WO2021008454A1 - 基于铁蛋白重链亚基的药物载体 - Google Patents

基于铁蛋白重链亚基的药物载体 Download PDF

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WO2021008454A1
WO2021008454A1 PCT/CN2020/101312 CN2020101312W WO2021008454A1 WO 2021008454 A1 WO2021008454 A1 WO 2021008454A1 CN 2020101312 W CN2020101312 W CN 2020101312W WO 2021008454 A1 WO2021008454 A1 WO 2021008454A1
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ferritin
seq
clathrin
amino acid
mutant polypeptide
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PCT/CN2020/101312
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English (en)
French (fr)
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柯天一
劳芳
姚德惠
欧阳芳幸
江冰
李彦良
温佰刚
成键伟
闫西冲
刘岩
李凡
李波
张荣坤
付叶
徐菊芳
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昆山新蕴达生物科技有限公司
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Priority to CN202080050897.2A priority Critical patent/CN114616241A/zh
Publication of WO2021008454A1 publication Critical patent/WO2021008454A1/zh

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

Definitions

  • the invention relates to the field of biomedicine. Specifically, the present invention relates to drug carriers based on ferritin heavy chain subunits. More specifically, the present invention relates to mutant polypeptides of ferritin heavy chain subunits, fusion proteins containing the polypeptides, clathrins containing the polypeptides, and their applications as drug carriers.
  • Ferritin is a large protein of about 450kDa, composed of 24 subunits self-assembled into a spherical cage structure, its internal and external dimensions are about 8 and about 12nm, respectively, the cage structure contains up to 4500 iron The iron nucleus of the atom.
  • Eukaryotic ferritin contains a heavy chain (H; 21 kDa) and a light chain (L; 19 kDa).
  • the H chain is responsible for the oxidation of Fe(II) to Fe(III) and includes catalytic iron oxidase sites, while the L chain plays a role in iron nucleation.
  • the H and L chains are assembled together into a 24-mer hybrid ferritin, where the ratio of the H chain to the L chain varies according to the tissue-specific distribution.
  • ferritin-based drug delivery systems Hybrid nanocarriers for vascular immunotargeting, Makan Khoshnejad et.al, Journal of Controlled Release 282 (2018) 13-24.
  • WO2015180325A1 describes that ferritin (H-ferritin) is self-assembled from only the H chain, which can be used as a drug carrier for tumor-specific drug delivery due to its ability to bind to the receptor TfR1 and target tumor cells.
  • the application does not involve mutants of ferritin subunits.
  • WO2018153372A1 teaches that H-ferritin can be used as a nano-drug carrier that can cross the blood-brain barrier.
  • WO2018073593A1 discloses that ferritin can be used as a drug carrier, and adriamycin can be enclosed in its inner cavity. This application modifies ferritin subunits so that ferritin subunits cannot assemble to form a ferritin cage without the presence of a nucleating agent.
  • Natural ferritin has an iron storage function. After it is used as a drug carrier and enters the body, if it still retains its natural iron storage function, it may affect the body's normal iron metabolism, and there is a risk of iron metabolism disorders. In addition, it has been reported that the natural iron oxidase activity may inhibit the development and maturation of human osteoblasts, and lead to reduced mineralization, osteopenia and osteoporosis (Zarjou A, Jeney V, Arosio P, Poli M, Zavacczki E, Balla G, Balla J.
  • Ferritin ferroxidase activity a potent inhibitor of osteogenesis. J Bone Miner Res. 2010, 25: 164-72).
  • ferritin used as a drug carrier still retains the function of storing iron, it may also reduce its drug loading and loading efficiency.
  • cysteine residues on the H subunit of ferritin there are three cysteine residues on the H subunit of ferritin (two are located on the outer surface of ferritin, and one is located on the triple symmetry axis of ferritin). Since the sulfhydryl group at the end of cysteine is an extremely active group, it is not only easy to be oxidized to form disulfide bonds, which makes ferritin form aggregates, but may also react with a variety of in vivo molecules, which may cause off-target, side effects, etc. Safety issues, on the other hand, reduce the effective concentration of the drug, resulting in insufficient efficacy. Therefore, the presence of cysteine with active sulfhydryl groups hinders the application of ferritin in drug carriers.
  • the present invention provides a ferritin heavy chain (H) subunit mutant polypeptide, which
  • the ferritin heavy chain (H) subunit mutant polypeptide is contained in positions corresponding to positions 27, 61, 62, and/or 65 of SEQ ID NO:1
  • the amino acid at position is substituted, and the positions corresponding to the 90th, 102nd, and 130th positions of SEQ ID NO:1 include amino acid residues other than cysteine.
  • the invention provides a fusion protein comprising the ferritin H subunit mutant polypeptide of the invention and at least one additional polypeptide portion.
  • the present invention provides a clathrin comprising at least one ferritin H subunit mutant polypeptide of the present invention or a fusion protein of the present invention.
  • the present invention provides a clathrin-active pharmaceutical ingredient (API) complex, which comprises the clathrin of the present invention, and a clathrin loaded in the clathrin or coupled to the clathrin Active Pharmaceutical Ingredient (API).
  • API clathrin-active pharmaceutical ingredient
  • the invention provides a pharmaceutical composition comprising the ferritin H subunit mutant polypeptide of the invention, the fusion protein of the invention, the clathrin of the invention, or the clathrin-API complex of the invention , And pharmaceutically acceptable excipients.
  • the invention provides the ferritin H subunit mutant polypeptide of the invention, the fusion protein of the invention, the clathrin of the invention, the clathrin-API complex of the invention or the pharmaceutical composition of the invention. Use in the preparation of medicines.
  • the present invention provides a method for treating and/or preventing diseases in a subject, the method comprising administering to the subject an effective amount of the ferritin H subunit mutant polypeptide of the present invention, the fusion protein of the present invention , The clathrin, clathrin-API complex of the present invention or the pharmaceutical composition of the present invention.
  • the present invention provides a method for preparing the clathrin-API complex of the present invention, the method comprising contacting the clathrin-API complex of the present invention with API, thereby obtaining the clathrin-API complex.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the ferritin H subunit mutant polypeptide of the invention or the fusion protein of the invention.
  • the present invention provides an expression construct comprising the polynucleotide of the present invention.
  • the present invention provides a host cell which contains the polynucleotide of the present invention or is transformed by the expression construct of the present invention, and is capable of expressing the ferritin H subunit mutant polypeptide or fusion protein of the present invention.
  • the present invention provides a method for producing the ferritin H subunit mutant polypeptide of the present invention or the fusion protein of the present invention, comprising:
  • step a) obtaining the mutant polypeptide or fusion protein expressed by the host cell from the culture obtained from step a);
  • step b) optionally further purifying the mutant polypeptide or fusion protein obtained from step b).
  • Figure 1 SDS-PAGE diagram showing the purified ferritin H subunit iron loading related site mutant after E. coli expression.
  • Figure 2 Native-PAGE image showing purified ferritin H subunit iron-loading related site mutants.
  • FIG. 1 Electron microscopy images showing purified ferritin H subunit iron-loading related site mutants.
  • Figure 4 Shows the comparison of iron content of different H-ferritin iron loading-related site mutants.
  • FIG. Shows the drug loading capacity of different ferritin iron loading related site mutants.
  • FIG. 7 SDS-PAGE chart showing the purified ferritin H subunit cysteine mutant after E. coli expression.
  • FIG. 1 Electron micrograph showing purified ferritin H subunit cysteine mutant.
  • Figure 9 Shows the comparison of receptor binding activity of different ferritin cysteine mutants.
  • Figure 10 Shows the iron content comparison of different H-ferritin cysteine mutants.
  • Figure 11 Shows the effect of administration of different H-ferritin cysteine mutants on white blood cells of cynomolgus monkeys.
  • Figure 12 Shows the effect of administration of different H-ferritin cysteine mutants on cynomolgus monkey red blood cells.
  • FIG. 1 Electron microscope image showing purified ferritin H subunit combination mutant.
  • Figure 14 Shows the results of stability analysis of H-ferritin combination mutants.
  • Figure 15 Shows the iron content comparison of different H-ferritin combination mutants.
  • FIG. 16 Shows the drug loading capacity of different ferritin combination mutants.
  • FIG. Shows the stability analysis of different ferritin combination mutants after loading.
  • FIG. 1 shows the non-specific binding analysis of different ferritin combination mutants.
  • Figure 19 Shows the results of reverse chromatography analysis of mutant samples with different ferritin combinations.
  • the term “and/or” encompasses all combinations of items connected by the term, and should be treated as if each combination has been individually listed herein.
  • “A and/or B” encompasses “A”, “A and B”, and “B”.
  • “A, B, and/or C” encompasses "A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and "A and B and C”.
  • “Ferritin” refers to an iron storage structure composed of two parts: a protein shell and an iron core.
  • the protein shell of ferritin is a clathrin structure (outer diameter 12nm, inner diameter 8nm) usually formed by self-assembly of 24 subunits, and the main component of the iron core is ferrihydrite.
  • the protein shell of ferritin without an iron core is also called “deferritin”.
  • “Ferritin” as used herein includes eukaryotic ferritin and prokaryotic ferritin, preferably eukaryotic ferritin, more preferably mammalian ferritin, such as human ferritin.
  • Eukaryotic ferritin usually includes a heavy chain H subunit and a light chain L subunit. In different tissues and organs of the body, the ratio of H and L subunits in ferritin molecules is different. However, through recombination, it is also possible to obtain "H ferritin (HFn)” assembled from only H subunits or "L ferritin (LFn)” assembled from L subunits only.
  • the number of polypeptides (subunits) assembled into a clathrin is not particularly limited as long as it can form the cage structure.
  • a clathrin can have a symmetrical structure or an asymmetrical structure, depending on its subunit composition.
  • a typical clathrin contains ferritin/deferritin.
  • hydrophilic amino acids include cysteine (C), aspartic acid (D), asparagine (N), glutamic acid (E), histidine (H), lysine (K), Glutamine (Q), Arginine (R), Glycine (G), Serine (S) and Threonine (T).
  • hydrophobic amino acids include alanine (A), phenylalanine (F), isoleucine (I), leucine (L), methionine (M), proline (P), valine (V), tyrosine (Y) and tryptophan (W).
  • Polypeptide “peptide”, and “protein” are used interchangeably in the present invention and refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are corresponding artificial chemical analogs of natural amino acids, as well as to polymers of natural amino acids.
  • the terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxyl And ADP-ribosylation.
  • polynucleotide refers to a macromolecule composed of multiple nucleotides connected by phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides.
  • the sequence of the polynucleotide of the present invention can be codon optimized for different host cells (such as E. coli), thereby improving the expression of the polypeptide. Methods of performing codon optimization are known in the art.
  • the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or cores at one or both ends of the protein or nucleic acid. Glycolic acid, but still has the activity described in the present invention.
  • methionine encoded by the start codon at the N-terminus of the polypeptide will be retained under certain practical conditions (for example, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide.
  • the coding nucleotide sequence may also include a start codon.
  • sequence identity between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences.
  • Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed by the online alignment tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/psa/). The sequence identity between two sequences can be evaluated using the Needleman-Wunsch algorithm, using default parameters.
  • expression construct refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in an organism. "Expression” refers to the production of a functional product.
  • the expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and/or the translation of RNA into a precursor or mature protein.
  • the "expression construct” of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or may be RNA (such as mRNA) that can be translated.
  • the nucleotide sequence of interest is operably linked to the regulatory sequence.
  • regulatory sequence and “regulatory element” can be used interchangeably and refer to the upstream (5' non-coding sequence), middle or downstream (3' non-coding sequence) of the coding sequence, and affect the transcription and RNA of the relevant sequence of interest. Processing or stability or translated nucleotide sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • operably linked means that a regulatory sequence is connected to a target nucleotide sequence so that the transcription of the target nucleotide sequence is controlled and regulated by the regulatory sequence.
  • Techniques for operably linking regulatory sequences to the nucleotide sequence of interest are known in the art.
  • active pharmaceutical ingredient or “active pharmaceutical ingredient” or “API (Active pharmaceutical ingredient)” refers to a substance in a medicine that has pharmacological activity or can directly affect the function of the body.
  • pharmaceutical active ingredients do not include pharmaceutical carriers or excipients.
  • “Pharmaceutically acceptable excipient” refers to any ingredients that are not pharmacologically active and non-toxic used in the formulation of pharmaceutical products, including but not limited to disintegrants, binders, fillers, buffers, Tonicity agent, stabilizer, antioxidant, surfactant or lubricant.
  • an effective amount or “therapeutically effective dose” refers to the amount of a substance, compound, material, or composition containing the compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of the disease.
  • the 27th, 61st, 62nd, and 65th amino acids in the H subunit of human ferritin are involved in iron loading.
  • glutamic acid at position 27, glutamic acid at position 62, and histidine at position 65 are considered to constitute the ferrite center of H ferritin; while glutamic acid at position 61 is considered to be H iron
  • the present inventors found that by mutating the amino acids at these specific positions in the ferritin heavy chain (H) subunit, the iron storage capacity of the formed ferritin can be reduced, so that the ferritin has a higher capacity when used as a drug carrier. The safety, and this mutation does not affect the assembly of ferritin and its binding to the TfR1 receptor.
  • the 90th and 102nd cysteines in the H subunit of wild-type human ferritin are located on the outer surface of H ferritin.
  • the 130th cysteine is located on the triple symmetry axis of ferritin, which may be involved in the loading of iron ions or drugs.
  • the present invention provides a ferritin heavy chain (H) subunit mutant polypeptide, which
  • the ferritin heavy chain (H) subunit mutant polypeptide is contained in positions corresponding to positions 27, 61, 62, and/or 65 of SEQ ID NO:1
  • the amino acid at position is substituted, and the positions corresponding to the 90th, 102nd, and 130th positions of SEQ ID NO:1 include amino acid residues other than cysteine.
  • the ferritin H subunit (also referred to as the starting ferritin H subunit) from which the mutant polypeptide of the present invention is derived may be the naturally occurring (wild-type) ferritin H subunit.
  • the wild-type human ferritin H subunit may include the amino acid sequence shown in SEQ ID NO:1.
  • the ferritin H subunit from which the mutant polypeptide of the present invention is derived may also be an artificially modified (modified) ferritin H subunit.
  • a variety of artificially modified ferritin H subunits are known in the art, which can be used to obtain the mutant polypeptide of the present invention.
  • Examples of known artificially engineered ferritin H subunits include, but are not limited to, those disclosed in WO2018153372A1, WO2014104768, WO2016051340A1, WO2014123399A1, WO2017039382A1, WO2016122259A1, CN109486827A, CN107286249A, CN104059156A, CN101942023A, CN104017088A, CN105801706A, and US7097841B2.
  • the ferritin H subunit derived from the mutant polypeptide of the present invention has at least part of the functional activity of the naturally occurring ferritin H subunit, and the partial functional activity includes, but is not limited to, the ability to assemble into a clathrin and/or The ability of clathrin to specifically bind to TfR1, etc.
  • the ferritin H subunit from which the mutant polypeptide of the present invention is derived contains at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least SEQ ID NO:1 An amino acid sequence of 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the ferritin H subunit from which the mutant polypeptide of the present invention is derived comprises one or more positions corresponding to the 90th, 102nd, and 130th positions of SEQ ID NO:1 Cysteine.
  • the ferritin H subunit from which the mutant polypeptide of the present invention is derived contains cysteine at positions corresponding to positions 90, 102, and 130 of SEQ ID NO:1 .
  • the ferritin H subunit polypeptide derived from the mutant polypeptide of the present invention includes the amino acid sequence shown in SEQ ID NO:1.
  • the mutant polypeptide comprises an amino acid substitution at a position corresponding to position 61 of SEQ ID NO:1.
  • the mutant polypeptide comprises amino acid substitutions at positions corresponding to positions 62 and 65 of SEQ ID NO:1.
  • the mutant polypeptide comprises amino acid substitutions at positions corresponding to the 61st, 62nd, and 65th positions of SEQ ID NO:1.
  • the mutant polypeptide comprises amino acid substitutions at positions corresponding to positions 27, 62, and 65 of SEQ ID NO:1.
  • the mutant polypeptide comprises amino acid substitutions at positions corresponding to the 27th, 61st, 62nd, and 65th positions of SEQ ID NO:1.
  • the amino acid substitution is a hydrophobic amino acid substitution or a wild-type ferritin light chain (L) subunit corresponding position.
  • hydrophobic amino acids for the key amino acids in the iron oxide center or iron ion channel can occupy the iron ion channel on the one hand to avoid iron loading, and on the other hand, it will facilitate the loading of hydrophobic drugs.
