Ferritin variants with increased stability, complexing ability and transferrin receptor affinity

Abstract

The present invention relates to a polypeptide comprising a transferrin receptor binding domain (TRBD) of a ferritin variant. The TRBD comprises one or more than one glutamine residue mutated to a glutamic acid residue and / or one or more than one asparagine residue mutated to an aspartic acid residue. The present invention also relates to a complex of the polypeptide and a tag or a drug, and an isolated cell delivery system comprising the polypeptide or the complex of the present invention, and the use of the system for prevention, treatment, diagnosis or diagnosis-treatment, in particular for treating cancer or an inflammatory disease.

Description

[0001] This invention relates to novel ferritin variants, such as peptides comprising a transferrin receptor-binding domain (TRBD). The TRBD comprises one or more glutamine residues mutated to glutamate residues and / or one or more asparagine residues mutated to aspartic acid residues. The invention also relates to complexes of such peptides with compounds, tags, or drugs, and to separate delivery systems comprising the peptides or complexes of the invention (alone or as a cellular system), and the use of said systems for prevention, treatment, or diagnosis, particularly for the treatment of cancer or inflammatory diseases. Background Technology

[0002] CD71 (transferrin receptor 1) is a membrane protein widely expressed on the surface of metabolically active cells. Increasing evidence demonstrates CD71's involvement in tumorigenesis and development, and its expression is significantly dysregulated in many cancers. Various anti-tumor therapeutic strategies have been designed to target CD71. Both transferrin and ferritin bind to CD71 and are internalized via clathrin-mediated cellular entry. Ferritin is stored intracellularly when transferrin cycles back to the cell surface. Ferritin possesses a cage-like structure composed of 24 protein subunits. It can be used to encapsulate active pharmaceutical ingredients and / or tags within its lumen.

[0003] The discovery of CD71 upregulation in malignant cells has made CD71 a valuable drug target for the treatment and diagnosis of cancer. Within this framework, ferritin-based nanocages have emerged as promising devices for delivering drugs or diagnostic molecules in cancer therapy. In particular, H-ferritin homopolymers exhibit excellent nanocage properties due to their unique assembly, transferrin receptor recognition properties, and high biocompatibility. By utilizing the natural recognition of CD71, ferritin nanocages can ensure convenient drug delivery and release properties. The encapsulation of small therapeutic molecules within ferritin cages has been explored and successfully performed in preclinical studies. CD45+ leukocytes, particularly activated macrophages, have been shown to absorb (not only via CD71) drug- or tagged ferritin in vitro and deliver these complexes to or into cells, preferably to or into tumor cells in vivo (WO 2016 / 207257 A1, WO 2016 / 207256 A1, WO2017 / 222398 A1).

[0004] To date, despite considerable efforts, no successful transferrin or ferritin-drug complex has entered clinical trials (Truffi M et al., Pharmacol Res. 2016 May; 107:57-65). The inventors have discovered that the affinity of ferritin for the CD71 receptor can be modulated by selectively introducing negatively charged mutations in the N-terminal region, particularly in the transferrin receptor-binding domain (TRBD). These mutations have been found to improve the properties of ferritin as a carrier, and particularly as a nanocage for drugs and / or tags. The TRBD variant peptides of the present invention particularly provide: (i) improved targeting of TRBD variant ferritin to CD71-expressing cells; (ii) enhanced binding of TRBD variant ferritin to CD71; (iii) improved encapsulation efficiency of drugs or tags in TRBD variant ferritin nanocages; (iv) improved loading of drugs or tags encapsulated in or attached to TRBD variant ferritin in cells, preferably in delivery cells; (v) improved targeting of drugs or tags encapsulated in or attached to TRBD variant ferritin to target tissues; (vi) improved delivery of drugs or tags encapsulated in or attached to TRBD variant ferritin to target tissues; (vii) improved delivery of drugs or tags encapsulated in or attached to TRBD variant ferritin to target tissues; (viii) Improved release of drugs or tags within or attached to TRBD variant ferritin at target tissues; (ix) Improved stability of TRBD variant ferritin nanocages; (x) Improved stability of drug encapsulation within TRBD variant ferritin nanocages; (xi) Improved protein recovery after loading TRBD variant ferritin nanocages; (xii) Increased cytotoxicity of tumor cells by drugs contained within or attached to TRBD variant ferritin nanocages; (xiii) Improved chemiphysical properties of TRBD variant ferritin nanocages; (xiv) Reduced tendency for dimer formation in TRBD variant ferritin nanocages; (xv) Reduced tendency for aggregation of TRBD variant ferritin nanocages. Summary of the Invention

[0005] In a first aspect, the present invention relates to polypeptides comprising a transferrin receptor-binding domain (TRBD) of a ferritin variant, wherein, within the TRBD, the ferritin variant comprises one or more glutamine residues mutated to glutamate residues and / or one or more asparagine residues mutated to aspartic acid residues, compared to the wild-type ferritin on which it is based.

[0006] In a second aspect, the present invention relates to ferritin variant polypeptides wherein at least one, at least two, at least three, or at least four, preferably four lysine residues, preferably at positions 54, 72, 87, and / or 144, of the lysine residues shown in SEQ ID NO.1 (human wild-type heavy chain ferritin) are missing or replaced by non-basic amino acids.

[0007] In a third aspect, the present invention relates to ferritin variant polypeptides wherein one or more cysteine ​​residues, particularly the cysteine ​​residues at positions 91, 103 and / or 131 shown in SEQ ID NO.1, are missing or substituted, preferably substituted with serine residues.

[0008] In a fourth aspect, the present invention relates to nucleic acids encoding polypeptides of the first, second, or third aspect.

[0009] In another respect, the present invention relates to a vector containing nucleic acids of the fourth aspect.

[0010] In a fifth aspect, the present invention relates to a conjugate comprising a polypeptide comprising a first aspect, a second aspect, or a third aspect and at least one label and / or at least one drug.

[0011] In a sixth aspect, the present invention relates to a complex comprising at least one polypeptide of the first, second or third aspect of the present invention and / or at least one conjugate of the fifth aspect.

[0012] In a seventh aspect, the present invention relates to a targeted delivery system comprising the separation of cells, wherein the cells comprise a polypeptide, a conjugate of the fifth aspect, or a complex of the sixth aspect of the invention.

[0013] In an eighth aspect, the present invention relates to pharmaceutical or diagnostic compositions comprising peptides of the first, second, or third aspect, conjugates of the fifth aspect, complexes of the sixth aspect, or isolated targeted delivery systems of the seventh aspect, as well as pharmaceutically acceptable carriers and / or suitable excipients.

[0014] In a ninth aspect, the present invention relates to polypeptides of the first, second or third aspect, conjugates of the fifth aspect, complexes of the sixth aspect, and isolated targeted delivery systems of the seventh aspect, which are used as pharmaceuticals.

[0015] In a tenth aspect, the present invention relates to polypeptides of the first, second, or third aspect, conjugates of the fifth aspect, complexes of the sixth aspect, isolated targeted delivery systems of the seventh aspect, or pharmaceutical or diagnostic compositions of the eighth aspect, for the treatment, prevention, or diagnosis of tumors, preferably solid tumors and / or their metastases, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, liver cancer, glioma / glioblastoma, or tumors with hypoxic areas; inflammatory diseases or ischemic sites, particularly at the site of skin wounds or organ infarction (heart) or ischemic retinal inflammatory diseases or ischemic sites; or for use in prophylactic or therapeutic vaccination, particularly for the prevention or treatment of infectious diseases or cancer.

[0016] In the eleventh aspect, the present invention relates to a method for treating, preventing, or diagnosing tumors, preferably solid tumors and / or metastatic tumors, particularly breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, liver cancer, glioma / glioblastoma, or tumors with hypoxic areas; inflammatory diseases or ischemic sites, particularly skin wounds or organ infarctions (heart) or ischemic retinal inflammatory diseases or ischemic sites; or methods for prophylactic or therapeutic vaccination, particularly by administering an effective amount of the polypeptide of the first, second, or third aspect, the conjugate of the fifth aspect, the complex of the sixth aspect, or the isolated targeted delivery system of the eighth aspect to a subject in need to prevent or treat infectious diseases or cancers.

[0017] Detailed description of the preferred implementation scheme

[0018] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which will be defined solely by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0019] The terms "peptide" or "polypeptide" are used interchangeably in the context of this invention to refer to a chain of at least two amino acids linked by peptide bonds. Therefore, the term "polypeptide" in the context of this invention also refers to an amino acid chain having more than 50, more than 100, or more than 150 amino acids.

