WO2022179536A1 - Ferritin heavy chain subunit mutant and application thereof - Google Patents

Abstract

The present invention relates to the field of biological medicines. Specifically, the present invention relates to a ferritin heavy chain subunit mutant and an application thereof. More specifically, the present invention relates to a ferritin heavy chain subunit mutant polypeptide, a fusion protein containing the polypeptide, a cage-shaped protein containing the polypeptide, and applications thereof as drug carriers.

Description

Ferritin heavy chain subunit mutants and their applications technical field

The present invention relates to the field of biomedicine. In particular, the present invention relates to ferritin heavy chain subunit mutants and uses thereof. More specifically, the present invention relates to ferritin heavy chain subunit mutant polypeptides, fusion proteins comprising said polypeptides, clathrin comprising said polypeptides, and their use as pharmaceutical carriers.

Background of the Invention

Ferritin is a large protein of approximately 450kDa, which self-assembles into spherical cage-like structures of 24 subunits with internal and external dimensions of about 8 and about 12 nm, respectively. The cage-like structure accommodates up to 4500 iron The iron core of an 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 a catalytic ferrooxidase site, while the L chain plays a role in iron nucleation. The H and L chains co-assemble into a 24-mer heteromeric ferritin, where the ratio of H chains to L chains varies according to tissue-specific distribution.

Due to ferritin's drug-encapsulating cage-like structure, remarkable stability, and small and uniform size, attempts have been made in the art to use ferritin as a drug carrier for drug delivery. The drug delivery method of ferritin can be to load the drug, such as the small molecule drug doxorubicin, in the cage-like cavity, for example, see H-ferritin–nanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection, Minmin Liang et al. .al, PNAS, vol 111(41), 2014, 14900-5. In addition, ferritin has the activity of binding to the receptor TfR1, which can target multiple tumor tissues with high TfR1 expression and can cross the blood-brain barrier . For example, WO2015180325A1 describes ferritin (H-ferritin), which is self-assembled from only H chains, and targets tumor cells due to its ability to bind to the receptor TfR1. WO2018153372A1 teaches that H-ferritin can be used as a nano-drug carrier capable of crossing the blood-brain barrier.

Another ferritin drug delivery method is to couple functional molecules on the outer surface of ferritin, which can carry more types of drug molecules compared with the inner package method that is encapsulated in the lumen of ferritin, and It is not limited by the internal space of ferritin, and there is no need to consider issues such as the release method of the drug in the cage reaching the target site, so the drug loading method has a broader application prospect.

The methods for coupling functional molecules on the outer surface of ferritin reported in the literature can be biologically, such as recombinant expression (refer to US20140234961A1), or chemically, such as Antibody–drug conjugates: targeting melanoma with cisplatin encapsulated in protein -cage nanoparticles based on human ferritin, Elisabetta Falvo et.al, Nanoscale, 2013, 5, 12278-12285. The method and process of recombinant expression are determined, and the product is clear. However, it has many steps, is greatly affected by the organism, has a long cycle and high cost. Every time a ferritin fusion expression drug is designed, it needs to go through the whole process of gene sequence design, protein expression purification, and impurity control. development. In addition, this method is difficult to simultaneously couple a variety of different functional molecules to the surface of ferritin, and its application scenarios and fields are limited. In contrast, it is more convenient and flexible to chemically couple drug molecules to the outer surface of ferritin, and various functional molecules can be coupled to the surface of ferritin with high throughput and rapidity through various chemical reactions, and simultaneously. Conjugate a variety of functional molecules.

The key to chemical conjugation is to provide optimized ferritin variants suitable for chemical conjugation scenarios. Because although wild-type ferritin already has good pH and temperature stability compared with other carrier proteins, and is easy to be prepared on a large scale by prokaryotic expression, it is still unavoidable in chemical reaction conditions. Problems such as polymerization, easy generation of impurities, low coupling efficiency (low bonding rate), and unstable coupling products. However, there is currently little research and development on ferritin mutants suitable for chemical conjugation scenarios, and there is an urgent need in this field.

Brief Description of Drawings

Figure 1. Shows the protein expression results of ferritin H subunit mutants.

Figure 2. Shows transmission electron microscopy results of ferritin H subunit mutant samples.

Figure 3. Shows the SEC profiles of Mut-HFn-212, Mut-HFn-241, Mut-HFn-242 and Mut-HFn-243.

Figure 4. Shows the TEM results of the mutants after conjugation with the small molecule drug SN38.

Figure 5. Shows the RP-HPLC profile of Mut-HFn-241 and Mut-HFn-243 conjugated small molecule drug SN38.

Figure 6. Shows SEC results of SN38 coupling products of Mut-HFn-212, Mut-HFn-233, Mut-HFn-241, Mut-HFn-242 and Mut-HFn-243.

Figure 7. Shows the TEM results of the mutants after conjugation with the small molecule drug CL2A-CM.

Figure 8. Shows SEC results of CM coupling products of Mut-HFn-212, Mut-HFn-233, Mut-HFn-241, Mut-HFn-242, Mut-HFn-243.

Figure 9. SN38 coupling products showing binding of Mut-HFn-212, Mut-HFn-203, Mut-HFn-233, Mut-HFn-241, Mut-HFn-242 and Mut-HFn-243 to TfR-1 Affinity determination results.

Figure 10. Shows the cell killing effect of Mut-HFn-241 and Mut-HFn-243 on MDA-MB-231 after conjugation with the small molecule drug SN38.

Figure 11. Shows the cell killing effect of Mut-HFn-241 and Mut-HFn-243 on HT-29 after conjugation with the small molecule drug SN38.

SUMMARY OF THE INVENTION

1. Definition

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are the terms and routine procedures widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "and/or" covers all combinations of the items linked by the term, as if each combination had been individually listed herein. For example, "A and/or B" covers "A", "A and B", and "B". For example, "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 consisting of a protein outer shell and an iron inner core. In nature, the protein shell of ferritin is a cage-like protein structure (12 nm in outer diameter and 8 nm in inner diameter) usually formed by self-assembly of 24 subunits, while the main component of the iron core is ferrihydrite. The protein coat of ferritin without an iron core is also called "apoferritin". As used herein, "ferritin" includes both eukaryotic and prokaryotic ferritin, preferably eukaryotic ferritin, more preferably mammalian ferritin, such as human ferritin. Eukaryotic ferritins generally include a heavy chain H subunit and a light chain L subunit. In different tissues and organs of the body, the proportion of H and L subunits contained in the ferritin molecule varies. However, by recombinant means, "H ferritin (HFn)" assembled only from H subunits or "L ferritin (LFn)" assembled from only L subunits can also be obtained.

The ferritin H subunits of the present invention include, but are not limited to, mammalian ferritin H subunits, such as human ferritin H subunits or equiferritin H subunits, preferably human ferritin H subunits. An exemplary wild-type human ferritin H subunit comprises the amino acid sequence set forth in SEQ ID NO:1.

"Cage protein", also known as "nano-cage", refers to a three-dimensional protein structure with an inner central cavity formed by multiple polypeptides (subunits) capable of self-assembly, that is, a cage-like structure. The number of polypeptides (subunits) assembled into a cage protein is not particularly limited as long as it can form the cage structure. Cage proteins can have symmetric or asymmetric structures, depending on their subunit composition. Typical clathrins contain ferritin/apo-ferritin.

"Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids. The term applies to amino acid polymers in which one or more of the amino acids is an artificial chemical analog of the corresponding natural amino acid, 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, hydroxylation and ADP-ribosylation.

As used herein, "polynucleotide" refers to a macromolecule in which multiple nucleotides are linked by phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (eg, E. coli) to improve the expression of the polypeptides. Methods for performing codon optimization are known in the art.

