WO2023142109A1 - 一种长效重组人生长激素及其应用 - Google Patents

一种长效重组人生长激素及其应用 Download PDF

Info

Publication number
WO2023142109A1
WO2023142109A1 PCT/CN2022/075176 CN2022075176W WO2023142109A1 WO 2023142109 A1 WO2023142109 A1 WO 2023142109A1 CN 2022075176 W CN2022075176 W CN 2022075176W WO 2023142109 A1 WO2023142109 A1 WO 2023142109A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
transferrin
growth hormone
binding
protein according
Prior art date
Application number
PCT/CN2022/075176
Other languages
English (en)
French (fr)
Inventor
韩照中
潘红芽
彭孟凡
Original Assignee
领诺(上海)医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 领诺(上海)医药科技有限公司 filed Critical 领诺(上海)医药科技有限公司
Priority to PCT/CN2022/075176 priority Critical patent/WO2023142109A1/zh
Publication of WO2023142109A1 publication Critical patent/WO2023142109A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This application relates to the field of biomedicine, in particular to a fusion protein comprising transferrin binding protein and growth hormone, and its use.
  • the present application provides a fusion protein, which comprises: a transferrin-binding protein, and growth hormone or a functionally active fragment thereof, and the transferrin-binding protein comprises a polypeptide capable of binding transferrin, an antibody or an antigen-binding fragment thereof.
  • the fusion protein described in this application realizes the prolongation of the half-life and the maximum bioavailability of the protein based on the process of combining transferrin and transferrin receptor and circulating inside and outside the cell.
  • the fusion protein is produced by prokaryotic Escherichia coli, the purity can reach more than 95% through SEC detection, the protein production is stable, and the molecular weight is as small as 37KD.
  • the EC50 of rhGH is 0.6nM, while the EC50 of VRS-317 is 6.8nM, which is about 10 times lower, and the biological activity of the fusion protein rhGH-VHH is maintained more Good, with small molecular weight and low immunogenicity.
  • the half-life of rhGH-VHH fusion protein administered at 150nmol/kg in mice is 11.5 hours, while the sogroya once-weekly formulation of Novanordisk, which is currently on the market, is 50nmol/kg in rats. The half-life is 5.3 hours.
  • Pfizer MOD-4023 which was submitted for BLA application in January 2021, once a week, has a half-life of 3.7 hours in rats administered at 75nmol/kg.
  • the rhGH-VHH fusion protein of this application has a pharmacokinetic In order to realize the long-acting dosage form, it also exhibits better characteristics.
  • the application provides a fusion protein, which comprises: transferrin binding protein, and growth hormone or its functionally active fragment, the transferrin binding protein comprises a polypeptide, antibody or antigen capable of binding transferrin Combine fragments.
  • the fusion protein has one or more of the following properties:
  • the transferrin is human transferrin.
  • the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragments include Fab, Fab', Fv fragments, F(ab') 2 , F(ab) 2 , scFv, VHH, di-scFv and/or dAb.
  • the transferrin binding protein in the fusion protein is VHH.
  • the transferrin binding protein in the fusion protein comprises CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the transferrin binding protein CDR2 in the fusion protein, and the CDR2 comprises the amino acid sequence shown in SEQ ID NO:2.
  • the transferrin binding protein CDR1 in the fusion protein, and the CDR1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the transferrin binding protein in the fusion protein contains VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.
  • the growth hormone or a functionally active fragment thereof in the fusion protein is human growth hormone or a functionally active fragment thereof.
  • the growth hormone or a functionally active fragment thereof in the fusion protein comprises the amino acid sequence shown in SEQ ID NO:10.
  • the transferrin binding protein in the fusion protein, is directly or indirectly linked to the growth hormone or a functionally active fragment thereof.
  • the N-terminal of the transferrin-binding protein is directly or indirectly linked to the C-terminal of the growth hormone or its functionally active fragment.
  • the C-terminus of the transferrin-binding protein is directly or indirectly linked to the N-terminus of the growth hormone or its functionally active fragment.
  • the transferrin-binding protein and the growth hormone or a functionally active fragment thereof are connected through a linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:9.
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the fusion protein is capable of binding transferrin and is capable of binding and/or activating growth hormone receptor.
  • the application provides one or more isolated nucleic acid molecules encoding the fusion proteins described herein.
  • 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, which comprises the fusion protein, the nucleic acid molecule, the carrier or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application provides the use of the fusion protein, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition in the preparation of a medicine, and the medicine is used for the prevention and/or treatment of diseases and/or conditions.
  • the diseases and/or conditions include diseases and/or conditions caused by growth hormone abnormalities.
  • the disease and/or condition comprises growth hormone deficiency.
  • the present application provides a method for preventing and/or treating diseases and/or conditions, which comprises administering the fusion protein, the nucleic acid molecule, the carrier, to a subject in need, The cells, and/or the pharmaceutical composition.
  • the diseases and/or conditions include diseases and/or conditions caused by growth hormone abnormalities.
  • the disease and/or condition comprises growth hormone deficiency.
  • the present application also provides a method for prolonging the half-life of growth hormone in vivo, which comprises administering the fusion protein.
  • Figures 1A-1C show the binding ability of transferrin-binding single domain antibody VHH to hTf (A) pH7.4; (B) pH6.0. VHH well combined with hTf significantly prolongs the half-life of recombinant ovalbumin OVA in mouse blood;
  • Figures 2A-2B show the SDS-PAGE (A) and SEC-HPLC (B) analysis of rhGH.
  • the theoretical molecular weight of rhGH is 22kD.
  • 3A-3B show the SDS-PAGE (A) and SEC-HPLC (B) analysis of rhGH-VHH.
  • the theoretical molecular weight of rhGH-VHH is 37 kD.
  • 4A-4B show the analysis of the binding ability of rhGH-VHH to human growth hormone receptor (hGHR) and human transferrin (hTf).
  • 5A-5B show the analysis of the binding activity of rhGH-VHH to transferrin (hTf)/transferrin receptor (hTfR1) complex or human growth hormone receptor (hGHR) on the cell membrane surface.
  • Figure 6 shows the establishment and identification of 293F-GAS-GHR stably transfected cell lines.
  • Figure 7 shows the biological activity assay of rhGH-VHH.
  • Figure 8 shows the development and validation of the pharmacokinetic ELISA method.
  • Figure 9 shows the comparison of metabolic half-lives of rhGH and rhGH-VHH in mouse blood.
  • Figures 10A-10B show the pharmacokinetic studies of rhGH-VHH in mice.
  • Figures 11A-11D show the growth phenotypes of GHRH-deficient mice.
  • 12A-12D show the therapeutic effect of rhGH in GHRH gene-deficient mice.
  • 13A-13D show the therapeutic effect of rhGH-VHH in GHRH gene-deficient mice.
  • fusion protein generally refers to a protein obtained by fusion of two or more proteins or polypeptides. Fusion proteins can be artificially produced by recombinant DNA techniques. For example, the 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 a fused nucleic acid molecule which can encode the fusion protein. Translation of the fusion gene may result in a single polypeptide, which may have the properties of at least one, or even each, of the two or more proteins or polypeptides prior to fusion.
  • transferrin generally refers to a glycoprotein capable of binding and transporting multivalent ions.
  • transferrin can be a single chain glycoprotein.
  • transferrin can have at least one ion binding site.
  • ion binding sites can have different affinities for iron ions.
  • the multivalent iron ion may be iron ion, chromium ion, manganese ion, cadmium ion or nickel ion thereof.
  • each molecule of transferrin can bind two atoms of ferric iron.
  • the transferrin can be Holo-transferrin that contains iron, or apo-transferrin that does not contain iron.
  • 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 can be specified in GenBank. AAH59367.1, AAH59367.1, or AAB22049.1.
  • the term "transferrin" may encompass functionally active fragments, homologues, analogs and/or variants thereof.