[0020] The term "amino acid" includes naturally occurring amino acids as well as amino acid derivatives. In the context of this specification, amino acids are identified using single-letter or three-letter codes (Hausman RE, Cooper GM (2004) The Cell: A Molecular Approach. Washington, DC: ASM Press. p. 51. ISBN 978-0-87893-214-6). Amino acids identified by the letter X correspond to any amino acid. Amino acids identified by the letter B correspond to D (asparagine) or N (aspartic acid). Amino acids identified by the letter Z correspond to E (glutamine) or Q (glutamic acid).

[0021] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and should be understood as polymeric or oligomeric macromolecules made of nucleotide monomers. A nucleotide monomer consists of a nucleobase, a pentose sugar (e.g., but not limited to ribose or 2'-deoxyribose), and one to three phosphate ester groups. Typically, polynucleotides are formed by phosphodiester bonds between the individual nucleotide monomers. In the context of this invention, nucleic acid molecules refer to, but are not limited to, ribonucleic acid (RNA) and its various forms (e.g., but not limited to ssRNA, LNA, etc.), deoxyribonucleic acid (DNA) and mixtures thereof, such as RNA-DNA hybrids. Nucleic acids can be synthesized, for example, chemically, such as according to the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). An “aptamer” is a nucleic acid that binds to a polypeptide with high affinity. Aptamers can be isolated from a wide variety of single-stranded RNA molecules using selection methods such as SELEmir146-a (see, for example, Jayaseena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981). Aptamers can also be synthesized and selected in their mirror forms, for example, as L-ribonucleotides (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). The forms isolated in this way have the advantage of not being degraded by naturally occurring ribonucleases, and therefore exhibit greater stability.

[0022] Regarding the comparison of polypeptide and nucleotide sequences, the term "identity" is used throughout the specification. Unless otherwise specified, when comparing two sequences and no reference sequence is specified for calculating the percentage of sequence identity, sequence identity is calculated with reference to the longer of the two sequences being compared. Unless otherwise specified, if a reference sequence is specified, sequence identity is determined based on the full length of the reference sequence shown in SEQ ID. For example, a polypeptide sequence of 200 amino acids may exhibit a maximum sequence identity percentage of 66.6% (200 / 300) compared to a reference polypeptide sequence of 300 amino acids, while a sequence of 150 amino acids may exhibit a maximum sequence identity percentage of 50% (150 / 300). If 15 of these 150 amino acids differ from the corresponding amino acids in the 300 amino acid-long reference sequence, the sequence identity level decreases to 45%. The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. The alignment can be performed using several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or the CLUSTAL algorithm (Thompson, JD, Higgins, D. G and Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), which can be obtained at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html. The preferred parameters used are the default parameters, as they are set at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The sequence identity (sequence matching) level can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). BLAST protein searches are performed using the BLASTP program with a score of 50 and a word length of 3.To obtain gap alignments for comparative purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, was used. When using the BLAST and GappedBLAST procedures, the default parameters for each procedure were used. Sequence matching analysis could be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl1:I54-I62) or Markov random fields. Structure-based alignments of multiple protein sequences and / or structures can also be performed using information from sequence database searches, available homologs with 3D structures, and user-defined constraints (Pei J, Grishin NV: PROMALS: towards accurate multiple sequence alignments of distantly related proteins. Bioinformatics 2007, 23:802-808; 3DCoffee@igs: a web server for combining sequences and structures into a multiple sequence alignment. Poirot O, Suhre K, Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul 1;32:W37-40.). Unless otherwise specifically stated, when percentages of sequence identity are mentioned in this application, these percentages are calculated relative to the full length of the longer sequence.

[0023] Several documents are referenced throughout this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer's specifications, operating instructions, etc.), whether mentioned above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that an invention does not precede these disclosures simply because it was invented earlier.

[0024] To carry out this invention, conventional methods of chemical, biochemical, and recombinant DNA techniques, as explained in the literature in this art, are employed unless otherwise stated (see, e.g., Molecular Cloning: A Laboratory Manual, 2). ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0025] Throughout the specification and the following claims, unless the context otherwise requires, the word “comprising” shall be understood to imply the inclusion of the stated integer or step or combination of integers or steps, but does not exclude any other integer or step or combination of integers or steps. As used in the specification and the appended claims, unless the context expressly indicates otherwise, when an element is not preceded by a quantifier, “a” and “the” include “one or more”.

[0026] The term "TRBD" refers to the N-terminal polypeptide fragment of a ferritin polypeptide of 15 to 40 amino acids, particularly about 20 amino acids, capable of specifically binding to CD71. Preferably, as a full-length ferritin polypeptide, TRBD has a binding affinity of at least 50% for CD71, more preferably at least 75%, and more preferably at least 90%. How to measure the binding affinity between two proteins is well known in the art. Preferably, the affinity between TRBD and CD71, and more preferably the affinity between TRBD and CD71 of the same species, is measured by surface plasmon resonance at room temperature. Preferably, the binding affinity K... D The values ​​are 100 nanomoles or less, 50 nanomoles or less, 20 nanomoles or less, or 5 nanomoles or less.

[0027] The elements of the invention will now be described. These elements are listed with particular embodiments; however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to the explicitly described embodiments.

[0028] This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed in the description of this application.

[0029] In a first aspect, the present invention relates to polypeptides comprising a transferrin receptor-binding domain (TRBD) of a ferritin variant, wherein, within the TRBD, the ferritin variant comprises, compared to the wild-type ferritin upon which it is based, one or more glutamine residues (E) mutated to a glutamate residue (Q) and / or one or more asparagine residues (D) mutated to an aspartic acid residue (N). The ferritin variant, particularly the TRBD of the ferritin variant, comprises at least the following amino acid sequence:

[0030] MTTASX1SZ1VRZ2BYHZ3DX2EAA (SEQ ID NO.81)

[0031] X1 = S or T, with T being preferred;

[0032] X2 = S or A, with S being the preferred choice;

[0033] Z1, Z2, and Z3 = Q or E; and

[0034] B = N or D;

[0035] At least one of Z2 and Z3 is E and / or B is D. The amino acid sequence may also contain one, two, or three amino acid substitutions other than Z, particularly Z2 and Z3, and / or B. M at position 1 may or may not be present. The TRBD of the ferritin variant is specified according to the amino acid sequence of SEQ ID NO. 81. In other words, the polypeptide according to the first aspect of the invention comprises the TRBD according to SEQ ID NO. 81. Preferably, the polypeptide of the first aspect of the invention is a ferritin polypeptide, i.e., a polypeptide having an amino acid sequence similar in structure to the wild-type ferritin polypeptide described below and / or sequence homologous to the wild-type ferritin polypeptide described below (e.g., having at least 80%, 85%, 90%, or 95% sequence identity with the wild-type ferritin polypeptide described below). According to a first aspect of the invention, a polypeptide comprising a TRBD variant of ferritin, wherein within the TRBD, compared to the wild-type ferritin on which it is based, the ferritin variant comprises one or more glutamine residues mutated to glutamate residues and / or one or more asparagine residues mutated to aspartic acid residues, the polypeptide is also referred to herein as a "TRBD variant ferritin polypeptide".

[0036] The transferrin receptor family includes transferrin receptor 1 (TfR-1) and transferrin receptor 2 (TfR-2). TfR-1 is expressed on all actively proliferating cells, while TfR-2 is primarily expressed on hepatocytes and erythroid precursor cells. It has been shown that transferrin receptor 1 mediates cellular uptake of ferritin through endocytosis. The names "TfR-1", "CD71", and "TFRC" are used interchangeably and refer to transferrin receptor 1.

[0037] Ferritin is a hollow, globular protein complex composed of 24 ferritin monomeric subunits assembled into a cage-like structure. Ferritin is the major intracellular iron storage protein. Ferritin is produced by almost all organisms and is present in every cell type. Ferritin genes are highly conserved across species. In vertebrates, two types of ferritin monomers exist: light (L) chain and heavy (H) chain types with molecular weights of 19 kDa and 21 kDa, respectively. Vertebrate ferritin 24-mers can be homooligomers composed of either L or H chains, or heterooligomers composed of both L and H chains (Theil EC, 1987, Annual Review of Biochemistry. 56 (1): 289–315). Typically, the inner and outer diameters of the ferritin complex are approximately 8 nm and 12 nm, respectively. Ferritin is internalized via endocytosis after binding to CD71. The interaction between ferritin and CD71 is mediated by the ferritin-H chain (Li L et al., Proc. Natl. Acad. Sci. USA 107 (8) (2010) 3505–3510). Ferritin is not abundant in plasma, but it can be readily produced in high yields as a recombinant protein in common protein expression systems such as Escherichia coli cells.