When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid Glycosides, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the initiation codon at the N-terminus of the polypeptide is retained in some practical situations (eg, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain the methionine encoded by the initiation codon at the N-terminus, the methionine containing the methionine is also covered at this time. sequence. Correspondingly, its encoding nucleotide sequence may also contain an initiation codon.

"Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of amino acids or nucleotides that are identical 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 a variety of 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/). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm, using default parameters.

As used in the present invention, "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. For example, expression of a nucleotide sequence can refer to transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of RNA into a precursor or mature protein. "Expression constructs" of the present invention may be linear nucleic acid fragments, circular plasmids, viral vectors, or, alternatively, may be RNA capable of translation (eg, mRNA). Typically, in an expression construct, the nucleotide sequence of interest is operably linked to regulatory sequences.

"Regulatory sequence" and "regulatory element" are used interchangeably and refer to a coding sequence upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) and affecting the transcription, RNA Processed or stable or translated nucleotide sequence. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

As used herein, the term "operably linked" refers to the linkage of a regulatory sequence to a nucleotide sequence of interest such that transcription of the nucleotide sequence of interest is controlled and regulated by the regulatory sequence. Techniques for operably linking regulatory sequences to nucleotide sequences of interest are known in the art.

As used herein, "pharmaceutical active ingredient" or "active pharmaceutical ingredient" or "API (Active pharmaceutical ingredient)" refers to a substance in a drug that has pharmacological activity or can directly affect the function of the body. Generally, a "pharmaceutically active ingredient" does not contain a pharmaceutical carrier or excipient.

"Pharmaceutically acceptable excipient" as used herein refers to any ingredient used in formulating a pharmaceutical product that is inactive and nontoxic, including but not limited to disintegrants, binders, fillers, buffers, Tonicity agents, stabilizers, antioxidants, surfactants or lubricants.

As used herein, an "effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of the disease.

2. Mutant polypeptides of ferritin heavy chain (H) subunit

In order to improve the properties of the ferritin heavy chain subunit as a drug carrier, the inventors have previously obtained improved mutants with mutations in the iron oxygen center and/or cysteine site (PCT/CN2020/101312).

As a drug carrier, ferritin can be chemically coupled to functional molecules (antibodies, tracer molecules, small peptides) through sulfhydryl (SH) groups on its surface to obtain ferritin-functional molecule conjugates. On the basis of the previous work, the inventors further modified the ferritin H subunit to provide a more flexible coupling site, reduce side reactions and improve the homogeneity of the coupling product, reduce the aggregation of ferritin, improve the The solubility of ferritin. Unexpectedly, the ferritin H subunit mutants of the present invention also have significantly improved chemical coupling reaction efficiency (bonding ratio).

Accordingly, in one aspect, the present invention provides a ferritin heavy chain (H) subunit mutant polypeptide comprising, relative to the wild-type ferritin H subunit, at position 98, position 98 corresponding to SEQ ID NO: 1 Amino acid substitutions at positions 108, and/or 156.

Without being bound by any theory, it is believed that substitution of amino acids at positions corresponding to positions 98, 108, and/or 156 of SEQ ID NO: 1 with more hydrophilic amino acids will increase ferritin hydrophilicity, reducing its aggregation.

In some embodiments, the amino acid at positions corresponding to positions 98, 108, and/or 156 of SEQ ID NO: 1 is substituted with a more hydrophilic amino acid, such as an amino acid with a carboxyl side chain . In some embodiments, the amino acid with a carboxyl side chain includes glutamic acid (E), aspartic acid (D), or histidine (H).

In some embodiments, the mutant polypeptide has an amino acid such as asparagine (N) substituted with aspartic acid (D) at the position corresponding to position 98 of SEQ ID NO: 1, also referred to as an N98D substitution . In some embodiments, the mutant polypeptide has an amino acid, eg, lysine (K), substituted with glutamic acid (E) at the position corresponding to position 108 of SEQ ID NO: 1, also referred to as a K108E substitution. In some embodiments, the mutant polypeptide has an amino acid, eg, arginine (R), substituted with histidine (H) at the position corresponding to position 156 of SEQ ID NO: 1, also referred to as a R156H substitution.

In some embodiments, the mutant polypeptide has an amino acid, eg, asparagine (N), substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO: 1, and at a position corresponding to SEQ ID NO: 1 An amino acid such as lysine (K) at position 108 of ID NO: 1 is substituted with glutamic acid (E).

In some embodiments, the mutant polypeptide has an amino acid, eg, asparagine (N), substituted with aspartic acid (D) at a position corresponding to position 98 of SEQ ID NO: 1, at a position corresponding to SEQ ID NO: 1 The amino acid at the position 108 of NO: 1, eg, lysine (K), is substituted with glutamic acid (E), and the amino acid at the position corresponding to the 156 position of SEQ ID NO: 1, eg, arginine (R) is substituted with histidine (H).

In some embodiments, the mutant polypeptide is comprised at positions corresponding to positions 27, 61, 62, and/or 65 of SEQ ID NO: 1 relative to the wild-type ferritin H subunit amino acid substitutions. Without being bound by any theory, based on previous findings, it is believed that amino acid substitutions at positions corresponding to positions 27, 61, 62 and/or 65 of SEQ ID NO: 1 can reduce the resulting The iron storage capacity of ferritin, so that the ferritin has higher safety when used as a drug carrier.

In some embodiments, the mutant polypeptide has a glutamic acid (E) substituted with a phenylalanine (F) at a position corresponding to position 27 of SEQ ID NO:1. In some embodiments, the mutant polypeptide has a glutamic acid (E) substituted for tryptophan (W) at a position corresponding to position 61 of SEQ ID NO:1. In some embodiments, the mutant polypeptide has an amino acid such as glutamic acid (E) substituted for lysine (K) at the position corresponding to position 62 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide has an amino acid, eg, histidine (H), substituted for glycine (G) at a position corresponding to position 65 of SEQ ID NO:1. In some embodiments, the mutant polypeptide has an amino acid, eg, glutamic acid (E), substituted for a lysine (K) at a position corresponding to position 62 of SEQ ID NO: 1, and at a position corresponding to SEQ ID NO: 1 An amino acid such as histidine (H) at position 65 of ID NO: 1 is substituted for glycine (G).

There are three sulfhydryl groups on the wild-type human ferritin H subunit, which are located in the Loop region between the second and third α-helices (the sulfhydryl group of the 90th cysteine in the wild-type human ferritin H subunit), and the third On the alpha helix (the sulfhydryl group of cysteine at position 102 of wild-type human ferritin H subunit), the fourth segment of the α helix is near the triple axis of symmetry (cysteine at position 130 of wild-type human ferritin H subunit). sulfhydryl). However, in the conjugation process, if there are multiple reaction sites, it is impossible to control the specific position of conjugation and the reaction ratio of functional molecules and HFn.

Thus, in some embodiments, the mutant polypeptide comprises reduced cysteine relative to the wild-type ferritin H subunit.

In some embodiments, the mutant polypeptide is substituted at least one of the cysteines at positions corresponding to positions 90, 102, and 130 of SEQ ID NO:1. In some embodiments, the cysteine is substituted with an amino acid selected from serine, threonine, asparagine, glutamine, glutamic acid, aspartic acid, lysine, arginine , histidine, alanine, glycine, preferably by serine or by the amino acid at the corresponding position of the wild-type ferritin light chain (L) subunit polypeptide. The amino acid sequence of an exemplary wild-type ferritin light chain (L) subunit polypeptide is set forth in SEQ ID NO:17.

In some embodiments, the mutant polypeptide comprises a cysteine at a position corresponding to position 90 of SEQ ID NO: 1 relative to the wild-type ferritin H subunit, and

The cysteine at the position corresponding to position 102 of SEQ ID NO: 1 is substituted, preferably by serine or alanine,

Optionally, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted, preferably by serine or alanine.