  • the term "transferrin binding protein” generally refers to a protein comprising a transferrin binding moiety, and optionally a scaffold or backbone moiety that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding protein to the antigen.
  • the 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, so long as they exhibit the desired antigen-binding activity.
  • an antigen binding protein is capable of specifically binding 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 the normal physiological function of iron transport.
  • antibody generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments.
  • immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes.
  • light chains can be classified as kappa or lambda, which can define the immunoglobulin classes: 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 may have structural units comprising tetramers, each tetramer may be composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD ), the N-terminus of each member may define a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • VL light chain variable region
  • VH heavy chain variable region
  • the term "antigen-binding fragment” generally refers to one or more parts of a full-length antibody that substantially retains the ability to bind to the same antigen (for example, CD38) to which the antibody binds, and is capable of interacting with the full-length antibody. Compete for specific binding to antigen. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is hereby incorporated by reference in its entirety. Can be obtained by recombinant DNA techniques or by intact antibodies Enzymatic or chemical cleavage of the antigen-binding fragments to generate antigen-binding fragments.
  • antigen-binding fragments include Fab, Fab', F(ab')2, (Fab)2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments , VHH, single-chain antibody (for example, scFv), chimeric antibody, diabody (diabody) and such polypeptides, it comprises at least a part of the antibody that is enough to confer polypeptide-specific antigen-binding ability.
  • Antigen-binding fragments of antibodies are obtained from a given antibody by conventional techniques (eg, 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.
  • 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). VHHs may also be referred to as Nanobodies (Nb) and/or Single Domain Antibodies.
  • 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.
  • a functionally active fragment of growth hormone may retain or partially retain the biologically active function of full-length growth hormone that causes cell proliferation.
  • growth hormone may include wild-type growth hormone, and may also include modified growth hormone.
  • the growth hormone may comprise homologues, analogs, derivatives and/or functional variants thereof.
  • the somatotropin can comprise full-length somatotropin, and the somatotropin can comprise a functionally active fragment of somatotropin.
  • 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 the Fab fragment.
  • a Fab' may include one or more cysteines from the antibody hinge region.
  • F(ab)2 generally refers to an antigen-binding fragment derived from a pair 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)).
  • 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), which can be It forms a precise complementarity with the antigenic determinant, so the hypervariable region is also called complementarity-determining region.
  • Fv fragment generally refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • scFv generally refers to a molecule formed by linking the heavy chain variable region and the light chain variable region of an antibody through a short peptide linker (linker), also known as a single-chain antibody.
  • sequence homology generally refers to sequence similarity or exchangeability 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 (such as blosum45 or blosum80) can be selected to optimize identity, similarity or homology scores.
  • 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.
  • 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 when aligned over sequences of substantial length. identity.
  • the amino group is connected to another carboxyl group in the polypeptide chain to make it a chain, but at the two ends of the protein, the remaining amino acid residues that do not form a peptide bond are respectively carrying free
  • N-terminal generally refers to the end of a polypeptide chain whose amino acid residues bear a free amino group.
  • the term “C-terminal” generally refers to the end of the polypeptide chain whose amino acid residue bears 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 tool into which a polynucleotide encoding a 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 carried by it can be expressed in the host cell.
  • vectors include: plasmids; bacteriophages; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal virus etc.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also
  • the term "pharmaceutical composition” generally refers to a preparation in a form that allows the biological activity of the active ingredients 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 .
  • these preparations can be sterile.
  • a pharmaceutically acceptable adjuvant generally means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications, Adjuvants with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable adjuvant can be one approved by a regulatory agency (such as the US Food and Drug Administration, China Food and Drug Administration, or the European Medicines Agency) or listed in a generally recognized pharmacopoeia (such as the US Pharmacopoeia, Chinese Pharmacopoeia, or European Pharmacopoeia). Pharmacopoeias) for use in animals, more particularly in humans.
  • the present application provides a fusion protein comprising: transferrin binding protein, and growth hormone or a functionally active fragment thereof.
  • the transferrin binding protein may comprise a polypeptide, antibody or antigen-binding fragment thereof capable of binding transferrin.
  • the fusion protein can have one or more properties.
  • the fusion protein is capable of extending the in vivo half-life of the growth hormone.
  • the fusion protein enables drug delivery across the blood-brain barrier.
  • the fusion protein can be delivered orally.
  • the fusion protein is capable of delivering the growth hormone to cells expressing the transferrin receptor.
  • the transferrin may be human transferrin.
  • the antibody capable of binding transferrin may include one or more selected from the group consisting of monoclonal antibody, chimeric antibody, humanized antibody and fully human antibody.
  • 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 can comprise at least one CDR in a VHH, and the VHH can comprise the amino acid sequence shown in SEQ ID NO:8.
  • the CDR of an antibody is also called complementarity determining region, which is a part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat/Chothia, etc. in combination. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • 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 or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) 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 comprise CDR1, CDR2 and CDR3, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, and 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.
  • the transferrin binding protein can comprise the framework region FR1 of VHH, and the FR1 can comprise the amino acid sequence shown in SEQ ID NO:4.
  • the transferrin binding protein can comprise the framework region FR2 of VHH, and the FR2 can comprise the amino acid sequence shown in SEQ ID NO:5.
  • the transferrin binding protein can comprise the framework region FR3 of VHH, and the FR3 can comprise the amino acid sequence shown in SEQ ID NO:6.
  • the transferrin binding protein can comprise the framework region FR4 of VHH, and the FR4 can 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.
  • the growth hormone or its functionally active fragment in the fusion protein may be human growth hormone or its functionally active fragment.
  • the growth hormone or its functionally active fragments can be modified so that it still retains the function and/or activity of the growth hormone.
  • the growth hormone or its functionally active fragment in the fusion protein may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the transferrin binding protein in the fusion protein, may be directly or indirectly linked to the growth hormone or its functionally active fragment.
  • the N-terminal of the transferrin-binding protein may be directly or indirectly linked to the C-terminal of the growth hormone or its functionally active fragment.
  • the C-terminal of the transferrin-binding protein may be directly or indirectly linked to the N-terminal of the growth hormone or its functionally active fragment.