[0038] In the TRBD variant ferritin polypeptide according to the first aspect of the invention, uncharged amino acids (glutamine or asparagine) in the wild-type sequence are replaced by negatively charged amino acids (glutamate or aspartate). The inventors unexpectedly found that this resulted in an increased affinity for CD71. Not wishing to be bound by any theory, the inventors believe that the negatively charged mutant may establish other interactions with the transferrin-binding portion of CD71, thereby leading to a more energy-favorable interaction between CD71 and the TRBD variant ferritin polypeptide.

[0039] The TRBD variant ferritin polypeptides according to the first aspect of the invention can be described as “isosteric mutants” because they exhibit the same or very similar geometry compared to their respective wild-type polypeptides. Mutants carrying other mutations in TRBD, compared to these “isosteric mutants,” have a different geometry from the wild type. Not wishing to be bound by any theory, the inventors believe that the resulting lack of surface complementarity with CD71 may lead to a reduced binding affinity for CD71.

[0040] In a preferred embodiment of the polypeptide according to the invention, the wild-type ferritin is mammalian ferritin. Mammalian ferritin can be ferritin from mice, rats, dogs, apes, particularly chimpanzees or humans. In a preferred embodiment, the mammalian ferritin is ferritin from mice, rabbits, rats, or humans.

[0041] In a preferred embodiment of the polypeptide according to the invention, the wild-type ferritin is human heavy chain ferritin.

[0042] In a preferred embodiment of the polypeptide according to the invention, wild-type ferritin has an amino acid sequence selected from SEQ ID NO: 1 corresponding to the human ferritin heavy chain polypeptide and SEQ ID NO: 2 corresponding to the mouse ferritin heavy chain polypeptide. In a preferred embodiment of the polypeptide according to the invention, wild-type ferritin has an amino acid sequence according to SEQ ID NO: 1.

[0043] In a preferred embodiment of the polypeptide according to the invention, the mutation is contained in the 20 N-terminal amino acids of wild-type ferritin.

[0044] Preferably, amino acid substitutions are selected in a manner that does not excessively alter the polypeptide conformation, as the lack of surface complementarity with TfR-1 will prevent the ferritin variant from binding to CD71. For example, a “small amino acid” should be substituted with another small amino acid. In the context of this invention, a “small amino acid” is preferably an amino acid with a molecular weight of less than 125 Daltons. Preferably, the small amino acid in the context of this invention is selected from the amino acids glycine, alanine, serine, cysteine, threonine, and valine, or derivatives thereof. As another example, an amino acid having a hydrophobic side chain should be substituted with another amino acid having a hydrophobic side chain.

[0045] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO.05 to SEQ ID NO.18, SEQ ID NO.20 to SEQ ID NO.33, SEQ ID NO.35 to SEQ ID NO.48 and SEQ ID NO.50 to SEQ ID NO.63, and may further comprise one, two or three amino acid substitutions other than the amino acid at position 11, 12 and / or 15.

[0046] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO. 05 to SEQ ID NO. 18, and may further comprise one, two, or three amino acid substitutions other than the 11th, 12th, and / or 15th amino acids. SEQ ID NO. 04 to SEQ ID NO. 18 correspond to single mutants, double mutants, triple mutants, or quadruple mutants of the human ferritin TRBD sequence.

[0047] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO.05, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.20, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.30, SEQ ID NO.35, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.45, SEQ ID NO.50, SEQ ID NO.56, SEQ ID NO.57 and SEQ ID NO.60, and may further comprise one, two or three amino acid substitutions other than the amino acid at positions 11, 12 and / or 15.

[0048] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO.05, SEQ ID NO.11, SEQ ID NO.20, SEQ ID NO.26, SEQ ID NO.35, SEQ ID NO.41, SEQ ID NO.50, and SEQ ID NO.56, and may further comprise one, two, or three amino acid substitutions other than the 11th amino acid. These sequences correspond to mutants Q11E and 2ECSE (Q11E Q15E).

[0049] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO.05, SEQ ID NO.20, SEQ ID NO.35, and SEQ ID NO.50, and may further comprise one, two, or three amino acid substitutions other than the 11th amino acid. SEQ ID NO.05, SEQ ID NO.20, SEQ ID NO.35, and SEQ ID NO.50 correspond to mutant Q11E.

[0050] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO.11, SEQ ID NO.26, SEQ ID NO.41, and SEQ ID NO.56, and may further comprise one, two, or three amino acid substitutions other than the 11th and 12th amino acids. SEQ ID NO.11, SEQ ID NO.26, SEQ ID NO.41, and SEQ ID NO.56 correspond to the mutant EDCSE (Q11E N12D).

[0051] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least the amino acid sequence selected from SEQ ID NO.12, SEQ ID NO.27, SEQ ID NO.42, and SEQ ID NO.57, and may further comprise one, two, or three amino acid substitutions other than the 11th and 15th amino acids. SEQ ID NO.12, SEQ ID NO.27, SEQ ID NO.42, and SEQ ID NO.57 correspond to mutant 2ECSE (Q11E Q15E).

[0052] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises at least an amino acid sequence selected from SEQ ID NO.15, SEQ ID NO.30, SEQ ID NO.45, and SEQ ID NO.60, and may further comprise one, two, or three amino acid substitutions other than the amino acids at positions 8, 11, and 15. SEQ ID NO.15, SEQ ID NO.30, SEQ ID NO.45, and SEQ ID NO.60 correspond to the mutant 3ECSE (Q8EQ11E Q15E).

[0053] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, contains at least SEQ ID NO.05 (mutant Q11E), and may also contain one, two or three amino acid substitutions other than the amino acid at position 11.

[0054] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, comprises SEQ ID NO. 11, and may further comprise one, two or three amino acid substitutions other than the amino acids at positions 11 and 12.

[0055] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, contains at least SEQ ID NO.12, and may also contain one, two or three amino acid substitutions other than the amino acids at positions 11 and 15.

[0056] In a preferred embodiment of the polypeptide according to the first aspect of the invention, the ferritin variant, particularly the TRBD of the ferritin variant, contains at least SEQ ID NO.15, and may also contain one, two or three amino acid substitutions other than the amino acids at positions 8, 11 and 15.

[0057] In a preferred embodiment of the polypeptide according to the invention, in addition to TRBD, the polypeptide further comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, and SEQ ID NO. 87, for example, 90%, 95%, 97%, 98%, 99%, or 100%. Preferably, this amino acid sequence is contained at the C-terminus of TRBD.

[0058] The polypeptides of the present invention substantially retain the properties of wild-type ferritin polypeptides in terms of complex formation (a cage-like structure composed of 24 ferritin monomer subunits) and iron uptake. The expression "substantially retains" means including embodiments in which the formation of the complex / 24-mer is improved compared to the wild type.

[0059] SEQ ID NO. 64 is a truncated concordant sequence based on the N-terminus of mammalian H-type ferritin. In SEQ ID NO. 64, the X at position 1 may or may not be present; if present, it represents any amino acid, preferably I; the X at position 2 represents any amino acid, preferably N; the X at position 9 represents any amino acid, preferably Y; the X at position 19 represents any amino acid, preferably C or Y, more preferably Y; the X at position 61 represents any amino acid, preferably F; the X at position 63 represents any amino acid, preferably Q; the X at position 70 represents any amino acid, preferably R or C, more preferably C; the X at position 85 represents any amino acid, preferably H; the X at position 89 represents any amino acid, preferably S or N, more preferably N; and the X at position 116 may or may not be present. If present, it represents any amino acid, preferably Y or H, more preferably H; X at position 119 represents any amino acid, preferably S or N, more preferably N; X at position 124 represents any amino acid, preferably S or A, more preferably A; X at position 143 represents any amino acid, preferably A or S, more preferably A; X at position 145 represents any amino acid, preferably M or L, more preferably L; X at position 157 represents any amino acid, preferably H or D, more preferably D; X at position 160 may be absent or any amino acid, preferably N; X at position 161 may be any amino acid, preferably E; X at position 162 may be any amino acid, preferably S.

