg/kg human transferrin was intraperitoneally injected the day before administration and every day during the study period.
- rhARSA The single tail vein dose of rhARSA-9056VHH and 9056VHH-rhARSA is 3MPK. Blood samples were collected at 2, 4, 8, 24, and 48 hours after administration to separate the serum.
- the 6.1PK method was used for quantitative analysis of the drug in the serum, and PKsovler software was used to calculate the PK parameters.
- PK studies were conducted in 6-8 week old male C57BL/6 mice. The animals were divided into groups according to their body weight, and 10 mg/Kg of human transferrin was intraperitoneally injected the day before administration and every day during the study period.
- the tail vein dose of rhARSA-9056VHH is 10MPK.
- blood samples were collected, serum was separated, and cerebrospinal fluid was collected; brain tissue was collected after perfusion with 20 ml PBS.
- the 6.1PK method was used to detect the drug content in serum, cerebrospinal fluid and brain tissue homogenate respectively.
- injection dose percentage % ID brain tissue drug concentration (ng/mg tissue)/10 mg/kg ⁇ 100%).
- Figures 12A-12C Figure 11A shows that the mean plasma concentration of hARSA is approximately 45ng/ml (4h), and the plasma concentration of 9056VHH-hARSA is approximately 5000ng/ml (4h), ⁇ 400ng/ml (24h), ⁇ 18ng/ ml(48h), which was significantly higher than the hARSA group.
- Figure 11B shows that hARSA-his is not detectable in brain tissue homogenate.
- the concentration of 9056VHH-hARSA in brain tissue homogenate is 4.6 ⁇ 10.8pg/mg brain tissue (4h), 0.7 ⁇ 4.2pg/mg brain tissue ( 24h), the efficiency of crossing the blood-brain barrier was much higher than that of the hARSA group.
- the experimental method is the same as 5.3.
- the tail vein administration doses of rhARSA-9056VHH are 45nmol/kg, 150nmol/kg, and 450nmol/kg respectively.
- the 6.1PK method was used to detect the drug content in serum, cerebrospinal fluid and brain tissue homogenate respectively.
- Figure 12A shows the plasma concentration of 9056VHH-hARSA at three doses of 45nmol/kg, 150nmol/kg, and 450nmol/kg
- Figure 12B shows the content of 9056VHH-hARSA in brain tissue homogenate.
- %ID average injection dose percentages
- mice were euthanized, and brain tissues were collected and fixed in 4% paraformaldehyde. Paraffin sections of the brain tissue were prepared as usual and cut into 5 ⁇ m sagittal sections (commissioned by Shanghai Ruibaohe Biotechnology Co., Ltd.). hARSA/CD31/Lamp1 immunofluorescence staining: deparaffinize, hydrate, and wash 3 times with PBS as usual.
- Blocking Block with 1% BSA in PBST (PBS+0.05% Tween 20) for 30 minutes. Wash 3 times with PBS. Immunofluorescence staining: primary anti-goat anti-human ARSA antibody (R&D, AF2486) or rabbit anti-mouse CD31 antibody (Abcam, Abcam, diluted 1:100 in 1% BSA PBST ab281583), incubate at room temperature for 1 hour. Wash 3 times with pre-cooled PBS.
- Drugs can cross the blood-brain barrier and enter brain tissue (anti-hARSA) and vascular endothelial cells (anti-CD31); at the same time, there are also drugs in vascular endothelial cells; Figure 13B. Drugs entering brain cells The drug (anti-hARSA,) co-localizes with the lysosomal membrane protein (anti-Lamp1).
- FIG. 14A-14C Figure 14A. Brain tissue drug concentration-time Curve; Figure 14B. Left ventricular drug concentration-time curve; Figure 14C. Brain tissue drug concentration/left ventricular drug concentration percentage: 20min to 30min after administration, the drug concentration brain/plasma ratio increased significantly, hARSA-9056VHH can be expressed in the brain Tissue accumulation and retention; the 10MPK group reached the peak at 96 hours, the brain/plasma ratio % was 53.3%; the 30MPK group reached the peak at 72 hours, the brain/plasma ratio % was 23.95%.
- mARSA-deficient mice The genotype of mARSA gene (GenBank: Gene ID 11883)-deficient mice is C57BL/6J-Arsa em1cyagen (KOCMP-11883-Arsa-B6J), which was verified by PCR and sequencing.
- mice 7.2.1 Animal grouping: Select mARSA -/- homozygous mice that are about 10-12 months old and in good health; match them by gender/age; weigh and record the mouse weight; mouse number, and fill in the cage Card information.
- hTf 10mg/kg intraperitoneally injected once a day to maintain a certain level of hTf in mice;
- Training period On the first day, a balance beam is used to allow the mice to enter the small dark box through the square balance beam. Each mouse is trained 3 Second-rate. Repeat the same steps as Day 1 on Day 2.
- Test period The third day is the test period. The mice are made to enter the small dark box through the square balance beam. The whole process is videotaped and the time of passing the balance beam is recorded.
- tissues, organs and kidneys were collected and stored at -80°C; the brain (including olfactory bulb, cerebellum, cerebrum, brainstem and part of the spinal cord) was cut sagittally, and the left brain and left kidney were quickly frozen in liquid nitrogen for TLC (dividing into cerebellum, cerebrum, brain trunk and part of the spinal cord), ELISA detection; the right brain and right kidney were fixed with 4% PFA for Alcian blue staining and immunofluorescence staining); and the peripheral nerve tissue-sciatic nerve, fixed with 4% PFA for Alcian blue staining .
- Developing agent chloroform: methanol: water 65:25:4 (v/v/v) (requires advance Prepare it well so that the steam inside the cylinder is saturated).
- Color development Soak the unfolded silica gel plate in the color development solution (10% CuSO4in 10% phosphoric acid solution) for about 3 minutes, take it out, blow dry with a hair dryer, and place it in the oven at 130°C for 10 minutes. If there are black spots or strips, Appear.
- Alcian blue staining Prepare frozen sections of brain tissue as usual, and cut 10 ⁇ m sagittal sections (entrusted to Shanghai Ruibaohe Biological Co., Ltd. to complete); prepare Alcian blue staining buffer (0.025M sodium acetate, pH 5.7, containing 0.3M MgCl 2 , 1% PFA) and 0.05% Alcian blue staining solution (Alcian blue 8GX, sigma, A9186-10G) were dissolved in the staining buffer.
- Alcian blue staining buffer 0.025M sodium acetate, pH 5.7, containing 0.3M MgCl 2 , 1% PFA
- Alcian blue staining solution Alcian blue 8GX, sigma, A9186-10G
- Frozen sections of brain tissue were dissolved at room temperature and fixed with 4% paraformaldehyde (PFA) for 1 minute; after sufficient washing with the staining buffer, incubated with 0.05% alcian blue staining solution at room temperature for 1 hour; after sufficient washing with the staining buffer, fast red counterstaining was performed for 2 minutes; wash thoroughly with tap water, seal the slide with glycerin; observe and take pictures under a microscope.
- PFA paraformaldehyde
- Detection of cerebroside sulfate substrate in kidneys Same as the above method for detecting cerebroside sulfate substrate in brain tissue. Analyze the Sulfatide/Galcer ratio by thin-layer chromatography and analyze the kidney cerebroside sulfate in the kidneys of mice in the treatment group and the untreated group. Glycoside substrate deposition.