  • the transferrin-binding protein in the fusion protein, can be connected with the growth hormone or its functionally active fragment through a linker.
  • the linker can 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 can bind transferrin, and can bind and/or activate growth hormone receptor.
  • the present application provides one or more isolated nucleic acid molecules encoding said fusion protein.
  • nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.
  • amplified in vitro such as by polymerase chain reaction (PCR) amplification
  • recombinantly produced by cloning e.g., recombinantly produced by cloning
  • purified iv
  • 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 produced recombinantly 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 nucleomonomers to the terminal 5'-hydroxyl groups of growing nucleotide polymer chains, with each addition being assisted by nucleophilic This is achieved by attacking the terminal 5'-hydroxyl of the growing chain at the 3'-position of the added monomer, usually a phosphorus derivative, such as phosphotriesters, phosphoramidites, and the like. 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 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 can include retroviruses, adenoviruses, and adeno-associated viruses.
  • the present application provides one or more vectors comprising the nucleic acid molecule.
  • the vector can comprise one or more nucleic acid molecules described herein.
  • Each vector may contain one or more such nucleic acid molecules.
  • 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 also contain expression control elements that permit proper 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, and the like.
  • the expression control sequences are regulatable elements.
  • the specific structure of the expression control sequence may 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 box, plus 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.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, 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 can be a host cell.
  • the cells may include a number of 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 fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or animal cells of human cells.
  • the vectors can be stably or transiently introduced into host cells by a variety of established techniques.
  • one method involves calcium chloride treatment, wherein the carrier is introduced by calcium precipitation.
  • Other salts such as calcium phosphate, can also be used in a similar manner.
  • electroporation ie, application of an 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 cell under the condition that the fusion protein can be expressed. For example, by using appropriate medium, appropriate temperature and incubation 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 nonionic Surfactant etc.
  • composition can be formulated together 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 Remington: The Science and Practice of Pharmacy, Nineteenth Edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995 by the technique disclosed.
  • the pharmaceutical composition can be formulated for oral administration, such as tablets, capsules, pills, powders, sustained-release preparations, solutions, and suspensions.
  • the pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosages.
  • the pharmaceutical composition may further include 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 contain a therapeutically effective amount of the fusion protein.
  • the therapeutically effective amount is that required to be able to prevent and/or treat (at least in part) the condition or disorder (e.g. growth hormone deficiency) and/or any complication thereof in a subject suffering from or at risk of developing dose.
  • the specific amount/concentration of the dosage may vary according to the method of administration and the needs of the patient, and may be determined based on, for example, patient volume, viscosity and/or body weight, and the like. It is to be understood that these specific dosages may be readily adjusted by those skilled in the art (eg, physician or pharmacist) based on the particular patient, formulation and/or disease conditions.
  • the present application provides the use of the fusion protein, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition in the preparation of a medicine, and the medicine is used 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 conditions, which comprises administering the fusion protein, the nucleic acid molecule, the carrier, to a subject in need, The cells, and/or the pharmaceutical composition.
  • the application provides the fusion protein, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition, which are used for preventing and/or treating diseases and / or illness.
  • the diseases and/or conditions may include diseases and/or conditions caused by growth hormone abnormalities.
  • the disease and/or condition may include growth hormone deficiency.
  • the present application also provides a method for prolonging the half-life of growth hormone in vivo, which comprises administering the fusion protein.
  • SLN9056 (SEQ ID NO:8) showed better stability than SLN0066 (SEQ ID NO:12) which did not bind hTF, at pH 7.4 (Fig. 1A) and pH 6.0 (Fig. Under the condition of 1B), it has a strong binding ability with hTf, and can significantly prolong the half-life of recombinant ovalbumin (OVA) fused with it in mouse blood (Fig. 1C).
  • OVA ovalbumin
  • SLN9056 is used to prepare the rhGH-VHH fusion protein in the following steps, and the amino acid sequence of the rhGH-VHH is shown in SEQ ID NO:11.
  • Both mammalian cell system and E. coli system were used for the production of recombinant protein rhGH and rhGH-VHH.
  • HEK293F cells are used for transient expression transfection of recombinant expression plasmids, and cell culture supernatants are used for purification of recombinant proteins.
  • the expression vector plasmid (pET28a) comprising DNA encoding rhGH and rhGH-VHH nucleotide sequences was transformed into Escherichia coli BL21 (DE3) strain.
  • Escherichia coli carrying the expression plasmid was cultured at 37°C, and 1 mM isopropyl ⁇ -D-1 thiogalactopyranoside (IPTG) (GENERAL-Reagent, 367-93-1) was used to induce the expression of the target protein.
  • IPTG isopropyl ⁇ -D-1 thiogalactopyranoside
  • Bacterial inclusion bodies were collected and dissolved in 2M urea and 25ml 100mM Tris buffer (pH 12.5) for the purification of recombinant proteins.
  • Ion-exchange column chromatography and molecular sieve chromatography were used for the purification of recombinant proteins, respectively.
  • the protein solution was first adjusted to the 25mM Tris buffer system (solution A) containing 5mM EDTA, 0.4M urea, and pH 8.5, and then loaded into the pre-prepared column with the same solution A at a flow rate of 5ml/min.
  • solution A 25mM Tris buffer system
  • the protein solution was loaded onto a Sephadex G75 chromatography column (GE Healthcare) pre-equilibrated with solution C at a flow rate of 1 ml/min, and the recombinant protein was eluted with solution C.
  • the purified recombinant protein was identified by SDS-PAGE and SEC-HPLC (Waters, CAT#E2695) respectively, the results are shown in Figure 2 (recombinant human growth hormone, rhGH) and Figure 3 (recombinant human growth hormone-VHH fusion protein, rhGH -VHH).
  • ELISA, FACS, and luciferase reporter gene methods were used to test the recombinant protein's ability to bind to transferrin, transferrin/transferrin receptor complex, human growth hormone receptor, and activate human growth hormone receptor.
  • a 96-well plate was coated with growth hormone receptor (GHR-Fc recombinantly expressed in HEK293 cells, 0.5 ⁇ g/well) and incubated overnight at 4°C, with PBST (phosphate buffered saline, 0.1% Tween-20, pH7.4) After washing 3 times, it was blocked with 200 ⁇ l 1% BSA/PBST for 1 hour at room temperature. Wash 3 times with PBST, add serially diluted rhGH or rhGH-VHH protein, and incubate at room temperature for 1 hour.
  • PBST phosphate buffered saline, 0.1% Tween-20, pH7.4
  • PBST 100 ⁇ l/well
  • biotinylated goat anti-human growth hormone antibody R&D, CAT#AF1201
  • R&D biotinylated goat anti-human growth hormone antibody
  • CAT#AF1201 biotinylated goat anti-human growth hormone antibody
  • 100 ⁇ l of PBST containing streptavidin-HRP Sigma, CAT#S5512-1MG
  • 100 ⁇ l TMB substrate solution was added after washing as before and incubated at room temperature for 15 minutes.
  • After adding 100 ⁇ l/well of the stop solution read the absorbance at 450 nm with a microplate reader. The results are shown in Figure 4A.
  • Figure 4A shows the analysis of the binding ability of the recombinant protein to the human growth hormone receptor (hGHR), comparing the in vitro binding activity of different concentrations of rhGH and rhGH-VHH with the purified hGHR-Fc recombinant protein , the results showed that rhGH-VHH and rhGH have similar human growth hormone receptor binding ability.