[0060] SEQ ID NO. 65 is a truncated common sequence at the N-terminus and C-terminus of the mammalian H-type ferritin sequence. In SEQ ID NO. 65, the X at position 1 may or may not be present; if present, it represents any amino acid, preferably I; the X at position 2 represents any amino acid, preferably N; the X at position 9 represents any amino acid, preferably Y; the X at position 19 represents any amino acid, preferably C or Y, more preferably Y; the X at position 61 represents any amino acid, preferably F; the X at position 63 represents any amino acid, preferably Q; the X at position 70 represents any amino acid, preferably R or C, more preferably C; the X at position 85 represents any amino acid, preferably H; the X at position 89 represents any amino acid, preferably S or N, more preferably N; the X at position 116 may or may not be present; if present, it represents any amino acid, preferably Y or H, more preferably H; the X at position 119 represents any amino acid, preferably S or N, more preferably N; the X at position 124 represents any amino acid, preferably S or A, more preferably A.

[0061] SEQ ID NO. 66 is a truncated N-terminal common sequence based on the mammalian H-type ferritin sequence. In SEQ ID NO. 66, X at position 1 represents any amino acid, preferably N; X at position 8 represents any amino acid, preferably Y; X at position 60 represents any amino acid, preferably F; X at position 62 represents any amino acid, preferably Q; X at position 84 represents any amino acid, preferably H; X at position 123 represents any amino acid, preferably S or A, more preferably A; X at position 159 may be absent or any amino acid, preferably N; X at position 160 may be any amino acid, preferably E; X at position 161 may be any amino acid, preferably S.

[0062] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO. 67 to SEQ ID NO. 70, for example, 90%, 95%, 97%, 98%, 99%, or 100%.

[0063] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity with the sequence comprising the amino acids at positions 2 to 118 of SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, or SEQ ID NO. 70.

[0064] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO. 67. In SEQ ID NO. 67, the 'I' at position 1 may or may not be present.

[0065] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity with the sequence comprising the amino acids at positions 2 to 118 of SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, or SEQ ID NO. 70.

[0066] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is constituted a sequence selected from SEQ ID NO. 05 to SEQ ID NO. 63, further comprising one, two, or three amino acid substitutions other than the amino acids at positions 11, 12, and / or 15; and has at least 90%, 95%, 97%, 98%, 99%, or 100% identity with sequences selected from SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, and SEQ ID NO. 87, or with sequences from SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, particularly SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, more particularly SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, most particularly SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, especially SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, most particularly SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, and most particularly SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO The sequence consisting of amino acids from position 1 to position 118 or from position 2 to position 118 of NO.80 has at least 90%, 95%, 97%, 98%, 99%, or 100% identity.

[0067] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is composed of a sequence selected from SEQ ID NO. 05 to SEQ ID NO. 18, further comprising one, two, or three amino acid substitutions other than the amino acids at positions 8, 11, 12, and / or 15, and a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO. 67.

[0068] Preferably, the polypeptide of the first aspect of the invention contains mutations in its amino acid sequence other than that of wild-type ferritin, in addition to TRBD. In a preferred embodiment, one, two, three, or four, preferably four lysine residues, are deleted or substituted with non-basic amino acids, relating to SEQ ID NO. 1 (human wild-type heavy chain ferritin). Substitution with non-basic amino acids is preferred over deletion. The non-basic amino acid can be an acidic amino acid, such as D or E, an uncharged polar amino acid, such as S, T, N, or Q, or an uncharged nonpolar amino acid. Preferably, substitution is with E or Q. Most preferably, K54 is substituted with E, K72 with E, K87 with Q, and K144 with E. The inventors unexpectedly found that these mutations improved purification efficiency, reduced contamination, particularly nucleic acid contamination, and even more particularly DNA and endotoxin contamination, increased stability, and improved encapsulation efficiency (Examples 3 to 8).

[0069] In some embodiments of the polypeptide of the first aspect of the invention, one, two, or three, preferably two or three, cysteine ​​residues, representing SEQ ID NO. 1 (human wild-type heavy chain ferritin), are deleted or substituted, preferably substituted with serine residues. The inventors unexpectedly discovered that these mutations reduce the aggregation of ferritin polypeptides into high molecular weight complexes and improve the formation of 24-mers (Examples 3 to 4). As described above, in some embodiments of the polypeptide of the first aspect of the invention, one or more lysine residues representing positions 54, 72, 87, and / or 144, and one or more cysteine ​​residues representing positions 91, 103, and / or 131, are deleted or substituted.

[0070] As claimed in the fifth aspect of the invention, in other embodiments, cysteine ​​residues are not mutated or deleted because they can be used for covalent conjugation with drugs or tags.

[0071] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or constitutes an amino acid sequence selected from SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity with one of SEQ ID NO. 71 to SEQ ID NO. 77. The polypeptide has at least the same affinity for TfR-1 as wild-type human heavy chain ferritin according to SEQ ID NO. 2 and / or at least the same ability to form 24-mers.

[0072] In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is composed of SEQ ID NO. 71. In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is composed of SEQ ID NO. 72. In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is composed of SEQ ID NO. 73. In a preferred embodiment of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is composed of SEQ ID NO. 74.

[0073] In even more preferred embodiments of the polypeptide according to the invention, the polypeptide comprises, substantially constitutes, or is composed of SEQ ID NO.75, SEQ ID NO.76, or SEQ ID NO.77, most preferably SEQ ID NO.77, or one of SEQ ID NO.75 to SEQ ID NO.77, preferably having an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO.77.

[0074] In a preferred embodiment of the polypeptide according to the invention, the affinity of TRBD for TfR-1 is increased by at least (≥) 1.5 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥30 times, ≥40 times, or ≥50 times, but less than (≤) 60 times, ≤50 times, ≤40 times, ≤30 times, ≤20 times, ≤10 times, or ≤5 times, compared to the affinity of TRBD for TfR-1 of wild-type ferritin. In this embodiment and the following embodiments, "TRBD of wild-type ferritin" refers to the TRBD of the human ferritin heavy chain polypeptide or the mouse ferritin heavy chain polypeptide according to SEQ ID NO. 1 or SEQ ID NO. 2, specifically the amino acids from position 1 to position 20. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 1.5 times compared to the affinity of TRBD for TR of wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 2-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 3-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 4-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 5-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 10-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 20-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 30-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 40-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by at least 50-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 60-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 50-fold compared to the affinity of wild-type ferritin for TfR-1. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 40-fold compared to the affinity of wild-type ferritin for TfR-1.In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 30-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 20-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 10-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by less than 5-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 1.5-fold to 50-fold compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 2 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 3 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 4 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 5 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 10 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 20 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 30 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 40 to 50 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 1.5 to 10 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, the affinity of TRBD for TfR-1 is increased by 2 to 20 times compared to the affinity of TRBD for TfR-1 from wild-type ferritin. In a preferred embodiment, TRBD has a 5- to 30-fold increased affinity for TfR-1 compared to wild-type ferritin.

[0075] The increased binding affinity of TRBD to TfR-1 is advantageous because it promotes the binding of TRBD variant ferritin peptides to TfR-1. This increases the amount of TRBD variant ferritin peptide bound to TfR-1 expressed on the cell surface at a given time and / or given ferritin concentration. Increased binding of TRBD variant ferritin peptides to TfR-1 also contributes to the loading of the active ingredient onto cells expressing TfR-1 if the active ingredient is conjugated to or encapsulated within oligomers of the TRBD variant ferritin peptide.

[0076] To function effectively, the active ingredient conjugated to or encapsulated in the TRBD variant ferritin peptide oligomers must ultimately be released. Unwilling to be bound by theory, the acidic pH of the (late) intracellular lumen can lead to the breakdown of the ferritin oligomers, thereby releasing the active ingredient encapsulated within them. The inventors have also found that the binding affinity of TRBD to TfR-1 should not be excessively increased to avoid completely preventing the dissociation of the TRBD variant ferritin peptide from TfR-1. For optimal results, the increase in affinity should not exceed two orders of magnitude.