In some embodiments, the mutant polypeptide comprises a cysteine at a position corresponding to position 102 of SEQ ID NO: 1 relative to the wild-type ferritin H subunit, and

The cysteine at the position corresponding to position 90 of SEQ ID NO: 1 is substituted, preferably by serine or glutamic acid,

Optionally, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted, preferably by serine or alanine.

In addition, it is also possible to make each iron The protein subunit retains only one site for chemical conjugation.

In some embodiments, the mutant polypeptide comprises one cysteine in the loop region, a cysteine at a position corresponding to position 102 of SEQ ID NO: 1, relative to the wild-type ferritin H subunit The amino acid is substituted and, optionally, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted. In some embodiments, except for one cysteine in the loop region and optionally a cysteine at a position corresponding to position 130 of SEQ ID NO: 1, the mutant polypeptide does not comprise an additional cysteine cysteine. In some preferred embodiments, the mutant polypeptide does not contain cysteine outside the loop region.

In some embodiments, the mutant polypeptide has cysteines substituted at positions corresponding to positions 90 and 102 of SEQ ID NO: 1 relative to the wild-type ferritin H subunit, optionally at The cysteine at the position corresponding to position 130 of SEQ ID NO:1 is substituted; and the mutant polypeptide is substituted at positions 79, 80, 81, 82, 83, 84, The amino acid at one of positions 85, 86, 87, 88, 89 and 91 was substituted with cysteine. In some embodiments, the mutant polypeptide has cysteines substituted at positions corresponding to positions 90, 102, and 130 of SEQ ID NO: 1 relative to the wild-type ferritin H subunit; and The amino acid of the mutant polypeptide at a position corresponding to one of positions 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 91 of SEQ ID NO: 1 is replaced by a cysteine replace. In some embodiments, the mutant polypeptide is substituted with cysteine (C) at an amino acid, eg, arginine (R), at a position corresponding to position 79 of SEQ ID NO:1. In some embodiments, the mutant polypeptide has an amino acid, eg, isoleucine (I), substituted with cysteine at a position corresponding to position 80 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide has an amino acid, eg, phenylalanine (F), substituted with cysteine at a position corresponding to position 81 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide is substituted with cysteine at an amino acid, eg, leucine (L), at a position corresponding to position 82 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide has a cysteine substituted for an amino acid, eg, glutamine (Q), at a position corresponding to position 83 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide has an amino acid, eg, aspartic acid (D), substituted with a cysteine at a position corresponding to position 84 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide has an amino acid, eg, isoleucine (I), substituted with a cysteine at a position corresponding to position 85 of SEQ ID NO:1. In some embodiments, the mutant polypeptide has an amino acid, eg, lysine (K), substituted with cysteine at a position corresponding to position 86 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide has an amino acid, such as lysine (K), substituted with cysteine at a position corresponding to position 87 of SEQ ID NO:1. In some embodiments, the mutant polypeptide has an amino acid, eg, proline (P), substituted with a cysteine at a position corresponding to position 88 of SEQ ID NO:1. In some embodiments, the mutant polypeptide is substituted with cysteine at an amino acid, eg, aspartic acid (D), at a position corresponding to position 89 of SEQ ID NO: 1. In some embodiments, the mutant polypeptide is substituted with a cysteine at an amino acid, eg, aspartic acid (D), at a position corresponding to position 91 of SEQ ID NO:1.

In some specific embodiments, the mutant polypeptide comprises the amino acid sequence set forth in any of SEQ ID NOs: 5-8.

In some embodiments, the mutant polypeptide is capable of assembling into a clathrin and/or capable of conferring the ability of the clathrin to specifically bind the TfR1 receptor upon assembly into a clathrin.

3. Polynucleotides, expression constructs, host cells and methods for preparing ferritin H subunit mutant polypeptides

In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding a recombinant ferritin H subunit polypeptide of the present invention.

In some embodiments, the polynucleotides of the present invention comprise, for example, a nucleotide sequence selected from one of SEQ ID NOs: 14-16.

In another aspect, the present invention provides an expression construct comprising a polynucleotide of the present invention operably linked to an expression control sequence.

Vectors for use in the expression constructs of the present invention include those that replicate autonomously in host cells, such as plasmid vectors; also include vectors capable of integrating into and replicating with host cell DNA. Many vectors suitable for the present invention are commercially available. In a specific embodiment, the expression construct of the invention is derived from pET22b from Novagen.

In another aspect, the present invention provides a host cell comprising or transformed with an expression construct of the present invention, wherein the host cell is capable of expressing a ferritin H subunit mutant polypeptide of the present invention.

Host cells useful for expressing the ferritin H subunit mutant polypeptides of the invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, Salmonella, and Pseudomonas and Streptomyces. In a preferred embodiment, the host cell is an Escherichia cell, preferably E. coli. In a specific embodiment of the present invention, the host cells used are Escherichia coli BL21(DE3) strain cells.

The recombinant expression constructs of the present invention can be introduced into host cells by one of a number of well-known techniques including, but not limited to: heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, lipid In vivo mediated transfection, calcium phosphate precipitation, protoplast fusion, particle bombardment, viral transformation and similar techniques.

In another aspect, the present invention provides a method of producing a ferritin H subunit mutant polypeptide of the present invention, comprising:

a) culturing the host cell of the invention under conditions that permit expression of the polypeptide;

b) obtaining a polypeptide expressed by said host cell from the culture obtained in step a); and

c) Optionally further purify the polypeptide from step b).

However, the ferritin H subunit mutant polypeptides of the present invention can also be obtained by chemical synthesis.

4. Polypeptide Conjugates

In another aspect, the present invention provides a polypeptide conjugate comprising a ferritin H subunit mutant polypeptide of the present invention and a functional moiety conjugated thereto through a sulfhydryl group of the ferritin H subunit mutant polypeptide.

In some embodiments, the functional moiety is selected from a therapeutic molecule, a detectable molecule, or a targeting molecule.

The therapeutic molecules include, but are not limited to, small molecule drugs, therapeutic polypeptides, therapeutic antibodies, and the like. Exemplary therapeutic small molecules include, but are not limited to, toxins, immunomodulators, antagonists, apoptosis inducers, hormones, radiopharmaceuticals, antiangiogenic agents, siRNAs, cytokines, chemokines, prodrugs, chemotherapeutics Wait. In some specific embodiments, the therapeutic molecule is 7-ethyl-10-hydroxycamptothecin (SN38). The structure of SN38 is shown in the following formula:

In some embodiments, the therapeutic molecule is the camptothecin toxoid gemitecan (CM). The structure of CM is shown in the following formula:

The detectable molecules include, but are not limited to, fluorescent molecules, luminescent chemicals, enzymes, radioisotopes, tags, and the like.

The targeting molecules include, but are not limited to, targeting antibodies, specific receptor ligands, and the like. For example, the targeting molecule can be an antibody that specifically targets a tumor antigen.

In some embodiments, the functional moiety is conjugated to the ferritin H subunit mutant polypeptide through a linker.

In some embodiments, the Polypeptide Conjugate is capable of assembling into a clathrin and/or capable of conferring the ability of the clathrin to specifically bind the TfR1 receptor upon assembly into a clathrin.

In some embodiments, the Polypeptide Conjugate is an isolated Polypeptide Conjugate, eg, that is not assembled into a clathrin. In some embodiments, the Polypeptide Conjugate is contained within a clathrin.

5. clathrin

Since the self-assembly ability and/or receptor binding ability of the wild-type ferritin H subunit is preserved, the ferritin H subunit mutant polypeptides of the present invention can independently assemble into caged proteins (i.e., H ferritin) in a suitable medium. / apoferritin), can also form clathrin with ferritin L subunit or other ferritin H subunit or other self-assembling polypeptides, and can confer specific targeting ability to the clathrin.