  • Suitable hydrophobic amino acids include phenylalanine, glycine, isoleucine, leucine, methionine, valine, alanine, tyrosine and tryptophan, preferably phenylalanine or Acid.
  • Wild-type ferritin light chain (L) subunit means the L subunit of ferritin that is assembled with a wild-type ferritin H subunit polypeptide in a natural state.
  • the human wild-type human ferritin L subunit polypeptide includes the amino acid sequence shown in SEQ ID NO: 36, also refer to NCBI Genbank Seq No: NP_000137.2.
  • the L ferritin formed by the wild-type ferritin light chain (L) subunit alone does not have the ability to store iron. Therefore, the substitution of amino acids at the corresponding position will help reduce the iron loading while maintaining the assembled cage ⁇ Like structure.
  • the amino acid at the position corresponding to the 27th position of SEQ ID NO:1 of the mutant polypeptide is substituted with phenylalanine. In some embodiments, the amino acid at the position corresponding to position 61 of SEQ ID NO:1 of the mutant polypeptide is substituted with tryptophan. In some embodiments, the amino acid at the position corresponding to the 62nd position of SEQ ID NO:1 of the mutant polypeptide is substituted with lysine. In some embodiments, the amino acid at the position corresponding to position 65 of SEQ ID NO:1 of the mutant polypeptide is substituted with glycine.
  • the mutant polypeptide at the position corresponding to the 62nd position of SEQ ID NO:1 is substituted with lysine, at the position corresponding to the 65th position of SEQ ID NO:1
  • the amino acid is replaced with glycine.
  • the amino acid of the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1 is substituted with tryptophan, at the position corresponding to the 62nd position of SEQ ID NO:1
  • the amino acid of is substituted with lysine, and the amino acid at position 65 corresponding to SEQ ID NO:1 is substituted with glycine.
  • the amino acid of the mutant polypeptide at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine, at the position corresponding to the 62nd position of SEQ ID NO:1
  • the amino acid at is substituted with lysine, and the amino acid at the position corresponding to the 65th position of SEQ ID NO:1 is substituted with glycine.
  • the amino acid at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine in the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1
  • the amino acid at position is substituted with tryptophan
  • the amino acid at position 62 corresponding to SEQ ID NO:1 is substituted with lysine
  • the amino acid is replaced with glycine.
  • the mutant polypeptide is a mutant of human ferritin H subunit, and compared to SEQ ID NO:1, it contains amino acid substitutions E27F, E61W, E62K and/or H65G.
  • the mutant polypeptide is a mutant of human ferritin H subunit, and compared to SEQ ID NO: 1, it includes a set of amino acid substitutions selected from:
  • the mutant polypeptide includes a hydrophilic amino acid other than cysteine at a position corresponding to position 90 of SEQ ID NO:1.
  • the hydrophilic amino acid may be selected from aspartic acid (D), asparagine (N), glutamic acid (E), histidine (H), lysine (K), glutamine (Q) , Arginine (R), Glycine (G), Serine (S) and Threonine (T). Since it is most similar to cysteine in space structure, serine is preferred.
  • the mutant polypeptide includes a hydrophilic amino acid other than cysteine at a position corresponding to position 102 of SEQ ID NO:1.
  • the hydrophilic amino acid may be selected from aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), glutamine (Q), arginine (R) , Glycine (G), Serine (S) and Threonine (T).
  • the hydrophilic amino acid is serine.
  • the mutant polypeptide includes a hydrophobic amino acid at a position corresponding to position 130 of SEQ ID NO:1.
  • the hydrophobic amino acid can be selected from alanine (A), phenylalanine (F), isoleucine (I), leucine (L), methionine (M), proline (P ), valine (V), tyrosine (Y) or tryptophan (W).
  • the hydrophobic amino acid is methionine.
  • the hydrophobic amino acid is tyrosine.
  • the mutant polypeptide includes a hydrophilic amino acid other than cysteine at a position corresponding to the 130th position of SEQ ID NO:1, and the hydrophilic amino acid may be selected from aspartame. Acid (D), Asparagine (N), Glutamic Acid (E), Histidine (H), Lysine (K), Glutamine (Q), Arginine (R), Glycine ( G), Serine (S) and Threonine (T).
  • the hydrophilic amino acid is histidine.
  • the hydrophilic amino acid is serine.
  • the mutant polypeptide includes serine at positions corresponding to positions 90, 102, and 130 of SEQ ID NO:1.
  • the mutant polypeptide includes serine at positions corresponding to the 90th and 102nd positions of SEQ ID NO:1, and includes the group at positions corresponding to the 130th position of SEQ ID NO:1. Acid.
  • the mutant polypeptide comprises serine at positions corresponding to the 90th and 102nd positions of SEQ ID NO:1, and comprises a methine at the positions corresponding to the 130th position of SEQ ID NO:1. Thionine.
  • the mutant polypeptide comprises serine at positions corresponding to the 90th and 102nd positions of SEQ ID NO:1, and contains serine at the positions corresponding to the 130th position of SEQ ID NO:1. Acid.
  • the amino acid of the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1 is substituted with tryptophan, at the position corresponding to the 62nd position of SEQ ID NO:1
  • the amino acid of is replaced with lysine, and the amino acid at the position corresponding to the 65th position of SEQ ID NO:1 is replaced with glycine, and the amino acid at the position corresponding to SEQ ID NO:1 is 90, 102 and 130 Serine is included at the position of the position.
  • the amino acid at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine in the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1
  • the amino acid at position is substituted with tryptophan
  • the amino acid at position 62 corresponding to SEQ ID NO:1 is substituted with lysine
  • the amino acid is substituted with glycine, and serine is included at positions corresponding to the 90th, 102nd, and 130th positions of SEQ ID NO:1.
  • the mutant polypeptide at the position corresponding to the 62nd position of SEQ ID NO:1 is substituted with lysine, at the position corresponding to the 65th position of SEQ ID NO:1
  • the amino acid of is replaced with glycine, and serine is included at positions corresponding to the 90th, 102th, and 130th positions of SEQ ID NO:1.
  • the amino acid of the mutant polypeptide at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine, at the position corresponding to the 62nd position of SEQ ID NO:1
  • the amino acid at position is substituted with lysine
  • the amino acid at position corresponding to SEQ ID NO:1 is substituted with glycine
  • the amino acid at position 90, 102, and position corresponding to SEQ ID NO:1 Serine is included at position 130.
  • the amino acid at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine in the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1
  • the amino acid at position is substituted with tryptophan
  • the amino acid at position 62 corresponding to SEQ ID NO:1 is substituted with lysine
  • the amino acid is substituted with glycine, and methionine is included at positions corresponding to the 90th, 102nd, and 130th positions of SEQ ID NO:1.
  • the amino acid at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine in the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1
  • the amino acid at position is substituted with tryptophan
  • the amino acid at position 62 corresponding to SEQ ID NO:1 is substituted with lysine
  • the amino acid is substituted with glycine
  • serine is included at positions corresponding to the 90th and 102nd positions of SEQ ID NO:1
  • methionine is included at the position corresponding to 130th positions of SEQ ID NO:1.
  • the mutant polypeptide at the position corresponding to the 62nd position of SEQ ID NO:1 is substituted with lysine, at the position corresponding to the 65th position of SEQ ID NO:1
  • the amino acid of is substituted with glycine, serine is included at positions corresponding to the 90th and 102nd positions of SEQ ID NO:1, and methionine is included at the positions corresponding to 130th position of SEQ ID NO:1.
  • the amino acid of the mutant polypeptide at the position corresponding to the 61st position of SEQ ID NO:1 is substituted with tryptophan, at the position corresponding to the 62nd position of SEQ ID NO:1
  • the amino acid of is replaced with lysine
  • the amino acid at the position corresponding to the 65th position of SEQ ID NO:1 is replaced with glycine, at the positions corresponding to the 90th and 102nd positions of SEQ ID NO:1 Contains serine, and contains methionine at the position corresponding to the 130th position of SEQ ID NO:1.
  • the amino acid of the mutant polypeptide at the position corresponding to the 27th position of SEQ ID NO:1 is substituted with phenylalanine, at the position corresponding to the 62nd position of SEQ ID NO:1
  • the amino acid at is replaced with lysine
  • the amino acid at the position corresponding to the 65th position of SEQ ID NO:1 is replaced with glycine, at the positions corresponding to the 90th and 102nd positions of SEQ ID NO:1 It contains serine at the position corresponding to the 130th position of SEQ ID NO:1 and contains methionine.
  • the mutant polypeptide comprises an amino acid sequence selected from one of SEQ ID NO: 2-5, 10-14, and 20-25.
  • the mutant polypeptide is capable of assembling into a clathrin and/or being capable of conferring the clathrin the ability to specifically bind to the TfR1 receptor after being assembled into the clathrin.
  • the mutant polypeptide is produced recombinantly, that is, the mutant polypeptide is a recombinant polypeptide.
  • the method for recombinantly producing the mutant polypeptide of the present invention is further described below.
  • ferritin H subunit mutant polypeptide can be fused with other sequences to give it additional characteristics without affecting its self-assembly activity or receptor binding activity.
  • fusions of ferritin H subunit polypeptides with other sequences include, but are not limited to, those disclosed in WO2014104768A1, WO2016051340A1, WO2014123399A1, WO2017039382A1, WO2016122259A1, CN109486827A, CN107286249A, CN104059156A, CN101942023A, CN104017088A, CN105801706A, US7097841B2.
  • the present invention provides a fusion protein comprising the ferritin H subunit mutant polypeptide of the present invention described above, and at least one additional polypeptide portion.
  • the ferritin H subunit mutant polypeptide of the present invention is directly connected to the at least one additional polypeptide portion or connected through a linker.
  • the additional polypeptide moiety is a polypeptide capable of imparting additional properties to the fusion protein.
  • the additional characteristics such as ease of expression or purification, different targeting properties, cytotoxicity, traceability, extended half-life in vivo, ease of assembly, etc.
  • the additional polypeptide moiety is a protein tag.
  • the protein tag includes but is not limited to His tag.
  • the additional polypeptide portion is a tracer polypeptide, such as GFP and the like.
  • the additional polypeptide moiety is an in vivo half-life extending polypeptide, such as serum albumin.
  • the additional polypeptide moiety is a targeting peptide, such as an antibody, protein ligand, and the like.
  • the additional polypeptide moiety is a therapeutic polypeptide.
  • Suitable therapeutic polypeptides include but are not limited to antibodies such as single-chain antibodies, cytotoxins, cytokines, antimicrobial peptides such as antiviral peptides or antibacterial peptides, tumor antigen fragments, and the like.
  • the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the ferritin H subunit mutant polypeptide or fusion protein of the present invention.
  • the polynucleotide of the present invention comprises, for example, a nucleotide sequence selected from one of SEQ ID NO: 6-9, 15-19, and 26-31.
  • the invention provides an expression construct comprising a polynucleotide of the invention operably linked to an expression control sequence.
  • the vectors used in the expression construct of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; they also include vectors that can integrate into host cell DNA and replicate together with host cell DNA. Many vectors suitable for the present invention are commercially available.
  • the expression construct of the present invention is derived from pET22b of Novagen.
  • the present invention provides a host cell containing the polynucleotide of the present invention or transformed with the expression construct of the present invention, wherein the host cell is capable of expressing the ferritin H subunit mutant polypeptide of the present invention or Fusion protein.
  • the host cell is a recombinant host cell.
  • Host cells that can be used to express the polypeptide or fusion protein of the present invention include prokaryotes, yeast, and higher eukaryotic cells.
  • Exemplary prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, Salmonella, and Pseudomonas and Streptomyces.
  • the host cell is an Escherichia cell, preferably Escherichia coli.
  • the host cell used is E. coli BL21 (DE3) strain cell.
  • the recombinant expression construct of the present invention can be introduced into host cells by one of many well-known techniques, such techniques include but are not limited to: heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, lipid Body-mediated transfection, calcium phosphate precipitation, protoplasmic fusion, particle bombardment, virus transformation and similar technologies.
  • the present invention provides a method for recombinantly producing the ferritin H subunit mutant polypeptide or fusion protein of the present invention, including:
  • step a) obtaining the ferritin H subunit mutant polypeptide or fusion protein expressed by the host cell from the culture obtained in step a);
  • step b) optionally further purifying the ferritin H subunit mutant polypeptide or fusion protein obtained from step b).
  • ferritin H subunit mutant polypeptide or fusion protein of the present invention can also be obtained by chemical synthesis.
  • ferritin H subunit mutant polypeptide or fusion protein of the present invention can be assembled into a clathrin (ie H-ferritin/deferritin) can also form a clathrin with ferritin L subunit or other self-assembling polypeptides, and can give the clathrin a specific targeting ability.
  • the present invention provides a cage protein comprising at least one ferritin H subunit mutant polypeptide or fusion protein of the present invention.
  • Exemplary cage proteins may include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 36, or 48 ferritin H subunit mutant polypeptides or fusion proteins of the present invention.
  • the cage protein comprises 24 ferritin H subunit mutant polypeptides or fusion proteins of the present invention.
  • the cage protein only comprises the ferritin H subunit mutant polypeptide or fusion protein of the present invention.
  • the clathrin is assembled from 24 ferritin H subunit mutant polypeptides or fusion eggs of the present invention.
  • the clathrin further comprises the L subunit of ferritin.
  • the cage protein comprises at least one ferritin H subunit mutant polypeptide or fusion protein of the present invention and at least one ferritin L subunit, preferably, the ferritin H subunit mutant polypeptide Or the ratio range of the fusion protein to the L subunit of ferritin can be, for example, 1:23-23:1.
  • the clathrin does not comprise the L subunit of ferritin.
  • the clathrin may also contain additional modifications, such as PEGylation modifications.
  • the clathrin of the present invention has a significantly reduced iron storage capacity compared to the corresponding clathrin comprising wild-type ferritin H subunit, for example, its iron storage capacity is reduced to that of the corresponding wild-type iron About 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or even lower of protein.
  • the iron storage capacity of different clathrins can be evaluated by measuring their iron content under the same conditions, for example, see the examples of this application.
  • the clathrin of the present invention has increased stability, especially when loaded with active pharmaceutical ingredients (API) Rear.
  • the clathrin of the present invention has reduced non-specific binding compared to the corresponding clathrin comprising wild-type ferritin H subunit, for example, reduced binding to serum proteins (e.g., albumin) Or globulin) non-specific binding.
  • serum proteins e.g., albumin
  • globulin e.g., albumin
  • the level of binding to albumin or globulin can be determined by the method in the examples of this application.
  • the clathrin of the present invention has comparable or higher loading capacity compared to the corresponding clathrin comprising wild-type ferritin H subunit.
  • the loading capacity refers to the ability to encapsulate substances such as API in the inner central cavity structure of the clathrin.
  • the loading capacity of clathrin can be determined by the method described in the examples of this application.
  • the clathrin of the present invention has comparable receptor binding capacity, such as TfR1 receptor binding capacity, compared to the corresponding clathrin containing wild-type ferritin H subunit.
  • the TfR1 receptor binding ability can be measured with reference to the method described in the Examples of the present application, for example.
  • the clathrin of the present invention has significantly reduced iron storage capacity, increased stability, and reduced non-specific binding, thereby having increased drug safety, and retaining suitable drug loading capabilities and TfR1-mediated tumor targeting capabilities And blood-brain barrier penetration ability, so it is particularly suitable as a drug carrier.
  • the present invention provides a clathrin-API complex, wherein the clathrin-API complex comprises the clathrin of the present invention, and is loaded inside or coupled to the clathrin To the pharmaceutical active ingredient (API) of the clathrin.
  • the clathrin-API complex comprises the clathrin of the present invention, and is loaded inside or coupled to the clathrin To the pharmaceutical active ingredient (API) of the clathrin.
  • the pharmaceutical active ingredient is not particularly limited, as long as it is suitable for being loaded into the clathrin of the present invention or coupled to the clathrin, for example, the API does not damage the clathrin
  • the cage structure and/or its size is suitable for being contained by the cage structure.
  • Examples of the API include, but are not limited to, alkylating agents, such as nitrosoureas; platinum, such as cisplatin, carboplatin and their derivatives; antimetabolites, such as thymidylate synthase inhibitors; tumor antibiotics Drugs such as doxorubicin, daunorubicin, daunorubicin; natural extracts such as plant alkaloids (vinblastine); hormones such as anti-estrogens (tamotifen); radiopharmaceuticals such as 64Cu, 235U; neurotransmitter drugs, such as carbachol, atropine, scopolamine, dopamine and its derivatives; dopamine receptor agonists, such as bromocriptine, pergolide, apomorphine and other ergot derivatives and non Ergot alkaloid derivatives; nerve center anticholinergic drugs, such as trihexyphenidyl, benztropine and procyclidine; cholinergic receptor agonist drugs, such as muscarinic
  • the coupling of API to the cage protein can be achieved by various methods, such as those known in the art, including but not limited to coupling API to the cage by reacting with free amino and carboxyl groups on the surface of the cage protein. Protein surface.