[0077] In a second aspect, the present invention relates to ferritin variant polypeptides wherein at least one, at least two, at least three, or at least four, preferably four lysine residues, preferably at positions 54, 72, 87, and / or 144, of the ferritin shown in SEQ ID NO. 1 (human wild-type heavy chain ferritin) are deleted or substituted with a non-basic amino acid. Substitution with a non-basic amino acid is preferred over deletion. The non-basic amino acid may be an acidic amino acid, such as D or E, an uncharged polar amino acid, such as S, T, N, or Q, or an uncharged nonpolar amino acid. The ferritin variant polypeptides are characterized by structural and functional homology with wild-type ferritin as defined above. The inventors have unexpectedly discovered that these mutations improve purification efficiency, reduce contamination, particularly nucleic acid contamination, and even more particularly DNA and endotoxin contamination, increase stability, and improve encapsulation efficiency (Examples 3 to 8). In some embodiments, the ferritin variant polypeptide of the second aspect contains mutations (i.e., deletions or substitutions) at positions 54 and 72, or 54 and 87, or 54 and 144, or 72 and 87, or 72 and 144, or 87 and 144, or 54, 72 and 87, or 54, 72 and 144, or 54, 87 and 144, or 72, 87 and 144, or 54, 72, 87 and 144. Preferably, the ferritin variant polypeptide contains mutations at positions 54, 72, 87 and / or 144. Most preferably, K54 is substituted with E, K72 is substituted with E, K87 is substituted with Q and K144 is substituted with E.

[0078] Preferably, the ferritin variant polypeptide of the second aspect of the present invention has a sequence according to SEQ ID NO.82 (mammal common sequence), SEQ ID NO.1 (human heavy chain ferritin) or SEQ ID NO.2 (mouse heavy chain ferritin), wherein at least one of positions 54, 72, 87 and / or 144, preferably all lysine residues are missing or replaced by non-basic amino acids, preferably replaced by E or Q, preferably replacing K at position 54 with E, replacing K at position 72 with E, replacing K at position 87 with Q and / or replacing K at position 144 with E. The sequences according to SEQ ID NO.82, SEQ ID NO.1 and SEQ ID NO.2 also contain one to five, for example, one, two, three, four or five, one to ten, for example, one, two, three, four or five, six, seven, eight, nine or ten, one to fifteen, one to twenty or one to twenty-five amino acid mutations other than the 54th, 72nd, 87th and / or 144th amino acid mutations.

[0079] In SEQ ID NO. 82, a mammalian common sequence, the X at position 6 can be any naturally occurring amino acid, preferably Pro; the X at position 14 can be any naturally occurring amino acid, preferably His; the X at position 16 can be any naturally occurring amino acid, preferably Asp; the X at position 21 may or may not be present, and if present, it represents any amino acid, preferably Ile; the X at position 22 represents any amino acid, preferably Asn; the X at position 30 can be any naturally occurring amino acid, preferably Tyr; the X at position 40 can be any naturally occurring amino acid, preferably Tyr or Cys, more preferably Tyr; the X at position 82 can be any naturally occurring amino acid, preferably Phe; the X at position 84 can be any naturally occurring amino acid, preferably Gln; the X at position 91 can be any naturally occurring amino acid, preferably Arg or Cys, more preferably Cys; the X at position 106 can be any naturally occurring amino acid, preferably His; and the X at position 110 can be any naturally occurring amino acid. The amino acid, preferably Asn or Ser, more preferably Asn, the X at position 137 can be any naturally occurring amino acid, preferably His or Tyr, more preferably His, the X at position 140 can be any naturally occurring amino acid, preferably Asn or Ser, more preferably Asn, the X at position 145 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala, the X at position 164 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ser, the X at position 166 can be any naturally occurring amino acid, preferably Met or Leu, more preferably Leu, the X at position 178 can be any naturally occurring amino acid, preferably Asp or His, more preferably Asp, the X at position 181 is absent or any naturally occurring amino acid, preferably Asn, the X at position 182 is absent or any naturally occurring amino acid, preferably Glu, the X at position 183 is absent or any naturally occurring amino acid, preferably Ser.

[0080] Preferably, the ferritin variant polypeptide of the second aspect of the invention has a sequence comprising replacing K54E or K72E or K87Q or K144E, or K54E and K72E, or K54E and K87Q, or K54E and K144E, or K72E and K87Q, or K72E and K144E, or K87Q and K144E, or K54E, K72E and K87Q, or K54E, K87Q and K144E, or K54E, K72E and K144E, or K72E, K87Q and K144E, or K54E, K72E and K87Q and K144E, preferably K54E, K72E, K87Q and K144E according to SEQ ID NO. 82, SEQ ID NO. 1 or SEQ ID NO. 2, wherein according to SEQ ID NO. 82, SEQ ID NO. 1, or SEQ ID NO. 2, the sequence comprising replacing K54E or K72E or K87Q or K144E, wherein ... The sequences of ID NO.1 and SEQ ID NO.2 also contain one to five, for example, one, two, three, four or five, one to ten, for example, one, two, three, four, five, six, seven, eight, nine or ten, one to fifteen, one to twenty or one to twenty-five amino acid mutations other than the 54th, 72nd, 87th and / or 144th amino acid mutations.

[0081] In a preferred embodiment, the ferritin variant polypeptide of the second aspect of the invention further comprises allelic mutations in TRBD, particularly mutations in Q8E, Q11E, N12D and / or Q15E, preferably Q11E or Q11E and Q15E. The allelic mutations are those relating to the human wild-type ferritin sequence according to SEQ ID NO.1 and described in the first aspect of the invention.

[0082] In some embodiments, in the ferritin variant polypeptide of the second aspect of the invention, one or more cysteine ​​residues, particularly the cysteine ​​residues at positions 91, 103 and / or 131, are deleted or mutated, preferably mutated to serine residues.

[0083] In the most preferred embodiment, the ferritin variant polypeptide of the second aspect of the present invention has a sequence according to SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 75, SEQ ID NO. 76 or SEQ ID NO. 77, or contains one to five, for example one, two, three, four or five, or one to ten, for example one, two, three, four, five, six, seven, eight, nine or ten amino acid mutations according to SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 75, SEQ ID NO. 76 or SEQ ID NO. 77. In a particularly preferred embodiment, the ferritin variant polypeptide of the second aspect of the present invention has a sequence according to SEQ ID NO. 77.

[0084] SEQ ID NO.86 is a mammalian common sequence, in which each X has the same meaning as shown above for SEQ ID NO.82.

[0085] In a third aspect, the present invention relates to ferritin variant polypeptides wherein one or more cysteine ​​residues, particularly those at positions 91, 103, and / or 131, as shown in SEQ ID NO. 1, are deleted or substituted, preferably substituted with serine residues. The ferritin variant polypeptides are characterized by structural and functional homology with wild-type ferritin as defined above. The inventors have unexpectedly discovered that these mutations reduce the aggregation of ferritin polypeptides into high molecular weight complexes and improve the formation of 24-mers (Examples 3 and 4).

[0086] Preferably, the ferritin variant polypeptide of the third aspect of the invention has a sequence according to SEQ ID NO. 82, SEQ ID NO. 1, or SEQ ID NO. 2, wherein all cysteine ​​residues at positions 91, 103, and / or 131 are mutated, preferably mutated to serine residues. The sequence according to SEQ ID NO. 82, SEQ ID NO. 1, and SEQ ID NO. 2 further comprises one to five, for example, one, two, three, four, or five, one to ten, for example, one, two, three, four, five, six, seven, eight, nine, or ten, one to fifteen, one to twenty, or one to twenty-five amino acid mutations other than positions 91, 103, and / or 131.

[0087] In SEQ ID NO.82, a mammalian common sequence, the X in the sequence has the meaning described above.

[0088] In a preferred embodiment, the ferritin variant polypeptide of the third aspect of the invention further comprises allelic mutations in TRBD, particularly mutations in Q8E, Q11E, N12D and / or Q15E, preferably Q11E or Q11E and Q15E. The allelic mutations are those relating to the human wild-type ferritin sequence according to SEQ ID NO.1 and described in the first aspect of the invention.

[0089] In a preferred embodiment, the ferritin variant polypeptide of the third aspect of the invention further includes mutations occurring at positions 54, 72, 87, and / or 144. Preferably, the mutations are substitutions, particularly substitutions to E at position 54, substitutions to E at position 72, substitutions to Q at position 87, and / or substitutions to E at position 144. These mutations are also described in the second aspect of the invention.

[0090] In the most preferred embodiment, the ferritin variant polypeptide of the third aspect of the present invention has a sequence according to SEQ ID NO. 75 or SEQ ID NO. 76 or a sequence according to SEQ ID NO. 76 containing one to five, for example one, two, three, four or five, or one to ten, for example one, two, three, four, five, six, seven, eight, nine or ten amino acid mutations other than position 91, position 103 and / or position 131.