Accordingly, in another aspect, the present invention provides a clathrin comprising at least one ferritin H subunit mutant polypeptide of the present invention and/or a polypeptide conjugate of the present invention.

Exemplary such clathrins may comprise, eg, 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 of the invention and/or polypeptide conjugates of the invention. In some preferred embodiments, the clathrin comprises 24 ferritin H subunit mutant polypeptides of the invention and/or polypeptide conjugates of the invention.

In some embodiments, the clathrin comprises only a ferritin H subunit mutant polypeptide of the invention and/or a Polypeptide Conjugate of the invention, eg, only a Polypeptide Conjugate of the invention. For example, in some preferred embodiments, the clathrin is assembled from 24 polypeptide conjugates of the invention.

In some embodiments, the clathrin comprises a plurality of Polypeptide Conjugates of the invention comprising the same or different functional moieties.

In some embodiments, the clathrin further comprises a ferritin L subunit. In some embodiments, the clathrin comprises at least one ferritin H subunit mutant polypeptide of the present invention and at least one ferritin L subunit, preferably, the ferritin H subunit mutant polypeptide and ferritin The ratio of L subunits may range, for example, from 1:23 to 23:1.

In some embodiments, the clathrin does not comprise a ferritin L subunit.

6. Conjugation method

Various methods for conjugating functional molecules to proteins via sulfhydryl groups are known in the art, and these can be applied to the present invention. Those skilled in the art can determine the appropriate conjugation method depending on the specific functional molecule and the selected linker. An exemplary method can be referred to Moon, S.J., et al., Antibody conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) for targeted cancer chemotherapy. J Med Chem, 2008.51(21):p.6916-26.

In one aspect, the present invention provides a method of preparing a clathrin of the present invention, the clathrin comprising at least one polypeptide conjugate of the present invention, the method comprising:

a) conjugating a functional molecule to the depolymerized ferritin H subunit mutant polypeptide of the invention, and

b) Reassembling the ferritin H subunit mutant polypeptide conjugated with the functional molecule into a caged protein.

In some embodiments, the functional molecule is conjugated to a depolymerized ferritin H subunit mutant polypeptide of the invention via a linker.

In some embodiments, the functional molecule is SN38. In some embodiments, wherein step a) comprises contacting a compound of the formula (SN38 with a linker) with a depolymerized ferritin H subunit mutant polypeptide of the invention,

In some embodiments, the functional molecule is a CM. In some embodiments, wherein step a) comprises contacting a compound of the formula (CM with a linker) with a depolymerized ferritin H subunit mutant polypeptide of the invention,

In one aspect, the present invention provides a method of preparing a clathrin of the present invention, the clathrin comprising at least one polypeptide conjugate of the present invention, the method comprising:

a) providing a clathrin comprising at least one ferritin H subunit mutant polypeptide of the invention, and

b) Conjugating a functional molecule to the ferritin H subunit mutant polypeptide of the invention in the clathrin.

In some embodiments, the functional molecule is conjugated to the ferritin H subunit mutant polypeptide of the invention in the clathrin via a linker.

In some embodiments, the functional molecule is SN38. In some embodiments, wherein step b) comprises contacting a compound of formula (SN38 with a linker) with the clathrin.

In some embodiments, the functional molecule is a CM. In some embodiments, wherein step b) comprises contacting a compound of formula (CM with a linker) with the clathrin

Seven, clathrin-API complex

In another aspect, the present invention provides a clathrin-API complex, wherein the clathrin-API complex comprises the clathrin of the present invention, and a pharmaceutical active ingredient ( API).

In some embodiments, the clathrin in the complex comprises a polypeptide conjugate of the invention comprising a ferritin H subunit mutant polypeptide of the invention and a therapeutic molecule. The clathrin of the present invention conjugated with a therapeutic molecule can simultaneously deliver different therapeutically active ingredients in two different ways.

In some embodiments, the clathrin in the complex comprises a polypeptide conjugate of the invention comprising a ferritin H subunit mutant polypeptide of the invention and a detectable molecule. The clathrin of the invention conjugated to a detectable molecule can be used to monitor (eg, monitor in real time) the delivery of a drug.

In some embodiments, the clathrin in the complex comprises a polypeptide conjugate of the invention comprising a ferritin H subunit mutant polypeptide of the invention and a targeting molecule. The clathrins of the invention conjugated with targeting molecules can target additional therapeutic targets in vivo.

The pharmaceutical active ingredient (API) loaded inside the clathrin is not particularly limited as long as it is suitable for loading in the clathrin of the present invention, for example, the API does not destroy the clathrin's properties. The cage-like structure and/or its size is suitable to be accommodated by the cage-like structure. Examples of such APIs include, but are not limited to, alkylating agents, platinums, antimetabolites, tumor antibiotics, natural extracts, hormones, radiopharmaceuticals, neurotransmitter drugs, dopamine agonists, neuronal Anticholinergics, choline receptor agonists, gamma secretase inhibitors, antioxidants, anesthetics.

8. Pharmaceutical compositions and their applications

In another aspect, the present invention provides a pharmaceutical composition comprising a ferritin H subunit mutant polypeptide of the present invention, a polypeptide conjugate of the present invention, a clathrin of the present invention, and/or a clathrin of the present invention A protein-API complex, and a pharmaceutically acceptable excipient.

In some embodiments, a pharmaceutical composition comprises a ferritin H subunit mutant polypeptide of the invention or a polypeptide conjugate of the invention, and optionally an effective amount of an API, wherein the ferritin H subunit mutant polypeptide . The polypeptide conjugates of the present invention are provided in the form of not assembled into caged proteins. The ferritin H subunit mutant polypeptide or polypeptide conjugate can self-assemble into a clathrin or a clathrin-API complex in vitro or after delivery into the body under suitable conditions.

In some embodiments, where the Polypeptide Conjugate of the present invention comprises a therapeutic molecule, the pharmaceutical composition comprising the Polypeptide Conjugate of the present invention may not comprise additional APIs.

The ferritin H subunit mutant polypeptides, polypeptide conjugates, clathrins, clathrin-API complexes and/or pharmaceutical compositions of the present invention may be used for the treatment and/or prevention of diseases depending on what they contain Therapeutic molecule or API. Moreover, since the clathrin of the present invention has tumor targeting ability and blood-brain barrier penetrating ability, it is especially suitable for treating tumors or brain diseases. Furthermore, if the polypeptide conjugate of the present invention comprises a targeting molecule, the ferritin H subunit mutant polypeptide, polypeptide conjugate, clathrin, clathrin of the present invention, depending on the target of the targeting molecule Protein-API complexes and/or pharmaceutical compositions can also be used for other diseases.

Examples of brain diseases include, but are not limited to, eg, brain tumors, Alzheimer's disease, Parkinson's disease, stroke, epilepsy, Huntington's disease, and amyotrophic lateral sclerosis. Examples of such tumors include, but are not limited to, for example, colorectal cancer, lung cancer, breast cancer, ovarian cancer, melanoma, gastric cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, and various hematopoietic cancers such as Hodgkin's disease, Non-Hodgkin's lymphoma, leukemia.

In another aspect, the present invention provides the use of the ferritin H subunit mutant polypeptide, polypeptide conjugate, clathrin, clathrin-API complex and/or pharmaceutical composition of the present invention in the manufacture of a medicament. In some embodiments, the medicament is used, for example, to treat tumors or brain disorders.

In another aspect, the present invention provides a method of treating and/or preventing a disease in a subject, the method comprising administering to the subject an effective amount of a mutant ferritin H subunit polypeptide, polypeptide conjugate of the present invention , clathrin, clathrin-API complexes and/or pharmaceutical compositions. The disease is as defined above, preferably a tumor or a brain disease.