  • hGHR human growth hormone receptor
  • a 96-well plate was coated with streptavidin (0.1 ⁇ g/well) and incubated overnight at 4°C, washed 3 times with PBST, and blocked with 200 ⁇ l of PBST containing 1% bovine serum albumin (BSA) at room temperature 1 Hour. Wash 3 times with PBST, add biotinylated human transferrin (1 ⁇ g/ml, 100 ⁇ l/well) and incubate at room temperature for 1 hour. Wash the plate 3 times with PBST, add serially diluted rhGH or rhGH-VHH protein, and incubate at room temperature for 1 hour.
  • streptavidin 0.1 ⁇ g/well
  • PBST bovine serum albumin
  • PBST containing mouse anti-human growth hormone monoclonal antibody (Abcam, CAT#Ab9821) was added to each well, incubated at room temperature for 1 hour and washed as before.
  • PBST containing HRP-conjugated goat anti-mouse Fc monoclonal antibody (Abcam, CAT#Ab9871) was added to each well, incubated at room temperature for 1 hour and washed as before.
  • Figure 4B shows the analysis of the binding ability of the recombinant protein to human transferrin (hTf).
  • hTf human transferrin
  • the 293F cell line stably expressing human transferrin receptor 1 (hTfR1, UniProt P02786) was used to detect the binding of the recombinant protein to the transferrin/transferrin receptor complex on the cell membrane surface.
  • FBS fetal Bovine serum
  • FIG. 5A shows the binding activity of the recombinant protein to the transferrin (hTf)/transferrin receptor (hTfR1) complex on the cell membrane surface.
  • the binding curves of different concentrations of rhGH and rhGH-VHH and HEK293F cells stably expressing hTfR1 (293F/hTfR1 stable cell line) were compared.
  • the results showed that rhGH-VHH can bind to human transferrin receptor.
  • the 293F cell line stably expressing human growth hormone receptor (hGHR, UniProt P10912) was used to detect the binding of recombinant protein to human growth hormone receptor on the cell membrane surface.
  • FBS fetal Bovine serum
  • Figure 5B shows the binding activity of the recombinant protein to the human growth hormone receptor (hGHR) on the cell membrane surface.
  • the binding curves of different concentrations of rhGH and rhGH-VHH and HEK293F cells stably expressing hGHR (293F/hGHR stable cell line) were compared, and the results showed that rhGH-VHH and rhGH have the same binding properties as human growth hormone receptor expressed on the cell membrane surface. binding ability.
  • the biological activity test of the recombinant protein was carried out in a cell line stably expressing the luciferase reporter gene.
  • the pSLN-0134(pNL2.2-SG-MinP)/GAS vector the luciferase gene (pNL2.2-SG - MinP, Promega, N104A) stable HEK293T cell line.
  • the 293T/GAS-luciferase stable cell line co-expressed the human growth hormone receptor (hGHR) and the plasmid containing the puromycin resistance gene, and the 293T/GAS/hGHR stable cell bank was obtained by puromycin pressure selection for 20 consecutive days , the monoclonal cell line clone#15 was obtained by limiting dilution method, and the results are shown in Figure 6.
  • Figure 6 shows the expression analysis of human growth hormone receptor (hGHR) in the stable cell bank.
  • hGHR antibody (Abcam, CAT#AF11210) and donkey anti-goat IgG-PE (Abcam, CAT#AF109) detection, using flow cytometry scanning analysis; and the stability of monoclonal stable cell lines and the response to human growth hormone
  • reactivity test the reactivity of cells of different generations (original 0, 12, 16 and 20 generations) to recombinant human growth hormone were compared.
  • DMEM medium Gibco, Cat NO.C11995500BT
  • 10% FBS and 2ug/ml puromycin was used for the cultivation of 293T/GAS/hGHR cell lines.
  • Cells with a density of about 75% were digested with 0.25% trypsin at 37°C for 5 minutes, the culture medium was diluted and blown to form a single cell suspension, and the DMEM medium containing 1% FBS was washed 3 times.
  • the washed cells were resuspended in DMEM medium containing 1% FBS and placed in a 96-well cell culture plate with a cell density of 20,000 cells/50 ⁇ l, and serially diluted samples (rhGH or rhGH-VHH ).
  • NanoGLO reagent Promega, N1110
  • rhGH and rhGH-VHH have similar biological activities, or the fusion of VHH has no effect on the biological activity of GH.
  • Recombinant proteins derived from mammalian cell expression system and Escherichia coli expression system have similar biological activities no matter at the protein level (ELISA) or at the cellular level (FACS and luciferase reporter gene test).
  • Mouse anti-human growth hormone monoclonal antibody (Abcam, CAT#Ab9821) was used to coat the 96-well plate, washed 3 times with PBST (Tween-20, 0.1%) and blocked with 200 ⁇ l of PBST containing 1% BSA for 1 hour at room temperature. After washing 3 times with PBST, PBST containing 1% mouse serum (as sample matrix) and serially diluted standards (rhGH or rhGH-VHH) was added. After incubation at room temperature for 1 hour, wash with PBST three times, add biotin-labeled goat anti-human growth hormone polyclonal antibody (R&D, CAT#AF1201), and incubate at room temperature for 1 hour.
  • HRP horseradish peroxidase
  • PK studies were performed in 6-8 week old male C57BL/6 mice. Animals were grouped according to body weight, and 10 mg/kg of human transferrin was subcutaneously injected the day before dosing and every day during the study.
  • the single subcutaneous doses of rhGH and rhGH-VHH are three doses of 50, 150, and 500 nmol/kg, respectively. 2, 4, 8, 24, 32 and 48 hours after administration (rhGH group), or 2, 6, 24, 30, 48, 54, 72, 78 and 96 hours after administration (rhGH-VHH group) Blood samples were used to separate serum, and the above-mentioned PK method was used for quantitative analysis of drugs in serum, and PK parameters were calculated by PKsovler software. The results shown in Fig.
  • FIG. 10A show that at a dose of 150 nmol/kg, the blood half-life of rhGH-VHH (11.5 hours) is significantly improved compared with rhGH (blood half-life less than 1 hour). In different dose groups, it was found that the blood half-life of rhGH was significantly prolonged by VHH binding to transferrin ( FIG. 10A ).
  • PK studies were performed in 6-8 week old male C57BL/6 mice. Animals were grouped according to body weight, and 10 mg/kg of human transferrin was subcutaneously injected the day before dosing and every day during the study.
  • the subcutaneous doses of rhGH-VHH are all three doses of 50, 150, and 500 nmol/kg, administered once every 48 hours (two days), and administered continuously for three times.
  • the GHRH gene (GenBank: M31658.1)-deficient mouse model was purchased from Nanjing Jicui Yaokang, and the genotype of the mice was verified by PCR and sequencing. Using a calibrated electronic balance and electronic digital calipers, measure the body weight (BW), body length (nose-anus distance, N-A), femur and tibia lengths of mice aged 1-5 weeks. The results showed that there was no significant difference between heterozygous mice and wild-type mice, but homozygous GHRH-deficient mice had significantly decreased body length, body weight, femur and tibia length (Figure 11). Homozygous mice were used for pharmacodynamic studies of recombinant proteins.
  • mice One-week-old male and female (5 each) homozygous GHRH gene-deficient mice were subcutaneously administered 150nmo/kg or 500nmol/kg per day, respectively, and treated continuously for 4 weeks, and the animal body weight (BW) and body length ( Nasal-anal distance, N-A). The mice were euthanized at the fifth week (day 44) after treatment, and serum was collected for the determination of IGF-1 levels. The results in Figure 12 show that daily administration can significantly improve the growth status of GHRH gene-deficient mice.
  • One-week-old male and female (5 each) homozygous GHRH gene-deficient mice were subcutaneously administered 150nmo/kg (twice a week), 500nmol/kg (twice a week) or 1500nmol/kg (once a week), respectively, The administration was continued for 4 weeks, and the body weight (BW) and body length (nose-anus distance, N-A) of the animals were measured twice a week. The mice were euthanized 5 weeks after treatment (day 44), and the serum was collected for the determination of IGF-1 levels.
  • the results in Figure 13 show that in GHRH gene-deficient mice, rhGH-VHH treatment group administered twice a week or weekly administration of 1500nmol/kg can achieve the same therapeutic effect as rhGH treatment group daily administration of 150nmol/kg.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Endocrinology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Diabetes (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本申请涉及一种融合蛋白，其包含：转铁蛋白结合蛋白，以及生长激素或其功能活性片段，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。本申请还提供了所述融合蛋白的制备方法及用途。