[0091] The active ingredient may be conjugated to a TRBD variant ferritin polypeptide of the first aspect of the invention or a polypeptide of the second or third aspect of the invention, or may be encapsulated in an oligomer of a TRBD variant ferritin polypeptide of the first aspect of the invention or a polypeptide of the second or third aspect of the invention. The term "active ingredient" includes therapeutic active ingredients and / or diagnostic active ingredients. Therefore, the term "active ingredient" refers to a therapeutic agent (also called a drug) and / or a diagnostic agent (also called a label). The inventors note that the TRBD variant ferritin polypeptide according to the invention represents a preferred construct for specifically delivering the active ingredient, particularly the encapsulated active ingredient, to cells expressing TfR-1. Furthermore, the inventors note that the ferritin variant according to the invention is capable of delivering the active ingredient, particularly the encapsulated active ingredient, to the cell nucleus.

[0092] The polypeptide according to the invention may contain other domains. In a preferred embodiment of the polypeptide according to the invention, the polypeptide further comprises an antigen-binding domain, particularly an antibody or antibody fragment.

[0093] As used in the context of this invention, the term "antibody" refers to a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably. Antibodies are protein molecules produced by plasma cells and used by the immune system to recognize and neutralize foreign substances such as bacteria and viruses. Antibodies recognize a unique portion of a foreign target, namely its antigen.

[0094] As used herein, the term "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments included in the term "antibody fragment" include fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain variable fragments (scFv), variable fragments (Fv), and V... H Structural domain, V L Domains, single-domain antibodies, nanobodies, IgNAR (immunoglobulin neoantigen receptor), di-scFv, bispecific T-cell adaptor (BITE), dual-parent retargeting (DART) molecules, trispecific antibodies, bispecific antibodies, single-chain bispecific antibodies, alternative scaffold proteins and their fusion proteins.

[0095] As used in this specification, the term "bispecific antibody" refers to a fusion protein or bivalent antibody that can bind to different antigens. A bispecific antibody consists of two single-protein chains, including a fragment of the antibody, i.e., a variable fragment. Bispecific antibodies contain a polypeptide chain (V...) H -V L 、 or V L -V H Light chain variable structural domains (V) on ) L heavy chain variable structural domains (V) connected H By using a short peptide linking two variable domains, these domains are forced to pair with complementary domains on another chain, thus creating two antigen-binding sites. Bispecific antibodies can target the same (monospecific) or different antigens (bispecific).

[0096] "Single-domain antibodies" are antibody fragments composed of a single monomeric variable domain of the antibody. Simply put, they contain only the variable heavy chain region of a single heavy chain antibody produced by camels or cartilaginous fish. Due to their different origins, they are also called VHH or VNAR (variable neoantigen receptor) fragments. Alternatively, single-domain antibodies can be obtained by monomerizing the variable domain of a conventional mouse or human antibody using genetic engineering. They exhibit a molecular weight of approximately 12 kDa to 15 kDa, making them the smallest antibody fragments capable of recognizing antigens. Other examples include nanobodies.

[0097] As used in the context of this specification, the term "antibody mimic" refers to a compound that is similar to but structurally unrelated to an antibody and can specifically bind to an antigen. Typically, an antibody mimic is an artificial peptide or protein having a molar mass of about 3 kDa to 20 kDa and containing one, two, or more than two exposed domains that specifically bind to the antigen. Examples include, in particular, LACI-D1 (lipoprotein-associated coagulation inhibitor); affilin, such as human-γB crystals or human ubiquitin; cysteine ​​protease inhibitors; Sac7D from Sulfolobusacidocaldarius; lipid carriers and anticalin derived from lipid carriers; DARPin (designed ankyrin repeat domain); the SH3 domain of Fyn; the Kunitz domain of protease inhibitors; monomers, such as the 10th type III domain of fibronectin; adnectin:knottin (cysteine ​​knottin); trimers; evibody, such as CTLA4-based binders; affinity bodies, such as the triple helix bundle of the Z-domain of protein A from Staphylococcus aureus; transbody, such as human transferrin; tetrahedra, such as monomeric or trimeric human type C lectin domains; microbes, such as trypsin inhibitor-II; affilin; and armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimics (aptamers), but this does not include artificial antibodies, antibody fragments, and fusion proteins composed of them. Their conventional advantages over antibodies include better solubility, tissue penetration, heat and enzyme stability, and relatively low production costs.

[0098] The term "antigen" is used to refer to a substance, preferably an immunogenic peptide containing at least one epitope, preferably an epitope that elicits a B cell or T cell response or a B cell and T cell response.

[0099] An epitope, also known as an antigenic determinant, is part of a substance recognized by the immune system, such as an immunogenic peptide. Preferably, this recognition is mediated by the binding of an antibody, B cell, or T cell to the epitope in question. In this context, the term "binding" preferably refers to specific binding. Epitopes typically consist of a chemically active surface group of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The term "epitaxe" includes conformational epitopes and non-conformational epitopes. The difference between conformational and non-conformational epitopes is that binding to a conformational epitope is lost in the presence of a denaturing solvent, while binding to a non-conformational epitope is not lost.

[0100] The immunogenic polypeptide according to the invention is preferably derived from pathogens selected from viruses, bacteria, and protozoa. However, in an alternative embodiment of the invention, the immunogenic polypeptide is a tumor antigen, i.e., a polypeptide or polypeptide fragment specifically expressed by cancer.

[0101] In a fourth aspect, the present invention relates to nucleic acids encoding polypeptides of the first, second, or third aspect.

[0102] In another respect, the present invention relates to a vector containing nucleic acids of the fourth aspect.

[0103] In a fifth aspect, the present invention relates to a conjugate comprising a polypeptide comprising a first aspect, a second aspect, or a third aspect and at least one tag and / or at least one drug.

[0104] As used in the context of this invention, the phrase "conjugate comprising a polypeptide and at least one tag and / or at least one pharmaceutical ingredient" refers to a composition in which one or more active ingredient molecules are covalently or non-covalently bound to a polypeptide of the first, second, or third aspect of this invention. The covalent or non-covalent binding between the polypeptide and the active ingredient can be direct or indirect. In the latter case, the active ingredient is linked to the polypeptide via a linker or spacer. Linkers or spacers are known to those skilled in the art, such as polyalanine linkers, polyglycine linkers, glycoside linkers, and (CH2) linkers. n Group linkers or peptide linkers, particularly peptide-based cleavable linkers (e.g., cathepsin-sensitive valine-citrulline sequences and p-aminobenzylcarbamate spacers). Therefore, those skilled in the art will be able to select appropriate linkers or spacers according to their respective applications.

[0105] In a sixth aspect, the present invention relates to a complex comprising at least one polypeptide of the first, second or third aspect of the present invention and / or at least one conjugate of the fifth aspect of the present invention.

[0106] As used in the context of this invention, the phrase "complex comprising a polypeptide and / or at least one conjugate" refers to a complex formed by one or more polypeptides of the first, second, or third aspect of this invention, one or more conjugates of the fifth aspect of this invention, or a complex formed by at least one polypeptide of the first, second, or third aspect of this invention and at least one conjugate of the fifth aspect of this invention. The complex is formed by covalent or non-covalent bonding between the polypeptide and / or conjugate. Covalent or non-covalent bonding can be direct or indirect. In a preferred embodiment, the complex is an oligomer, particularly a 24-mer, formed by non-covalent bonding between the polypeptide and / or conjugate.

[0107] In a preferred embodiment of the composite according to the invention, the composite further comprises at least one label and / or at least one drug.

[0108] The terms "drug" or "therapeutic agent" are used synonymously in the context of this invention and refer to any compound, preferably a prodrug, that modifies or regulates cellular activity in a patient or that can be activated to modify or regulate cellular activity. Examples of the active ingredient include so-called "small molecules" and peptides. As used in the context of this invention, the term "small molecule" refers to a hydrocarbon or a pharmaceutically active radioisotope with a molecular weight of less than 1,500 g / mol. Preferably, the drug may include an anticancer drug, a pharmaceutically active radioisotope, or ferrohydrate.

[0109] As used in the context of this invention, the term "prodrug" refers to any active ingredient that, upon administration, is metabolized or otherwise converted into a component (or drug) that is biologically active or more active in at least one property. A prodrug is chemically modified to be less active or inactive relative to a drug, but the modification results in the production of the corresponding drug through metabolic or other biological processes after administration of the prodrug to a patient. For example, a prodrug may have altered metabolic stability or transport properties, fewer side effects or lower toxicity, or improved taste relative to an active drug (see, for example, Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). Prodrugs can be synthesized using reactants other than the corresponding drug.