  • the API can be coupled to the clathrin via a linker such as a chemical linker or a peptide linker.
  • the API can also be coupled to the surface of the clathrin in a non-covalent manner, such as coupling via van der Waals forces, hydrogen bonds, ionic bonds, and the like.
  • the invention provides a pharmaceutical composition comprising the ferritin H subunit mutant polypeptide of the invention, the fusion protein of the invention, the clathrin of the invention, or the clathrin-API complex of the invention And pharmaceutically acceptable excipients.
  • the pharmaceutical composition comprises the ferritin H subunit mutant polypeptide of the present invention or the fusion protein of the present invention, and an effective amount of API, wherein the ferritin H subunit mutant polypeptide or fusion protein is not Provided in the form of assembled clathrin.
  • the ferritin H subunit mutant polypeptide or fusion protein can self-assemble into a clathrin-API complex under suitable conditions in vitro or after being delivered to the body.
  • the pharmaceutical composition including the fusion protein of the present invention may not include an additional API.
  • the ferritin H subunit mutant polypeptide, fusion protein, clathrin, clathrin-API complex or pharmaceutical composition of the present invention can be used to treat and/or prevent diseases depending on the therapeutic polypeptide or API.
  • the clathrin of the present invention has tumor targeting ability and blood-brain barrier penetration ability, it is particularly suitable for treating tumors or brain diseases.
  • brain diseases include, but are not limited to, for example, brain tumors, Alzheimer's disease, Parkinson's disease, stroke, epilepsy, Huntington's disease, and amyotrophic lateral sclerosis.
  • tumor include, but are not limited to, for example, colorectal cancer, lung cancer, breast cancer, ovarian cancer, melanoma, stomach cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, and various hematopoietic cancers such as Hodgkin's disease, Non-Hodgkin's lymphoma, leukemia.
  • the present invention provides the ferritin H subunit mutant polypeptide of the present invention, the fusion protein of the present invention, the clathrin of the present invention, the clathrin-API complex of the present invention, or the pharmaceutical composition of the present invention Use in the preparation of medicines.
  • the drug is used, for example, to treat tumors or brain diseases.
  • the present invention provides a method of treating and/or preventing diseases in a subject, the method comprising administering to the subject an effective amount of the ferritin H subunit mutant polypeptide of the present invention, the fusion of the present invention Protein, the clathrin of the present invention, the clathrin-API complex or the pharmaceutical composition of the present invention.
  • the disease is as defined above, preferably a tumor or a brain disease.
  • the ferritin H subunit mutant polypeptide of the present invention, the fusion protein of the present invention, the clathrin of the present invention, the clathrin-API complex of the present invention, or the pharmaceutical composition of the present invention can be obtained by those of ordinary skill in the art. It is administered by any appropriate method known (see, for example, Remington: The Science and Practice of Pharmacy, "21st Edition, 2005).
  • the pharmaceutical composition can be administered, for example, intravenously, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, and joint Administration is intra-, intra-synovial, intrathecal, oral, topical or inhalation routes.
  • the present invention provides a method for preparing the clathrin-API complex of the present invention, the method comprising making the ferritin H subunit mutant polypeptide of the present invention, the fusion protein of the present invention or the present invention
  • the clathrin is contacted with API, thereby obtaining a clathrin-API complex.
  • the method includes:
  • depolymerization refers to a process in which the tightly closed globular structure of a clathrin protein is opened to separate all or part of the subunits from each other under certain conditions, such as protein denaturation conditions. , Such as a buffer solution containing a high concentration of urea.
  • “reassembly” refers to a process in which depolymerized clathrins, that is, separated subunits, re-assemble into clathrins by changing conditions, such as replacing them with physiologically compatible conditions.
  • the API will be encapsulated in the clathrin to form a clathrin-API complex.
  • the physiologically compatible conditions are, for example, in a physiological buffer solution.
  • the method further includes a step of depolymerizing the clathrin of the present invention before step a).
  • the clathrin of the present invention is depolymerized by the presence of a high concentration (e.g., at least 6M, preferably 8M) of urea.
  • a high concentration e.g., at least 6M, preferably 8M
  • the clathrin is reassembled by gradually reducing the urea concentration (e.g., gradient dialysis).
  • the method includes:
  • the non-depolymerization conditions include placing the clathrin and API in a physiologically acceptable buffer.
  • physiologically acceptable buffers include but are not limited to PBS solution, physiological saline, pure water, HEPES buffer and the like.
  • the API binds to the clathrin through non-covalent or covalent interactions.
  • the non-covalent interactions include van der Waals forces, hydrogen bonds, ionic bonds, and the like.
  • the covalent interaction includes a reaction such as a condensation reaction with free amino and carboxyl groups on the surface of the clathrin.
  • the API shuttles to the inner central cavity of the clathrin by passive diffusion.
  • the API can enter the inner cavity of the clathrin by diffusion without depolymerization of the clathrin.
  • the amino acid sequence of the H subunit mutant was designed, and the site of the H subunit that may be involved in iron loading was mutated. Glutamate at position 27 (E27), glutamate at position 62 (E62), and histidine at position 65 (H65) are thought to be involved in the ferrite center of H-ferritin, while the Glutamic acid (E61) is an important amino acid that may bind to iron ions in the iron channel of H-ferritin. All amino acid positions refer to SEQ ID NO:1.
  • the inventors made different combinations of mutations to the amino acids at these positions.
  • the amino acids at the site can be replaced with hydrophobic amino acids in order to occupy the iron ion channel or facilitate the loading of hydrophobic drugs (for example, E27 and E61 can be replaced by F or W).
  • hydrophobic drugs for example, E27 and E61 can be replaced by F or W.
  • it is also considered to replace the amino acid at the position with the corresponding amino acid on the L subunit (for example, E62 and H65 are replaced by K and G, respectively).
  • the specific design is shown in Table 1.
  • the resulting subunit mutants were named Mut1 (SEQ ID NO: 2), Mut2 (SEQ ID NO: 3), Mut3 (SEQ ID NO: 4), and Mut4 (SEQ ID NO: 5).
  • the coding sequence was optimized for E. coli.
  • the codon-optimized nucleotide sequences of Mut1-4 are shown in SEQ ID NO: 6-9, respectively.
  • the above four mutants encode plasmids with nucleotide sequences.
  • the wild-type plasmid was constructed in the same way.
  • ferritin H subunit polypeptide is obtained through protein purification.
  • the characterization of peptide purification is shown in Figure 1 for SDS-PAGE analysis.
  • TFR1 human origin
  • protein stabilizer purchased from Huzhou Yingchuang Biotechnology Co., Ltd., PR-SS-002
  • Anti-TFR1 antibody mouse source
  • protein stabilizer purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.: 11020-MM02
  • the ELISA plate was washed 3 times with 1 ⁇ PBST and 2 times with 1 ⁇ PBS.
  • Anti-mouse IgG is diluted with HRP coupling stabilizer (1:5000) and added at 100 ⁇ L/well. Incubate for 1h in a 37°C incubator.
  • the ELISA plate was washed 3 times with 1 ⁇ PBST and 3 times with 1 ⁇ PBS.
  • H-ferritin at a concentration of 1 mg/ml was dissolved in 8M urea (Sinnopharm Chemical Reagent Co. Ltd.), and was gently shaken at room temperature to ensure complete dissolution.
  • Doxorubicin (DOX) is added to the solution to a final concentration of 1 mg/ml. After incubating for 10 minutes in the dark, the mixed solution was transferred to a dialysis bag (with a molecular weight cut-off of 3000Da, Termo Scientific).
  • the dialysate was a gradient urea buffer containing 1mg/ml DOX (7M, 5M, 3M, 2M, 1M and 0M, Each concentration 4h), let DOX slowly enter the reassembled ferritin cage at 4°C.
  • H-ferritin The product was then dialyzed with physiological saline overnight to stop the reaction and remove free DOX.
  • the final concentration of DOX coated in the H-ferritin cage was determined using Superdex 20010/300 GL column (GE company) connected to the SEC system (Amersham Parmacia Biotech) (mobile phase: 50mM Tris buffer, pH7.0).
  • H-ferritin has an absorption peak at 280 nm under UV conditions, while DOX has an absorption peak at 485 nm. The smaller the ratio, the higher the adriamycin loading.
  • the amino acid sequence of the H subunit mutant was designed. Cysteine is mutated to serine, which is the closest in structure and physicochemical properties, thereby replacing the physiologically active SH group with inactive OH.
  • the cysteine at the position of the triple symmetry axis is mutated to a hydrophobic amino acid (favorable for hydrophobic drug loading).
  • a mutant with all cysteines mutated to hydrophobic methionine was constructed to compare the effects of different mutants.
  • the coding sequence was optimized for E. coli.
  • the codon-optimized nucleotide sequence of each mutant is shown in SEQ ID NO: 15-19, respectively.
  • a plasmid containing the nucleotide sequences encoding the above five mutants was obtained.
  • the polypeptide purification method includes the following steps: resuspend the induced expression of E. coli cells in 20 mM Tris (pH 8.0) buffer, and then lyse and disrupt the cells by ultrasonic; centrifugation (1500 rpm, 10 min) to remove E.
  • TFR1 human origin
  • protein stabilizer purchased from Huzhou Yingchuang Biotechnology Co., Ltd., PR-SS-002
  • ELISA plate was washed 3 times with 1 ⁇ PBST and 2 times with 1 ⁇ PBS.
  • Anti-TFR1 antibody (mouse source) (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.: 11020-MM02) diluted with protein stabilizer to 1 ⁇ g/mL (1:1000), added at 100 ⁇ L/well, and incubated in a 37°C incubator 1h.
  • the ELISA plate was washed 3 times with 1 ⁇ PBST and 2 times with 1 ⁇ PBS.
  • Anti-mouse IgG is diluted with HRP coupling stabilizer (1:5000) and added at 100 ⁇ L/well. Incubate for 1h in a 37°C incubator.
  • the ELISA plate was washed 3 times with 1 ⁇ PBST and 3 times with 1 ⁇ PBS.
  • the iron ions are all oxidized to trivalent iron form, diluted with ultrapure water to make the volume constant to 10 mL, and 10 microliters of sample are taken and tested with ICP-OES 730 (Agilent), and the Fe content is determined based on the standard peak of trivalent iron.
  • the mutant of the present invention has a significant decrease in iron content compared with the wild-type control.
  • the iron content is significantly reduced, so that the ferritin of the present invention, as a drug carrier, has a reduced influence on the body's own iron metabolism after entering the body, thereby improving the safety of medication.
  • H-ferritin at a concentration of 1 mg/ml was dissolved in 8M urea (Sinnopharm Chemical Reagent Co. Ltd.), and was gently shaken at room temperature to ensure complete dissolution.
  • Doxorubicin (DOX) is added to the solution to a final concentration of 1 mg/ml. After incubating for 10 minutes in the dark, the mixed solution was transferred to a dialysis bag (with a molecular weight cut-off of 3kDa, Termo Scientific).
  • the dialysate was a gradient urea buffer containing 1 mg/ml DOX (7M, 5M, 3M, 2M, 1M and 0M, Each concentration 4h), let DOX slowly enter the reassembled ferritin cage at 4°C.
  • the product was dialyzed with physiological saline overnight to terminate the reaction and remove free DOX.
  • the final concentration of DOX coated in the H-ferritin cage was determined using Superdex 20010/300 GL column (GE company) connected to the SEC system (Amersham Parmacia Biotech) (mobile phase: 50mM Tris buffer, pH7.0).
  • H-ferritin has an absorption peak at 280 nm under UV conditions, while DOX has an absorption peak at 485 nm. The smaller the ratio, the higher the adriamycin loading.
  • CysKO-1, CysKO-3, CysKO-4, CysKO-5 score the same. These mutants have their own advantages: CysKO-1-HFn has the best stability; CysKO-5-HFn has the lowest iron content; CysKO-3-HFn and CysKO-5-HFn have better binding activity with Trf1. it is good.
  • Test product Test the test product CysKO-1-HFn, CysKO-5-HFn, WT-HFn and CysKO-1-HFn-DOX with DOX, CysKO-5-HFn-DOX The effect of WT-HFn-DOX on the blood system of cynomolgus monkeys.
  • the mutant ferritin caused the reduction of red blood cells in cynomolgus monkeys to varying degrees in both the no-load state and the drug-loaded state (DOX), but it can be recovered to varying degrees after the end of the administration.
  • the degree of impact is less than that of the wild type, and the safety is more it is good.
  • Blood coagulation function During the experiment, the blood coagulation function, cardiotoxicity index (cTNI, cTNT, BNP, NT-proBNP, CK-MB and SOD) and urinalysis were not obvious during the test period. Abnormal changes.
  • Histopathological examination euthanasia (D44) at the end of the dosing period, animal gross anatomy and histopathological examination showed no abnormal pathological changes related to the test article.
  • the amino acid sequence of the H subunit mutant was designed, and the H subunit may be involved in iron loading sites and cysteine The acid was mutated. Glutamate at position 27 (E27), glutamate at position 62 (E62), and histidine at position 65 (H65) are thought to be involved in the ferrite center of H-ferritin, while the Glutamic acid (E61) is an important amino acid that may bind to iron ions in the iron channel of H-ferritin. Cysteines at positions 90, 102 and 130 are believed to affect the stability of ferritin and cause safety issues such as off-target. All amino acid positions refer to SEQ ID NO:1.
  • the inventors made different combinations of mutations to the amino acids at these positions.
  • glutamic acid at position 27 (E27), glutamic acid at position 62 (E62), and histidine at position 65 (H65)
  • hydrophobic amino acids in order to occupy iron ion channels or contribute to hydrophobicity Drug loading (for example, E27 and E61 can be replaced by F or W).
  • E27 and E61 can be replaced by F or W.
  • it is also considered to replace the 27th glutamic acid (E27), the 62nd glutamic acid (E62) and the 65th histidine (H65) with the L subunit.
  • the corresponding amino acids above for example, E62 and H65 are replaced by K and G, respectively).
  • the 90th, 102nd, and 130th cysteines were mutated to serine, which has the closest structural and physicochemical properties, thereby replacing the physiologically active -SH group with the inactive -OH group.
  • the position of the triple axis of symmetry was changed.
  • the cysteine was mutated to a hydrophobic amino acid as a control. The specific design is shown in Table 14.
  • the resulting subunit mutants were named CysKO-1-Mut2-HFn (SEQ ID NO: 20), CysKO-1-222-HFn (SEQ ID NO: 21), CysKO-1-Mut1-HFn (SEQ ID NO: 22), CysKO-2-Mut2-HFn (SEQ ID NO: 23), CysKO-5-Mut2-HFn (SEQ ID NO: 24) and CysKO-5-222-HFn (SEQ ID NO: 25).
  • the coding sequence was optimized for E. coli.
  • the codon-optimized nucleotide sequences are shown in SEQ ID NO: 26-31, respectively.
  • Add a BamH1 restriction site before the optimized nucleotide sequence add a stop codon and HindIII restriction site sequence after the sequence, synthesize the whole gene, and after the sequencing is correct, connect it to the pET22b expression plasmid vector to obtain
  • the above 6 mutants encode plasmids with nucleotide sequences.
  • the control plasmid was constructed in the same way.
  • ferritin H subunit polypeptide is obtained through protein purification, for example, refer to CN102127166A.
  • the protein sample (20 ⁇ L, 0.1 mg/mL) was added dropwise to the treated copper mesh, stained with 1% uranyl acetate for 1 minute, and then imaged with JEM-1400 80 kv TEM (JEOL, Japan). Transmission electron microscopy results ( Figure 13) also showed that the mutant H subunit polypeptide can self-assemble to form a 24-mer clathrin with an outer diameter of 12 nm, just like the wild-type H subunit.
  • mutant of the present invention enters the body as a drug carrier, compared with wild-type and some other mutants, the combination of 1-2, 5-6 interacts with protein molecules in the blood in the bloodstream.
  • the possibility of aggregates is lower, that is, non-specific binding ability is weaker and specificity is better.
  • TFR1 human origin
  • protein stabilizer purchased from Huzhou Yingchuang Biotechnology Co., Ltd., PR-SS-002
  • Anti-TFR1 antibody mouse source
  • protein stabilizer purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.: 11020-MM02
  • the ELISA plate was washed 3 times with 1 ⁇ PBST and 2 times with 1 ⁇ PBS.