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Abstract
一种融合蛋白,其包含:转铁蛋白结合蛋白,以及芳香基硫酸酯酶A(Arylsulfatase A,ARSA)或其功能活性片段,所述转铁蛋白结合蛋白包含能够结合转铁蛋白的多肽、抗体或其抗原结合片段。以及所述融合蛋白的制备方法及用途。
Description
本申请涉及生物医药领域,具体的涉及一种包含转铁蛋白结合蛋白和芳基硫酸酯酶A的融合蛋白,及其用途。
芳基硫酸酯酶A(Arylsulfatase A或ARSA,也称酰基硫酸酯酶A、脑苷硫酸酯酶/cerebroside-sulfatase或芳香基硫酸酯酶A)属于溶酶体水解酶的一种,它可以分解脑硫脂(sulfatide),即分解"脑苷3硫酸"(cerebroside 3-sulfate)为脑苷脂及硫酸盐。ARSA主要在溶酶体内发挥生物学作用,参与多聚磷脂的分解代谢。同时,在细胞胞浆内,ARSA酶和α-synuclein的直接结合有效阻止了后者的蛋白聚集和胞外分泌。因此,ARSA酶的缺失或活性降低,是异染性脑白质病(Metachromatic Leukodystrophy,或MLD,一种严重影响生命的溶酶体储积症)的直接病因,也与多种退行性神经官能症比如帕金森综合征(Parkinson’s disease)的发生、发展有关。因此,ARSA酶的替代治疗,特别是中枢神经系统靶向的酶替代治疗,有希望为这些严重影响人类生命和健康、目前尚无有效治疗手段的疾病治疗提供必要的解决方案。
发明内容
本申请提供了一种融合蛋白,其包含:转铁蛋白结合蛋白,以及芳香基硫酸酯酶A(Arylsulfatase A,ARSA)或其功能活性片段。本申请所述的融合蛋白采用靶向转铁蛋白的单域抗体技术,构建与单域抗体融合的蛋白或多肽片段,保持与转铁蛋白良好的结合活性,同时不影响转铁蛋白和转铁蛋白受体的结合,具备延长半衰期和穿越血脑屏障的功能。
一方面,本申请提供了一种融合蛋白,其包含:转铁蛋白结合蛋白,以及芳香基硫酸酯酶A(Arylsulfatase A,ARSA)或其功能活性片段,所述转铁蛋白结合蛋白包含能够结合转铁蛋白的多肽、抗体或其抗原结合片段。
在某些实施方式中,所述融合蛋白具有下述性质中的一种或多种:1)能够延长所述ARSA的体内半衰期;2)能够跨越血脑屏障;3)能够通过口服递送;以及4)能够将所述ARSA递送至表达转铁蛋白受体的细胞。
在某些实施方式中,所述转铁蛋白为人转铁蛋白。
在某些实施方式中,所述抗体选自下组中的一种或多种:单域抗体、单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中,所述抗原结合片段包括Fab,Fab’,Fv片段,F(ab’)2,F(ab)2,scFv,VHH,di-scFv和/或dAb。
在某些实施方式中,所述转铁蛋白结合蛋白为VHH。
在某些实施方式中,所述转铁蛋白结合蛋白包含CDR3,且所述CDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中,所述转铁蛋白结合蛋白包含CDR2,且所述CDR2包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中,所述转铁蛋白结合蛋白包含CDR1,且所述CDR1包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中,所述转铁蛋白结合蛋白包含VHH,且所述VHH包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中,所述ARSA为人ARSA。
在某些实施方式中,所述ARSA或其功能活性片段包含ARSA或其功能活性片段的变体。
在某些实施方式中,所述ARSA或其功能活性片段包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中,在所述融合蛋白中,所述转铁蛋白结合蛋白和所述ARSA或其功能活性片段直接或间接相连。
在某些实施方式中,在所述融合蛋白中,所述转铁蛋白结合蛋白的N端和所述ARSA或其功能活性片段的C端直接或间接相连。
在某些实施方式中,在所述融合蛋白中,所述转铁蛋白结合蛋白的C端和所述生ARSA或其功能活性片段的N端直接或间接相连。
在某些实施方式中,在所述融合蛋白中,所述转铁蛋白结合蛋白和所述ARSA或其功能活性片段通过连接子相连。
在某些实施方式中,所述连接子包含肽接头。
在某些实施方式中,所述连接子包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中,所述融合蛋白包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中,所述融合蛋白能够结合转铁蛋白,且能够催化降解芳香基硫酸酯酶A的底物硫酸脑苷脂。
另一方面,本申请还提供了一种或多种分离的核酸分子,其编码所述融合蛋白。
另一方面,本申请提供了一种载体,其包含本申请所述的核酸分子。
另一方面,本申请提供了一种细胞,其包含所述的核酸分子,和/或所述的载体。
另一方面,本申请提供了一种药物组合物,其包含所述的融合蛋白,所述的核酸分子,所述的载体或所述的细胞,以及任选地药学上可接受的载体。
另一方面,本申请提供了所述的融合蛋白,所述的核酸分子,所述的载体,所述的细胞,和/或所述的药物组合物在制备药物中的用途,所述药物用于预防和/或治疗疾病和/或病症。
在某些实施方式中,所述疾病和/或病症包括ARSA异常引起的疾病和/或病症。
在某些实施方式中,所述疾病和/或病症包括由硫酸脑苷脂底物沉积导致的神经系统疾病。
在某些实施方式中,所述疾病和/或病症包括异染性脑白质病(Metachromatic Leukodystrophy,MLD)。
在某些实施方式中,所述疾病和/或病症包括神经退行性疾病。
在某些实施方式中,所述疾病和/或病症包括帕金森综合症(Parkinson’s disease)。
另一方面,本申请提供了一种预防和/或治疗疾病和/或病症的方法,其包括向有需要的受试者施用所述的融合蛋白,所述的核酸分子,所述的载体,所述的细胞,和/或所述的药物组合物。
在某些实施方式中,所述疾病和/或病症包括ARSA异常引起的疾病和/或病症。
在某些实施方式中,所述疾病和/或病症包括由硫酸脑苷脂底物沉积导致的神经系统疾病。
在某些实施方式中,所述疾病和/或病症包括异染性脑白质病(Metachromatic Leukodystrophy,MLD)。
在某些实施方式中,所述疾病和/或病症包括神经退行性疾病。
在某些实施方式中,所述疾病和/或病症包括帕金森综合症(Parkinson’s disease)。
另一方面,本申请还提供了所述的融合蛋白,所述的核酸分子,所述的载体,所述的细胞,和/或所述的药物组合物,其用于预防和/或治疗疾病和/或病症。
在某些实施方式中,所述疾病和/或病症包括ARSA异常引起的疾病和/或病症。
在某些实施方式中,所述疾病和/或病症包括由硫酸脑苷脂底物沉积导致的神经系统疾病。
在某些实施方式中,所述疾病和/或病症包括异染性脑白质病(Metachromatic Leukodystrophy,MLD)。
在某些实施方式中,所述疾病和/或病症包括神经退行性疾病。
在某些实施方式中,所述疾病和/或病症包括帕金森综合症(Parkinson’s disease)。
另一方面,本申请还提供了一种递送药物穿越血脑屏障的方法,其包括施用所述的融合
蛋白。
另一方面,本申请还提供了一种延长ARSA在体内半衰期的方法,其包括施用所述的融合蛋白。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的,本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地,本申请的附图和说明书中的描述仅仅是示例性的,而非为限制性的。
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下:
图1显示的是结合转铁蛋白的单域抗体VHH显著延长重组卵清蛋白在小鼠血液中的半衰期和增强穿越血脑屏障转运能力。
图2显示的是rhARSA相关重组蛋白的SDS-PAGE图。
图3显示的是rhARSA相关重组蛋白与hTf的结合检测。
图4显示的是FACS检测rhARSA相关重组蛋白与hTf/hTfR1的结合结果。
图5A显示的是VHH与hARSA蛋白融合不影响M6PR结合。图5B显示mARSA-/-成纤维细胞摄取9056VHH-hARSA量是hARSA的5.57倍(10μM),部分可被M6P阻断。图5C显示的是细胞摄取9056VHH-hARSA有时间依赖性。
图6显示的是激光共聚焦显微镜观察mARSA-/-成纤维细胞摄取ARSA重组蛋白后,细胞内定位。
图7A-7B显示的是体外重组ARSA酶比活测定(底物pNCS)。
图8显示的是mARSA-/-成纤维细胞分别加10μM,3μM,0μM hARSA,9056VHH-hARSA和hARSA-9056VHH培养48h后,阿利新蓝/固红染色结果。
图9A-9B显示的是重组ARSA酶体外血浆稳定性结果。
图10显示的是hARSA、hARSA-9056VHH和9056VHH-hARSA在小鼠血液中代谢半衰期。
图11A-11B显示的是hARSA和9056VHH-ARSA在野生C57bl/6小鼠中穿越血脑屏障效率。
图12A-12C显示的是递增剂量下9056VHH-ARSA在野生C57bl/6小鼠中穿越血脑屏障效率。
图13显示的是MLD模型小鼠hARSA-9056VHH静脉注射,30MPK给药后2小时药物分布和细胞器定位。
图14A-14C显示的是食蟹猴中重组hARSA-9056VHH穿越血脑屏障的PK研究结果。
图15显示的是hARSA-9056VHH治疗后,mARSA缺陷小鼠的运动能力评估。
图16A-16F显示的是hARSA-9056VHH治疗后,TLC检测mARSA缺陷小鼠的脑组织中硫酸脑苷脂底物水平。
图17显示的是hARSA-9056VHH治疗后,ABS染色检测mARSA缺陷小鼠的脑组织中硫酸脑苷脂底物水平。