Polypeptides of the present invention, ferritin H subunit mutant polypeptides, polypeptide conjugates, clathrin, clathrin-API complexes and/or pharmaceutical compositions of the present invention can be prepared by any method known to those of ordinary skill in the art. Administration is carried out by a suitable method (see, e.g., Remington: The Science and Practice of Pharmacy," 21st Ed., 2005). The pharmaceutical composition may be administered, for example, intravenously, intramuscularly, intraperitoneally, Administration by intraluminal, intrathecal, oral, topical or inhalation routes.

9. Method for preparing clathrin-API complex

In another aspect, the present invention provides a method of preparing a clathrin-API complex of the present invention, the method comprising making a ferritin H subunit mutant polypeptide of the present invention, a polypeptide conjugate of the present invention and/or Or the clathrin of the present invention is contacted with API, thereby obtaining a clathrin-API complex.

In some embodiments, the method includes:

a) contacting the depolymerized clathrin of the invention with an API; and

b) Reassembling the clathrin, thereby obtaining a clathrin-API complex.

As used in the present invention, "depolymerization" refers to a process in which the tightly closed globular structure of a clathrin is opened to separate all or part of its subunits from each other under certain conditions, such as protein denaturation conditions , such as buffered solutions containing high concentrations of urea.

As used herein, "reassembly" refers to the process by which depolymerized clathrins, ie, separated subunits, re-self-assemble into clathrins by changing conditions, eg, by replacing them with physiologically compatible conditions. During the reassembly process of clathrin, the API will be encapsulated within it, thereby forming a clathrin-API complex. The physiologically compatible conditions are, for example, physiological buffer solutions.

In some embodiments, the method further comprises the step of depolymerizing the clathrin of the invention prior to step a). In some embodiments, wherein the clathrin of the invention is depolymerized by the presence of high concentrations (eg, at least 6M, preferably 8M) of urea. In some embodiments, wherein the clathrin is reassembled by stepwise decreasing urea concentration (eg, gradient dialysis).

In some embodiments, the method includes:

a) contacting a clathrin of the invention with an API under non-depolymerizing conditions, thereby allowing the API to bind to the clathrin and/or be loaded into the inner central cavity of the clathrin,

b) Obtaining clathrin-API complexes.

In some embodiments, the non-depolymerizing conditions include placing the clathrin and API in a physiologically acceptable buffer. Suitable physiologically acceptable buffers include, but are not limited to, PBS solution, physiological saline, purified water, HEPES buffer, and the like.

In some embodiments, the API is shuttled to the interior central cavity of the clathrin by passive diffusion. By placing the clathrin and API in a physiologically acceptable buffer, the API can diffuse into the inner lumen of the clathrin without depolymerization of the clathrin.

Example

A further understanding of the present invention may be obtained by reference to some specific examples given herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way. Obviously, various modifications and changes can be made to the present invention without departing from the essence of the present invention, therefore, these modifications and changes are also within the scope of protection claimed in this application.

Example 1. Construction of human H-ferritin with improved bonding rate and water solubility

1.1 Design of ferritin H subunit mutation

The amino acid sequence of the H subunit mutant was designed according to the wild-type amino acid sequence of the human ferritin H subunit (SEQ ID NO: 1; see PDB: 3AJO_A), and the sites in the H subunit that may be involved in iron loading were mutated, The ferric center site involving glutamic acid at position 62 (E62) and histidine at position 65 (H65). At the same time, in order to increase the hydrophilicity of ferritin and reduce aggregation, the lysine (K108) with an amino side chain at position 108 was replaced with an amino acid with a carboxyl side chain; the asparagine at position 98 (N98) was replaced with an amino acid with a carboxyl side chain; amino acid substitution with a carboxyl side chain; and/or substitution of arginine with an amino side chain at position 156 (R156) with an amino acid with a carboxyl side chain. All amino acid positions refer to SEQ ID NO:1. At the same time, in order to increase the homogeneity of the ferritin-coupled drug, two of the three active sulfhydryl sites of the H subunit (positions 90 and 130) were mutated into other amino acids, and only the cysteine at position 102 was retained. amino acid as the coupling site.

The specific design of the mutants is shown in Table 1. The resulting subunit mutants were named Mut-HFn-240 (SEQ ID NO:5), Mut-HFn-241 (SEQ ID NO:6), Mut-HFn-242 (SEQ ID NO:7) and Mut-HFn, respectively -243 (SEQ ID NO: 8).

In addition, mutants Mut-HFn-212 (SEQ ID NO: 2), Mut-HFn-233 (SEQ ID NO: 3) and Mut-HFn-203 (SEQ ID NO: 4) were used as controls, which did not The hydrophilic site and iron loading related site were improved, only one Cys was retained as the chemical coupling site, and the other two Cys on the outer surface were mutated (C102S and C130S).

Table 1. Design of HFn mutants

1.2 Preparation and purification of H-ferritin

After obtaining the mutated amino acid sequence, its coding sequence was codon-optimized for E. coli. The codon-optimized nucleotide sequences of wild-type ferritin and the 5 mutants are shown in SEQ ID NOs: 9-16, respectively. The construction methods of wild-type HFn, Mut-HFn-212, Mut-HFn-233, and Mut-HFn-242 are as follows: select the commonly used vector pET-30a(+) for expressing foreign proteins in E. coli, kanamycin resistance ( Kan+), select Nde I and Hind III restriction sites to embed the target gene. The successful construction of the expression vector was confirmed by enzyme digestion map and gene sequencing.

The construction methods of Mut-HFn-240, Mut-HFn-241, and Mut-HFn-242 are as follows: selecting the commonly used vector pET-28a(+) for expressing foreign proteins in E. coli, kanamycin resistance (Kan+), selecting Nco I and Xho I restriction sites are inserted into the target gene. The successful construction of the expression vector was confirmed by enzyme digestion map and gene sequencing.

After the above-mentioned expression vector was successfully constructed, E.coli BL21 (DE3) was selected as the host bacteria, and the recombinant plasmid containing the target gene was transformed into the competent cells of the host bacteria, and positive clones were screened by the resistance plate containing kanamycin to determine recombinant strains.

The recombinant strain was inoculated in 1L LB medium/2L shake flask, cultured at 37°C in a low-speed shake flask to an OD600 of about 1.0, and 0.5mM IPTG was added to induce expression for 3 to 4 hours, and then centrifuge to collect the bacterial slurry. Add 25 mL of 20 mM Tris-HCl buffer for each 30 ml of bacterial slurry collected by centrifugation to resuspend evenly, crush it in a high-pressure homogenizer at 1000 bar for 0.5 to 2 min, and centrifuge the lysate at 5000 r/min for 30 min. Take the supernatant and send it to SDS-PAGE. The loading volume was 10 μl (sample:Loading buffer=1:1). The expression results of each protein are shown in Figure 1. All proteins were expressed in the supernatant.

The protein purification method includes the following steps: the above-mentioned high-pressure homogenized and broken cell lysate is centrifuged (1500rpm, 10min) to remove Escherichia coli cell fragments; the supernatant is heated at 72° C. for 15 minutes; impurity proteins are precipitated, and the precipitate is removed by centrifugation; The supernatant was separated and purified on a Superdex 200pg column of size exclusion chromatography; the purity was identified by SDS-PAGE electrophoresis; the protein concentration was determined by BCA. All samples were more than 96% pure.

Example 2. Characterization of ferritin H subunit mutants

2.1. TEM results of mutant HFn

Sample preparation method: ferritin samples (20 μL, 0.1 mg/mL) were placed on a common carbon support membrane for 1 min, then washed twice with 2% uranyl acetate, and finally stained with 2% uranyl acetate for one minute. FEI Tecnai Spirit (120kV) 3) Electron microscope observation conditions: use transmission electron microscope (FEI Tecnai Spirit (120kV)) to observe, observation conditions: HT100Kv. The results of transmission electron microscopy (Fig. 2) showed that both the mutant H subunit polypeptide and the wild-type H subunit polypeptide could form a uniform and regular cage-like protein structure with a diameter of about 12-19 nm.