  • Anti-mouse IgG is diluted with HRP coupling stabilizer (1:5000) and added at 100 ⁇ L/well. Incubate for 1h in a 37°C incubator.
  • the ELISA plate was washed 3 times with 1 ⁇ PBST and 3 times with 1 ⁇ PBS.
  • H-ferritin at a concentration of 1 mg/ml was dissolved in 8M urea (Sinnopharm Chemical Reagent Co. Ltd.), and was gently shaken at room temperature to ensure complete dissolution.
  • Doxorubicin (DOX) is added to the solution to a final concentration of 1 mg/ml. After incubating for 10 minutes in the dark, the mixed solution was transferred to a dialysis bag (with a molecular weight cut-off of 3000Da, Termo Scientific).
  • the dialysate was a gradient urea buffer containing 1mg/ml DOX (7M, 5M, 3M, 2M, 1M and 0M, Each concentration 4h), let DOX slowly enter the reassembled ferritin cage at 4°C.
  • H-ferritin The product was then dialyzed with physiological saline overnight to stop the reaction and remove free DOX.
  • the final concentration of DOX coated in the H-ferritin cage was determined using Superdex 20010/300 GL column (GE company) connected to the SEC system (Amersham Parmacia Biotech) (mobile phase: 50mM Tris buffer, pH7.0).
  • H-ferritin has an absorption peak at 280 nm under UV conditions, while DOX has an absorption peak at 485 nm. The smaller the ratio, the higher the adriamycin loading.
  • the inventors also investigated the results of HFn and CysK0-1-222-HFn (combination 2) related to globulin at 37°C and 50°C, respectively. Analyze the purity of HFn protein by size exclusion high performance liquid chromatography, and observe whether globulin and ferritin form aggregates (the small peaks before the characteristic peaks of globulin and ferritin are aggregate peaks).
  • Preparation of mobile phase Weigh 36.34g of Trizma base, add 2L of water, adjust the pH to 7.20 with hydrochloric acid, filter with a 0.2um microporous membrane, and sonicate it for 10 minutes to get it.
  • HFn Dilute the protein stock solution with a concentration of 13.3 mg/mL to a concentration of 2 mg/mL. Sampling 200 ⁇ L at 0h/4h/24h/48h into the injection vial, the injection volume is: 80 ⁇ L
  • CysKO-1-222-HFn will dilute the protein stock solution with a concentration of 3.1 mg/mL to a concentration of 2 mg/mL. Sampling 200 ⁇ L at 0h/4h/24h/48h into the injection vial, the injection volume is: 80 ⁇ L
  • Globulin Dilute the protein stock solution with a concentration of 50 mg/mL to a concentration of 2 mg/mL. Sampling 200 ⁇ L into the injection vial at 0h/4h/24h/48h respectively, the injection volume is: 80 ⁇ L
  • the testing equipment and parameters are as follows:
  • Detection wavelength 280nm, 254nm, 485nm
  • Test product H-ferritin-DOX, wild-type H-ferritin-DOX with the above 9 different mutation sequences, and H-ferritin empty carrier, wild-type H-ferritin empty carrier and free carrier with 9 different mutation sequences DOX.
  • the dose of the ferritin test substance in the experimental group was 100 mg/kg, and the test substance was administered on D1, D8, D15, D22, and D29 respectively. The whole test was administered 5 times.
  • the dose of free DOX is 5 mg/kg.
  • the route of administration is intravenous drip, the administration period is 4 weeks, once a week, and the recovery period is 2 weeks.
  • the number of animals in each group is 2 cynomolgus monkeys.
  • Dead/dying During the experiment, no animals in each experimental group were dead/dying.
  • Body weight, body temperature, ECG parameters and waveforms, blood pressure were not obvious Abnormal changes.
  • Blood coagulation function During the experiment, the blood coagulation function, cardiotoxicity index (cTNI, cTNT, BNP, NT-proBNP, CK-MB and SOD) and urinalysis were not obvious during the test period. Abnormal changes.
  • Histopathological examination euthanasia (D44) at the end of the dosing period, animal gross anatomy and histopathological examination showed no abnormal pathological changes related to the test article.
  • each test product has no obvious toxic and side effects to animals, and the maximum tolerated dose of animals is 200 mg/kg.

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Abstract

涉及生物医药领域。具体而言，涉及基于铁蛋白重链亚基的药物载体。更具体地，涉及铁蛋白重链亚基的突变体多肽、包含所述多肽的融合蛋白、包含所述多肽的笼状蛋白，以及它们作为药物载体的应用。

Description

基于铁蛋白重链亚基的药物载体 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及基于铁蛋白重链亚基的药物载体。更具体地，本发明涉及铁蛋白重链亚基的突变体多肽、包含所述多肽的融合蛋白、包含所述多肽的笼状蛋白，以及它们作为药物载体的应用。
发明背景
铁蛋白(Ferritin)是一种大约450kDa的大蛋白，由24个亚基自组装成球形笼状结构，其内部和外部尺寸分别为大约8和大约12nm，笼状结构容纳包含多达4500个铁原子的铁核。真核生物铁蛋白包含重链(H；21kDa)和轻链(L；19kDa)。H链负责Fe(II)氧化成Fe(III)并包括催化性铁氧化酶位点，而L链在铁成核中发挥作用。H和L链共同组装成24聚体的杂聚体铁蛋白，其中H链与L链的比例根据组织特异性分布而不同。
由于其具有可包裹药物的笼状结构、显著的稳定性、较小且均一的尺寸，本领域已经尝试将铁蛋白用作药物载体以递送药物，例如可见Ferritin-based drug delivery systems:Hybrid nanocarriers for vascular immunotargeting,Makan Khoshnejad et.al,Journal of Controlled Release 282(2018)13-24。
WO2015180325A1描述了仅由H链自组装成的铁蛋白(H-铁蛋白)，由于其能够与受体TfR1结合而靶向肿瘤细胞，从而可以用作肿瘤特异性给药的药物载体。该申请没有涉及铁蛋白亚基的突变体。WO2018153372A1则教导了H-铁蛋白可用作能够穿越血脑屏障的纳米药物载体。该申请提及了铁蛋白亚基的突变体，然而仅仅教导这些突变体没有影响与TfR1的结合以及TfR1介导的转运作用。WO2018073593A1公开了铁蛋白可以作为药物载体，其内腔可包入阿霉素。该申请通过修饰铁蛋白亚基，使得铁蛋白亚基在没有成核试剂存在的情况下不能组装形成铁蛋白笼。
现有技术没有教导在用作药物载体时，需要改变铁蛋白的储铁能力或如何改变铁蛋白的储铁能力。天然铁蛋白具有储铁功能，在其用作药物载体而进入体内后，如其仍旧保留天然储铁功能，可能会影响机体正常铁代谢，存在铁代谢紊乱的风险。另外，有报道天然的铁氧化酶活性可能抑制人类成骨细胞的发育和成熟，并导致矿化降低、骨质减少和骨质疏松(Zarjou A,Jeney V,Arosio P,Poli M,Zavaczki E,Balla G,BallaJ.Ferritin ferroxidase activity:a potent inhibitor of osteogenesis.J BoneMiner Res.2010,25:164-72)。另一方面，若用作药物载体的铁蛋白还保留储铁的功能，也可能降低其药物包载量和包载效率。
此外，铁蛋白的H亚基上存在3个半胱氨酸残基(有2个位于铁蛋白的外表面，1个位于铁蛋白的三重对称轴的位置)。由于半胱氨酸末端的巯基是异常活跃的基团，其 不仅容易被氧化形成二硫键，使得铁蛋白形成聚集体，还可能与多种体内分子发生反应，从而一方面引发脱靶、副作用等安全性问题，另一方面降低了药物的有效浓度，造成药效不足。因此，具有活跃巯基的半胱氨酸的存在阻碍了铁蛋白在药物载体方面的应用。
因此，本领域仍然需要新的基于铁蛋白的药物载体，其具有降低的储铁能力，和/或能够消除半胱氨酸带来的负面影响，从而具有提高的安全性和/或增加的药物包装能力，同时保留肿瘤靶向和血脑屏障穿透能力。
发明简述
在一方面，本发明提供一种铁蛋白重链(H)亚基突变体多肽，其
i)包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和/或第65位的位置处的氨基酸取代，和/或
ii)在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含除半胱氨酸外的氨基酸残基。
在一些实施方案中，所述铁蛋白重链(H)亚基突变体多肽包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和/或第65位的位置处的氨基酸取代，且在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含除半胱氨酸外的氨基酸残基。
在一方面，本发明提供一种融合蛋白，其包含本发明的铁蛋白H亚基突变体多肽，以及至少一个额外的多肽部分。
在一方面，本发明提供一种笼状蛋白，其包含至少一个本发明的铁蛋白H亚基突变体多肽或本发明的融合蛋白。
在一方面，本发明提供一种笼状蛋白-药物活性成分(API)复合物，其包含本发明的笼状蛋白，以及装载在所述笼状蛋白内或与所述笼状蛋白偶联的药物活性成分(API)。
在一方面，本发明提供一种药物组合物，其包含本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白或本发明的笼状蛋白-API复合物，以及药学上可接受的赋形剂。
在一方面，本发明提供本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白、本发明的笼状蛋白-API复合物或本发明的药物组合物在制备药物中的用途。
在一方面，本发明提供一种在对象中治疗和/或预防疾病的方法，所述方法包括给所述对象施用有效量的本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白、笼状蛋白-API复合物或本发明的药物组合物。
在一方面，本发明提供一种制备本发明的笼状蛋白-API复合物的方法，所述方法包括使本发明的笼状蛋白与API接触，由此获得笼状蛋白-API复合物。
在一方面，本发明提供一种分离的多核苷酸，其包含编码本发明的铁蛋白H亚基突变体多肽或本发明的融合蛋白的核苷酸序列。
在一方面，本发明提供一种表达构建体，其包含本发明的多核苷酸。
在一方面，本发明提供一种宿主细胞，其包含本发明的多核苷酸或由本发明的表达构建体转化，且能够表达本发明的铁蛋白H亚基突变体多肽或融合蛋白。
在一方面，本发明提供一种产生本发明的铁蛋白H亚基突变体多肽或本发明的融合蛋白的方法，包括：
a)在允许所述突变体多肽或融合蛋白表达的条件下培养本发明的宿主细胞；
b)从得自步骤a)的培养物获得由所述宿主细胞表达的所述突变体多肽或融合蛋白；及
c)任选进一步纯化得自步骤b)的突变体多肽或融合蛋白。
附图简述
图1.示出大肠杆菌表达后纯化的铁蛋白H亚基铁装载相关位点突变体的SDS-PAGE图。
图2.示出纯化的铁蛋白H亚基铁装载相关位点突变体的Native-PAGE图像。
图3.示出纯化的铁蛋白H亚基铁装载相关位点突变体的电镜图像。
图4.示出不同H-铁蛋白铁装载相关位点突变体的铁含量比较。
图5.示出不同铁蛋白铁装载相关位点突变体的受体结合活性比较。
图6.示出不同铁蛋白铁装载相关位点突变体的药物装载能力。
图7.示出大肠杆菌表达后纯化的铁蛋白H亚基半胱氨酸突变体的SDS-PAGE图。
图8.示出纯化的铁蛋白H亚基半胱氨酸突变体的电镜图像。
图9.示出不同铁蛋白半胱氨酸突变体的受体结合活性比较。
图10.示出不同H-铁蛋白半胱氨酸突变体的铁含量比较。
图11.示出施用不同H-铁蛋白半胱氨酸突变体对食蟹猴白细胞的改变。
图12.示出施用不同H-铁蛋白半胱氨酸突变体对食蟹猴红细胞的改变。
图13.示出纯化的铁蛋白H亚基组合突变体的电镜图像。
图14.示出H-铁蛋白组合突变体的稳定性分析结果。
图15.示出不同H-铁蛋白组合突变体的铁含量比较。
图16.示出不同铁蛋白组合突变体的药物装载能力。
图17.示出不同铁蛋白组合突变体的装载药物后的稳定性分析。