图18显示的是hARSA-9056VHH治疗后,TLC/ABS染色检测mARSA缺陷小鼠的坐骨神经中硫酸脑苷脂底物水平。
图19显示的是hARSA-9056VHH治疗后,mARSA缺陷小鼠肾脏中硫酸脑苷脂底物水平。
以下由特定的具体实施例说明本申请发明的实施方式,熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中,术语“融合蛋白”通常指由两个或更多个蛋白或多肽融合得到的蛋白。融合蛋白可通过重组DNA技术人工制备。例如,编码所述两个或更多个蛋白或多肽的基因或核酸分子可彼此连接而形成融合基因或融合的核酸分子,该融合基因或融合的核酸分子可编码所述融合蛋白。所述融合基因的翻译可以产生单一多肽,其可以具有融合前的所述两个或更多个蛋白或多肽中至少一个、甚至每一个的性质。
在本申请中,术语“转铁蛋白”通常指能与能与多价离子结合和运输的糖蛋白。例如,转铁蛋白可以是一种单链糖蛋白。例如,转铁蛋白可以有至少一个离子结合点。例如,离子结合位点可以与铁离子有不同的亲和力。例如,多价铁离子可以是铁离子、铬离子、锰离子、镉离子或镍离子。例如,转铁蛋白的每个分子可以结合两个三价铁原子。例如,转铁蛋白可以是含铁的Holo-转铁蛋白,或不含铁的apo-转铁蛋白。例如,转铁蛋白可以是小鼠转铁蛋白。例如,小鼠转铁蛋白的氨基酸序列可以是GenBank中规定的。EDL21066.1,AAL34533.1,
或AAL34533.1。例如,转铁蛋白可以是人类转铁蛋白。例如,人转铁蛋白的氨基酸序列可以是GenBank中规定的。AAH59367.1,AAH59367.1,或AAB22049.1。在本申请中,术语“转铁蛋白”可以包含其功能活性片段、同源物、类似物和/或其变体。
在本申请中,术语“转铁蛋白结合蛋白”通常是指包含转铁蛋白结合部分的蛋白质,并且可选择地允许抗原结合部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。例如,本申请所述的转铁蛋白结合蛋白可以包括但不限于抗体、抗原结合片段(Fab、Fab'、F(ab)2、Fv片段、F(ab')2、VHH、scFv、二scFv和/或dAb)、免疫缀合物、多特异性抗体、抗体片段、抗体衍生物、抗体类似物或融合蛋白,只要它们能显示出所需的抗原结合活性。例如,抗原结合蛋白能够与转铁蛋白特异性结合。例如,所述转铁蛋白结合蛋白可以不干扰转铁蛋白和转铁蛋白受体1之间的相互作用。例如,所述转铁蛋白结合蛋白可能不影响Tf/TfR1的结合。例如,转铁蛋白结合蛋白可以保持铁运输的正常生理功能。
在本申请中,术语“抗体”通常是指包含一个或多个基本上由免疫球蛋白基因或免疫球蛋白基因片段编码的多肽的蛋白质。例如,免疫球蛋白基因可以包括κ、λ、α、γ、δ、ε和μ恒定区基因,以及无数的免疫球蛋白可变区基因。例如,轻链可被分类为κ或λ,其可以分别定义免疫球蛋白类型:Igκ和Igλ。重链可被分类为γ、μ、α、δ或ε,其依次分别定义免疫球蛋白类别:IgG、IgM、IgA、IgD和IgE。例如,抗体可具有包含四聚体的结构单元,每个四聚体可由两对相同的多肽链组成,每对具有一条“轻”链(约25kD)和一条“重链”(约50-70kD),每个成员的N末端可以界定约100至110个或更多个氨基酸的可变区,其主要负责抗原识别。例如,术语“轻链可变区(VL)”和“重链可变区(VH)”通常分别指轻链和重链的可变区区域。抗体可作为完整免疫球蛋白存在或作为通过用各种肽酶消化或从头表达产生的许多充分表征的片段存在。在本申请中,所述抗体也可仅包含重链可变区。例如,所述抗体可以为单域抗体。
在本发明中,术语“抗原结合片段”通常是指全长抗体的一个或多个部分,所述部分基本上保持结合抗体所结合的相同抗原(例如,CD38)的能力,能够与全长抗体竞争对抗原的特异性结合。通常参见,Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版,Raven Press,N.Y.(1989),并且将其全文通过引用并入本申请。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合片段。在一些情况下,抗原结合片段包括Fab、Fab'、F(ab')2、(Fab)2、Fd、Fv、dAb和互补决定区(CDR)片段、VHH、单链抗体(例如,scFv)、嵌合抗体、双抗体(diabody)和这样的多肽,其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。可使用本领域技术人员已知的常规技术(例如,重组DNA技术或酶促或化学
断裂法)从给定的抗体获得抗体的抗原结合片段,并且以与对于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。例如,胃蛋白酶可以消化铰链区中二硫键以下的抗体以产生F(ab')2。
在本申请中,术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体(参见Vanlandschoot P.等人,2011,Antiviral Research 92,389-407)。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。
在本申请中,术语“功能活性片段”通常是指具有全长蛋白质或核酸的部分区域,但保留或部分保留全长蛋白质或核酸的生物活性或功能的片段。例如,功能活性片段可以保留或部分保留全长蛋白质结合另一种分子的能力。例如,芳基硫酸酯酶A(ARSA)的功能活性片段,可以保留或部分保留全长ARSA的引起细胞增殖的生物活性功能。
在本申请中,术语“芳基硫酸酯酶A”与“ARSA”可以互换使用,该术语可包含野生型的ARSA,也可包含经修饰的ARSA。例如,所述ARSA可以包含其同源物、类似物、衍生物和/或其功能性变体。例如,所述ARSA可以包含全长ARSA,所述ARSA可以包含ARSA的功能活性片段。在本申请中,所述ARSA可包含人ARSA。
在本申请中,术语“Fab”通常指由VL、VH、CL和CH1结构域组成的抗体片段。
在本申请中,术语“Fab'”通常是指与Fab片段相比在CH1结构域的羧基末端具有几个额外的残基的抗体片段。例如,Fab'可包括来自抗体铰链区的一个或多个半胱氨酸。
在本申请中,术语“F(ab)2”通常是指由半胱氨酸相连接的成对的Fab片段所得到的抗原结合片段。
在本申请中,术语“dAb片段”通常是指由VH结构域组成的抗体片段(Ward等人,Nature341:544-546(1989))。
在本申请中,术语“互补决定区CDR”通常是指轻链可变区(VL)与重链可变区(VH)的3个高变区(HVR),该部位因在空间结构上可与抗原决定簇形成精密的互补,故高变区又称互补性决定区。
在本申请中,术语“Fv片段”通常是指由抗体的单臂的VL和VH结构域组成的抗体片段。
在本申请中,术语“scFv”通常是指是由抗体重链可变区和轻链可变区通过短肽连接子(linker)连接而成的分子,又称为单链抗体。
在本申请中,涉及的蛋白质、多肽和/或氨基酸序列,还应理解为至少包含以下的范围:与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中,所述变体可以为,例如在所述蛋白质和/或所述多肽的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如,所述功能性变体可包含已经通过至少1个,例如1-30个、1-20个或1-10个,又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如,所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60%,70%,80%,90%,或100%的生物学活性(例如抗原结合能力)。例如,所述取代可以为保守取代。
如本申请中所用,术语“序列同源性”通常是指两个或多个多核苷酸序列之间或两个或多个多肽序列之间的序列相似性或可交换性。当使用程序(例如Emboss Needle或BestFit)来测定两个不同氨基酸序列之间的序列同一性、相似性或同源性时,可以使用默认设置,或者可以选择合适的评分矩阵(诸如blosum45或blosum80)来最优化同一性、相似性或同源性得分。在一些实施方案中,同源的多核苷酸是在严格条件下杂交的那些序列,并且相较于那些序列具有至少60%,至少65%,至少70%,至少80%,至少90%,至少95%,至少97%,至少98%,至少99%,甚至100%的序列同一性。当对准具有相当长度的序列时,同源的多肽具有至少80%,或至少90%,或至少95%,或至少97%,或至少98%的序列同一性,或具有至少99%的序列同一性。
通常情况下,在多肽链中,氨基与多肽链中的另一个羧基相连可以使其成为一个链,但是在蛋白质的两个末端,分别剩余没有成肽键的氨基酸残基,分别是携带游离的氨基的多肽链末端和携带羧基的多肽链末端。在本申请中,术语“N端”通常是指氨基酸残基携带游离的氨基的多肽链的末端。在本申请中,术语“C端”通常是指氨基酸残基携带游离的羧基的多肽链的末端。
在本申请中,术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本发明中,术语“载体”通常指的是,可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞,使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说,载体包括:质粒;噬菌体;柯斯质粒;人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件,包括启
动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分,如病毒颗粒、脂质体或蛋白外壳,但不仅仅只有这些物质。