Description

一种长效重组人生长激素及其应用 技术领域
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生长激素的融合蛋白，及其用途。
背景技术
目前，国内市场上生长激素分为短效型和长效型，从短效到长效的迭代实现更好的治疗效果的同时带来也是患者依从性的提高。2014年金赛药业推出自主研发的全球首支聚乙二醇长效生长激素，得以将矮小症患儿的生长激素注射频率由每天一次拉至每周一次，从一年365天次注射用药到52次的改变。至今，国内尚未有第二款长效生长激素获批上市。但是，由于长效剂型的价格相对较高，并未达到“后来居上”的效果，短效水针剂仍然是国内的主流方案，据相关数据统计，2020年第一至第三季度，国内样本医院生长激素销售中，普通水针占73％，粉针占26％，长效水针占1％。同时据Frost&Sullivan报告显示，我国重组生长激素销售总额59亿元，其中长效生长激素销售额在6亿左右。因此，目前长效剂型并未能走进大多数的生长激素缺乏的患儿使用，中国仍有近300万的儿童患有生长激素缺乏症。
因此，亟需开发更高效且成本更低的长效制剂，以提高患者依从性和生活质量并降低医护成本。
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，以及生长激素或其功能活性片段，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。本申请所述的融合蛋白基于转铁蛋白和转铁蛋白受体结合并在细胞内外循环的过程实现半衰期的延长以及蛋白的最大生物利用度。所述融合蛋白用原核大肠杆菌生产，通过SEC检测纯度可达到95％以上，蛋白生产稳定，分子量小为37KD。相较于长春金赛增用PEG耦联成本更低，蛋白更稳定。相较于天境生物TJ101杂合FC融合rhGH蛋白，分子量103KD，用哺乳细胞生产，大肠杆菌生产成本更低。而且，于大肠杆菌生产的rhGH-VHH融合蛋白在细胞模型上体外活性(EC50 2nM)与天然rhGH(EC50 1nM)相比，EC50下降约为1倍。相较于Versartis的VRS-317(XTEN1-rhGH-XTEN2)分子量119kD，rhGH的EC50为0.6nM，而VRS-317EC50为6.8nM，下降约10倍，融合蛋白rhGH-VHH的生物学活性保持的更好，且分子量小免疫原性低。另外从药代动力学研究分析，rhGH-VHH融合蛋白，给药150nmol/kg在小鼠体内的 半衰期为11.5小时，而目前已上市的Novanordisk的sogroya每周一次制剂的在大鼠的50nmol/kg半衰期为5.3小时，于2021年1月提交BLA申请的Pfizer MOD-4023每周一次制剂分子在大鼠75nmol/kg给药半衰期为3.7小时，本申请的rhGH-VHH融合蛋白在药代动力学方面为实现长效剂型也展现了较优的特性。
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，以及生长激素或其功能活性片段，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)能够延长所述生长激素的体内半衰期；
(2)能够跨越血脑屏障；
(3)能够通过口服递送；以及
(4)能够将所述生长激素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，所述融合蛋白中的转铁蛋白结合蛋白为VHH。
在某些实施方式中，所述融合蛋白中的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，所述融合蛋白中的转铁蛋白结合蛋白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，所述融合蛋白中的转铁蛋白结合蛋白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，所述融合蛋白中的转铁蛋白结合蛋白含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，所述融合蛋白中的生长激素或其功能活性片段为人生长激素或其功能活性片段。
在某些实施方式中，所述融合蛋白中的生长激素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生长激素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端和所述生长激素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端和所述生长激素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生长激素或其功能活性片段通过连接子相连。
在某些实施方式中，在所述融合蛋白中，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:11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，且能够结合和/或活化生长激素受体。
另一方面，本申请提供了一种或多种分离的核酸分子，其编码本申请所述的融合蛋白。
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/或病症包括生长激素异常引起的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括生长激素缺乏症。
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。
在某些实施方式中，所述疾病和/或病症包括生长激素异常引起的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括生长激素缺乏症。
另一方面，本申请还提供了一种延长生长激素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。
附图说明
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1A-1C显示的是结合转铁蛋白的单域抗体VHH与hTf的结合能力(A)pH7.4；(B)pH6.0。与hTf结合良好的VHH显著延长重组卵清蛋白OVA在小鼠血液中的半衰期；
图2A-2B显示的是rhGH的SDS-PAGE(A)与SEC-HPLC(B)分析。rhGH的理论分子量为22kD。
图3A-3B显示的是rhGH-VHH的SDS-PAGE(A)与SEC-HPLC(B)分析。rhGH-VHH的理论分子量为37kD。
图4A-4B显示的是rhGH-VHH与人生长激素受体(hGHR)和人转铁蛋白(hTf)的结合能力分析。
图5A-5B显示的是rhGH-VHH与细胞膜表面转铁蛋白(hTf)/转铁蛋白受体(hTfR1)复合物或人生长激素受体(hGHR)结合活性的分析。
图6显示的是293F-GAS-GHR稳转细胞株的建立与鉴定。
图7显示的是rhGH-VHH的生物学活性测定。
图8显示的是药代动力学ELISA方法的开发与验证。
图9显示的是rhGH和rhGH-VHH在小鼠血液中代谢半衰期对比。
图10A-10B显示的是rhGH-VHH在在小鼠体内的药代动力学研究。
图11A-11D显示的是GHRH缺陷小鼠的生长表型。
图12A-12D显示的是rhGH在GHRH基因缺陷小鼠中的治疗效果。
图13A-13D显示的是rhGH-VHH在GHRH基因缺陷小鼠中的治疗效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员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、镉离子或其镍离子。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，生长激素的功能活性片段，可以保留或部分保留全长生长激素的引起细胞增殖的生物活性功能。
在本申请中，术语“生长激素”可以包含野生型的生长激素，也可以包含经修饰的生长激素。例如，所述生长激素可以包含其同源物、类似物、衍生物和/或其功能性变体。例如，所述生长激素可以包含全长生长激素，所述生长激素可以包含生长激素的功能活性片段。
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,Nature 341:544-546(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。
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。
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，以及生长激素或其功能活性片段。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，所述融合蛋白能够具有一种或多种性质。例如，所述融合蛋白能够延长所述生长激素的体内半衰期。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生长激素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，所述融合蛋白中的生长激素或其功能活性片段可以为人生长激素或其功能活性片段。在本申请中，可以对所述生长激素或其功能活性片段进行改造，使其仍保留生长激素的功能和/或活性。
在本申请中，所述融合蛋白中的生长激素或其功能活性片段可包含SEQ ID NO:10所示 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生长激素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端可以和所述生长激素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端可以和所述生长激素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生长激素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，且能够结合和/或活化生长激素受体。
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(例如生长激素缺乏症)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/或病症可包括生长激素异常引起的疾病和/或病症。
在本申请中，所述疾病和/或病症可包括生长激素缺乏症。
另一方面，本申请还提供了一种延长生长激素在体内半衰期的方法，其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发明的各个技术方案，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
1.1噬菌体展示文库的建立
用自人血浆中纯化的人转铁蛋白免疫2-3岁龄亚洲驼两只，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(10 8)个独立VHH序列。
1.2 VHH文库的淘选
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、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，用于在大肠杆菌中感染扩整，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。
1.3重组卵清蛋白(VHH-OVA)
特异性VHH序列中，SLN9056(SEQ ID NO:8)与不结合hTF的SLN0066(SEQ ID NO:12)相比，表现出更好的稳定性，在pH 7.4(图1A)和pH 6.0(图1B)条件下，与hTf均具有较强的结合能力，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)。
将SLN9056用于制备下述步骤中的rhGH-VHH融合蛋白，所述rhGH-VHH的氨基酸序列如SEQ ID NO:11所示。
实施例2融合蛋白的生产
2.1蛋白表达
哺乳动物细胞系统和大肠杆菌系统均用于重组蛋白rhGH和rhGH-VHH的生产。哺乳动物细胞系统中，HEK293F细胞用于重组表达质粒的瞬时表达转染，细胞培养上清用于重组蛋白的纯化。在大肠杆菌表达系统中，包含编码rhGH和rhGH-VHH核苷酸序列DNA的表达 载体质粒(pET28a)转化到埃希氏大肠杆菌BL21(DE3)菌株中。携带表达质粒的大肠杆菌在37℃下培养，1mM异丙基β-D-1硫代吡喃半乳糖苷(IPTG)(GENERAL-Reagent，367-93-1)用于诱导表达目的蛋白。收集细菌包涵体，溶解于2M尿素、25ml 100mM Tris缓冲液(pH 12.5)中，用于重组蛋白的纯化。