[0110] The terms "label" or "diagnostic agent" are used interchangeably herein and refer to any kind of compound suitable for diagnostic purposes. Preferred compounds are selected from fluorescent dyes, radioactive isotopes, and contrast agents. Contrast agents are dyes or other substances that help visualize abnormal sites in the body. In one embodiment, the term "label" refers to a compound containing a chelating agent that forms a complex with a divalent or trivalent metal cation. Preferred radioactive isotopes / fluorescent emission isotopes are selected from alpha radiation emission isotopes, gamma radiation emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, and fluorescent isotopes such as... 65 Tb, fluorescent emission isotopes such as 18 F, 51 Cr 67 Ga、 68 Ga、 111 In、 99m Tc, 140 La、 175 Yb、 153 Sm、 166 Ho、 88 Y、 89 Zr、90 Y、 149 Pm, 177 Lu、 47 Sc、 142 Pr、 159 Gd, 212 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101m15 Rh、 119 Sb、 128 Ba、 123 I, 124 I, 131 I, 197 Hg, 211 At、 169 Eu、 203 Pb, 212 Pb, 64 Cu、 67 Cu、 188 Re、 186 Re、 198 Au and 199 Ag and the above-mentioned proteins, peptides, small molecule inhibitors, antibodies or other compounds, such as 18 F-fluorodeoxyglucose (F-fluorodeoxyglucose) 18 F-FDG) or 64 Conjugates and combinations thereof. Preferred fluorescent dyes are selected from the following categories of dyes: xanthines (e.g., fluorescein), acridines (e.g., acridine yellow), oxazines (e.g., oxazine 1), cyanines (e.g., Cy7 / Cy3), styryl dyes (e.g., Dye-28), coumarins (e.g., Alexa Fluor 350), porphyrins (e.g., chlorophyll B), metal ligand complexes (e.g., PtOEPK), fluorescent proteins (e.g., APC, R-phycoerythrin), nanocrystals (e.g., quantum dot 705), perylenes (e.g., Lumogen RedF300), and phthalocyanines (e.g., IRDYE™ 700DX), as well as conjugates and combinations thereof or dyes emitting fluorescence. 65 Tb. Preferred contrast agents are selected from paramagnetic agents, such as Gd, Eu, W, and Mn, preferably chelated with a chelating agent. Other options are superparamagnetic iron (Fe) complexes and particles, compounds containing high atomic numbers, i.e., iodine for computed tomography (CT), microbubbles, and carriers such as liposomes containing these contrast agents.

[0111] At least one label and / or at least one drug (i.e., at least one active ingredient) is covalently or non-covalently bound to a TRBD variant ferritin polypeptide according to the first aspect of the invention or a polypeptide according to the second or third aspect of the invention, or is encapsulated in a complex according to the sixth aspect of the invention. Thus, even in the absence of a covalent or non-covalent bond between the protein and the active ingredient, the term "complex" includes the enclosure of the active ingredient within a cage. The formation of the complex allows the active ingredient to be transported into the cell when ferritin is intracellularly inoculated. Therefore, it is preferred that the active ingredient binds to the iron-binding protein in a manner that does not interfere with the transport mechanism. This can be readily tested by those skilled in the art using uptake assays known in the art and described in the Examples section below. If the complex containing the active ingredient is taken up by the cell and transported to the target site in the body, it is preferred that the complex is sufficiently stable to survive the transport from the cell to the target site in the body. Therefore, it is preferable to deliver the complex, rather than the active ingredient alone, to the cell or to the cell at the target site. This property also reduces the potential harmful effects of the active ingredient on the cell to which the active ingredient is delivered, such as cytotoxicity.

[0112] By utilizing the binding / dissociation properties of ferritin macromolecules, the active ingredient can be encapsulated within the lumen of ferritin oligomers (physical confinement). The active ingredient remains in situ through non-covalent interactions with amino acid residues on the lumen surface.

[0113] If the active ingredient is covalently coupled to a TRBD variant ferritin polypeptide or a polypeptide according to the second or third aspect of the invention, such coupling is preferably via amino acid residues known to be located in surface regions that do not participate in the binding of ferritin to TfR-1. The TRBD variant ferritin polypeptide used in the context of this invention can form stable, non-covalently bound complexes with a variety of active ingredients. If the active ingredient is a peptide, such as an antigenic peptide, it is preferable that it is not expressed as a fusion with an iron-binding protein, because in this case, releasing the peptide from the iron-binding protein would require endosomal processing of the entire ferritin peptide fusion protein.

[0114] The inventors have demonstrated that, regardless of the conjugation / adsorption / binding method, once the TRBD variant ferritin peptides or polypeptides according to the second or third aspect of the present invention, and their conjugates and complexes, are loaded into a suitable cellular system with tumor-targeting properties, such as activated macrophages, they become privileged carriers of drugs and tags. The purification process for these TRBD variant ferritin peptides or polypeptides according to the second or third aspect of the present invention is simple, rapid, inexpensive, and safe, providing significant added value.

[0115] The following preferred embodiments further illustrate the fifth and sixth aspects of the invention.

[0116] In a preferred embodiment, the drug and / or label are selected from proteins, nucleic acids, chemical non-protein, non-nucleic acid compounds with a molecular weight of less than 1.5 kDa, more preferably less than 1 kDa, viruses, and radioisotopes that emit α or β radiation and γ radiation in amounts that cause cell damage.

[0117] If the drug is a nucleic acid, it is preferably a miRNA, siRNA, chemically modified RNA, LNA, ssRNA, DNAzyme, or a nucleic acid encoding a drug-active protein, such as an antibody, antibody mimic, cytokine, prodrug converting enzyme, immunogenic peptide, etc.

[0118] In a preferred embodiment, the label is selected from fluorescent dyes, radioactive isotopes / fluorescent emission isotopes, detectable peptides or nucleic acids encoding detectable peptides, and contrast agents.

[0119] In a preferred embodiment, the fluorescent dye is selected from the following categories of fluorescent dyes: xanthanids, acridines, oxazines, anthocyanins, styryl dyes, coumarins, porphyrins, metal-ligand-complexes, fluorescent proteins, nanocrystals, perylenes, and phthalocyanines, as well as conjugates and combinations thereof.

[0120] In a preferred embodiment, the radioactive isotope / fluorescent emission isotope is selected from alpha emission isotopes, gamma emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, fluorescent isotopes such as 65Tb, and fluorescent emission isotopes such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, and 142 Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au, and 199Ag, as well as the above combined with proteins, peptides, small molecule inhibitors, antibodies, or other compounds (e.g., 18 F-FDG, 89 Zr oxide or 64 Combinations and combinations of Cu-porfirin.

[0121] In a preferred embodiment, the detectable peptide is an autofluorescent protein, preferably a green fluorescent protein or any structural variant thereof having altered adsorption and / or emission spectra.

[0122] In a preferred embodiment, the contrast agent includes a paramagnetic agent, preferably selected from Gd, Eu, W and Mn, or iron hydride.

[0123] In a preferred embodiment, the label contains a chelating agent that forms a complex with a divalent or trivalent metal cation.

[0124] In a preferred embodiment of the targeted delivery system of the present invention, the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA), and 6-hydrazylpyridine-3-carboxylic acid (HYNIC).

[0125] In a preferred embodiment, the drug is selected from anticancer drugs, anti-atherosclerotic drugs, and anti-inflammatory drugs or immunomodulatory drugs (e.g., TRL agonists, STING agonists, mimicking viral or bacterial infections).

[0126] In a preferred embodiment, the anticancer drug is a cell-inhibiting drug, a cytotoxic drug, or a prodrug thereof.

[0127] Preferred anticancer drugs are selected from those that induce apoptosis, autophagy, or necrosis. These drugs can be any drug that effectively induces apoptosis, autophagy, or necrosis even in cells with abnormal cell proliferation. These drugs are preferably used in combination with one or more ferritins.