图18.A-H示出不同铁蛋白组合突变体的非特异性结合分析。
图19.示出反向色谱法分析不同铁蛋白组合突变体样品的结果。
发明详述
一、定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语 和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
“铁蛋白”是指由蛋白外壳和铁内核两部分构成的储铁结构。天然情况下，铁蛋白的蛋白外壳是通常由24个亚基自组装形成的笼状蛋白结构(外径12nm，内径8nm)，而铁内核的主要成分为水铁矿。不含铁内核的铁蛋白的蛋白外壳也称为“去铁蛋白”。本文所述“铁蛋白”包括真核生物铁蛋白和原核生物铁蛋白，优选真核生物铁蛋白，更优选哺乳动物铁蛋白，例如人铁蛋白。真核生物铁蛋白通常包括重链H亚基和轻链L亚基。在机体不同组织和器官中，铁蛋白分子中含有H和L亚基的比例有所不同。然而，通过重组方式，也可以获得仅由H亚基组装成的“H铁蛋白(HFn)”或仅由L亚基组装成的“L铁蛋白(LFn)”。
“笼状蛋白”，也称作“纳米笼”，是指由多个能够自组装的多肽(亚基)形成的具有内部中央空腔的三维蛋白结构，即笼状结构。组装成笼状蛋白的多肽(亚基)数目没有特别限制，只要其能够形成所述笼状结构。笼状蛋白可以具有对称结构，也可以具有非对称结构，取决于其亚基组成。典型的笼状蛋白包含铁蛋白/去铁蛋白。
如本文所用，“亲水氨基酸”包括半胱氨酸(C)、天冬氨酸(D)、天冬酰胺(N)、谷氨酸(E)、组氨酸(H)、赖氨酸(K)、谷氨酰胺(Q)、精氨酸(R)、甘氨酸(G)、丝氨酸(S)和苏氨酸(T)。
如本文所用，“疏水氨基酸”包括丙氨酸(A)、苯丙氨酸(F)、异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、脯氨酸(P)、缬氨酸(V)、酪氨酸(Y)和色氨酸(W)。
“多肽”、“肽”、和“蛋白质”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然氨基酸的聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白质”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。
如本文所用，“多核苷酸”是指多个核苷酸通过磷酸二酯键连接而成的大分子，其中所述核苷酸包括核糖核苷酸和脱氧核糖核苷酸。本发明的多核苷酸的序列可以针对不同的宿主细胞(如大肠杆菌)进行密码子优化，从而改善多肽的表达。进行密码子优化的方法是本领域已知的。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。此外，本领域技术人员清楚多肽N端由起始密码子编码的甲硫氨酸在某些实际情况下(例如在特定表达系统表达时)会被保留，但不实质影响多肽的功能。因此，本申请说明书和权利要求书中在描述具体的多肽氨基酸序列时，尽管其可能不包含N端由起始密码子编码的甲硫氨酸，然而此时也涵盖包含该甲硫氨酸 的序列。相应地，其编码核苷酸序列也可以包含起始密码子。
两个多肽序列或两个多核苷酸序列之间的“序列相同性”指的是所述序列之间的相同的氨基酸或核苷酸的百分比。评估多肽或多核苷酸序列之间的序列相同性水平的方法是本领域已知的。序列相同性可以使用已知的各种序列分析软件评估。例如，序列相同性可以通过EMBL-EBI的在线比对工具来评估(https://www.ebi.ac.uk/Tools/psa/)。两个序列之间的序列相同性可以使用Needleman-Wunsch算法，使用默认参数来评估。
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，可以是能够翻译的RNA(如mRNA)。通常，在表达构建体中，感兴趣的核苷酸序列与调控序列可操作地连接。
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关感兴趣的序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。
如本文中所用，术语“可操作地连接”指调控序列与目的核苷酸序列连接，使得目的核苷酸序列的转录被所述调控序列控制和调节。用于将调控序列可操作地连接于目的核苷酸序列的技术为本领域已知的。
如本文所用，“药物活性成分”或“活性药物成分”或“API(Active pharmaceutical ingredient)”指的是药物中具有药理活性或能够直接影响机体功能的物质。通常而言，“药物活性成分”并不包含药物载体或赋形剂。
本文使用的“药学上可接受的赋形剂”是指在配制药物产品中所用的没有药理活性且无毒的任意成分，包括但不限于崩解剂、粘合剂、填充剂、缓冲剂、张力剂、稳定剂、抗氧化剂、表面活性剂或润滑剂。
如本文所用，“有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病的症状所必需的量。
二、铁蛋白重链(H)亚基突变体多肽
不束缚于任何理论，据认为人铁蛋白H亚基中的第27位、第61位、第62位、和第65位氨基酸参与铁的装载。例如，第27位的谷氨酸、第62位的谷氨酸、第65位的组氨酸据认为是构成H铁蛋白的铁氧中心；而第61位的谷氨酸据认为是H铁蛋白的铁离子通道中结合铁离子的重要氨基酸。本发明人发现，通过突变铁蛋白重链(H)亚基中这些特定位置的氨基酸，可以降低所形成的铁蛋白的储铁能力，从而使得所述铁蛋白在用作药物载体时具有更高的安全性，并且这样的突变并不影响铁蛋白组装以及其与TfR1 受体的结合。
此外，野生型人铁蛋白H亚基中的第90位和第102位的半胱氨酸位于H铁蛋白的外表面。而第130位的半胱氨酸则位于铁蛋白的三重对称轴，其可能涉及铁离子或药物的装载。如本申请实施例所公开的，令人惊奇地发现，将所述三个半胱氨酸突变后，可以显著减少铁蛋白聚集、减少氧化反应、减少非特异性结合，从而提高其作为药物载体的安全性。
因此，在一个方面，本发明提供一种铁蛋白重链(H)亚基突变体多肽，其
i)包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和/或第65位的位置处的氨基酸取代；和/或
ii)在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含除半胱氨酸之外的氨基酸残基。
在一些实施方案中，所述铁蛋白重链(H)亚基突变体多肽包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和/或第65位的位置处的氨基酸取代，且在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含除半胱氨酸之外的氨基酸残基。
本发明的突变体多肽所衍生自的铁蛋白H亚基(也可称作起始铁蛋白H亚基)可以是天然存在的(野生型)铁蛋白H亚基。例如，野生型人铁蛋白H亚基可以包含SEQ ID NO:1所示氨基酸序列。然而，本发明的突变体多肽所衍生自的铁蛋白H亚基也可能是经过人工改造的(经修饰的)铁蛋白H亚基。本领域已知多种经人工改造的铁蛋白H亚基，其都可用来获得本发明的突变体多肽。已知的人工改造的铁蛋白H亚基的实例包括但不限于WO2018153372A1、WO2014104768、WO2016051340A1、WO2014123399A1、WO2017039382A1、WO2016122259A1、CN109486827A、CN107286249A、CN104059156A、CN101942023A、CN104017088A、CN105801706A、US7097841B2中所公开的那些。本发明的突变体多肽所衍生自的铁蛋白H亚基至少具有天然存在的铁蛋白H亚基的部分功能活性，所述部分功能活性包括但不限于组装成笼状蛋白的能力和/或使笼状蛋白特异性结合TfR1的能力等。
在一些实施方案中，本发明的突变体多肽所衍生自的铁蛋白H亚基包含与SEQ ID NO:1具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。在一些实施方案中，本发明的突变体多肽所衍生自的铁蛋白H亚基在对应于SEQ ID NO:1的第90位、第102位、和第130位的一或多个位置处包含半胱氨酸。在一些实施方案中，本发明的突变体多肽所衍生自的铁蛋白H亚基在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含半胱氨酸。在一些具体实施方案中，本发明的突变体多肽所衍生自的铁蛋白H亚基多肽包含SEQ ID NO:1所示氨基酸序列。
在一些实施方案中，所述突变体多肽包含在对应于SEQ ID NO:1的第61位的位置处的氨基酸取代。
在一些实施方案中，所述突变体多肽包含在对应于SEQ ID NO:1的第62位和第65 位的位置处的氨基酸取代。
在一些实施方案中，所述突变体多肽包含在对应于SEQ ID NO:1的第61位、第62位、和第65位的位置处的氨基酸取代。
在一些实施方案中，所述突变体多肽包含在对应于SEQ ID NO:1的第27位、第62位、和第65位的位置处的氨基酸取代。
在一些优选实施方案中，所述突变体多肽包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和第65位的位置处的氨基酸取代。
在一些实施方案中，所述氨基酸取代是被疏水性氨基酸取代或被野生型铁蛋白轻链(L)亚基相应位置处的氨基酸取代。
用疏水氨基酸取代铁氧中心或铁离子通道的关键氨基酸，一方面可以占据铁离子通道，避免铁的装载，另一方面将有利于疏水药物的装载。合适的疏水氨基酸包括苯丙氨酸、甘氨酸、异亮氨酸、亮氨酸、甲硫氨酸、缬氨酸、丙氨酸、酪氨酸和色氨酸，优选为苯丙氨酸或色氨酸。
“野生型铁蛋白轻链(L)亚基”意指在天然状态下与野生型铁蛋白H亚基多肽组装成铁蛋白的L亚基。例如，人野生型人铁蛋白L亚基多肽包含SEQ ID NO:36所示氨基酸序列，也可参见NCBI Genbank Seq No:NP_000137.2。
野生型铁蛋白轻链(L)亚基单独形成的L铁蛋白不具有储铁能力，因此用其相应位置处的氨基酸进行取代，将在有利于在减少铁装载的同时保持所组装成的笼状结构。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸。在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸。在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸。在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸。
在一些实施方案中，所述突变体多肽是人铁蛋白H亚基的突变体，且相比于SEQ ID NO:1，包含氨基酸取代E27F、E61W、E62K和/或H65G。
在本文中，当描述氨基酸取代时，示例性的表述“E27F”表示的是在第27位的氨基酸E被F取代。其它氨基酸取代也类似地表述。
在一些实施方案中，所述突变体多肽是人铁蛋白H亚基的突变体，且相比于SEQ ID NO:1，包含选自以下的一组氨基酸取代：
(i)E61W；
(ii)E62K和H65G；
(iii)E27F、E61W、E62K和H65G；
(iv)E27F、E62K和H65G；和
(v)E61W、E62K和H65G。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第90位的位置处包含除半胱氨酸之外的亲水氨基酸。所述亲水氨基酸可以选自天冬氨酸(D)、天冬酰胺(N)、谷氨酸(E)、组氨酸(H)、赖氨酸(K)、谷氨酰胺(Q)、精氨酸(R)、甘氨酸(G)、丝氨酸(S)和苏氨酸(T)。由于与半胱氨酸空间结构最相似，优选为丝氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第102位的位置处包含除半胱氨酸之外的亲水氨基酸。所述亲水氨基酸可以选自天冬氨酸(D)、谷氨酸(E)、组氨酸(H)、赖氨酸(K)、谷氨酰胺(Q)、精氨酸(R)、甘氨酸(G)、丝氨酸(S)和苏氨酸(T)。在一些优选实施方案中，所述亲水氨基酸是丝氨酸。
在一些优选实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第130位的位置处包含疏水氨基酸。所述疏水氨基酸可以选自丙氨酸(A)、苯丙氨酸(F)、异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、脯氨酸(P)、缬氨酸(V)、酪氨酸(Y)或色氨酸(W)。在一些具体实施方案中，所述疏水氨基酸是甲硫氨酸。在一些具体实施方案中，所述疏水氨基酸是酪氨酸。在另一些实施方式中，所述突变体多肽在对应于SEQ ID NO:1的第130位的位置处包含除半胱氨酸之外的亲水氨基酸，所述亲水氨基酸可以选自天冬氨酸(D)、天冬酰胺(N)、谷氨酸(E)、组氨酸(H)、赖氨酸(K)、谷氨酰胺(Q)、精氨酸(R)、甘氨酸(G)、丝氨酸(S)和苏氨酸(T)。在一些优选实施方案中，所述亲水氨基酸是组氨酸。在一些优选实施方案中，所述亲水氨基酸是丝氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含丝氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含组氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含甲硫氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含酪氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含丝氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含丝氨酸。(对应于实施例组合1)
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含丝氨酸。(对应于实施例组合2)
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含丝氨酸。(对应于实施例组合3)
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含甲硫氨酸。(对应于实施例组合4)
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含甲硫氨酸。(对应于实施例组合5)
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含甲硫氨酸。(对应于实施例组合6)
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应 于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含甲硫氨酸。
在一些实施方案中，所述突变体多肽在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸，在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含甲硫氨酸。
在一些具体实施方案中，所述突变体多肽包含选自SEQ ID NO:2-5、10-14、和20-25之一的氨基酸序列。
在一些实施方案中，所述突变体多肽能够组装成笼状蛋白和/或能够在组装成笼状蛋白后赋予所述笼状蛋白特异性结合TfR1受体的能力。
在一些实施方案中，所述突变体多肽通过重组产生，即所述突变体多肽是重组多肽。下文进一步描述了重组产生本发明的突变体多肽的方法。
本领域已有报道，铁蛋白H亚基突变体多肽可以与其他序列融合，赋予其额外的特性但是不影响其自组装活性或受体结合活性。铁蛋白H亚基多肽与其它序列融合的实例包括但不限于WO2014104768A1、WO2016051340A1、WO2014123399A1、WO2017039382A1、WO2016122259A1、CN109486827A、CN107286249A、CN104059156A、CN101942023A、CN104017088A、CN105801706A、US7097841B2中公开的那些。
因此，在另一方面，本发明提供一种融合蛋白，其包含上文所述的本发明的铁蛋白H亚基突变体多肽，和至少一个额外的多肽部分。在一些实施方案中，在所述融合蛋白中，本发明的铁蛋白H亚基突变体多肽与所述至少一个额外的多肽部分直接连接或通过接头连接。
在一些实施方案中，所述额外的多肽部分是能够赋予所述融合蛋白额外的特性的多肽。例如所述额外的特性例如易于表达或纯化、不同的靶向性、细胞毒性、示踪性、延长的体内半衰期、易组装性等。
在一些实施方案中，所述额外的多肽部分是蛋白标签。所述蛋白标签包括但不限于His标签。在一些实施方案中，所述额外的多肽部分是示踪多肽，例如GFP等。在一些实施方案中，所述额外的多肽部分是体内半衰期延长多肽，例如血清白蛋白。在一些实施方案中，所述额外的多肽部分是靶向肽，例如抗体、蛋白配体等。在一些实施方案中，所述额外的多肽部分是治疗性多肽。合适的治疗性多肽包括但不限于抗体如单链抗体、细胞毒素、细胞因子、抗微生物肽如抗病毒肽或抗菌肽、肿瘤抗原片段等。
三、多核苷酸、表达构建体、宿主细胞和多肽制备方法
在另一方面，本发明提供一种分离的多核苷酸，其包含编码本发明的铁蛋白H亚基突变体多肽或融合蛋白的核苷酸序列。