2.2蛋白纯化
离子交换柱层析和分子筛层析分别用于重组蛋白的纯化。离子交换柱层析过程中，首先将蛋白质溶液调整至包含5mM EDTA、0.4M尿素、pH 8.5的25mM Tris缓冲液体系(溶液A)中，然后以5ml/min的流速加载到用同样溶液A预先平衡的DEAE-Sepharose阴离子交换柱(GE Healthcare)。用5个柱体积的溶液A洗涤层析柱，然后用3个柱体积的溶液B(在溶液A中加入0.1M的NaCl)洗涤，使用0.1–0.5M NaCl梯度的溶液A以5ml/min的流速洗脱重组蛋白，在5mM EDTA、pH 8.5的25mM Tris缓冲液(溶液C)中透析去除尿素，5000rpm离心去除沉淀后，上清经过超滤浓缩(Millipore，10KDa)后用于凝胶过滤柱层析。凝胶过滤纯化过程中，蛋白质溶液以1ml/min的流速装载到预先用溶液C平衡的Sephadex G75层析柱(GE Healthcare)，用溶液C洗脱重组蛋白。
2.3重组蛋白的纯度鉴定
纯化的重组蛋白分别用SDS-PAGE和SEC-HPLC(Waters，CAT#E2695)进行纯度鉴定，结果如图2(重组人生长激素，rhGH)和图3(重组人生长激素-VHH融合蛋白，rhGH-VHH)。
实施例3重组人生长激素的生物化学特性分析
重组蛋白分别用ELISA、FACS、luciferase报道基因方法测试与转铁蛋白、转铁蛋白/转铁蛋白受体复合物、人生长激素受体的结合能力以及活化人生长激素受体的能力。
3.1重组蛋白与生长激素受体结合能力的ELISA检测
96孔板用生长激素受体(HEK293细胞重组表达的GHR-Fc，0.5μg/孔)包被并在4℃孵育过夜，用PBST(磷酸盐缓冲液，0.1％Tween-20，pH7.4)洗涤3次后，在室温用200μl1％BSA/PBST封闭1小时。用PBST洗涤3次，加入系列稀释的rhGH或者rhGH-VHH蛋白，室温孵育1小时。用PBST洗涤板3次后，加入包含生物素化的山羊抗人生长激素抗体(R&D，CAT#AF1201)的PBST(100μl/孔)并在室温孵育1小时。用PBST洗涤3次后向每个孔中加入100μl包含链霉亲和素-HRP(Sigma，CAT#S5512-1MG)的PBST并在室温孵育1小时。然后如前洗涤后加入100μl TMB底物溶液并在室温孵育15分钟。加入100μl/孔终止溶液液后，用酶标仪读取450nm吸光值。结果如图4A所示，图4A显示的是重组蛋白与人人生长激素受体(hGHR)的结合能力分析，比较了不同浓度rhGH和rhGH-VHH与纯化 的hGHR-Fc重组蛋白的体外结合活性，结果显示，rhGH-VHH与rhGH有相似的人生长激素受体结合能力。
3.2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/孔)包被并在4℃孵育过夜，用PBST洗涤3次后，在室温下用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rhGH或rhGH-VHH蛋白，室温孵育1小时。PBST洗涤3次后向每个孔中加入包含鼠抗人生长激素的单抗(Abcam，CAT#Ab9821)的PBST，室温下孵育1小时后如前洗涤。PBST洗涤3次后向每个孔中加入包含HRP偶联的羊抗鼠Fc单克隆抗体(Abcam，CAT#Ab9871)的PBST，室温下孵育1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100μl/孔终止溶液后，用酶标仪读取450nm吸光值。结果如图4B所示，图4B显示的是重组蛋白与人转铁蛋白(hTf)的结合能力分析。比较了不同浓度rhGH和rhGH-VHH与纯化的天然hTf蛋白的体外结合活性，结果显示，rhGH-VHH能够与人转铁蛋白结合，rhGH不能与人转铁蛋白结合。
3.3重组蛋白与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/转铁蛋白受体复合物的结合。以每孔0.5x10 6的密度将293F/hTfR1细胞铺入96孔U型底血清板，4℃条件下1000rpm离心5分钟，用200ul 1×PBS(pH7.4)洗涤后用200ul含2％胎牛血清(FBS，Gibco，Cat NO.10099-141C)的PBS(封闭液)重悬并封闭细胞。4℃孵育30min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，包含或不包含天然转铁蛋白(Sigma，Cat NO.T3309)。4℃孵育30分钟后依次加入鼠抗VHH的抗体(Genscript)和AF647标记的羊抗鼠抗体(Abcam，CAT#ab150115)。4℃孵育30min后离心、洗涤并重悬于封闭液后，用流式细胞仪分析细胞与重组蛋白的结合。结果如图5A所示，图5A显示的是重组蛋白与细胞膜表面转铁蛋白(hTf)/转铁蛋白受体(hTfR1)复合物的结合活性。比较了不同浓度rhGH和rhGH-VHH与稳定表达hTfR1的HEK293F细胞(293F/hTfR1稳定细胞系)的结合曲线，结果显示，rhGH-VHH能够通过人转铁蛋白的介导，结合于细胞膜表面的人转铁蛋白受体。
3.4重组蛋白与细胞膜表面人生长激素受体结合的FACS检测
稳定表达人生长激素受体(hGHR,UniProt P10912)的293F细胞系用于检测重组蛋白与细胞膜表面人生长激素受体的结合。以每孔0.5x10 6的密度将293F/hGHR细胞铺入96孔U 型底血清板，4℃条件下1000rpm离心5分钟，用200ul 1×PBS(pH7.4)洗涤后用200ul含2％胎牛血清(FBS，Gibco，Cat NO.10099-141C)的PBS(封闭液)重悬并封闭细胞。4℃孵育30min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，包含或不包含天然转铁蛋白(Sigma，Cat NO.T3309)。4℃孵育30分钟后依次加入鼠抗人生长激素的抗体(Abcam，CAT#Ab9821)和荧光标记的羊抗鼠抗体(Abcam，CAT#ab150115)。4℃孵育30min后离心、洗涤并重悬于封闭液后，用流式细胞仪分析细胞与重组蛋白的结合。结果如图5B所示，图5B显示的是重组蛋白与细胞膜表面人生长激素受体(hGHR)结合活性。比较了不同浓度rhGH和rhGH-VHH与稳定表达hGHR的HEK293F细胞(293F/hGHR稳定细胞系)的结合曲线，结果显示，rhGH-VHH和rhGH具备同样与表达于细胞膜表面的人生长激素受体的结合能力。
实施例4重组蛋白的生物学活性分析
重组蛋白的生物学活性测试在稳定表达荧光素酶(luciferase)报道基因的细胞株中进行。
4.1 293T/GAS-luminescence/hGHR的稳转细胞株的建立
首先构建pSLN-0134(pNL2.2-SG-MinP)/GAS载体：基因组中插入干扰素激活位点(IFN-γactivated site，GAS)调控元件的荧光素酶(luciferase)基因(pNL2.2-SG-MinP，Promega,N104A)的稳定HEK293T细胞株。在293T/GAS-luciferase稳定细胞株共表达人生长激素受体(hGHR)和包含嘌呤霉素抗性基因的质粒，连续20天的嘌呤霉素压力筛选获得293T/GAS/hGHR的稳转细胞库，有限稀释法得到单克隆细胞株clone#15，结果如图6所示，图6显示的是稳转细胞库中人生长激素受体(hGHR)的表达分析，具体为hGHR阳性细胞利用山羊抗hGHR抗体(Abcam,CAT#AF11210)和驴抗山羊IgG-PE(Abcam,CAT#AF109)检测，利用流式细胞仪扫描分析；以及单克隆稳转细胞株的稳定性与对人生长激素处理的反应性测试，分别比较了不同代次(原始0、12代、16代、20代)细胞对重组人生长激素的反应性。
4.2重组蛋白的生物学功能检测
包含10％FBS和2ug/ml嘌呤霉素的DMEM培养基(Gibco，Cat NO.C11995500BT)用于293T/GAS/hGHR细胞株的培养。密度约75％的细胞经0.25％胰蛋白酶37度消化5分钟，培养基稀释、吹打成为单细胞悬液，含1％FBS的DMEM培养基洗涤3次。洗涤后的细胞重悬于含1％FBS的DMEM培养基中并置于96孔细胞培养板中，细胞密度为20000个细胞/50μl，加入培养基为介质的系列稀释样品(rhGH或rhGH-VHH)。在37℃、5％CO2培养4小时后，加入NanoGLO试剂(Promega，N1110)定量检测luciferase表达。结果如图7所示，rhGH与rhGH-VHH具有相类似的生物学活性，或者VHH的融合对GH的生物学活性没有影响。来源于哺乳动物细胞表达系统和埃希氏大肠杆菌表达系统的重组蛋白，无论是在蛋 白水平(ELISA)还是在细胞水平(FACS和luciferase报告基因测试)，均具有类似的生物学活性。
实施例5重组蛋白在小鼠中的药代动力学(PK)研究
5.1 PK方法的建立
小鼠抗人生长激素单克隆抗体(Abcam，CAT#Ab9821)用于包被96孔板，PBST(Tween-20，0.1％)洗涤3次后用200μl包含1％BSA的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(rhGH或rhGH-VHH)的PBST。室温孵育1小时后用PBST洗涤3次，加入生物素标记的山羊抗人生长激素多克隆抗体(R&D，CAT#AF1201)，室温孵育1小时。经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1小时。然后如前洗涤，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8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。
5.2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皮下注射10mg/kg的人转铁蛋白。rhGH和rhGH-VHH的单次皮下给药剂量分别为50，150，500nmol/kg三个剂量。分别在给药后2、4、8、24、32及48小时后(rhGH组)，或2、6、24、30、48、54、72、78及96小时后(rhGH-VHH组)采集血样分离血清，用上述PK方法做血清中的药物定量分析，用PKsovler软件计算PK参数。图9显示的结果表明，在150nmol/kg的剂量下，rhGH-VHH的血液半衰期(11.5小时)比rhGH(血液半衰期小于1小时)有明显提高。在不同剂量组中，均发现通过VHH结合转铁蛋白显著延长rhGH的血液半衰期(图10A)。
5.3多次剂量给药的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皮下注射10mg/kg的人转铁蛋白。rhGH-VHH的皮下给药剂量均为50，150，500nmol/kg三个剂量，每48小时(两天)给药一次，连续给药三次。分别在首次给药后2、6、24、48、54(二次给药后6小时)、72(二次给药后24小时)、96(二次给药后48小时)、102(三次给药后6小时)、120(三次给药后24小时)小时，以及首次给药后第7天(三次给药后72小时)、第9天(三次给药后120小时)采集血样分离血清，用于上述PK方法做血液中的药物定量分析，用PKsovler软件计算PK参数。结果如图10B。
实施例6重组蛋白在小鼠中的药代动力学(PK)研究
6.1 GHRH缺陷小鼠模型
GHRH基因(GenBank:M31658.1)缺陷小鼠模型购自南京集萃药康，小鼠的基因型经PCR和测序验证。使用校准的电子天平和电子数显卡尺，测量1-5周龄小鼠的体重(BW)、体长(鼻-肛门距离，N-A)、股骨和胫骨长度。结果表明，杂合子小鼠与野生型小鼠没有明显差别，但纯合子GHRH缺陷小鼠在体长、体重、股骨和胫骨长度方面均有明显降低(图11)。纯合子小鼠用于重组蛋白的药效学研究。
6.2 rhGH的药效学研究
一周龄雄性和雌性(各5只)纯合子GHRH基因缺陷小鼠分别每日皮下给药150nmo/kg或500nmol/kg，连续处理4周，每周2次测量动物体重(BW)及体长(鼻-肛门距离，N-A)。治疗后第5周(第44天)将小鼠安乐死处理，收集血清用于IGF-1水平的测定。图12的结果表明，每日给药能显著改善GHRH基因缺陷小鼠的生长状况。
6.3 rhGH-VHH的药效学研究
一周龄雄性和雌性(各5只)纯合子GHRH基因缺陷小鼠分别皮下给药150nmo/kg(每周两次)、500nmol/kg(每周两次)或1500nmol/kg(每周一次)，连续给药4周，每周2次测量动物体重(BW)及体长(鼻-肛门距离，N-A)。治疗后第5周(第44天)将小鼠安乐死，收集血清用于IGF-1水平的测定。图13的结果表明，在GHRH基因缺陷小鼠中，rhGH-VHH治疗组每周给药两次或每周给药1500nmol/kg可以达到rhGH治疗组每日给药150nmol/kg类似的治疗效果。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (31)