2.2 SEC results of mutant HFn

Take 1ml of protein samples with a protein concentration of 1mg/ml respectively, put them in a clean 1.5ml EP tube, and take 10 microliters of samples to pass through a gel filtration chromatography column (Agilent Technologies 1260 Infinity II) in a high performance liquid chromatography system (Agilent Technologies 1260 Infinity II). Agilent Advance Bio SEC 300A 2.7um 7.8*300nm, column number: ARD-007 flow rate: 0.5ml/min) SEC analysis of ferritin monomer and aggregate peaks, mobile phase: 50mM Tris buffer, pH7.2. Detection wavelength: 280nm Column temperature: 25℃

The results of SEC are shown in Table 2, and Figure 3 shows the SEC profiles of Mut-HFn-212, Mut-HFn-241, Mut-HFn-242 and Mut-HFn-243 in detail. Under the same preparation conditions, the particle size of Mut-HFn-212 was slightly larger, and aggregation was easy to occur.

Table 2

sample	Particle size (nm)
Mut-HFn-212	19.23
Mut-HFn-233	20.31
Mut-HFn-203	20.98
Mut-HFn-240	14.73
Mut-HFn-241	16.97

Mut-HFn-242	16.89
Mut-HFn-243	17.076

Example 3. Conjugation of mutant human H-ferritin and drugs

3.1 Coupling with Mal-PEG2-VC-PABC-SN-38

3.1.1 Coupling method

The camptothecin-like toxoid SN-38 (Mal-PEG2-VC-PABC-SN-38) linked to the linker was used as the coupling object, and chemical coupling reaction was carried out with the mutant obtained in Example 1.

Mal-PEG2-VC-PABC-SN-38 was synthesized and prepared by Shanghai Ruizhi Chemical, and its structure is as follows:

Coupling step: Mut-HFn-212, Mut-HFn-233, Mut-HFn-203, Mut-HFn-241, Mut-HFn-242, and Mut-HFn-243 prepared in Example 1 were replaced by desalting columns solution to 5mM EDTA, 75mM PB pH 8.0. Mal-PEG2-VC-PABC-SN-38 was dissolved in DMF. The ferritin protein solution was mixed with a vortex shaker at 37°C while adding DMF, then the above-mentioned DMF-dissolved Mal-PEG2-VC-PABC-SN-38 was added, and the solution was left to stand at 37°C for 20 min. The samples were centrifuged at 10,000 g for 2.0 min, then desalted with AKTA and exchanged in a solution of 10 mM Tris-HCl pH 7.0. The desalted samples were concentrated and prepared into protein concentrations of 20, 30, and 40 mg/mL.

3.1.2 Detection of coupling products

3.1.2.1 TEM morphology detection

The TEM sample preparation method of the mutant HFn after coupling with the small molecule drug is the same as that in Example 1. The TEM results are shown in Figure 4. After coupling, each mutant still has a cage-like structure with a diameter of 12-19 nm.

3.1.2.1 RP-HPLC detection method

The coupled product prepared in 3.1.1 was subjected to RP-HPLC (Agilent, 1260 Infinity II) liquid chromatography column (ACQUITY

C18 1.7μm 2.1×100mm) for analysis, detection wavelengths of 280nm and 363nm, gradient elution, mobile phase: A phase: 0.1% TFA/water; B phase: acetonitrile, column temperature 20 ℃, flow rate 0.4Ml/min, feed Sample size 2ul.

The gradient elution conditions were:

time (min)	Flow rate (ml/min)	M.P.A (%)	M.P.B (%)
0	0.4	62.0	38.0
7.0	0.4	55.0	45.0

SN-38 has absorption at 363nm and 280nm, mainly at 363nm, while ferritin has absorption peak at 280nm. The results showed that all mutants had overlapping peak times of SN-38 small molecule (at 363 nm) and ferritin (at 280 nm), indicating that SN-38 was coupled to the ferritin mutant. Figure 5 shows the specific RP-HPLC profiles of Mut-HFn-241 and Mut-HFn-243, respectively.

At the same time, according to the absorption peak areas of SN-38 and ferritin in the characteristic absorption band, combined with the concentration of the stock solution at the time of injection, the drug loading (bonding rate) of each mutant ferritin was calculated. In addition, the results of drug loading and endotoxin after conjugation are shown in Table 3.

Wherein drug loading = the amount of HF subunits coupled with SN-38/total amount of HFn subunits * 100%.

Table 3: Parameters of HFn mutant SN-38 conjugated products

The results showed that the mutants showed good drug loading properties, and the drug loading (bonding ratio) of Mut-HFn-241, 242 and 243 was higher than that of Mut-HFn-212, Mut-HFn-233 and Mut-HFn-203 higher. Moreover, Mut-HFn-212, Mut-HFn-233 and Mut-HFn-203 are easy to precipitate, while Mut-HFn-241, 242 and 243 are more stable and less prone to precipitation. Compared with the control 212 mutant, the binding rate of the mutant of the present invention to SN-38 was significantly increased, reaching 15-16/ferritin molecule, while the 233 binding rate was only 5/ferritin molecule. The binding ratio of up to 15 far exceeds the drug loading capacity of the ADC drug-to-antibody ratio (DAR) of 8, which makes the effective dose of the ferritin conjugated drug of the present invention lower, and has great potential to surpass ADC.

3.1.2.2 SEC detection method

The coupling products of Mut-HFn-212, Mut-HFn-233, Mut-HFn-241, Mut-HFn-242 and Mut-HFn-243 prepared in Section 3.1.1 were prepared by SEC (TSK gel G4000SWxl 7.8×300mm, 8μm) Detection wavelengths were 280nm and 363nm, column temperature was 30°C, isocratic elution was performed, and the mobile phase was: 0.1M PB&0.2M NaCl&5% IPA, pH 7.0. The flow rate was 0.4Ml/min, and the injection volume was 30ul.

The SEC results are shown in Figure 6. The peaks of ferritin and the small molecule drug appear at the same position, indicating that the two form a coupled product. Mut-HFn-241, Mut-HFn-242 and Mut-HFn-243 produced fewer aggregates (small or no aggregate peaks) than Mut-HFn-212 and Mut-HFn-233.

3.2 Coupling with CM

3.2.1 Coupling method

The camptothecin-like toxin gemitecan (CM) linked to the linker was used as the coupling object, and Mut-HFn-212, Mut-HFn-233, Mut-HFn-241, Mut-HFn- 242. Mut-HFn-243 is chemically coupled. Coupling steps are the same as 3.1.1.

CL2A-CM was synthesized and prepared by Suzhou Lianning Biotechnology Co., Ltd., and its structure is as follows:

3.2.2 Detection of coupling products

3.2.2.1 TEM morphology detection

The TEM sample preparation method of the mutant HFn prepared in Section 3.2.1 after coupling with small molecule drugs is the same as that in Example 1. The TEM results are shown in Figure 7. After coupling, each mutant still has a cage-like structure with a diameter of 12- 19nm.

3.1.2.2 SEC detection method

The coupling products of Mut-HFn-212, Mut-HFn-233, Mut-HFn-241, and Mut-HFn-243 prepared in Section 3.2.2 were detected by SEC (TSK gel G4000SWxl 7.8×300mm, 8μm), and the method was the same as that in 3.1. 2.2.

The SEC results are shown in Figure 8, which indicated that CM could also be successfully coupled to the ferritin mutant. Compared with Mut-HFn-212 and Mut-HFn-233, Mut-HFn-241 and Mut-HFn-243 have less aggregates and more uniform coupling products.