在本申请中,术语“药物组合物”通常是指以允许活性成分的生物学活性有效的形式的制剂,并且其不含有对所述制剂待施用的受试者有不可接受的毒性的另外成分。例如,这些制剂可为无菌的。
在本申请中,术语“药学上可接受”通常是指在合理医学判断的范围内,适宜用于与人和动物的组织接触而无过度的毒性、刺激、过敏反应或者其他问题或并发症,具有合理的收益/风险比的佐剂。例如,药学上可接受的佐剂可以指由管理机构批准(如美国食品药品管理局、中国食品药品管理局或欧洲药品局)或者列于普遍认可的药典中(如美国药典、中国药典或欧洲药典)的用于动物(更特别地用于人)的那些佐剂。
在本申请中,术语“包含”通常是指包括、总括、含有或包涵的含义。在某些情况下,也表示“为”、“由……组成”的含义。
在本申请中,术语“约”通常是指在指定数值以上或以下0.5%-10%的范围内变动,例如在指定数值以上或以下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%、或10%的范围内变动。
发明详述
一方面,本申请提供一种融合蛋白,其包含:转铁蛋白结合蛋白,以及芳香基硫酸酯酶A(Arylsulfatase A,ARSA)或其功能活性片段。例如,所述转铁蛋白结合蛋白可包含能够结合转铁蛋白的多肽、抗体或其抗原结合片段。
在本申请中,所述融合蛋白可具有一种或多种性质。例如,所述融合蛋白能够延长所述ARSA的体内半衰期。例如,所述融合蛋白能够跨越血脑屏障,实现药物的递送。例如,所述融合蛋白能够通过口服递送。例如,所述融合蛋白能够将所述ARSA递送至表达转铁蛋白受体的细胞。
在本申请中,所述转铁蛋白可以为人转铁蛋白。
在本申请中,所述能够结合转铁蛋白的抗体可以包括选自下组的一种或多种:单域抗体、单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在本申请中,所述能够结合转铁蛋白的抗原结合片段可以包括Fab,Fab’,Fv片段,F(ab’)2,F(ab)2,scFv,VHH,di-scFv和/或dAb。
在本申请中,所述转铁蛋白结合蛋白可包含能够结合转铁蛋白的VHH。
例如,所述转铁蛋白结合蛋白可包含VHH中的至少一个CDR,且所述VHH可包含SEQ ID NO:8所示的氨基酸序列。
在本申请中,抗体的CDR又称互补决定区,是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定,如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知,具体可参见,例如,http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构,用不同的编码系统确定出CDR区。使用不同的编码系统,CDR区可能存在差别。在本申请中,所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列;也涵盖其变体,所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个,又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入;也涵盖其同源物,所述同源物可以为与所述CDR的氨基酸序列具有至少约85%(例如,具有至少约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或更高的)序列同源性的氨基酸序列。在某些实施方式中,本申请所述转铁蛋白结合蛋白的CDR可通过Kabat编码系统定义。
例如,所述转铁蛋白结合蛋白可以包含CDR3,且所述CDR3包含SEQ ID NO:3所示的氨基酸序列。
例如,所述转铁蛋白结合蛋白可以包含CDR2,且所述CDR2可包含SEQ ID NO:2所示的氨基酸序列。
例如,所述转铁蛋白结合蛋白可以包含CDR1,且所述CDR1可包含SEQ ID NO:1所示的氨基酸序列。
例如,所述转铁蛋白结合蛋白可以包含CDR1,CDR2和CDR3,且所述CDR1可包含SEQ ID NO:1所示的氨基酸序列,所述CDR2可包含SEQ ID NO:2所示的氨基酸序列,且所述CDR3可包含SEQ ID NO:3所示的氨基酸序列。
例如,所述转铁蛋白结合蛋白可包含VHH的框架区FR1,且所述FR1可包含SEQ ID NO:4所示的氨基酸序列。例如,所述转铁蛋白结合蛋白可包含VHH的框架区FR2,且所述FR2可包含SEQ ID NO:5所示的氨基酸序列。例如,所述转铁蛋白结合蛋白可包含VHH的框架区FR3,且所述FR3可包含SEQ ID NO:6所示的氨基酸序列。例如,所述转铁蛋白结合蛋白可包含VHH的框架区FR4,且所述FR4可包含SEQ ID NO:7所示的氨基酸序列。
在本申请中,所述转铁蛋白结合蛋白可包含能够结合转铁蛋白的VHH,且所述VHH包含SEQ ID NO:8所示的氨基酸序列。
在本申请中,所述融合蛋白中的ARSA或其功能活性片段可以为人ARSA或其功能活性片段。在本申请中,可以对所述ARSA或其功能活性片段进行改造,使其仍保留ARSA的功能和/或活性。
在本申请中,所述融合蛋白中的ARSA或其功能活性片段可包含SEQ ID NO:10所示的氨基酸序列。
本申请所述“ARSA或其功能活性片段”还可包含其变体、截短体、同源物、类似物。例如,所述ARSA或其功能活性片段还可包含与SEQ ID NO:10所示的氨基酸序列具有至少约70%,至少约71%,至少约72%,至少约73%,至少约74%,至少约75%,至少约76%,至少约77%,至少约78%,至少约79%,至少约80%,至少约81%,至少约82%,至少约83%,至少约84%,至少约85%,至少约86%,至少约87%,至少约88%,至少约89%,至少约90%,至少约91%,至少约92%,至少约93%,至少约94%,至少约95%,至少约96%,至少约97%,至少约98%,至少约99%,至少约99.9%%的序列同源性的氨基酸序列。例如,所述ARSA或其功能活性片段可包含与SEQ ID NO:10相比,具有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、25个、30个或更多个氨基酸突变的氨基酸序列。
在本申请中,在所述融合蛋白中,所述转铁蛋白结合蛋白可以和所述ARSA或其功能活性片段直接或间接相连。
在本申请中,在所述融合蛋白中,所述转铁蛋白结合蛋白的N端可以和所述ARSA或其功能活性片段的C端直接或间接相连。
在本申请中,在所述融合蛋白中,所述转铁蛋白结合蛋白的C端可以和所述ARSA或其功能活性片段的N端直接或间接相连。
在本申请中,在所述融合蛋白中,所述转铁蛋白结合蛋白可以和所述ARSA或其功能活性片段通过连接子相连。例如,所述连接子可包含柔性接头。例如,所述连接子可包含肽接头。
在本申请中,在所述融合蛋白中,所述连接子可以包含SEQ ID NO:9所示的氨基酸序列。
在本申请中,所述融合蛋白可包含SEQ ID NO:11所示的氨基酸序列。
在本申请中,所述融合蛋白能够结合转铁蛋白,且能够催化降解芳香基硫酸酯酶A的底物硫酸脑苷脂。
核酸分子、载体、细胞、药物组合物、制备方法
另一方面,本申请提供一个或多个分离的核酸分子,其编码所述的融合蛋白。
本申请所述的核酸分子可以为分离的。例如,其可以是通过以下方法产生或合成的:(i)在体外扩增的,例如通过聚合酶链式反应(PCR)扩增产生的,(ii)通过克隆重组产生的,(iii)纯化的,例如通过酶切和凝胶电泳分级分离,或者(iv)合成的,例如通过化学合成。
重组DNA和分子克隆技术包括由Sambrook,J.,Fritsch,E.F.和Maniatis,T.Molecular Cloning:A Laboratory Manual;Cold Spring Harbor Laboratory Press:Cold Spring Harbor,(1989)(Maniatis)和由T.J.Silhavy,M.L.Bennan和L.W.Enquist,Experiments with Gene Fusions,Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.(1984)以及由Ausubel,F.M.等,Current Protocols in Molecular Biology,pub.by Greene Publishing Assoc.and Wiley-Interscience(1987)描述的那些技术。简而言之,可从基因组DNA片段、cDNA和RNA制备所述核酸,所有这些核酸可直接从细胞中提取或通过各种扩增方法(包括但不限于PCR和RT-PCR)重组产生。
核酸的直接化学合成通常涉及将3'-封闭的和5'-封闭的核苷酸单体依次添加至生长中的核苷酸聚合物链的末端5'-羟基,其中每次添加通过亲核攻击所添加的单体的3'-位上的生长链的末端5'-羟基来实现,所述单体通常是磷衍生物,诸如磷酸三酯、亚磷酰胺等。参见,例如,Matteuci等,Tet.Lett.521:719(1980);属于Caruthers等的美国专利第4,500,707号;和属于Southern等的美国专利第5,436,327号和第5,700,637号;在另一方面,本申请提供了包含本申请的分离的多核苷酸的载体。所述载体可以是任何线性核酸、质粒、噬菌体、粘粒、RNA载体、病毒载体等。病毒载体的非限制性实例可包括逆转录病毒、腺病毒和腺相关病毒。
另一方面,本申请提供一个或多个载体,其包含所述的核酸分子。例如,所述载体可以包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外,所述载体中还可包含其他基因,例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外,所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的,例如,可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中,所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化,但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列,例如TATA盒、加帽序列、CAAT序列等。例如,5’非转录表达控制序列可包含启动子区,启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。所述载体可以包括,例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。