  1. 融合蛋白，其包含：转铁蛋白结合蛋白，以及生长激素或其功能活性片段，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。
  2. 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)能够延长所述生长激素的体内半衰期；
    (2)能够跨越血脑屏障；
    (3)能够通过口服递送；以及
    (4)能够将所述生长激素递送至表达转铁蛋白受体的细胞。
  3. 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。
  4. 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：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  5. 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。
  6. 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。
  7. 根据权利要求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。
  8. 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。
  9. 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。
  10. 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。
  11. 根据权利要求1-10中任一项所述的融合蛋白，其中所述生长激素为人生长激素。
  12. 根据权利要求1-11中任一项所述的融合蛋白，其中所述生长激素或其功能活性片段包含SEQ ID NO:10所示的氨基酸序列。
  13. 根据权利要求1-12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生长激素或其功能活性片段直接或间接相连。
  14. 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的N端和所述生长激素或其功能活性片段的C端直接或间接相连。
  15. 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的C端和所述生长激素或其功能活性片段的N端直接或间接相连。
  16. 根据权利要求1-15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生长激 素或其功能活性片段通过连接子相连。
  17. 根据权利要求16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。
  18. 根据权利要求1-17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。
  19. 根据权利要求1-18中任一项所述的融合蛋白，其能够结合转铁蛋白，且能够结合和/或活化生长激素受体。
  20. 一种或多种分离的核酸分子，其编码权利要求1-19中任一项所述的融合蛋白。
  21. 载体，其包含权利要求20所述的核酸分子。
  22. 细胞，其包含权利要求20所述的核酸分子，和/或权利要求21所述的载体。
  23. 药物组合物，其包含权利要求1-19中任一项所述的融合蛋白，权利要求20所述的核酸分子，权利要求21所述的载体或权利要求22所述的细胞，以及任选地药学上可接受的载体。
  24. 制备权利要求1-19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22所述的细胞。
  25. 权利要求1-19中任一项所述的融合蛋白，权利要求20所述的核酸分子，权利要求21所述的载体，权利要求22所述的细胞，和/或权利要求23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。
  26. 根据权利要求25所述的用途，其中所述疾病和/或病症包括生长激素异常引起的疾病和/或病症。
  27. 根据权利要求25-26中任一项所述的用途，其中所述疾病和/或病症包括生长激素缺乏症。
  28. 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1-19中任一项所述的融合蛋白，权利要求20所述的核酸分子，权利要求21所述的载体，权利要求22所述的细胞，和/或权利要求23所述的药物组合物。
  29. 根据权利要求28所述的方法，其中所述疾病和/或病症包括生长激素异常引起的疾病和/或病症。
  30. 根据权利要求28-29中任一项所述的方法，其中所述疾病和/或病症包括生长激素缺乏症。
  31. 一种延长生长激素在体内半衰期的方法，其包括施用权利要求1-19中任一项所述的融合蛋白。
PCT/CN2022/075176 2022-01-30 2022-01-30 一种长效重组人生长激素及其应用 WO2023142109A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/075176 WO2023142109A1 (zh) 2022-01-30 2022-01-30 一种长效重组人生长激素及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/075176 WO2023142109A1 (zh) 2022-01-30 2022-01-30 一种长效重组人生长激素及其应用