The drug loading (bonding ratio) of the above samples is shown in Table 4 below:

Table 4: Bonding ratio of HFn mutant CM coupling products

Example 4 TfR-1 affinity experiment

experimental method:

1. Sample: the SN-38 coupling product prepared in Example 3.

2. Dilution and coating: Dilute each ferritin sample with coating solution (carbonic acid buffer, pH9.0) to 0.04 μg/mL, mix well and add it to the ELISA plate, 100 μL/well, three samples per sample. Duplicate wells were covered with sealing film at 4°C overnight.

3. Blocking: The ELISA plate was washed 3 times with 1×PBST and 1×PBS each. 300 μL/well of blocking solution (5% nonfat dry milk) was added, and incubated at 37° C. for 2 h.

4. Dilute human TfR-1 with protein stabilizer (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., PR-SS-002) to 1 μg/mL (1:200), 100 μL/well, cover with sealing film, Incubate at 37°C for 2h.

5. Wash the ELISA plate three times with 1×PBST and 1×PBS each. Anti-TFR1 antibody (human source) (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.: 11020-MM02) was diluted with protein stabilizer to 0.1 μg/mL (1:1000), 100 μL/well, covered with sealing film, 37 Incubate at ℃ for 1.5h.

6. Wash the ELISA plate three times with 1×PBST and 1×PBS each. Anti-mouse IgG was diluted 1:5000 with coupling stabilizer, 100 μL/well, covered with sealing film, and incubated at 37°C for 30 min.

7. Wash the ELISA plate three times with 1×PBST and 1×PBS each. Add TMB one-step chromogenic solution, 100 μL/well in the dark, mix and incubate for 30 min on a shaker in the dark at room temperature, and immediately measure the OD 652nm with a microplate reader. Use Graphpad 6.0 software to analyze the raw data, and make a curve graph, the ordinate is the absorption value at 652 nm, and the abscissa is the H ferritin (HFn) sample coating concentration.

The affinity results of the coupled products are shown in FIG. 9 . The results showed that the binding affinities of the coupling products of Mut-HFn-241/242/243 and 233 were higher than those of the coupling products of Mut-HFn-212 and Mut-HFn-203, among which the coupling products of Mut-HFn-241/242/243 The co-product has a stronger affinity.

Example 5 Conjugate drug efficacy test

1. MDA-MB-231 cell model

Using the coupling products of SN-38 prepared in Example 3 with Mut-HFn-241 and Mut-HFn-243 in the BALB/c of human triple negative breast cancer MDA-MB-231 (ATCC:CRM-HTB-26 ^™ ) Pharmacodynamic experiments were carried out in nude mice subcutaneously transplanted tumor models to study the application of the conjugated drugs of the present invention in cancer treatment.

24 qualified tumor-bearing BALB/c nude mice were screened and randomly divided into 4 groups, with 6 mice in each group, and were given sterile water for injection, commercially available irinotecan hydrochloride injection (CPT-11, 60 mg/kg), Mut -HFn-241-SN38 (2.5 mg/kg) and Mut-HFn-243-SN38 (2.5 mg/kg). It was administered by tail vein injection once a week for four consecutive weeks. The day of the first tumor mass inoculation was the 0th day, the tumor volume was measured on the 16th day, and the tumor volume was measured twice a week thereafter.

The experimental results are shown in Figure 10. The results showed that Mut-HFn-241-SN38 and Mut-HFn-243-SN38 showed significant tumor-suppressive effects in the BALB/c nude mouse subcutaneous xenograft model of human colon cancer HT-29, which was comparable to high-dose commercial The sale of Yilikang hydrochloride injection (60mg/kg) is equivalent.

2. HT-29 cell model

The coupling products of SN-38 prepared in Example 3 with Mut-HFn-241 and Mut-HFn-243 were transplanted subcutaneously in BALB/c nude mice tumor model of human colon cancer HT-29 (ATCC:HTB 3B ^TM ). Pharmacodynamic experiments were carried out to study the application of the composition of the present invention in cancer treatment.

Twenty-four qualified tumor-bearing BALB/c nude mice were screened and randomly divided into 4 groups, with 6 mice in each group, respectively given sterile water for injection, commercially available irinotecan hydrochloride injection (60 mg/kg), Mut-HFn-241 -SN38 and Mut-HFn-243-SN38 (30 mg/kg). It was administered by tail vein injection once a week for four consecutive weeks. The day of the first tumor mass inoculation was the 0th day, the tumor volume was measured on the 13th day, and the tumor volume was measured twice a week thereafter.

The experimental results are shown in Figure 11. The Mut-HFn-241-SN-38 and Mut-HFn-243-SN-38 of the present invention show significant tumor-suppressing effect in the BALB/c nude mouse subcutaneous transplanted tumor model of human colon cancer HT-29. The high dose of commercially available Iricang hydrochloride injection (60mg/kg) is equivalent.

sequence listing

SEQ ID NO: 1 wild-type H subunit amino acid sequence

SEQ ID NO:2 Mut-HFn-212 amino acid sequence (C102S; C130S;)

SEQ ID NO:3 Mut-HFn-233 amino acid sequence K86C C90E C102A C130A

SEQ ID NO:4 Mut-HFn-203 amino acid sequence I80C C90S C102S C130S

SEQ ID NO: 5 Mut-HFn-240 (K108E; C90E; C130A; E62K; H65G):

SEQ ID NO: 6 Mut-HFn-241 (K108E; N98D; C90E; C130A; E62K; H65G):

SEQ ID NO: 7 Mut-HFn-242 (K108E; N98D; R156H; C90E; C130A; E62K; H65G):

SEQ ID NO: 8 Mut-HFn-243 (K108E; N98D; R156H; C102A; C130A; E62K; H65G)

SEQ ID NO:9 wild type H subunit nucleotide sequence

SEQ ID NO: 10 Nucleotide sequence of Mut-HFn-212

SEQ ID NO: 11 Nucleotide sequence of Mut-HFn-233

SEQ ID NO: 12 Nucleotide sequence of Mut-HFn-203

SEQ ID NO: 13 Nucleotide sequence of Mut-HFn-240

SEQ ID NO: 14 Nucleotide sequence of Mut-HFn-241

SEQ ID NO: 15 Nucleotide sequence of Mut-HFn-242

SEQ ID NO: 16 Nucleotide sequence of Mut-HFn-243

SEQ ID NO: 17 Wild-type ferritin light chain (L) subunit

Claims (38)