另一方面,本申请提供一种细胞,所述细胞包含所述的融合蛋白,所述的核酸分子,或所述的载体。所述细胞可以是宿主细胞。例如,所述细胞可以包括如下许多细胞类型,如大肠杆菌或枯草菌等原核细胞,如酵母细胞或曲霉菌等真菌细胞,如S2果蝇细胞或Sf9等昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK293细胞或人细胞的动物细胞。
例如,可通过多种已建立的技术将所述载体稳定地或瞬时引入宿主细胞。例如,一种方法涉及氯化钙处理,其中通过钙沉淀引入载体。也可以按照类似方法使用其它盐,例如磷酸钙。另外,可以用电穿孔(即,施加电流以增加细胞对核酸的渗透性)。转化方法的其它实例包括显微注射、DEAE葡聚糖介导的转化和在乙酸锂存在下的热休克。脂质复合物、脂质体和树状聚合物也可用于转染宿主细胞。
另一方面,本申请提供一种制备所述的融合蛋白的方法,其可以包括在使得所述融合蛋白能够表达的条件下培养所述的细胞。例如,可通过使用适当的培养基、适当的温度和培养时间等,这些方法是本领域普通技术人员所了解的。
另一方面,本申请提供一种组合物,所述组合物包含所述的融合蛋白,或所述的核酸分子,以及任选地药学上可接受的载体。
例如,所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中,可按照本领域的常规技术手段将所述组合物与药学上可接受的载体或稀释剂以及任何其他已知的辅剂和赋形剂配制在一起,例如按照Remington:The Science and Practice of Pharmacy,第十九版,Gennaro编辑,Mack Publishing Co.,Easton,PA,1995中公开的技术进行操作。
在本申请中,所述药物组合物可被配制用于口服给药的形式,如片剂,胶囊剂,丸剂,粉剂,缓释制剂,溶液剂,混悬剂。所述药物组合物可以是适合精确剂量单次给药的单位剂量形式。所述药物组合物可以进一步包含常规的药物载体或赋形剂。此外,所述药物组合物可以包括其他药物或药剂,载体,佐剂等。
本申请所述的药物组合物可以包含治疗有效量的所述融合蛋白。所述治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的病症或病症(例如神经退行性疾病)和/或其任何并发症而所需的剂量。所述剂量的具体量/浓度可以根据施用方法和患者需要而变化,并且可以基于例如患者体积,粘度和/或体重等来确定。应当理解的是,基于特定患者,制剂和/或疾病的状况,本领域技术人员(例如,医生或药剂师)可以方便地调整
这些特定剂量。
用途
另一方面,本申请提供了所述的融合蛋白,所述的核酸分子,所述的载体,所述的细胞,和/或所述的药物组合物在制备药物中的用途,所述药物用于预防和/或治疗疾病和/或病症。
另一方面,本申请提供了一种预防和/或治疗疾病和/或病症的方法,其包括向有需要的受试者施用所述的融合蛋白,所述的核酸分子,所述的载体,所述的细胞,和/或所述的药物组合物。
另一方面,本申请提供了所述的融合蛋白,所述的核酸分子,所述的载体,所述的细胞,和/或所述的药物组合物,其用于预防和/或治疗疾病和/或病症。
在本申请中,所述疾病和/或病症可包括ARSA异常引起的疾病和/或病症。
在本申请中,所述疾病和/或病症可包括由硫酸脑苷脂底物沉积导致的神经系统疾病。
在本申请中,所述疾病和/或病症可包括异染性脑白质病(Metachromatic Leukodystrophy,MLD)。
在本申请中,所述疾病和/或病症可包括神经退行性疾病。
在本申请中,所述疾病和/或病症可包括帕金森综合症(Parkinson’s disease)。
另一方面,本申请还提供了一种递送药物穿越血脑屏障的方法,其可包括施用所述的融合蛋白。
另一方面,本申请还提供了一种延长ARSA在体内半衰期的方法,其可包括施用所述的融合蛋白。
不欲被任何理论所限,下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等,而不用于限制本申请发明的范围。
实施例
实施例1转铁蛋白特异性单域抗体VHH的发现
采用人血浆中纯化的人转铁蛋白免疫2-3岁龄亚洲羊驼两只,至特异性血清效价达10000以上(ELISA方法)。取200毫升外周血分离、纯化单核细胞(PBMC),提取mRNA、反转录为cDNA,并用VHH特异性引物扩增编码VHH的核苷酸序列,克隆入噬菌体展示载体建立VHH噬菌体展示文库。根据近100个随机挑选的克隆DNA序列测定结果推算,所建噬菌体展示文库的有效容量约1亿(109)个独立VHH序列。
VHH文库的淘选用液相方法:1-10微克生物素化的人转铁蛋白与1012CFU(克隆形成单
位)的噬菌体在包含1%牛血清白蛋白、0.05%吐温20的磷酸盐缓冲液(PBST-1%BSA)中孵育1小时,加入用PBST-1%BSA预先洗涤、封闭的、链霉亲和素streptavidin偶联的磁珠(Invitrogen,Dynabeads M-280 streptavidin)。室温孵育1小时后,用PBST洗涤磁珠10次。结合磁珠的噬菌体用10微克/毫升的胰酶解离,在大肠杆菌中感染扩增,用于下一轮的淘筛,或挑取单克隆用于ELISA方法挑选与人转铁蛋白特异结合、但不影响转铁蛋白与人转铁蛋白受体结合的VHH序列。得到的多个特异性VHH序列中,SLN9056(其CDR1的氨基酸序列如SEQ ID NO:1所示,CDR2的氨基酸序列如SEQ ID NO:2,CDR3的氨基酸序列如SEQ ID NO:3所示,VHH的氨基酸序列如SEQ ID NO:8所示)表现出更好的人转铁蛋白结合活性(pH7.4,pH6.0)(图1A-B),能显著延长与之融合的重组卵清蛋白(OVA)在小鼠血液中的半衰期(图1C)。小鼠以10mg/kg的剂量静脉注射IV纯化重组蛋白(如VHH-OVA-His),给药前一天开始每天腹腔注射IP注射10mg/kg人转铁蛋白。在给药后的不同时间点取样,20mL PBS灌注后采集大脑,冷冻于液氮中。ELISA定量测定脑组织匀浆中VHH-OVA-His的含量。兔抗OVA多克隆抗体(Sigma C6534-2mL)包被,生物素化兔抗VHH抗体(GeneScript,A02015-200)进行检测。与阴性对照(SLN0066,氨基酸序列为SEQ ID NO:12)相比),结合转铁蛋白的VHH显著提高了脑组织内试验品的水平(对于所选的VHH,注射剂量的平均%ID(injection dose)百分比,给药4小时后为~1.03%至~3.5%)(图1D)。
实施例2重组蛋白的生产
2.1蛋白表达和纯化鉴定
构建重组质粒以生产9056VHH-hARSA-his或hARSA-9056VHH-his蛋白(hARSA由SEQ ID NO:10所示的氨基酸序列组成;9056VHH的CDR1的氨基酸序列如SEQ ID NO:1所示,CDR2的氨基酸序列如SEQ ID NO:2,CDR3的氨基酸序列如SEQ ID NO:3所示,VHH的氨基酸序列如SEQ ID NO:8所示;连接子由SEQ ID NO:9所示的氨基酸序列组成)。测序确认质粒序列后,重组质粒为载体,lipofectamine或PEI为转染试剂由,瞬时转染HEK293F细胞,批量生产重组蛋白。在30ml–100ml的摇瓶培养基中培养5-7天。离心去除细胞,上清液用Ni-NTA琼脂糖纯化蛋白。在未还原(NR)或还原条件(R)下,4-12%梯度的SDS-PAGE凝胶分析纯化的蛋白(图2)。
实施例3重组蛋白的生物学特性
3.1重组蛋白与转铁蛋白结合能力的ELISA检测
96孔板用链霉亲和素(0.1μg/100μl/孔)包被,4℃孵育过夜;PBST洗涤3次后,室温
下用200μl包含1%牛血清白蛋白(BSA)的PBST封闭1小时。用PBST洗涤3次,加入生物素化的人转铁蛋白(1μg/ml,100μl/孔)后室温孵育1小时。用PBST洗涤板3次,加入系列稀释的hARSA或者hARSA-9056VHH蛋白,室温孵育1小时。PBST洗涤3次后向每个孔中加入加入0.125ug/ml生物素标记的山羊抗人Anti-hARSA抗体(R&D,BAF2485)(100μl/孔),室温孵育1小时。PBST洗涤3次,每孔加入100μl链霉亲和素-HRP(Sigma,CAT#S5512-1MG),室温孵育0.5小时后如前洗涤。加入100μl/孔TMB底物溶液后在室温孵育15分钟。加入终止溶液100μl/孔终止反应,酶标仪读取450nm吸光值。结果如图3所示,结果显示hARSA-9056VHH能够与人转铁蛋白结合,hARSA不能与人转铁蛋白结合。
3.2重组蛋白与转铁蛋白结合、不影响细胞膜表面转铁蛋白/转铁蛋白受体复合物结合的FACS检测
稳定表达人转铁蛋白受体1(hTfR1,UniProt P02786)的293F细胞系用于检测重组蛋白与细胞膜表面转铁蛋白/转铁蛋白受体的结合。以每孔0.5×106细胞密度将293F/hTfR1细胞铺入96孔板,4℃,1000rpm离心5分钟,200μl 1×PBS(pH7.4)洗涤后用200ul含2%BSA的PBS(封闭液)重悬并封闭细胞。4℃孵育30分钟后如上洗涤,重悬于包含系列稀释重组蛋白样品的封闭液中,包含或不包含天然人转铁蛋白(Sigma,Cat NO.T3309)。4℃孵育30分钟后依次加入鼠抗VHH(Genscript)和AF647标记的山羊抗小鼠抗体(Abcam,CAT#ab150115)。4℃孵育30分钟后离心、洗涤并重悬于封闭液,流式细胞仪分析细胞与重组蛋白的结合。结果如图4所示,结果显示hARSA-9056VHH能够和人转铁蛋白(hTf)结合,且不影响转铁蛋白与细胞膜表面的人转铁蛋白受体的结合。