Publications (1)

Publication Number Publication Date
WO2023142109A1 true WO2023142109A1 (zh) 2023-08-03

Family

ID=87470262

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/075176 WO2023142109A1 (zh) 2022-01-30 2022-01-30 一种长效重组人生长激素及其应用

Country Status (1)

Country Link
WO (1) WO2023142109A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102268094A (zh) * 2002-08-30 2011-12-07 比奥雷克西斯药物公司 被修饰的转铁蛋白抗体的融合蛋白
CN105555310A (zh) * 2013-07-08 2016-05-04 南京金斯瑞生物科技有限公司 一种提高蛋白血清半衰期的组合物和方法
WO2018004294A2 (ko) * 2016-06-30 2018-01-04 주식회사 크레아플래닛 인간 성장호르몬 변이 단백질 또는 이의 트랜스페린 융합 단백질을 유효성분으로 포함하는 약학적 조성물
WO2019074498A1 (en) * 2017-10-11 2019-04-18 Nanjing Legend Biotech Co., Ltd. COMPOSITIONS AND METHODS FOR INCREASING THE HALF-LIFE OF A PROTEIN IN A SERUM
CN110054700A (zh) * 2018-01-18 2019-07-26 分子克隆研究室有限公司 长效治疗性融合蛋白

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102268094A (zh) * 2002-08-30 2011-12-07 比奥雷克西斯药物公司 被修饰的转铁蛋白抗体的融合蛋白
CN105555310A (zh) * 2013-07-08 2016-05-04 南京金斯瑞生物科技有限公司 一种提高蛋白血清半衰期的组合物和方法
WO2018004294A2 (ko) * 2016-06-30 2018-01-04 주식회사 크레아플래닛 인간 성장호르몬 변이 단백질 또는 이의 트랜스페린 융합 단백질을 유효성분으로 포함하는 약학적 조성물
WO2019074498A1 (en) * 2017-10-11 2019-04-18 Nanjing Legend Biotech Co., Ltd. COMPOSITIONS AND METHODS FOR INCREASING THE HALF-LIFE OF A PROTEIN IN A SERUM
CN110054700A (zh) * 2018-01-18 2019-07-26 分子克隆研究室有限公司 长效治疗性融合蛋白

Similar Documents

Publication Publication Date Title
US11466085B2 (en) Anti-PD-L1 nanobody, coding sequence and use thereof
EP1888641B1 (en) Serum albumin binding proteins
WO2021042694A1 (zh) 抗vegf单域抗体及其应用
CA2663042A1 (en) Serum albumin binding proteins with long half-lives
TW201805308A (zh) 結合β-ＫＬＯＴＨＯ，ＦＧＦ受體及其複合物之人類抗原結合蛋白
KR20110127224A (ko) 개선된 항-혈청 알부민 결합 변이체
CA2786660A1 (en) Liver targeting molecules
BRPI1013341B1 (pt) Variantes de domínio variável único de imunoglobulina anti-albumina de soro, ligante multiespecífico e composição que compreendem as ditas variantes, bem como ácidos nucleicos e vetor
US20220251154A1 (en) Fusion protein and use thereof
WO2021155635A1 (zh) 抗cd3和cd123双特异性抗体及其用途
EP4198055A1 (en) Antibody of il-11 and use thereof
JP2016027801A (ja) 改良された抗血清アルブミン結合変異体
WO2021082573A1 (zh) 抗il-4r单域抗体及其应用
WO2023093831A1 (zh) 包含SIRPα突变体的融合蛋白
WO2023142109A1 (zh) 一种长效重组人生长激素及其应用
CN116120438A (zh) 靶向新型冠状病毒rbd结构域的纳米抗体及其衍生蛋白
EP3808847A1 (en) Apj antibody, fusion protein thereof with elabela, and pharmaceutical compositions and use thereof
CN116554343A (zh) 一种长效重组人生长激素及其应用
WO2023207574A1 (zh) 一种穿越血脑屏障的重组hARSA
WO2023227134A1 (zh) 长效il5纳米抗体及用途
TW201809013A (zh) 用於藥物遞送之抗體融合蛋白
CN115873113B (zh) 靶向CaSR的纳米抗体及其制备方法和应用
WO2023097605A1 (en) Transferrin binding antibodies and use thereof
EP4389769A1 (en) Anti-ptk7 single-domain antibody and application thereof
CN116082504A (zh) 靶向人lilrb2的纳米抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22922935

Country of ref document: EP

Kind code of ref document: A1