在一些优选实施方式中，本发明的多核苷酸包含例如选自SEQ ID NO:6-9、15-19、和26-31之一的核苷酸序列。
在另一方面，本发明提供了一种表达构建体，其包含与表达调控序列可操作地连接的本发明的多核苷酸。
用于本发明的表达构建体的载体包括那些在宿主细胞中自主复制的载体，如质粒载体；还包括能够整合到宿主细胞DNA中并和宿主细胞DNA一起复制的载体。可商购获得许多适于本发明的载体。在一个具体实施方案中，本发明的表达构建体衍生自Novagen公司的pET22b。
在另一方面，本发明提供一种宿主细胞，其含有本发明的多核苷酸或以本发明的表达构建体转化，其中所述宿主细胞能够表达本发明的铁蛋白H亚基突变体多肽或融合蛋白。优选地，所述宿主细胞是重组宿主细胞。
可用于表达本发明的多肽或融合蛋白的宿主细胞包括原核生物、酵母和高等真核细胞。示例性的原核宿主包括埃希氏菌属(Escherichia)、芽孢杆菌属(Bacillus)、沙门氏菌属(Salmonella)以及假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)的细菌。在优选的实施方案中，宿主细胞是埃希氏菌属细胞，优选是大肠杆菌。在本发明的一个具体实施方案中，所使用的宿主细胞为大肠杆菌BL21(DE3)菌株细胞。
可以通过许多已熟知的技术之一将本发明的重组表达构建体导入宿主细胞，这样的技术包括但不限于：热激转化，电穿孔，DEAE-葡聚糖转染，显微注射，脂质体接介导的转染，磷酸钙沉淀，原生质融合，微粒轰击，病毒转化及类似技术。
在另一方面，本发明提供了一种重组产生本发明的铁蛋白H亚基突变体多肽或融合蛋白的方法，包括：
a)在允许铁蛋白H亚基突变体多肽或融合蛋白表达的条件下培养本发明的宿主细胞；
b)从得自步骤a)的培养物获得由所述宿主细胞表达的铁蛋白H亚基突变体多肽或融合蛋白；及
c)任选进一步纯化得自步骤b)的铁蛋白H亚基突变体多肽或融合蛋白。
然而，本发明的铁蛋白H亚基突变体多肽或融合蛋白也可以通过化学合成的方法获得。
四、笼状蛋白
由于保留了野生型铁蛋白H亚基的自组装能力和/或受体结合能力，本发明的铁蛋白H亚基突变体多肽或融合蛋白在重组产生后，可以独自组装成笼状蛋白(即H铁蛋白/去铁蛋白)，也可以与铁蛋白L亚基或其他自组装多肽形成笼状蛋白，并且能够赋予所述笼状蛋白特异性靶向能力。
因此，在另一方面，本发明提供一种笼状蛋白，其包含至少一个本发明的铁蛋白H亚基突变体多肽或融合蛋白。
示例性的所述笼状蛋白可以包含例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、36、或48个本发明的铁蛋白H亚基突变体多肽或融合蛋白。在一些优选实施方案中，所述笼状蛋白包含24个本发明的铁蛋白H亚基突变体多肽或融合蛋白。
在一些实施方案中，所述笼状蛋白仅包含本发明的铁蛋白H亚基突变体多肽或融合蛋白。例如，在一些优选实施方案中，所述笼状蛋白由24个本发明的铁蛋白H亚基突变体多肽或融合蛋组装形成。
在一些实施方案中，所述笼状蛋白还包含铁蛋白L亚基。在一些实施方案中，所述笼状蛋白包含至少一个本发明的铁蛋白H亚基突变体多肽或融合蛋白和至少一个铁蛋白L亚基，优选地，所述铁蛋白H亚基突变体多肽或融合蛋白与铁蛋白L亚基的比例范围例如可以是1:23-23:1。
在一些实施方案中，所述笼状蛋白不包含铁蛋白L亚基。
在一些实施方案中，所述笼状蛋白还可以包含额外的修饰，例如PEG化修饰。
在一些实施方案中，与相应的包含野生型铁蛋白H亚基的笼状蛋白相比，本发明的笼状蛋白具有显著降低的储铁能力，例如其储铁能力降低至相应的野生型铁蛋白的大约60％、大约50％、大约40％、大约30％、大约20％、大约10％、大约5％或甚至更低。不同笼状蛋白的储铁能力可以通过在相同条件下测定其铁含量来评估，例如参见本申请实施例。
在一些实施方案中，与相应的包含野生型铁蛋白H亚基的笼状蛋白相比，本发明的笼状蛋白具有增加的稳定性，特别是在装载药物活性成分(Active pharmaceutical ingredient,API)后。
在一些实施方案中，与相应的包含野生型铁蛋白H亚基的笼状蛋白相比，本发明的笼状蛋白具有减少的非特异性结合，例如，减少的与血清中的蛋白质(例如白蛋白或球蛋白)的非特异性结合。与白蛋白或球蛋白结合水平可以通过本申请实施例的方法进行测定。
在一些实施方案中，与相应的包含野生型铁蛋白H亚基的笼状蛋白相比，本发明的笼状蛋白具有相当的或者更高的装载能力。在一些具体实施方案中，所述装载能力是指将物质如API包封在所述笼状蛋白的内部中央空腔结构的能力。笼状蛋白的装载能力可以通过如本申请实施例描述的方法测定。
在一些实施方案中，与相应的包含野生型铁蛋白H亚基的笼状蛋白相比，本发明的笼状蛋白具有相当的受体结合能力，例如TfR1受体结合能力。TfR1受体结合能力例如可以参照本申请实施例的方法进行测定。
五、作为药物载体的应用
本发明的笼状蛋白具有显著降低的储铁能力，增加的稳定性，减少的非特异性结合，从而具有增加的药物安全性，并且保留了合适的药物装载能力以及TfR1介导的肿瘤靶 向能力和血脑屏障穿透能力，因此特别适合于作为药物载体。
因此，在另一方面，本发明提供一种笼状蛋白-API复合物，其中所述笼状蛋白-API复合物包含本发明的笼状蛋白，以及装载在所述笼状蛋白内部或偶联至所述笼状蛋白的药物活性成分(API)。
所述药物活性成分(API)并没有特别限制，只要其适合于装载于本发明的所述笼状蛋白中或偶联至所述笼状蛋白，例如，所述API不会破坏笼状蛋白的笼状结构和/或其大小适合于被所述笼状结构容纳。所述API的实例包括但不限于烷基化剂，如亚硝基脲类；铂类，如顺铂、卡铂及其衍生物；抗代谢类，如胸苷酸合成酶抑制剂；肿瘤抗生素类药物如阿霉素、道诺霉素、柔红霉素；天然提取物，如植物碱(长春花碱)；激素类，如抗雌激素类(他莫替芬)；放射性药物如64Cu、235U；神经递质类药物，如碳酰胆碱、阿托品、东莨菪碱、多巴胺及其衍生物；多巴胺受体激动剂，如溴隐亭、培高利特、阿扑吗啡等麦角碱类衍生物及非麦角碱类衍生物；神经中枢抗胆碱药，如苯海索、苯扎托品及丙环定；胆碱受体激动剂类药物，如毒蕈碱、毛果芸香碱；γ分泌酶抑制剂如双氟酮类；抗氧剂，如褪黑激素；麻醉剂，如蒽胺。此外，除了融合表达外，治疗性多肽例如抗体也可以通过例如化学修饰的方式偶联至所述笼状蛋白。在一些具体实施方案中，所述API是阿霉素。
将API与所述笼状蛋白偶联可以通过各种方法实现，例如本领域已知的那些方法，包括但不限于通过与笼状蛋白表面游离的氨基、羧基发生反应将API偶联至笼状蛋白表面。在一些实施方式中，API可通过接头例如化学接头或肽接头与笼状蛋白偶联。在一些实施方式中，API还可以通过非共价方式偶联至笼状蛋白表面，例如通过范德华力、氢键、离子键等偶联。
在另一方面，本发明提供一种药物组合物，其包含本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白或本发明的笼状蛋白-API复合物，以及药学上可接受的赋形剂。
在一些实施方案中，药物组合物包含本发明的铁蛋白H亚基突变体多肽或本发明的融合蛋白，以及有效量的API，其中所述铁蛋白H亚基突变体多肽或融合蛋白以未组装成笼状蛋白的形式提供。所述铁蛋白H亚基突变体多肽或融合蛋白，可以在体外或在递送至体内后，在适合条件下，自组装为笼状蛋白-API复合物。
在一些实施方案中，在本发明的融合蛋白包含治疗性多肽的情况下，包含本发明的融合蛋白的药物组合物可以不包含额外的API。
本发明的铁蛋白H亚基突变体多肽、融合蛋白、笼状蛋白、笼状蛋白-API复合物或药物组合物可以用于治疗和/或预防的疾病取决于其所包含的治疗性多肽或API。并且，由于本发明的笼状蛋白具有肿瘤靶向能力和血脑屏障穿透能力，特别适合于治疗肿瘤或脑部疾病。
脑部疾病的实例包括但不限于，例如脑瘤、阿尔茨海默病、帕金森病、脑卒中、癫痫、亨廷顿病及肌萎缩侧索硬化症。所述肿瘤的实例包括但不限于，例如结直肠癌、肺 癌、乳腺癌、卵巢癌、黑色素瘤、胃癌、胰腺癌、膀胱癌、肾癌、前列腺癌以及各种造血系统癌如Hodgkin氏疾病、非Hodgkin氏淋巴癌、白血病。
在另一方面，本发明提供本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白、本发明的笼状蛋白-API复合物或本发明的药物组合物在制备药物中的用途。在一些实施方案中，所述药物例如用于治疗肿瘤或脑部疾病。
在另一方面，本发明提供一种在对象中治疗和/或预防疾病的方法，所述方法包括给所述对象施用有效量的本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白、笼状蛋白-API复合物或本发明的药物组合物。所述疾病如上文所定义，优选是肿瘤或脑部疾病。
本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白、本发明的笼状蛋白、本发明的笼状蛋白-API复合物或本发明的药物组合物可通过本领域普通技术人员已知的任何适当方法进行施用(参见例如，Remington：The Science and Practice of Pharmacy，”第21版，2005)。药物组合物例如可通过静脉内、肌肉内、腹腔内、脑脊髓内、皮下、关节内、滑膜腔内、鞘内、口服、局部或吸入途径施用。
六、制备笼状蛋白-API复合物的方法
在另一方面，本发明提供一种制备本发明的笼状蛋白-API复合物的方法，所述方法包括使本发明的铁蛋白H亚基突变体多肽、本发明的融合蛋白或本发明的笼状蛋白与API接触，由此获得笼状蛋白-API复合物。
在一些实施方案中，所述方法包括：
a)使解聚的本发明的笼状蛋白与API接触；和
b)重组装所述笼状蛋白，由此获得笼状蛋白-API复合物。
如本发明所用，“解聚”指的是在一定条件下，笼状蛋白的紧密闭合球状结构被打开，使其全部亚基或部分亚基相互分离的过程，所述条件例如是蛋白变性条件，例如含高浓度脲的缓冲溶液。
如本发明所用，“重组装”指的是通过改变条件例如更换成生理学相容条件，使解聚的笼状蛋白，即分离的亚基重新自组装成笼状蛋白的过程。在笼状蛋白的重组装过程中，API将会被包裹在其内部，从而形成笼状蛋白-API复合物。所述生理学相容条件例如是在生理缓冲溶液中。
在一些实施方案中，所述方法在步骤a)之前还包括使本发明的笼状蛋白解聚的步骤。在一些实施方案中，其中通过在高浓度(例如至少6M，优选8M)的脲存在下使本发明的笼状蛋白解聚。在一些实施方案中，其中通过逐步降低脲浓度(例如梯度透析)来重组装所述笼状蛋白。
在一些实施方案中，所述方法包括：
a)在非解聚条件下，使本发明的笼状蛋白与API接触，由此允许API偶联至所述笼状蛋白，和/或装载至所述笼状蛋白的内部中央空腔，
b)获得笼状蛋白-API复合物。
在一些实施方式中，所述非解聚条件包括使所述笼状蛋白和API置于生理可接受的缓冲液中。合适的生理可接受的缓冲液包括但不限于PBS溶液、生理盐水、纯水、HEPES缓冲液等。
在一些实施方案中，API通过非共价或共价相互作用结合至所述笼状蛋白。所述非共价相互作用例如范德华力、氢键、离子键等。所述共价相互作用包括与笼状蛋白表面的游离氨基、羧基发生反应如缩合反应。
在一些实施方案中，API通过被动扩散穿梭至所述笼状蛋白的内部中央空腔。通过使所述笼状蛋白和API至于生理可接受的缓冲液中，API可以在笼状蛋白不解聚的情况下通过扩散的方式进入笼状蛋白的内部内腔中。
实施例
通过参考在此给出的一些具体实施例可获得对本发明的进一步的理解，这些实施例仅用于说明本发明，其无意于对本发明的范围做出任何限制。显然，可以对本发明作出多种改动和变化而不脱离本发明的实质，因此，这些改动和变化同样在本申请要求保护的范围内。
实施例1、构建具有改变的储铁能力的突变的人H-铁蛋白
1.1铁蛋白H亚基突变的设计
根据人铁蛋白H亚基的野生型氨基酸序列(SEQ ID NO:1；参见PDB:3AJQ_A)设计H亚基突变体的氨基酸序列，对H亚基中可能涉及铁装载的位点进行了突变。第27位的谷氨酸(E27)、第62位的谷氨酸(E62)和第65位的组氨酸(H65)据认为是参与H-铁蛋白的铁氧中心，而第61位的谷氨酸(E61)则是H-铁蛋白中铁离子通道中可能与铁离子结合的重要氨基酸。所有的氨基酸位置均参考SEQ ID NO:1。
发明人对这几个位点的氨基酸进行了不同组合的突变。所述位点的氨基酸可以替换成疏水性氨基酸，以期占据铁离子通道或者利于疏水性药物的装载(例如E27和E61可以被F或W取代)。同时，为了尽可能保持笼状结构，也考虑将所述位点的氨基酸替换成L亚基上对应的氨基酸(例如E62和H65分别被K和G取代)。具体设计见表1。所得亚基突变体分别命名为Mut1(SEQ ID NO:2)、Mut2(SEQ ID NO:3)、Mut3(SEQ ID NO:4)、Mut4(SEQ ID NO:5)。
表1、突变体的设计
Figure PCTCN2020101312-appb-000001
获得突变的氨基酸序列后，针对大肠杆菌对其编码序列进行了密码子优化。Mut1-4的密码子优化的核苷酸序列分别示于SEQ ID NO:6-9。在优化的核苷酸序列前加NdeI酶切位点，在该序列之后加终止密码子和BamH1酶切位点序列，通过全基因合成，测序正确后，连接至pET22b表达质粒载体中，获得含有上述4个突变体编码核苷酸序列的质粒。野生型的按照同样方式进行质粒构建。
1.2 H-铁蛋白的制备
上述质粒用BL21(DE3)大肠杆菌作为宿主细胞诱导表达后、经过蛋白纯化获得铁蛋白H亚基多肽。多肽纯化的表征见图1的SDS-PAGE分析。
此外，还采用Native-PAGE(非变性聚丙烯酰胺凝胶)法检测了突变的H亚基多肽在非变性条件下的状态。结果表明(图2)，制备获得的H亚基多肽主要以单体的形式存在，分子量与野生型相同，说明铁蛋白H亚基突变体与野生型H亚基一样，能够自组装形成24聚体的天然笼形结构的H铁蛋白。
然后，将蛋白样品(20μL，0.1mg/mL)滴加到处理后的铜网中，用1％的乙酸铀酰染色1分钟，然后用JEM-1400 80kv TEM(JEOL，Japan)成像。透射电镜结果(图3)同样表明了突变的H亚基多肽与野生型H亚基一样能够自组装形成外径在12nm的24聚体笼状蛋白(即H-铁蛋白(Hfn))，分别称作WT-HFn、Mut1-HFn、Mut2-HFn、Mut3-HFn、Mut4-HFn。
1.3含铁量测定
实验步骤：分别测定前述制备的蛋白样品(WT-HFn、Mut1-HFn、Mut2-HFn)的蛋白浓度，而后稀释为1mg/ml的蛋白浓度，取1ml，置于干净的聚四氟乙烯烧杯中，加入浓硝酸2mL，120度加热赶酸到2mL，使铁蛋白完全裂解，释放出铁离子，并将铁离子都氧化为3价铁形式，用超纯水稀释定定容到10mL，取10微升样品用ICP-OES 730(Agilent)测试，根据三价铁元素的标准峰测定Fe元素含量。
结果如表2和图4所示，突变体H-铁蛋白的储铁能力均明显下降，Mut2-HFn突变体下降最明显，效果最好。此外，令人惊讶的是，Mut2的蛋白表达量似乎显著提高，且铁含量显著更低。
表2、不同突变体含铁量结果
蛋白 蛋白浓度(mg/ml) 1mg铁蛋白铁含量(μg/mg)
WT-HFn 1.77 2.8
Mut1-HFn 1.7 0.28
Mut2-HFn 2.88 0.14
1.4稳定性分析实验
分别取1ml蛋白浓度为1mg/ml的蛋白样品(WT-HFn、Mut1-HFn、Mut2-HFn)，置 于干净的1.5ml EP管中，在37度恒温箱中放置一周。一周后取出，取10微升样品在高效液相层析系统通过凝胶过滤层析柱SEC分析铁蛋白单体和聚体峰，流动相：50mM Tris buffer，pH7.0。
结果如表3和表4所示：37度放置一周后Mut2-HFn和野生型的稳定性显著比Mut1-HFn好，聚体无明显增加。
表3
Figure PCTCN2020101312-appb-000002
表4
Figure PCTCN2020101312-appb-000003
1.5与Trf1受体结合活性
使用包被液(碳酸缓冲液，pH9.0)将各组铁蛋白稀释到20微克/毫升，将稀释的样品混匀后，按照实验设计加入到酶标板中，100μL/孔，每个样品三个复孔，放至4℃，过夜。然后将酶标板用1×PBST洗3次，1×PBS洗2次。加入封闭液(5％脱脂奶粉)300μL/孔进行封闭。37℃培养箱中孵育2h。之后将酶标板用1×PBST洗3次，1×PBS洗2次。TFR1(人源)用蛋白稳定剂(购自湖州英创生物科技有限公司，PR-SS-002)稀释至2μg/mL(1：100)，按100μL/孔添加。37℃培养箱中孵育2h。将酶标板用1×PBST洗3次，1×PBS洗2次。抗-TFR1抗体(鼠源)(购买自北京义翘神州科技有限公司：11020-MM02)用蛋白稳定剂稀释至1μg/mL(1:1000)，按100μL/孔添加。37℃培养箱中孵育1h。将酶标板用1×PBST洗3次，1×PBS洗2次。anti-mouse IgG用HRP偶联稳定剂稀释(1:5000)，按100μL/孔添加。37℃培养箱中孵育1h。将酶标板用1×PBST洗3次，1×PBS洗3次。避光加入TMB一步显色液，100μL/孔，立即用酶标仪测定OD 652nm，模式为kinetic，5min/次，连续测定60min。用Graphpad 6.0软件分析原始数据，选取时间点15分钟和30分钟做成柱状图，纵坐标为吸收652nm值，横坐标为H铁蛋白(HFn)样品包被浓度。BSA和无结合活性的L铁蛋白(LFn)作为对照。
结果如表5和图5所示，突变的铁蛋白的受体结合活性与对照相比，无显著变化。
表5、Trf1受体结合活性