3.3重组9056VHH-hARSA蛋白与M6P受体结合能力的ELISA检测
M6P受体(HEK293F细胞重组表达的胞外段M6PR-Fc,0.5μg/孔)包被96孔板,4℃孵育过夜。PBST(磷酸盐缓冲液,0.1%Tween-20,pH7.4)洗涤3次,200μl 1%BSA/PBST室温封闭1小时。PBST洗涤3次,加入系列稀释的rhARSA或者rhARSA-9056VHH蛋白,室温孵育1小时。PBST洗涤板3次,加入0.125ug/ml生物素标记的山羊抗人Anti-hARSA抗体(R&D,BAF2485)(100μl/孔),室温孵育1小时。PBST洗涤3次,每孔加入100μl链霉亲和素-HRP(Sigma,CAT#S5512-1MG),室温孵育1小时。PBST洗涤3次,加入100μl TMB底物溶液,室温孵育15分钟。终止后,酶标仪读取450nm吸光值,Graph Pad 8.0软件分析数据。结果如图5A所示,rhARSA-9056VHH和rhARSA有相似的M6P受体结合能力。
3.4mARSA-/-小鼠皮肤成纤维细胞摄取重组蛋白
mARSA-/-小鼠皮肤成纤维细胞取自C57BL/6J-Arsaem1cyagen(KOCMP-11883-Arsa-B6J)
小鼠,该模型鼠购自赛业(苏州)生物科技有限公司。按常规进行组织块培养,培养基采用高糖DMEM(含4.0mM L-Glutamine,Sodium Pyruvate)(HyClone,SH30243.01),10%胎牛血清(Gibco,10099-141C)。待细胞爬出成片,生长良好,传代备用。
3.4.1剂量依赖细胞摄取实验
1)取生长良好的P1mARSA-/-小鼠皮肤成纤维细胞,接种细胞至96孔板,2×104细胞/孔,培养过夜。
2)分别配制2倍浓度药物的9056VHH-hARSA、hARSA(20μM起始,10倍梯度稀释)和10mM M6P溶液(D-甘露糖-6-磷酸二钠盐水合物(J&K,530168));不加M6P组用空白培养液代替。弃去96孔板中培养液,先加入10mM M6P孵育15分钟,再加入等体积相应各组药物(终浓度10μM起始,10倍梯度稀释),继续培养24小时。
3)收集细胞裂解液:细胞裂解液加如1×蛋白酶抑制剂,混匀备用;细胞弃上清,PBS洗涤3次;每孔加入60μl细胞裂解液,充分裂解细胞后收集至1.5ml EP管中;12000rpm离心,取上清,测定总蛋白浓度,-80度保存待检测。
ELISA检测细胞裂解液中药物含量:山羊抗人Anti-hARSA抗体(R&D,AF2485)0.25μg/ml包被96孔酶联板,PBST(Tween-20,0.1%)洗涤3次后用200μl含1%BSA的PBST室温封闭1小时。PBST洗涤3次后,加入系列稀释检测样品(以系列稀释标准品rhARSA、9056VHH-rhARSA为标准曲线)。室温孵育1小时后用PBST洗涤3次,加入0.125μg/ml生物素标记的山羊抗人Anti-hARSA抗体(R&D,BAF2485),室温孵育1小时。PBST洗涤3次,加入链霉亲和素标记的辣根过氧化物酶(HRP)1:5000(Sigma,CAT#S5512-1MG)并在室温孵育1小时。PBST洗涤3次,加100μl TMB底物溶液孵育15分钟,终止后读取450nm吸光度。根据标准曲线计算各样品中药物浓度,细胞摄取量=样品中药物浓度/总蛋白浓度。如图5B显示mARSA-/-成纤维细胞摄取9056VHH-hARSA量是hARSA的5.57倍(10μM),部分可被M6P阻断。
3.4.2时间依赖细胞摄取实验
1)同3.4.1实验,配制20μM药物,稀释10倍至2μM(终浓度1μM);弃去96孔板中培养液,先加入10mM M6P孵育15min,再加入相应各组药物,继续培养48小时、24小时、6小时、2小时和0小时,收集细胞裂解液,12000rpm离心,取上清,测定总蛋白浓度,-80度保存待检测。
2)同3.4.1实验采用ELISA检测细胞裂解液中药物含量,计算各时间点药物的细胞摄取量=样品中药物浓度/总蛋白浓度。如图5C显示mARSA-/-成纤维细胞摄取9056VHH-rhARSA
具有时间依赖性。
3.5 mARSA-/-小鼠皮肤成纤维细胞摄取重组蛋白细胞内定位
3.5.1药物孵育
同3.4实验,培养mARSA-/-小鼠皮肤成纤维细胞。接种生长状态良好的细胞至加有盖玻片的24孔板中,培养细胞生长至70-80%汇合。弃去旧培养液,加入含2μM 9056VHH-hARSA或hARSA培养液,继续培养24小时。
3.5.2免疫荧光染色和激光共聚焦显微镜观察
1)固定:弃去培养液,预冷PBS洗涤细胞3次;4%PBS pH 7.4多聚甲醛室温固定10分钟。预冷PBS洗涤细胞3次。
2)破膜:含0.1%Triton X-100 PBS室温孵育样品10分钟。预冷PBS洗涤细胞3次。
3)封闭:含1%BSA PBST(PBS+0.05%Tween 20)封闭30分钟。预冷PBS洗涤细胞3次。
4)免疫荧光染色:用1%BSA PBST 1:100稀释一抗山羊抗人ARSA抗体(R&D,AF2486),室温孵育1小时。预冷PBS洗细胞3次。用1%BSA PBST 1:100稀释PE标记二抗驴抗山羊IgG(R&D,F0107)和溶酶体标记抗体CD107a(LAMP-1)(eBio1D4B(1D4B))(Alexa Fluor 488,eBioscienceTM,53-1071-82)共孵育,室温避光1小时。预冷PBS洗涤细胞3次。
5)封片剂Gold Antifade Reagent with DAPI(CST,#8961)封片,4℃避光保存,激光共聚焦显微镜观察、拍片。
结果如图6所示:大量9056VHH-hARSA(A)和hARSA(B)重组蛋白均能被mARSA-
/-小鼠皮肤成纤维细胞内吞,并转运至溶酶体(箭头)。
实施例4重组蛋白的生物学活性检测
4.1体外酶活性检测(pNCs人工底物)6
配制溶液:
1)p-硝基邻苯二酚(p-nitrocatechol,p-NC,TCI,Lot.N5V4L-RT)标准溶液:pNC,1mM溶解于终止液中(MW=155.11,5mg in 32.23mL).
2)终止液:0.2M NaOH(MW=40,0.8g in 100mL)。
3)反应缓冲液Assay Buffer:50mM NaOAc,0.5M NaCl,pH 4.5(NaOAc,NaCl,HOAc调
节pH至4.5).
4)p-硝基邻苯二酚硫酸酯(p-nitrocatechol sulfate,p-NCs,sigma,Lot#MKCN3971)储存液:pNCS,2mM溶于反应缓冲液中(MW=311.35,10mg in 16mL)。
实验步骤:
1)p-NC标准曲线:分别将pNC标准品0,20,40,60,80,100μL加入96孔板中;用终止液调整总体积至100μL/孔。对应pNC含量分别为0,20,40,60,80,100nmol/孔。
2)用反应缓冲液稀释rhARSA或9056VHH-hARSA至200nM(终浓度100nM);反应体系中加或不加hTf。
3)用反应缓冲液稀释pNCS至20mM(终浓度10mM)。
4)分别取75μL 200nM rhARSA或9056VHH-hARSA与75μL 2mM pNCS充分混匀;75μl反应缓冲液和75μl底物混合液作为空白对照;37℃分别反应0,5分钟、10分钟、15分钟和30分钟。
5)加入150μL 0.2M NaOH终止反应。
6)每个反应取200μL至新96孔板,酶标仪读取515nm吸光度值。
7)根据标准曲线,绘制酶活反应时效关系曲线并计算酶比活。
结果如图7所示:CHO K1(7A)表达体系生产的重组酶蛋白均保持良好的酶活;(7B)重组酶与人转铁蛋白结合不影响酶活性。
4.2重组蛋白降解体外培养mARSA-/-小鼠皮肤成纤维细胞内硫酸脑苷脂底物沉积
同实验3.4,体外培养mARSA-/-小鼠皮肤成纤维细胞,待细胞生长状态良好,将mARSA-
/-成纤维细胞接种至24孔板,培养至细胞80-90%汇合;分别加入9056VHH-hARSA 10μM,3μM,0μM作用48小时;弃去培养液后参照试剂盒(阿尔新蓝-核固红染色试剂盒(pH1.0),碧云天,C0153S说明书加入阿利新蓝染液,室温1小时;蒸馏水洗涤3次后,加入固红复染5分钟,自来水洗涤3次;显微镜下观察。结果如图8所示:mARSA-/-成纤维细胞分别加10μM,3μM,0μM 9056VHH-hARSA培养48h后,阿利新蓝/固红染色,3组细胞均能见到胞浆内不同程度湖蓝色点、块状染色,蓝染物质是含硫酸根的硫酸酯代谢产物;10μM组细胞胞浆内蓝染物质为弥漫、细小点状,块状很少;3组细胞胞浆内蓝染物质点、块状大小依次为:10μM组<3μM组<0μM组;3组细胞胞浆内蓝染物质量依次为:10μM组<3μM组=0μM组。9056VHH-hARSA 10μM组在细胞内发挥了一定程度降解底物的作用。
实施例5体外血浆稳定性检测
5.1小鼠血浆稳定性检测
1)样品制备:将标准品蛋白在小鼠血浆中配置成一定的浓度:6μg/ml,并分别在37℃恒温保湿条件下孵育1小时、4小时、8小时、24小时和48小时,然后将样品在液氮中快速冷冻并保存于-80℃下直至使用。不含血浆的蛋白样品作阳性对照。
2)ELISA检测:SLN7134(anti-9056VHH,领诺自制)1μg/ml,100μL/孔包被96孔板,4℃过夜。1%BSA TBST,每孔200μL,37℃封闭1小时。1%BSA TBST稀释蛋白,制作标准曲线,起始浓度200ng/ml,2倍梯度稀释,每孔100μL,室温孵育1小时。1%血浆稀释样品至合适的浓度范围,100倍、200倍、300倍稀释,每孔100ul,室温孵育1小时。1%BSA TBST稀释SLN7165-Biotin(生物素化抗人ARSA抗体,领诺自制),终浓度为0.5μg/ml,每孔加入100μL室温孵育1小时。1%BSA TBST 1:5000稀释SA-HRP(Sigma,CAT#S5512-1MG),每孔加入100μL室温0.5小时。每孔加入100μL TMB,室温避光显色15分钟后每孔加入100μL的硫酸终止反应。酶标仪设置波长450nm,读板,softmax软件分析数据。结果如图9(A)所示:rhARSA-9056VHH-his在小鼠血浆中稳定性良好。