1. A ferritin heavy chain (H) subunit mutant polypeptide, relative to wild-type ferritin H subunit, included at position 98, 108, and/or 156 corresponding to SEQ ID NO: 1 amino acid substitution at the position.
2. The mutant polypeptide of claim 1, relative to the wild-type ferritin H subunit, the mutant polypeptide is at a position corresponding to the 98th, 108th, and/or 156th position of SEQ ID NO: 1 Amino acids are substituted with more hydrophilic amino acids such as amino acids with carboxyl side chains.
3. The mutant polypeptide of claim 1, wherein the amino acid with a carboxyl side chain is selected from the group consisting of glutamic acid (E), aspartic acid (D), or histidine (H).
4. The mutant polypeptide of any one of claims 1-3, wherein an amino acid such as an asparagine (N) at a position corresponding to position 98 of SEQ ID NO: 1 is replaced by an aspartic acid ( D) replace.
5. The mutant polypeptide of any one of claims 1-3, wherein an amino acid such as a lysine (K) at a position corresponding to position 108 of SEQ ID NO: 1 is replaced by a glutamic acid (E) in the mutant polypeptide. )replace.
6. The mutant polypeptide of any one of claims 1-3, wherein an amino acid such as an arginine (R) at a position corresponding to position 156 of SEQ ID NO: 1 is replaced by a histidine (H) in the mutant polypeptide )replace.
7. The mutant polypeptide of any one of claims 1-3, wherein an amino acid such as an asparagine (N) at a position corresponding to position 98 of SEQ ID NO: 1 is replaced by an aspartic acid ( D) Substitution, and the amino acid at the position corresponding to position 108 of SEQ ID NO: 1, such as lysine (K), is substituted with glutamic acid (E).
8. The mutant polypeptide of any one of claims 1-3, wherein an amino acid such as an asparagine (N) at a position corresponding to position 98 of SEQ ID NO: 1 is replaced by an aspartic acid ( D) Substitution in which the amino acid at the position corresponding to position 108 of SEQ ID NO: 1 is replaced by glutamic acid (E), for example, lysine (K), and at position 156 corresponding to SEQ ID NO: 1 An amino acid such as arginine (R) is substituted with histidine (H) at the position of .
9. The mutant polypeptide of any one of claims 1-8, wherein with respect to the wild-type ferritin H subunit, the mutant polypeptide is comprised at position 62 and/or 65 corresponding to SEQ ID NO: 1 Amino acid substitution at position.
10. 9. The mutant polypeptide of claim 9, wherein the amino acid of the mutant polypeptide at the position corresponding to position 62 of SEQ ID NO: 1, such as glutamic acid (E), is substituted for lysine (K).
11. 9. The mutant polypeptide of claim 9, wherein an amino acid, such as histidine (H), at a position corresponding to position 65 of SEQ ID NO: 1 is substituted for glycine (G) in the mutant polypeptide.
12. The mutant polypeptide of claim 9, wherein the mutant polypeptide has an amino acid such as glutamic acid (E) substituted for lysine (K) at a position corresponding to position 62 of SEQ ID NO: 1, and in An amino acid such as histidine (H) at the position corresponding to position 65 of SEQ ID NO: 1 is substituted for glycine (G).
13. 12. The mutant polypeptide of any one of claims 1-12, wherein the mutant polypeptide comprises reduced cysteine relative to wild-type ferritin H subunit.
14. The mutant polypeptide of claim 13, wherein at least one of the cysteines at positions corresponding to positions 90, 102 and 130 of SEQ ID NO:1 is substituted in the mutant polypeptide.
15. The mutant polypeptide of claim 14, wherein the cysteine is substituted with an amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, glutamic acid, aspartic acid, lysine, Arginine, histidine, alanine, glycine are preferably substituted by serine or by amino acids at the corresponding positions in the wild-type ferritin light chain (L) subunit polypeptide.
16. The mutant polypeptide of claim 14, wherein relative to the wild-type ferritin H subunit, the mutant polypeptide comprises a cysteine at a position corresponding to position 90 of SEQ ID NO: 1, and

    The cysteine at the position corresponding to position 102 of SEQ ID NO: 1 is substituted, preferably by serine or alanine,

    Optionally, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted, preferably by serine or alanine.
17. The mutant polypeptide of claim 14, wherein relative to the wild-type ferritin H subunit, the mutant polypeptide comprises a cysteine at a position corresponding to position 102 of SEQ ID NO: 1, and

    The cysteine at the position corresponding to position 90 of SEQ ID NO: 1 is substituted, preferably by serine or glutamic acid,

    Optionally, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted, preferably by serine or alanine.
18. The mutant polypeptide of claim 13, wherein relative to the wild-type ferritin H subunit, the mutant polypeptide comprises a cysteine in the loop region at a position corresponding to position 102 of SEQ ID NO: 1 The cysteine of is substituted, and optionally, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted.
19. The mutant polypeptide of claim 18, wherein except for one cysteine in the loop region and optionally a cysteine at a position corresponding to position 130 of SEQ ID NO: 1, the mutant polypeptide does not Contains additional cysteine.
20. 19. The mutant polypeptide of claim 18, wherein the mutant polypeptide does not contain cysteine outside the loop region.
21. The mutant polypeptide of claim 18, wherein relative to the wild-type ferritin H subunit, the mutant polypeptide has cysteine substitutions at positions corresponding to positions 90 and 102 of SEQ ID NO: 1, optionally ground, the cysteine at the position corresponding to position 130 of SEQ ID NO: 1 is substituted; and the mutant polypeptide is substituted at positions 79, 80, 81, 82, 83 corresponding to SEQ ID NO: 1 The amino acid at one of positions , 84, 85, 86, 87, 88, 89 and 91 was substituted with cysteine.
22. The mutant polypeptide of claim 18, wherein relative to the wild-type ferritin H subunit, the mutant polypeptide has cysteine substitutions at positions corresponding to positions 90, 102 and 130 of SEQ ID NO: 1 and the amino acid of the mutant polypeptide at a position corresponding to one of positions 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 91 of SEQ ID NO: 1 is halved Cystine substitution.
23. The mutant polypeptide of claim 1, wherein the mutant polypeptide comprises an amino acid sequence selected from one of SEQ ID NOs: 5-8.
24. The mutant polypeptide of any one of claims 1-23, wherein the mutant polypeptide is capable of being assembled into a clathrin and/or capable of conferring to the clathrin specific binding to the TfR1 receptor after being assembled into a clathrin ability.
25. A polypeptide conjugate comprising the ferritin H subunit mutant polypeptide of any one of claims 1-24, and a functional moiety conjugated thereto through a sulfhydryl group of the ferritin H subunit mutant polypeptide.
26. 26. The Polypeptide Conjugate of claim 25, wherein the functional moiety is selected from the group consisting of a therapeutic molecule, a detectable molecule or a targeting molecule.
27. 27. The polypeptide conjugate of claim 26, wherein the therapeutic molecule is selected from the group consisting of small molecule drugs, therapeutic polypeptides, and therapeutic antibodies, eg, the therapeutic molecule is SN38 or CM.
28. 27. The Polypeptide Conjugate of claim 26, wherein the detectable molecule is selected from the group consisting of fluorescent molecules, luminescent chemicals, enzymes, isotopes, labels.
29. The Polypeptide Conjugate of claim 26, wherein the targeting molecule is a targeting antibody.
30. 29. The polypeptide conjugate of any one of claims 25-29, wherein the functional moiety is conjugated to the ferritin H subunit mutant polypeptide through a linker.
31. The Polypeptide Conjugate of any one of claims 25-30, which is capable of assembling into a clathrin and/or capable of conferring specific binding to the TfR1 receptor after being assembled into a clathrin Ability.
32. A clathrin comprising at least one ferritin H subunit mutant polypeptide of any one of claims 1-24 and/or at least one ferritin H subunit mutant polypeptide of any one of claims 25-31 of polypeptide conjugates.
33. 32. The clathrin of claim 32, comprising 24 of said ferritin H subunit mutant polypeptides and/or said polypeptide conjugate.
34. 32. The clathrin of claim 32, formed from the assembly of 24 of said polypeptide conjugates.
35. 33. The clathrin of claim 32, said clathrin comprising a plurality of said Polypeptide Conjugates, said plurality of Polypeptide Conjugates comprising the same or different functional moieties.
36. A clathrin-API complex, wherein the clathrin-API complex comprises the clathrin of any one of claims 32-35, and a pharmaceutically active ingredient (API) loaded inside the clathrin ).
37. A pharmaceutical composition comprising the ferritin H subunit mutant polypeptide of any one of claims 1-24, the polypeptide conjugate of any one of claims 25-31, any one of claims 32-35 The clathrin of claim 36 and/or the clathrin-API complex of claim 36, and a pharmaceutically acceptable excipient.
38. The ferritin H subunit mutant polypeptide of any one of claims 1-24, the polypeptide conjugate of any one of claims 25-31, the clathrin of any one of claims 32-35/claims Use of the clathrin-API complex of 36 and/or the pharmaceutical composition of claim 36 in the manufacture of a medicament.

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