Figure PCTCN2020101312-appb-000004
1.6突变的H铁蛋白的API装载能力
浓度为1mg/ml的H铁蛋白溶解在8M脲中(Sinnopharm Chemical Reagent Co.Ltd.)，并在室温条件下以轻轻震荡的方式保证完全溶解。阿霉素(DOX)被加入到该溶液中，其 终浓度为1mg/ml。避光孵育10min后，混合溶液转移到透析袋中(截留分子量为3000Da，Termo Scientific)，透析液为含有1mg/ml的DOX的梯度脲缓冲液(7M、5M、3M、2M、1M和0M，每个浓度4h)，让DOX在4℃条件缓慢进入重新组装的铁蛋白笼中。产物然后采用生理盐水透析过夜，终止反应并去除游离的DOX。DOX包被到H铁蛋白笼中的终浓度采用Superdex 20010/300 GL柱(GE公司)连接SEC系统(Amersham Parmacia Biotech)来测定(流动相：50mM Tris buffer,pH7.0)。H铁蛋白在280nm的UV条件有吸收峰，而DOX在485nm下有吸收峰。比值越小，表明阿霉素装载量越高。
结果如图6显示，突变的铁蛋白的API装载能力与对照相比，无显著变化。
1.7突变体综合评估
对突变体评分共涉及4项参数，每项分别设置0分和1分：对照自身效果好的给分1，效果差的给分0；突变体与对照相比无显著差异，或比对照效果好的评分给1分，突变体与对照相比效果变差的给0分。结果见表6所示，综合考虑，Mut2表现最优。
表6
Figure PCTCN2020101312-appb-000005
实施例2、构建半胱氨酸突变的人H-铁蛋白
2.1铁蛋白H亚基突变的设计
根据人铁蛋白H亚基的野生型氨基酸序列(SEQ ID NO:1；参见PDB:3AJQ_A)设计H亚基突变体的氨基酸序列。将半胱氨酸突变为结构和理化性质与其最相近的丝氨酸，由此将生理活跃的SH基团换成不活跃的OH。三重对称轴位置的半胱氨酸突变为疏水性氨基酸(对疏水性药物装载有利)。作为对照，构建全部半胱氨酸突变为疏水性甲硫氨酸的突变体，以对比不同突变体的效果。
表7、突变体的设计(所有的氨基酸位置均参考SEQ ID NO:1)
Figure PCTCN2020101312-appb-000006
获得突变的氨基酸序列后，针对大肠杆菌对其编码序列进行了密码子优化。各突变体的密码子优化的核苷酸序列分别示于SEQ ID NO:15-19。在优化的核苷酸序列前加起始密码子和NdeI酶切位点，在该序列之后加终止密码子和BamH1酶切位点序列，通过 全基因合成，测序正确后，连接至pET22b表达质粒载体中，获得含有上述5个突变体编码核苷酸序列的质粒。
2.2 H-铁蛋白的制备
上述质粒用BL21(DE3)大肠杆菌作为宿主细胞诱导表达后、经过蛋白纯化获得铁蛋白H亚基多肽。多肽纯化方法包括以下步骤：将诱导表达后的大肠杆菌菌体用20mM Tris(pH8.0)缓冲液重悬后，超声裂解破碎菌体；离心(1500rpm，10min)去除大肠杆菌菌体碎片；上清液72℃加热15分钟；沉淀杂蛋白，离心去除沉淀；上清液在排阻色谱Superdex200pg柱上分离纯化；SDS-PAGE电泳鉴定纯度；BCA测定蛋白浓度。多肽纯化的表征见图7的SDS-PAGE分析。
然后，将蛋白样品(20μL，0.1mg/mL)滴加到处理后的铜网中，用1％的乙酸铀酰染色1分钟，然后用JEM-1400 80 kv TEM(JEOL，Japan)成像。透射电镜结果(图8)表明，突变的H亚基多肽与野生型H亚基多肽均可形成均匀、规则的笼状蛋白结构，直径为大约12nm。
2.3稳定性分析实验
分别取1ml蛋白浓度为1mg/ml的蛋白样品(WT-HFn、CysKO-1-HFn、CysKO-2-HFn、CysKO-3-HFn、CysKO-4-HFn、CysKO-5-HFn)，置于干净的1.5ml EP管中，每种蛋白设置两个管，即分为两组，每组6管。第一组样品立即取10微升样品，用HPLC-SEC分析铁蛋白单体和聚体峰，流动相：50mM Tris缓冲液，pH7.0。第二组样品在37度恒温箱中放置一周后取出，用同样的方法检测。
结果显示：CysKO-2-HFn突变体稳定性较差，经过长时间储存后聚集体含量增加，其余四种突变体稳定性良好(参见下表8-9)。
表8
Figure PCTCN2020101312-appb-000007
表9
Figure PCTCN2020101312-appb-000008
2.4与Trf1受体结合活性
使用包被液(碳酸缓冲液，pH9.0)将各组铁蛋白稀释到20微克/毫升，将稀释的样品混匀后，按照实验设计加入到酶标板中，100μL/孔，每个样品三个复孔，放至4℃冰箱，过夜。然后将酶标板用1×PBST洗3次，1×PBS洗2次。加入封闭液(5％脱脂奶粉)300μL/孔进行封闭。37℃培养箱中孵育2h。之后将酶标板用1×PBST洗3次，1×PBS洗2次。TFR1(人源)用蛋白稳定剂(购自湖州英创生物科技有限公司，PR-SS-002)稀释至2μg/mL(1：100)，按100μL/孔添加。37℃培养箱中孵育2h。将酶标板用1×PBST洗3次，1×PBS洗2次。抗-TFR1抗体(鼠源)(购买自北京义翘神州科技有限公司：11020-MM02)用蛋白稳定剂稀释至1μg/mL(1:1000)，按100μL/孔添加，37℃培养箱中孵育1h。将酶标板用1×PBST洗3次，1×PBS洗2次。anti-mouse IgG用HRP偶联稳定剂稀释(1:5000)，按100μL/孔添加。37℃培养箱中孵育1h。将酶标板用1×PBST洗3次，1×PBS洗3次。避光加入TMB一步显色液，100μL/孔，立即用酶标仪测定OD 652nm。用Graphpad 6.0软件分析原始数据，选取时间点15分钟和30分钟做成柱状图，纵坐标为吸收652nm值，横坐标为H铁蛋白(HFn)样品包被浓度。BSA和无结合活性的L铁蛋白(LFn)作为对照。
结果如表10和图9所示，半胱氨酸突变的铁蛋白的受体结合活性与对照(野生型)相比，无显著变化。
表10
Figure PCTCN2020101312-appb-000009
2.5含铁量测定
实验步骤：分别测定实施例1制备的蛋白样品(WT-HFn、CysKO-1-HFn、CysKO-2-HFn、CysKO-3-HFn、CysKO-4-HFn、CysKO-5-HFn)的蛋白浓度，而后稀释为1mg/ml的蛋白浓度，取1ml，置于干净的聚四氟乙烯烧杯中，加入浓硝酸2mL，120度加热赶酸到2mL，使铁蛋白完全裂解，释放出铁离子，并将铁离子都氧化为3价铁形式，用超纯水稀释定定容到10mL，取10微升样品用ICP-OES 730(Agilent)测试，根据三价铁元素的标准峰测定Fe元素含量。
结果如表11和图10所示，出乎意料的是，虽然突变位点并不位于铁氧中心，但本发明的突变体与野生型对照相比，含铁量均有明显下降。含铁量明显降低使得本发明的铁蛋白作为药物载体在进入机体后，对机体自身铁代谢的影响降低，提高用药的安全性。
表11
Figure PCTCN2020101312-appb-000010
Figure PCTCN2020101312-appb-000011
2.6突变的H铁蛋白的药物装载能力
浓度为1mg/ml的H铁蛋白溶解在8M脲中(Sinnopharm Chemical Reagent Co.Ltd.)，并在室温条件下以轻轻震荡的方式保证完全溶解。阿霉素(DOX)被加入到该溶液中，其终浓度为1mg/ml。避光孵育10min后，混合溶液转移到透析袋中(截留分子量为3kDa，Termo Scientific)，透析液为含有1mg/ml的DOX的梯度脲缓冲液(7M、5M、3M、2M、1M和0M，每个浓度4h)，让DOX在4℃条件下缓慢进入重新组装的铁蛋白笼中。产物采用生理盐水透析过夜，终止反应并去除游离的DOX。DOX包被到H铁蛋白笼中的终浓度采用Superdex 20010/300 GL柱(GE公司)连接SEC系统(Amersham Parmacia Biotech)来测定(流动相：50mM Tris buffer,pH7.0)。H铁蛋白在280nm的UV条件有吸收峰，而DOX在485nm下有吸收峰。比值越小，表明阿霉素装载量越高。
结果如表12显示，半胱氨酸突变的铁蛋白的药物装载能力与对照相比，无显著变化。
2.7装载阿霉素后铁蛋白的稳定性
实验方法：采用本实施例前述实验方法，即分别取1ml蛋白浓度为1mg/ml的铁蛋白-阿霉素样品(已装载阿霉素的WT-HFn、CysKO-1-HFn、CysKO-2-HFn、CysKO-3-HFn、CysKO-4-HFn、CysKO-5-HFn样品)，置于干净的1.5ml EP管中。取10微升样品，用HPLC-SEC分析铁蛋白单体和聚体峰，流动相：50mM Tris缓冲液，pH7.0。
实验结果：除CysKO-2-HFn外，另外4种突变体在装载DOX后，与野生型相比，单体的纯度明显提高，这说明半胱氨酸突变能够提高药物装载后铁蛋白的稳定性(参见表12)。
表12：
Figure PCTCN2020101312-appb-000012
2.8突变体综合评估
对突变体评分共涉及4项参数，每项分别设置0分和1分：对照自身效果好的给分1，效果差的给分0；突变体与对照相比无显著差异，或比对照效果好的评分给1分，突变体与对照相比效果变差的给0分。结果见表13。
表13
Figure PCTCN2020101312-appb-000013
CysKO-1、CysKO-3、CysKO-4、CysKO-5评分相同。这几种突变体各有优势：CysKO-1-HFn的稳定性最好；CysKO-5-HFn的含铁量最低；CysKO-3-HFn和CysKO-5-HFn与Trf1结合的活性保持得更好。
2.9在食蟹猴中的毒性检测
实验目的：
观察本实施例的突变体给药后，对动物是否产生明显的毒副作用。
实验方法：
(1)供试品：测试供试品铁蛋白空载体CysKO-1-HFn、CysKO-5-HFn、WT-HFn和载带DOX的CysKO-1-HFn-DOX，CysKO-5-HFn-DOX、WT-HFn-DOX对于食蟹猴血液系统的影响。
(2)给药方法：以给药前三天(D-3)检测的指标为基础参照值。分别于D1给予100mg/kg剂量的铁蛋白供试品，于D8、D15、D22、D29给予200mg/kg剂量的供试品，整个试验共给药5次。给药途径为静脉滴注，最后一次给药后恢复期2周，每组食蟹猴动物数为2只。
(3)观察指标：临床观察、体重、血压、心电图、摄食量、临床病理。
实验结果：
白细胞的结果如图11所示。从结果来看，突变铁蛋白在空载状态下和载药状态(DOX)下均不同程度引起食蟹猴白细胞降低，但影响程度小于野生型，安全性更好。
红细胞的结果如图12所示。从结果来看，突变铁蛋白在空载状态下和载药状态(DOX)下均不同程度引起食蟹猴红细胞降低，但给药结束后可不同程度恢复，影响程度小于野生型，安全性更好。
血生化：与给药前(D-3)比较，200mg/kg剂量重复静脉输注给予食蟹猴后可见ALT升高，Alb和CHO降低，提示后续试验中关注动物肝脏相关指标的改变；Fe和FER 升高，考虑可能与供试品的药理学作用相关。
凝血功能、心脏毒性指标、尿液分析：试验期间，动物各时间段的凝血功能、心脏毒性指标(cTNI、cTNT、BNP、NT-proBNP、CK-MB和SOD)和尿液分析均未见明显异常改变。
组织病理学检查：给药期结束安乐死(D44)，动物大体解剖及组织病理学检查均未见供试品相关的异常病理改变。
实施例3、构建组合突变的人H-铁蛋白
3.1铁蛋白H亚基突变的设计
根据人铁蛋白H亚基的野生型氨基酸序列(SEQ ID NO:1；参见PDB:3AJQ_A)设计H亚基突变体的氨基酸序列，对H亚基中可能涉及铁装载的位点和半胱氨酸进行了突变。第27位的谷氨酸(E27)、第62位的谷氨酸(E62)和第65位的组氨酸(H65)据认为是参与H-铁蛋白的铁氧中心，而第61位的谷氨酸(E61)则是H-铁蛋白中铁离子通道中可能与铁离子结合的重要氨基酸。第90、102和130位半胱氨酸被认为影响铁蛋白稳定性，引发脱靶等安全性问题。所有的氨基酸位置均参考SEQ ID NO:1。
发明人对这几个位点的氨基酸进行了不同组合的突变。例如，第27位的谷氨酸(E27)、第62位的谷氨酸(E62)和第65位的组氨酸(H65)可以替换成疏水性氨基酸，以期占据铁离子通道或者利于疏水性药物的装载(例如E27和E61可以被F或W取代)。同时，为了尽可能保持笼状结构，也考虑将第27位的谷氨酸(E27)、第62位的谷氨酸(E62)和第65位的组氨酸(H65)替换成L亚基上对应的氨基酸(例如E62和H65分别被K和G取代)。此外第90、102和130位半胱氨酸突变为结构和理化性质与其最相近的丝氨酸，由此将生理活跃的-SH基团换成不活跃的-OH基，此外还将三重对称轴位置的半胱氨酸突变成疏水氨基酸作为对照。具体设计见表14。所得亚基突变体分别命名为CysKO-1-Mut2-HFn(SEQ ID NO:20)、CysKO-1-222-HFn(SEQ ID NO:21)、CysKO-1-Mut1-HFn(SEQ ID NO:22)、CysKO-2-Mut2-HFn(SEQ ID NO:23)、CysKO-5-Mut2-HFn(SEQ ID NO:24)和CysKO-5-222-HFn(SEQ ID NO:25)。
表14、突变体的设计
Figure PCTCN2020101312-appb-000014
Figure PCTCN2020101312-appb-000015
获得突变的氨基酸序列后，针对大肠杆菌对其编码序列进行了密码子优化。密码子优化的核苷酸序列分别示于SEQ ID NO:26-31。在优化的核苷酸序列前加BamH1酶切位点，在该序列之后加终止密码子和HindIII酶切位点序列，通过全基因合成，测序正确后，连接至pET22b表达质粒载体中，获得含有上述6个突变体编码核苷酸序列的质粒。对照按照同样方式进行质粒构建。
3.2H-铁蛋白的制备
上述质粒用BL21(DE3)大肠杆菌作为宿主细胞诱导表达后、经过蛋白纯化获得铁蛋白H亚基多肽，例如参照CN102127166A。
将蛋白样品(20μL，0.1mg/mL)滴加到处理后的铜网中，用1％的乙酸铀酰染色1分钟，然后用JEM-1400 80 kv TEM(JEOL，Japan)成像。透射电镜结果(图13)同样表明了突变的H亚基多肽与野生型H亚基一样能够自组装形成外径在12nm的24聚体笼状蛋白。
3.3稳定性分析实验
分别配制蛋白浓度为2mg/ml的9种铁蛋白溶液和相同浓度的人血清白蛋白溶液。分别移取300μL的9种铁蛋白溶液于9个1.5mL EP管中，各EP管再加入同体积的人血清白蛋白溶液，混匀，即得1mg/mL的混合样品。以上混合样品做两组重复。其中一组样品取样10微升，立即在高效液相层析系统通过凝胶过滤层析柱SEC分析铁蛋白单体和聚体峰，流动相：50mM Tris buffer,pH7.0(“处理前”)。另外一组样品在37度恒温箱中放置一周后用同样的方法测定(“处理后”)。
结果如图14所示，组合1和组合2、组合5和组合6、对照1的突变体蛋白与人血清白蛋白37度共孵育1周后，蛋白聚体量与处理前相比无明显增加，而对照3(WT-HFn组)、对照2(222-HFn组)、组合3(CysKO-1-Mut1-HFn)和组合4(CysKO-2-Mut2-HFn)的单体与其他突变体相比，有不同程度的降低，单体比例不足90％。这可能提示着本发明的突变体作为药物载体进入机体后，与野生型和部分其他突变体相比，组合1-2、5-6在血流中与血液中的蛋白类分子发生相互作用产生聚集体的可能性更低，即非特异性结合能力较弱，特异性更好。
3.4含铁量测定
实验步骤：分别测定本实施例前述制备的蛋白样品的蛋白浓度，而后稀释为1mg/ml的蛋白浓度，取1ml，置于干净的聚四氟乙烯烧杯中，加入浓硝酸2mL，120度加热赶 酸到2mL，使铁蛋白完全裂解，释放出铁离子，并将铁离子都氧化为3价铁形式，用超纯水稀释定定容到10mL，取10微升样品用ICP-OES 730(Agilent)测试，根据三价铁元素的标准峰测定Fe元素含量。
结果如表15和图15所示，除对照1外，所有的突变体铁蛋白的装铁能力均大大减弱，即突变后的铁蛋白不影响机体铁代谢，能够提高其作为药物载体的生物安全性。具体为：相同发酵条件和纯化条件得到的突变体铁蛋白铁含量显著少于对照1和对照3。
表15、不同突变体含铁量结果
蛋白 蛋白浓度(mg/ml) 1mg铁蛋白铁含量(μg/mg)
WT-HFn(对照3) 2.02 2.9
222-HFn(对照2) 2.3 0.23
对照1 1.98 2.83
组合1 2.7 0.14
组合2 2.98 0.25
组合3 2.11 0.29
组合4 1.93 0.13
组合5 2.33 0.14
组合6 2.09 0.26
3.5与Trf1受体结合活性
使用包被液(碳酸缓冲液，pH9.0)将各组铁蛋白稀释到20微克/毫升，将稀释的样品混匀后，按照实验设计加入到酶标板中，100μL/孔，每个样品三个复孔，放至4℃，过夜。然后将酶标板用1×PBST洗3次，1×PBS洗2次。加入封闭液(5％脱脂奶粉)300μL/孔进行封闭。37℃培养箱中孵育2h。之后将酶标板用1×PBST洗3次，1×PBS洗2次。TFR1(人源)用蛋白稳定剂(购自湖州英创生物科技有限公司，PR-SS-002)稀释至2μg/mL(1：100)，按100μL/孔添加。37℃培养箱中孵育2h。将酶标板用1×PBST洗3次，1×PBS洗2次。抗-TFR1抗体(鼠源)(购买自北京义翘神州科技有限公司：11020-MM02)用蛋白稳定剂稀释至1μg/mL(1:1000)，按100μL/孔添加。37℃培养箱中孵育1h。将酶标板用1×PBST洗3次，1×PBS洗2次。anti-mouse IgG用HRP偶联稳定剂稀释(1:5000)，按100μL/孔添加。37℃培养箱中孵育1h。将酶标板用1×PBST洗3次，1×PBS洗3次。避光加入TMB一步显色液，100μL/孔，立即用酶标仪测定OD 652nm，模式为kinetic，5min/次，连续测定60min。用Graphpad 6.0软件分析原始数据，选取时间点15分钟和30分钟做成柱状图，纵坐标为吸收652nm值，横坐标为H铁蛋白(HFn)样品包被浓度。BSA和无结合活性的L铁蛋白(LFn)作为对照。