5.2人血浆稳定性检测
1)同5.1实验制备重组蛋白在人血浆溶液中37度孵育样品,并用5.1ELISA方法检测样品中蛋白浓度,softmax软件分析数据。结果如图9(B)所示:rhARSA-9056VHH-his在人血浆中稳定性良好。
实施例6重组蛋白在小鼠中的药代动力学(PK)研究
6.1 PK检测方法的建立:山羊抗人Anti-hARSA抗体(R&D,AF2485)用于包被96孔酶联板,PBST(Tween-20,0.1%)洗涤3次后用200μl 1%BSA PBST室温封闭1小时。PBST洗涤3次后,加入包含1%小鼠血清(作为样品基质)和系列稀释标准品(rhARSA或rhARSA-9056VHH)样品100μl/孔。室温孵育1小时后PBST洗涤3次,加入生物素标记的山羊抗人Anti-hARSA抗体(R&D,BAF2485),室温孵育1小时。PBST洗涤3次后加入链霉亲和素标记的辣根过氧化物酶(HRP)(Sigma,CAT#S5512-1MG)并在室温孵育30分钟。PBST洗涤3次,加100μl TMB底物溶液孵育15分钟,加入终止溶液后读取450nm吸光度。图10显示PK方法的验证结果,用三个标准品浓度(高、中、低)验证的结果表明,此方法的精确性(CV%<20%)和准确度(RE%+/-25%)均符合样品检测要求。
6.2单一剂量给药的PK研究:PK研究在6-8周龄雄性C57BL/6小鼠中进行。根据动物体重分组,于给药前一天及研究期间的每天腹腔注射10mg/kg的人转铁蛋白。rhARSA、
rhARSA-9056VHH和9056VHH-rhARSA的单次尾静脉给药剂量为3MPK。分别在给药后2、4、8、24、48小时后采集血样分离血清,用6.1PK方法做血清中的药物定量分析,用PKsovler软件计算PK参数。图10显示的结果表明,在3MPK剂量下,rhARSA-9056VHH血液半衰期(5.29小时)、9056VHH-rhARSA血液半衰期(1.50小时)比rhARSA(血液半衰期小于0.76小时)有明显提高。通过9056VHH结合转铁蛋白显著延长rhARSA的血液半衰期。
6.3单一剂量给药的穿越血脑屏障PK研究:PK研究在6-8周龄雄性C57BL/6小鼠中进行。根据动物体重分组,于给药前一天及研究期间的每天腹腔注射10mg/Kg的人转铁蛋白。rhARSA-9056VHH尾静脉给药剂量为10MPK。分别在给药后4、24、48小时,采集血样分离血清,采集脑脊液;20ml PBS灌流后采集脑组织。用6.1PK方法分别检测血清、脑脊液和脑组织匀浆中的药物含量。Graph Pad 8.0软件分析数据,计算穿越血脑屏障效率(注射剂量百分比%ID=脑组织药物浓度(ng/mg组织)/10mg/kg×100%)。结果如图12A-12C所示,图11A显示hARSA血药浓度均值大约45ng/ml(4h),9056VHH-hARSA血药浓度大约5000ng/ml(4h),~400ng/ml(24h),~18ng/ml(48h),明显高于hARSA组。图11B显示在脑组织匀浆中均检测不到hARSA-his,9056VHH-hARSA在脑组织匀浆中的浓度为4.6~10.8pg/mg脑组织(4h),0.7~4.2pg/mg脑组织(24h),穿越血脑屏障效率远高于hARSA组。
6.4递增剂量给药的穿越血脑屏障PK研究:实验方法同5.3,rhARSA-9056VHH尾静脉给药剂量分别为45nmol/kg,150nmol/kg,450nmol/kg。用6.1PK方法分别检测血清、脑脊液和脑组织匀浆中的药物含量。Graph Pad 8.0软件分析数据,计算穿越血脑屏障效率(注射剂量百分比%ID=脑组织药物浓度(nmol/mg组织)/注射剂量nmol/kg×100%)。结果如图12A-12C所示:其中图12A为45nmol/kg,150nmol/kg,450nmol/kg三个剂量下9056VHH-hARSA血药浓度;图12B为9056VHH-hARSA在脑组织匀浆中的含量,给药后4小时,45nmol/kg-4h(3MPK);450nmol/kg-4h(10MPK);450nmol/kg-4h(30MPK)各组的平均注射剂量百分比(%ID)分别是:0.503%,0.25%,0.15%;图12C显示9056VHH-hARSA在CSF中的含量较低。
6.5 MLD小鼠递增剂量给药后脑组织药物分布研究:实验方法同5.3,rhARSA-9056VHH尾静脉给药剂量分别为150nmol/kg,450nmol/kg。给药后2小时,安乐死小鼠,采集脑组织,4%多聚甲醛固定。按常规制备脑组织石蜡切片,5μm矢状切,(委托上海睿宝和生物有限公司完成)。hARSA/CD31/Lamp1免疫荧光染色:按常规脱蜡、水化、PBS洗涤3次。封闭:含1%BSA PBST(PBS+0.05%Tween 20)封闭30分钟。PBS洗涤3次。免疫荧光染色:用1%BSA PBST 1:100稀释一抗山羊抗人ARSA抗体(R&D,AF2486)或兔抗小鼠CD31抗体(Abcam,
ab281583),室温孵育1小时。预冷PBS洗3次。用1%BSA PBST 1:100稀释PE标记二抗驴抗山羊IgG(R&D,F0107)和溶酶体标记抗体CD107a(LAMP-1)(eBio1D4B(1D4B))(Alexa Fluor 488,eBioscienceTM,53-1071-82)共孵育,室温避光1小时。预冷PBS洗涤3次。封片剂Gold Antifade Reagent with DAPI(CST,#8961)封片,4℃避光保存,激光共聚焦显微镜观察、拍片。结果如图13所示:图13A.药物可以穿越血脑屏障进入脑组织(anti-hARSA),血管内皮细胞(anti-CD31);同时也有药物在血管内皮细胞中;图13B.进入脑细胞的药物(anti-hARSA,)与溶酶体膜蛋白(anti-Lamp1)共定位。
6.6 PET/CT检测食蟹猴体内124I标记单一剂量给药的穿越血脑屏障PK:
6.6.1动物体内实验、样品采集
每组1只食蟹猴(雄性),静脉输注10MPK或30MPK 124I-供试品,即刻开始进行脑部PET/CT动态扫描2h0-2h),随后选取7个时间点:给药后6h、10h、24h、48h、72h、96h、144h进行PET/CT静态扫描。
6.6.2数据分析:计算124I-供试品入脑效率(Kp=脑(放射剂量)/血液(放射剂量)比值。结果如图14A-14C所示:图14A.脑组织药物浓度-时间曲线;图14B.左心室药物浓度-时间曲线;图14C.脑组织药物浓度/左心室药物浓度百分比:给药后20min~30min,药物浓度脑/血浆比有明显增加,hARSA-9056VHH可在脑组织积聚滞留;10MPK组在96小时达峰,脑/血浆比%为53.3%;30MPK组在72小时达峰,脑/血浆比%为23.95%。
实施例7重组蛋白在mARSA缺陷小鼠MLD模型中的药效学研究
7.1 mARSA缺陷小鼠:mARSA基因(GenBank:Gene ID 11883)缺陷小鼠基因型为C57BL/6J-Arsaem1cyagen(KOCMP-11883-Arsa-B6J),经PCR和测序验证。
7.2 rhARSA-9056VHH的体内药效学研究:
7.2.1动物分组:挑选出约10-12月龄、健康状态良好的mARSA-/-纯合子小鼠;按性别/年龄进行匹配;称量并记录小鼠体重;小鼠编号,并填写笼卡信息。hTf 10mg/kg腹腔注射,每日一次,以维持小鼠体内一定的hTf水平;
7.2.2给药:给药频率:BIW×4或BIW×8次;给药剂量:10MPK或30MPK,给药前一天药物与hTf按摩尔比1:1预混4度过夜;给药当天早上称重,计算给药体积;尾静脉注射给药,并记录给药时间。
每日记录:称量并记录小鼠体重;观察并记录小鼠状态、活动量等。
7.2.3小鼠行为学评估:平衡木法
1)训练期:第1天,釆用平衡木,使小鼠通过方形平衡木进入小暗盒。每只小鼠训练3
次。第2天重复第1天相同的步骤。
2)测试期:第3天为测试阶段,使小鼠通过方形平衡木进入小暗盒,全程录像,记录通过平衡木时间。
3)比较各组小鼠通过平衡木的时间。结果如图15所示:30MPK,BIW×8组给药4周后,小鼠通过平衡木时间比给药前明显减少,是给药前的83.53%,P<0.001(Dunnett’s multiple comparisons)。
7.2.4样品采集:第32天(末次给药后8天),安乐死小鼠,收集小鼠尿液0.2ml,(安乐死前4h);-20℃保存待测。小鼠眼眶窦静脉丛穿刺采集全血,入血清分离管,静置10min后,3000g离心5min,收集上清入1.5ml离心管,-80℃保存。同时采集组织脏器肾脏,-80℃保存;脑(包括嗅球、小脑、大脑、脑干和部分脊髓)矢状切,左脑、左肾液氮速冻,用于TLC(分小脑、大脑、脑干和部分脊髓)、ELISA检测;右脑、右肾使用4%PFA固定,用于阿利新蓝染色和免疫荧光染色);以及外周神经组织-坐骨神经,4%PFA固定,用于阿利新蓝染色。
7.2.5样品检测:
7.2.5.1脑组织中硫酸脑苷脂底物检测:
1)薄层色谱分析sulfatide/GalCer比值:称取各组小鼠大脑100mg,小脑20mg,脑干延髓20mg,按质量体积比,分别加入1mL或200μl TBS pH7.4和钢珠进行匀浆,功率60HZ 60s;补液至2mL匀浆液用低温离心13000rpm,2h;弃上清,组织碎片沉淀用于脂质萃取。沉淀中加入5mL CH3Cl/CH3OH(2:1),60℃萃取,4h;离心,转移溶剂上清,沉淀再用5mLC/M(1:1),60℃萃取,4h;合并两次的提取物(粗提),混匀,取一半进行后续的操作,留一半作为对照;取约5mL提取液,旋干,可见白色贴壁物质,用5mL MeOH充分溶解。加入250μL 4N NaOH,在37℃孵育2hr,然后用40μL 100%乙酸中止。蒸干溶剂,加入1mL MeOH溶解。加入1mL 0.3M乙酸铵溶液,将所有的混合物上样。脱盐:约1mL ODS填料(MeOH预先活化)填柱(日本YMC,AAG12S50),用6mL混合溶剂:C/M/0.1M KCl 6:96:94平衡柱子。将混合物样品加入柱中,先用6mL H2O洗涤脱盐,再用1mL MeOH和6mL C/M(1:1)洗脱,洗脱过程中有白色浑浊出现,随后逐渐消失。浓缩:旋蒸仪(上海博楚仪器设备有限公司,LOOYE)蒸干含有脂类分子的洗脱液,最后用500μL的C/M 2:1充分溶解,用于TLC分析。上载一定体积的样品至硅胶板,sulfatide(Matreya LLC;Cat.NO.:1049-50mg;Lot No.:24534),GalCer(Matreya LLC;Cat.NO.:1066-10mg;Lot No.:23332)为标准品展开时间约10分钟。展开剂:氯仿:甲醇:水65:25:4(v/v/v)(需要提前