结果如图16所示，突变的铁蛋白的受体结合活性与对照相比，无显著变化。
3.6突变的H铁蛋白的API装载能力
浓度为1mg/ml的H铁蛋白溶解在8M脲中(Sinnopharm Chemical Reagent Co.Ltd.)，并在室温条件下以轻轻震荡的方式保证完全溶解。阿霉素(DOX)被加入到该溶液中，其终浓度为1mg/ml。避光孵育10min后，混合溶液转移到透析袋中(截留分子量为3000Da， Termo Scientific)，透析液为含有1mg/ml的DOX的梯度脲缓冲液(7M、5M、3M、2M、1M和0M，每个浓度4h)，让DOX在4℃条件缓慢进入重新组装的铁蛋白笼中。产物然后采用生理盐水透析过夜，终止反应并去除游离的DOX。DOX包被到H铁蛋白笼中的终浓度采用Superdex 20010/300 GL柱(GE公司)连接SEC系统(Amersham Parmacia Biotech)来测定(流动相：50mM Tris buffer,pH7.0)。H铁蛋白在280nm的UV条件有吸收峰，而DOX在485nm下有吸收峰。比值越小，表明阿霉素装载量越高。
结果如表16显示，突变的铁蛋白的API装载能力与对照相比，无显著变化。
3.7装载阿霉素后铁蛋白的稳定性
实验方法：分别取1ml蛋白浓度为1mg/ml的铁蛋白-阿霉素样品(如前述装载)，置于干净的1.5ml EP管中。取10微升样品，用HPLC-SEC分析铁蛋白单体和聚体峰，流动相：50mM Tris缓冲液，pH7.0。
实验结果如表16和图17所示，铁蛋白装载阿霉素药物后，组合1-2、5-6的单体比例明显高于WT及组合2-3，与对照1-2相比也略高。
表16
Figure PCTCN2020101312-appb-000016
3.8突变体综合评估
对突变体评分共涉及4项参数，每项分别设置0分和1分：对照自身效果好的给分1，效果差的给分0；突变体与对照相比无显著差异，或比对照效果好的评分给1分，突变体与对照相比效果变差的给0分。结果见表17：组合1，2，5，6评分最高。将组合1，2，5，6四个组合突变体再次评分：组合1，2，5，6四个突变体的差异主要体现在稳定性上，四个突变体稳定性比较结果为：组合2>组合1>组合5>组合6。因此组合2突变体突变效果最佳。
表17
Figure PCTCN2020101312-appb-000017
Figure PCTCN2020101312-appb-000018
3.9突变体与球蛋白/白蛋白的非特异性结合
发明人还考察HFn、CysK0-1-222-HFn(组合2)在37℃、50℃条件下分别与球蛋白相关作用的结果。采用体积分子排阻的高效液相色谱分析法分析HFn的蛋白质的纯度，观察球蛋白和铁蛋白是否形成聚体(球蛋白和铁蛋白特征峰前的小峰为聚体峰)。
样品信息
Figure PCTCN2020101312-appb-000019
色谱信息
色谱柱型号：Agilent Advance Bio SEC 300A 2.7
色谱柱编号：ARD-007
检测波长：280nm
柱温：25℃
样品盘温度：室温
洗脱方式：等度洗脱
实验过程
流动相的配制：称取Trizma base36.34g，加水2L，用盐酸调pH至7.20，用0.2um的微孔滤膜过滤，超声10min中，即得。
蛋白处理过程
1.HFn：将浓度为13.3mg/mL蛋白原液稀释至浓度为2mg/mL。分别在0h/4h/24h/48h取样200μL至进样小瓶中，进样量为：80μL
2.CysKO-1-222-HFn将浓度为3.1mg/mL蛋白原液稀释至浓度为2mg/mL。分别在0h/4h/24h/48h取样200μL至进样小瓶中，进样量为：80μL
3.球蛋白：将浓度为50mg/mL蛋白原液稀释至浓度为2mg/mL。分别在0h/4h/24h/48h 取样200μL至进样小瓶中，进样量为：80μL
CysKO-1-222-HFn与球蛋白的处理过程与进样计划
Figure PCTCN2020101312-appb-000020
实验结果(如图18所示)表明：在多个时间点，不同浓度配比情况下，野生型HFn均会与铁蛋白发生结合形成聚体，而突变体CysKO-1-222-HFn与野生型HFn相比，不与球蛋白结合产生聚体或产生聚体的量明显减少，说明突变后的CysKO-1-222-HFn与血清中的蛋白的非特异性结合降低，有效的提高了铁蛋白载体的药物有效浓度和安全性。在与白蛋白的结合实验中亦观察到相同的结果。
3.10反向色谱法分析不同铁蛋白组合突变体样品的成分性质
用50mM Tris-HCl，pH8.0含15％葡萄糖的缓冲液将待测突变铁蛋白CysKO-1-222-HFn/CysKO-1-Mut2-HFn和WT-HFn稀释至蛋白终浓度为4mg/mL，而后60℃水浴0min、20min后上样。
检测仪器及参数如下：
仪器：UPLC-UV
色谱柱ACQUITY UPLC BEH C18 1.7μm 2.1*100mm
进样量：1μl
流速：0.5mL/min
检测波长：280nm,254nm,485nm
柱温：35℃
流动相A：0.1％TFA(水配制)
流动相B：0.1％TFA(MeOH/CAN 1：4配制)
洗脱梯度：
Figure PCTCN2020101312-appb-000021
0分钟和20分钟的洗脱结果参见图19。由图可知，CysKO-1-222-HFn和CysKO-1-Mut2-HFn与WT-HFn相比，洗脱峰更集中，CysKO-1-Mut2-HFn的洗脱峰基本呈单峰，说明其亚基或氨基酸片段疏水性质更均一。
3.11在食蟹猴中的毒性检测
实验目的：
观察本发明的突变体给药后，对动物是否产生明显的毒副作用。
实验方法:
(1)供试品：上述9个不同突变序列的H铁蛋白-DOX、野生型H铁蛋白-DOX，以及9个不同突变序列的H铁蛋白空载体、野生型H铁蛋白空载体和游离DOX。铁蛋白供试品实验组的剂量为100mg/kg，分别于D1、D8、D15、D22、D29给予供试品，整个试验共给药5次。游离DOX的给药剂量为5mg/kg。给药途径为静脉滴注，给药期4周，每周1次，恢复期2周，每组食蟹猴动物数为2只。
(2)观察指标：临床观察、体重、血压、心电图、摄食量、临床病理，所有动物于末次药后2周(D44)实施安乐死并进行大体解剖观察，其中，大体异常组织及主要组织脏器进行组织病理学检查。
(3)实验结果:
死亡/濒死：试验期间，各实验组动物均未见死亡/濒死。
临床观察：试验期间，各实验动物一般临床观察和给药局部观察均未见供试品相关的异常。
体重、体温、心电图参数和波形、血压：试验期间，各实验组动物各时间段的体重、体温、心电图参数(心率、P-R间期、Q-T间期和QRS时限)和波形、血压均未见明显异常改变。
凝血功能、心脏毒性指标、尿液分析：试验期间，动物各时间段的凝血功能、心脏毒性指标(cTNI、cTNT、BNP、NT-proBNP、CK-MB和SOD)和尿液分析均未见明显异常改变。
组织病理学检查：给药期结束安乐死(D44)，动物大体解剖及组织病理学检查均未见供试品相关的异常病理改变。
综上所述，在本试验条件下，各供试品对动物无明显的毒副作用，动物最大耐受剂量为200mg/kg。

Claims (39)

  1. 一种铁蛋白重链(H)亚基突变体多肽，其
    i)包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和/或第65位的位置处的氨基酸取代，和/或
    ii)在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含除半胱氨酸外的氨基酸残基。
    例如，所述铁蛋白H亚基突变体多肽包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和/或第65位的位置处的氨基酸取代，且在对应于SEQ ID NO:1的第90位、第102位、和第130位的位置处包含除半胱氨酸之外的氨基酸残基。
  2. 权利要求1的铁蛋白H亚基突变体多肽，其包含在对应于SEQ ID NO:1的第61位的位置处的氨基酸取代。
  3. 权利要求1的铁蛋白H亚基突变体多肽，其包含在对应于SEQ ID NO:1的第62位、和第65位的位置处的氨基酸取代。
  4. 权利要求1的铁蛋白H亚基突变体多肽，其包含在对应于SEQ ID NO:1的第61位、第62位、和第65位的位置处的氨基酸取代。
  5. 权利要求1的铁蛋白H亚基突变体多肽，其包含在对应于SEQ ID NO:1的第27位、第62位、和第65位的位置处的氨基酸取代。
  6. 权利要求1的铁蛋白H亚基突变体多肽，其包含在对应于SEQ ID NO:1的第27位、第61位、第62位、和第65位的位置处的氨基酸取代。
  7. 权利要求1-6中任一项的铁蛋白H亚基突变体多肽，其中所述氨基酸取代是被疏水性氨基酸取代或被野生型铁蛋白轻链(L)亚基多肽相应位置处的氨基酸取代。
  8. 权利要求1-7中任一项的铁蛋白H亚基突变体多肽，其中在对应于SEQ ID NO:1的第27位的位置处的氨基酸被取代为苯丙氨酸，在对应于SEQ ID NO:1的第61位的位置处的氨基酸被取代为色氨酸，在对应于SEQ ID NO:1的第62位的位置处的氨基酸被取代为赖氨酸，和/或在对应于SEQ ID NO:1的第65位的位置处的氨基酸被取代为甘氨酸。
  9. 权利要求1-8中任一项的铁蛋白H亚基突变体多肽，其衍生自人铁蛋白H亚基，且相比于SEQ ID NO:1，包含氨基酸取代E27F、E61W、E62K和/或H65G。
  10. 权利要求9的铁蛋白H亚基突变体多肽，其包含选自以下的一组氨基酸取代：
    (i)E61W；
    (ii)E62K和H65G；
    (iii)E27F、E61W、E62K和H65G；
    (iv)E27F、E62K和H65G；和
    (v)E61W、E62K和H65G。
  11. 权利要求1-10中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1 的第90位和第102位的位置处包含除半胱氨酸之外的亲水氨基酸，优选丝氨酸。
  12. 权利要求1-11中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1的第130位的位置处包含除半胱氨酸之外的亲水氨基酸，例如组氨酸或丝氨酸。
  13. 权利要求1-11中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1的第130位的位置处包含疏水氨基酸，例如甲硫氨酸或酪氨酸。
  14. 权利要求1-10中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1的第90位、第102位和第130位的位置处包含丝氨酸。
  15. 权利要求1-10中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含组氨酸。
  16. 权利要求1-10中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸取代，在对应于SEQ ID NO:1的第130位的位置处包含甲硫氨酸。
  17. 权利要求1-10中任一项的铁蛋白H亚基突变体多肽，其在对应于SEQ ID NO:1的第90位和第102位的位置处包含丝氨酸，在对应于SEQ ID NO:1的第130位的位置处包含酪氨酸。
  18. 权利要求1的铁蛋白H亚基突变体多肽，其包含选自SEQ ID NO:2-5、10-14、和20-25之一的氨基酸序列。
  19. 一种融合蛋白，其包含权利要求1-18中任一项的铁蛋白H亚基突变体多肽，以及至少一个额外的多肽部分。
  20. 权利要求19的融合蛋白，其中权利要求1-18中任一项的铁蛋白H亚基突变体多肽与所述至少一个额外的多肽部分直接连接或通过接头连接。
  21. 权利要求19或20的融合蛋白，其中所述额外的多肽部分是蛋白标签，例如His标签；示踪多肽，例如GFP；体内半衰期延长多肽，例如血清白蛋白；靶向肽，例如蛋白配体；治疗性多肽，例如抗体、细胞毒素、细胞因子、抗微生物肽如抗病毒肽或抗菌肽、肿瘤抗原片段。
  22. 一种笼状蛋白，其包含至少一个权利要求1-18中任一项的铁蛋白H亚基突变体多肽或权利要求19-21中任一项的融合蛋白，优选地，所述笼状蛋白包含至少一个权利要求1-18中任一项的铁蛋白H亚基突变体多肽或权利要求19-21中任一项的融合蛋白与至少一个铁蛋白轻链(L)亚基，更优选地，所述铁蛋白H亚基突变体多肽或融合蛋白与铁蛋白L亚基的比例为1:23-23:1。
  23. 权利要求21的笼状蛋白，其包含24个权利要求1-18中任一项的铁蛋白H亚基突变体多肽或权利要求19-21中任一项的融合蛋白。
  24. 一种笼状蛋白-药物活性成分(API)复合物，其包含权利要求22-23中任一项的笼状蛋白，以及装载在所述笼状蛋白内或与所述笼状蛋白偶联的药物活性成分(API)。
  25. 权利要求24的笼状蛋白-API复合物，其中所述API选自烷基化剂，如亚硝基 脲类；铂类，如顺铂、卡铂及其衍生物；抗代谢类，如胸苷酸合成酶抑制剂；肿瘤抗生素类药物如阿霉素、道诺霉素、柔红霉素；天然提取物，如植物碱(长春花碱)；激素类，如抗雌激素类(他莫替芬)；放射性药物如64Cu、235U；神经递质类药物，如碳酰胆碱、阿托品、东莨菪碱、多巴胺及其衍生物；多巴胺受体激动剂，如溴隐亭、培高利特、阿扑吗啡等麦角碱类衍生物及非麦角碱类衍生物；神经中枢抗胆碱药，如苯海索、苯扎托品及丙环定；胆碱受体激动剂类药物，如毒蕈碱、毛果芸香碱；γ分泌酶抑制剂如双氟酮类；抗氧剂，如褪黑激素；麻醉剂，如蒽胺。
  26. 一种药物组合物，其包含权利要求1-18中任一项的铁蛋白H亚基突变体多肽、权利要求19-21中任一项的融合蛋白或权利要求22-23中任一项的笼状蛋白或权利要求24-25中任一项的笼状蛋白-API复合物，以及药学上可接受的赋形剂。
  27. 权利要求1-18中任一项的铁蛋白H亚基突变体多肽或权利要求19-21中任一项的融合蛋白或权利要求22-23中任一项的笼状蛋白或权利要求24-25的笼状蛋白-API复合物或权利要求26的药物组合物在制备药物中的用途。
  28. 权利要求27的用途，其中所述药物用于治疗和/或预防脑部疾病和/或肿瘤，所述脑部疾病例如是脑瘤、阿尔茨海默病、帕金森病、脑卒中、癫痫、亨廷顿病及肌萎缩侧索硬化症，所述肿瘤例如是结直肠癌、肺癌、乳腺癌、卵巢癌、黑色素瘤、胃癌、胰腺癌、膀胱癌、肾癌、前列腺癌以及各种造血系统癌如Hodgkin氏疾病、非Hodgkin氏淋巴癌、白血病。
  29. 一种制备权利要求24-25中任一项的笼状蛋白-API复合物的方法，所述方法包括使权利要求1-18中任一项的铁蛋白H亚基突变体多肽、权利要求19-21中任一项的融合蛋白或权利要求22-23中任一项的笼状蛋白与API接触，由此获得笼状蛋白-API复合物。
  30. 权利要求29的方法，其包括以下步骤：
    a)使解聚的权利要求22-23中任一项的笼状蛋白与API接触；和
    b)重组装所述笼状蛋白，由此获得笼状蛋白-API复合物。
  31. 权利要求30的方法，所述方法在步骤a)之前还包括使权利要求22-23中任一项的笼状蛋白解聚的步骤。
  32. 权利要求30或31的方法，其中通过在至少6M，优选8M的脲存在下使所述笼状蛋白解聚。
  33. 权利要求32的方法，其中通过逐步降低脲浓度(例如梯度透析)来重组装所述笼状蛋白。
  34. 权利要求29的方法，其包括以下步骤：
    a)在非解聚条件下，使权利要求22-23中任一项的笼状蛋白与API接触，由此允许API偶联至所述笼状蛋白和/或装载至所述笼状蛋白的内部中央空腔，
    b)获得笼状蛋白-API复合物。
  35. 权利要求35的方法，其中所述非解聚条件包括使所述笼状蛋白与API至于生 理可接受的缓冲液中。
  36. 分离的多核苷酸，其包含编码权利要求1-18中任一项的铁蛋白H亚基突变体多肽或权利要求19-21中任一项的融合蛋白的核苷酸序列，例如选自SEQ ID NO:8-13之一的核苷酸序列。
  37. 表达构建体，其包含权利要求36的多核苷酸。
  38. 宿主细胞，其包含权利要求36的多核苷酸或由权利要求36的表达构建体转化，且能够表达所述铁蛋白H亚基突变体多肽或融合蛋白。
  39. 产生权利要求1-18中任一项的铁蛋白H亚基突变体多肽或权利要求19-21中任一项的融合蛋白的方法，包括：
    a)在允许所述铁蛋白H亚基突变体多肽或融合蛋白表达的条件下培养权利要求38的宿主细胞；
    b)从得自步骤a)的培养物获得由所述宿主细胞表达的所述铁蛋白H亚基突变体多肽或融合蛋白；及
    c)任选进一步纯化得自步骤b)的铁蛋白H亚基突变体多肽或融合蛋白。
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