配好,使展缸内部蒸汽饱和)。显色:将展开好的硅胶板浸泡在显色液(10%CuSO4in 10%磷酸溶液)中,约3min,取出,用吹风机吹干,置于烘箱中130℃烘10min,有黑色斑点或条带出现。凝胶成像仪扫描,图像灰度分析,计算各组小鼠脑组织sulfatide/GalCer比值,mean+SD,统计分析治疗组与未治疗组小鼠大脑、小脑和脑干组织中sulfatide/GalCer比值的差异。如图16所示:mARSA-/-小鼠静脉注射给药(BIW)治疗4周,显著降低了大脑(图16A,图16B)、小脑(图16C,图D)和脑干(图16E,图16F)中sulfatide底物积聚,并且表现出剂量相关性(p值分别为0.027,0.026和0.059,One-way ANOVA)。
2)阿利新蓝染色:按常规制备脑组织冰冻切片,10μm矢状切(委托上海睿宝和生物有限公司完成);配制阿利新蓝染色缓冲液(0.025M sodium acetate,pH 5.7,含0.3M MgCl2,1%PFA)、0.05%阿利新蓝染色液(阿利新蓝8GX,sigma,A9186-10G)溶于染色缓冲液中。脑组织冰冻切片室温溶解后4%多聚甲醛(PFA)固定1分钟;染色缓冲液充分洗涤后,0.05%阿利新蓝染色液室温孵育1小时;染色缓冲液充分洗涤后,固红复染2分钟;自来水充分洗涤后,甘油封片;显微镜下观察拍照。结果如图17:mARSA-/-小鼠静脉注射给药(BIW)治疗4周,10MPK(B)和30MPK(C)组大脑(B1,C1,D)、小脑(B3,C3,E)和脑干(B2,C2,F)中底物sulfatide积聚数量无明显差别,平均灰度密度均降低,并且表现出剂量相关性。
7.2.5.2坐骨神经硫酸脑苷脂底物检测:同上实验检测坐骨神经中硫酸脑苷脂底物方法,通过阿利新蓝染色和薄层色谱分析sulfatide/Cholesterol比值,分析治疗组与未治疗组小鼠硫酸脑苷脂底物沉积情况。结果如图18所示:A.TLC结果显示mARSA-/-小鼠静脉注射给药(BIW)治疗4周,10MPK和30MPK组坐骨神经底物sulfatide积聚均明显降低,并且表现出剂量相关性,P值均小于0.0001(One-way ANOVA);B.ABS染色结果与TLC结果一致,10MPK和30MPK组底物sulfatide积聚均明显降低,并且表现出剂量相关性,P值分别为0.033,0.032(One-way ANOVA)。
7.2.5.3肾脏硫酸脑苷脂底物检测:同上实验检测脑组织中硫酸脑苷脂底物方法,通过和薄层色谱分析Sulfatide/Galcer比值,分析治疗组与未治疗组小鼠肾脏肾脏硫酸脑苷脂底物沉积情况。结果如图19所示:30MPK,BIW×4组给药2周后,硫酸脑苷脂底物水平比为给药组降低,但无统计学差异;30MPK,BIW×8组给药4周后,硫酸脑苷脂底物水平显著降低,P=0.0169(One-way ANOVA)。
Claims (40)
- 融合蛋白,其包含:转铁蛋白结合蛋白,以及芳香基硫酸酯酶A(Arylsulfatase A,ARSA)或其功能活性片段,所述转铁蛋白结合蛋白包含能够结合转铁蛋白的多肽、抗体或其抗原结合片段。
- 根据权利要求1所述的融合蛋白,其具有下述性质中的一种或多种:1)能够延长所述ARSA的体内半衰期;2)能够跨越血脑屏障;3)能够通过口服递送;以及4)能够将所述ARSA递送至表达转铁蛋白受体的细胞。
- 根据权利要求1-2中任一项所述的融合蛋白,其中所述转铁蛋白为人转铁蛋白。
- 根据权利要求1-3中任一项所述的融合蛋白,其中所述抗体选自下组中的一种或多种:单域抗体、单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
- 根据权利要求1-4中任一项所述的融合蛋白,其中所述抗原结合片段包括Fab,Fab’,Fv片段,F(ab’)2,F(ab)2,scFv,VHH,di-scFv和/或dAb。
- 根据权利要求1-5中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白为VHH。
- 根据权利要求求1-6中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白包含CDR3,且所述CDR3包含SEQ ID NO:3所示的氨基酸序列。
- 根据权利要求1-7中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白包含CDR2,且所述CDR2包含SEQ ID NO:2所示的氨基酸序列。
- 根据权利要求1-8中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白包含CDR1,且所述CDR1包含SEQ ID NO:1所示的氨基酸序列。
- 根据权利要求1-9中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白包含VHH,且所述VHH包含SEQ ID NO:8所示的氨基酸序列。
- 根据权利要求1-10中任一项所述的融合蛋白,其中所述ARSA为人ARSA。
- 根据权利要求1-11中任一项所述的融合蛋白,其中所述ARSA或其功能活性片段包含ARSA或其功能活性片段的变体。
- 根据权利要求1-12中任一项所述的融合蛋白,其中所述ARSA或其功能活性片段包含SEQ ID NO:10所示的氨基酸序列。
- 根据权利要求1-13中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白和所述ARSA或其功能活性片段直接或间接相连。
- 根据权利要求1-14中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白的N端和所述ARSA或其功能活性片段的C端直接或间接相连。
- 根据权利要求1-14中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白的C端和所述生ARSA或其功能活性片段的N端直接或间接相连。
- 根据权利要求1-16中任一项所述的融合蛋白,其中所述转铁蛋白结合蛋白和所述ARSA或其功能活性片段通过连接子相连。
- 根据权利要求17所述的融合蛋白,其中所述连接子包含肽接头。
- 根据权利要求17-18中任一项所述的融合蛋白,其中所述连接子包含SEQ ID NO:9所示的氨基酸序列。
- 根据权利要求1-19中任一项所述的融合蛋白,其包含SEQ ID NO:11所示的氨基酸序列。
- 根据权利要求1-20中任一项所述的融合蛋白,其能够结合转铁蛋白,且能够催化降解芳香基硫酸酯酶A的底物硫酸脑苷脂。
- 一种或多种分离的核酸分子,其编码权利要求1-21中任一项所述的融合蛋白。
- 载体,其包含权利要求22所述的核酸分子。
- 细胞,其包含权利要求22所述的核酸分子,和/或权利要求23所述的载体。
- 药物组合物,其包含权利要求1-21中任一项所述的融合蛋白,权利要求22所述的核酸分子,权利要求23所述的载体或权利要求24所述的细胞,以及任选地药学上可接受的载体。
- 制备权利要求1-21中任一项所述的融合蛋白的方法,其包括在所述融合蛋白表达的条件下,培养权利要求24所述的细胞。
- 权利要求1-21中任一项所述的融合蛋白,权利要求22所述的核酸分子,权利要求23所述的载体,权利要求24所述的细胞,和/或权利要求25所述的药物组合物在制备药物中的用途,所述药物用于预防和/或治疗疾病和/或病症。
- 根据权利要求27所述的用途,其中所述疾病和/或病症包括ARSA异常引起的疾病和/或病症。
- 根据权利要求28所述的用途,其中所述疾病和/或病症包括由硫酸脑苷脂底物沉积导致的神经系统疾病。
- 根据权利要求27-28中任一项所述的用途,其中所述疾病和/或病症包括异染性脑白质病(Metachromatic Leukodystrophy,MLD)。
- 根据权利要求27-30中任一项所述的用途,其中所述疾病和/或病症包括神经退行性疾病。
- 根据权利要求27-31中任一项所述的用途,其中所述疾病和/或病症包括帕金森综合症。
- 一种预防和/或治疗疾病和/或病症的方法,其包括向有需要的受试者施用权利要求1-21中任一项所述的融合蛋白,权利要求22所述的核酸分子,权利要求23所述的载体,权利要求24所述的细胞,和/或权利要求25所述的药物组合物。
- 根据权利要求30所述的方法,其中所述疾病和/或病症包括ARSA异常引起的疾病和/或病症。
- 根据权利要求33-34中任一项所述的方法,其中所述疾病和/或病症包括由硫酸脑苷脂底物沉积导致的神经系统疾病。
- 根据权利要求33-35中任一项所述的方法,其中所述疾病和/或病症包括异染性脑白质病。
- 根据权利要求33-36中任一项所述的方法,其中所述疾病和/或病症包括神经退行性疾病。
- 根据权利要求33-37中任一项所述的方法,其中所述疾病和/或病症包括帕金森综合症。
- 一种延长ARSA在体内半衰期的方法,其包括施用权利要求1-21中任一项所述的融合蛋白。
- 一种穿越血脑屏障递送ARSA入脑的方法,其包括施用权利要求1-21中任一项所述的融合蛋白。
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