[0128] In a preferred embodiment, the anticancer drug is selected from apoptosis-inducing drugs, alkylating agents, antimetabolites, antibiotics, epothilone, nuclear receptor agonists and antagonists, antiandrogens, antiestrogens, platinum compounds, hormones, antihormones, interferons, cyclin-dependent kinase (CDK) inhibitors, cyclooxygenase and / or lipoxygenase inhibitors, biogenic fatty acids, biogenic fatty acid derivatives including prostaglandin hormones and leukotrienes, protein kinase inhibitors, protein phosphatase inhibitors, lipid kinase inhibitors, platinum coordination complexes, ethyleneimine, methylmelamine, triazine, vinca alkaloids, pyrimidine analogs, purine analogs, alkanol sulfonates, folic acid analogs, anthraquinones, substituted urea, and methylhydrazine derivatives, enediyne antibiotics, microtubule polymerization inhibitors such as maytansine or aurestatin derivatives, immune checkpoint inhibitors, and inhibitors of tumor-specific proteins or markers, preferably Rho-GDP-dissociation inhibitors, more preferably Grp94, or AXL inhibitors.

[0129] In a preferred embodiment, the anticancer drug is selected from sulfadiazine, arubicin, amprolium, aminoglutamine, aurestatin, L-asparaginase, azathioprine, banoanthraquinone, bendamustine, bleomycin, busulfan, leucovorin, carboplatin, capecitabine, carmustine, celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, actinomycin dapsone, daunorubicin, dibromopropionine, diethylstilbestrol, docetaxel, doxorubicin, endyne, epirubicin, epothilone B, epothilone D, estradiol phosphate sodium, estrogen, ethinylestradiol, haloperidol, etoposide, fulapine, fluorouracil, fludarabine, fluorouracil, flumethasone, flumethasone, flutamide. Species estradiol, furazolidone, gemcitabine, gonadotropin-releasing hormone analogs, hexamethylmelamine, hydroxyurea, hydroxymethylfurantoin, progesterone caproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon alpha, irinotecan, leuprorelin, lomustine, letopecan, sulfamidosulfamethoxazole, dichloromethyldiethylamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mipaleline, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitoxazine, mitotane, mitoxantrone, photomycin, nalidixic acid, nifuratel, nifurofenazine, furaprazine, nifuratemo, nimustine, nimoprazole, nitrofurantoin, nitrogen mustard, oxaquinic acid, pentamivir, pentostatin, phenapyridine , sulfadiazine, piperobromane, prednimustine, prednisone, (2S,3S,5R)-1-methyl-5-nonyl-2-benzyl-3-pyrrolidone, methylbenzylhydrazine, pyrimethamine, raltitrexate, rapamycin, rofecoxib, rosiglitazone, sulfasalazine, acridine yellow hydrochloride, semustine, streptozotocin, sulfaurea, sulfacetamide, sulfachlorpyridazine, sulfadiazine, sulfapentene, sulfadiazine, sulfadiazine, sulfadiazine, sulfadiazine, sulfaguanidine, sulfaguanoxidine, sulfamethoxazole, sulfamethoxazole, compound sulfamethoxazole, sulfamethoxypyrimidine, sulfamethoxypyrazine, sulfadiazine, sulfonamide, sulfapeline, sulfabenzylazole, sulfathiazole, sulfadimethoxazole, sulfadiazine, salinomycin Tamoxifen, paclitaxel, teniposide, teniposide, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triamcinolone, trimethoprim, trimethoprim, trefocillin, UCN-01, vincristine, vinblastine, vinorelbine, vinorelbine, and zorubicin, preferably selected from aurostatin, banroanthraquinone, bendamustine, chlorambucil, chaliceamycin, cyclophosphamidedynemycin A, maytansine, melphalan, maytansine, and neo-carcinogen, most preferably banroanthraquinone, bendamustine, chlorambucil, cyclophosphamide, pyrrole-benzoheptatriene, and melphalan.

[0130] In a preferred embodiment, the anticancer drug is a proliferation inhibitor protein, preferably a cell cycle inhibitor or an antibody or antibody-like binding protein that specifically binds to a proliferation-promoting protein or nucleic acid, preferably an antibody or antibody-like binding protein that encodes a proliferation inhibitor protein or specifically binds to a proliferation-promoting protein or siRNA or DNA ribozyme.

[0131] In a preferred embodiment, the immunomodulatory drug activates or inhibits the activity of immune cells. These can be natural or synthetic ligands, including antibodies, or antagonists of pattern recognition receptors, particularly Toll-like receptors, NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs). Physiologically, these receptors recognize a class of signals known as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).

[0132] Preferred examples of antibodies used in the context of this invention are single-chain antibodies, antibody fragments, nanobodies, light or heavy chains, variable light or heavy chains, or bispecific antibodies. Preferred antibody fragments include fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain variable fragments (scFv), variable fragments (Fv), and V... H Structural domain, V L Domains, single-domain antibodies, nanobodies, IgNAR (immunoglobulin neoantigen receptor), di-scFv, bispecific T-cell adaptor (BITE), dual-parent retargeting (DART) molecules, trispecific antibodies, bispecific antibodies, single-chain bispecific antibodies, and their fusion proteins.

[0133] In a preferred embodiment, the virus is an oncolytic virus.

[0134] In a preferred embodiment, alpha or beta radiation of a damaging gamma radiation amount is also emitted. The emitted radioactive isotope is selected from lutetium-177, ytterbium-90, iodine-131, samarium-153, phosphorus-32, cesium-131, palladium-103, radium-233, iodine-125, and boron-10, or a damaging alpha radiation amount, preferably selected from actinium-225, bismuth-213, lead-212, and polonium-212. Also preferred are complexes of the above compounds and isotopes linked to nanoparticles (e.g., gold, silver, graphene) or such nanoparticles.

[0135] In a preferred embodiment, the drug is a hypoxia-activated prodrug, preferably selected from benzotriazine N-oxide, apaquinone (E09), telazamine (TPN), SN30000, PR-104A, TH-302, TH-4000, and AQ4N.

[0136] In a preferred embodiment, the drug is an antigen or a nucleic acid encoding an antigen.

[0137] In a preferred embodiment, the bond between the TRBD variant ferritin polypeptide or polypeptide and the active ingredient in the conjugate according to the second or third aspect of the invention is covalent and / or non-covalent; and / or the oligomer of the TRBD variant ferritin polypeptide embeds / encapsulates the active ingredient contained in the complex. In one embodiment, covalent and / or non-covalent coupling is carried out indirectly through a linker or spacer. If covalent bonding is required, the active ingredient modified by a specific active linker portion reacting with the thiol, amino, or carboxyl group of the TRBD variant ferritin polypeptide is directly or indirectly covalently coupled to the TRBD variant ferritin polypeptide using the relevant thiol, amino, or carboxyl group.

[0138] According to the second or third aspect of the invention, a TRBD variant ferritin polypeptide or polypeptide can be linked to a cysteine ​​thiol reactive active ingredient with a peptide-based cleavable linker (e.g., a cathepsin-sensitive valine-citrulline sequence and a p-aminobenzylcarbamate spacer). As a prominent example, the antimitotic agent monomethylaurestatin E (MMAE) has been used. The peptide-based linker stably binds the protein to the cytotoxic compound, thus preventing the drug from being released from the protein under physiological conditions, helping to prevent toxicity to healthy cells and ensuring dose efficiency. The resulting ferritin active ingredient adduct is able to attach to a selected receptor type, namely ferritin's TfR-1. Once bound, the ferritin active ingredient adduct is internalized via cellularity, thereby being selectively absorbed by the target cell. The drug-containing vesicle fuses with lysosomes and lysosomal cysteine ​​proteases; in particular, cathepsin B initiates the cleavage of the valine-citrulline linker, and MMAE is no longer bound to ferritin and is released directly into the tumor environment.

[0139] Alternatively, DM1-SMCC, a potent maytansine derivative with a linker that specifically binds to lysine residues, forms a covalent complex with ferritin in reactions that have successfully described antibody responses. Specifically, ferritin can react with DM1-SMCC, thereby providing a covalent protein-drug adduct that can be cleaved intracellularly and release the active drug in a time-dependent manner. DM1's inhibition of microtubule dynamics induces mitotic arrest and cell death.

[0140] In the context of this invention, the term "full load" refers to the maximum amount of ferritin chelated with the active ingredient that can be absorbed by cells of an active delivery system.

[0141] Different pharmaceutically active substances, labels, or pharmaceutically active substances and labels are also envisioned to be contained in a complex according to a third aspect of the invention. For example, one type of active ingredient may bind (non-covalently) to one or more TRBD variant ferritin polypeptides according to a second or third aspect of the invention, while another type is encapsulated in the complex. This method utilizes the different release rates of the active ingredient in the complex delivered to the target tissue and / or cells. For example, by utilizing the reactivity of the associated thiol, amino, or carboxyl groups, the active ingredient may be covalently linked to the ferritin molecule on the surface or lumen of the 24-mer. The types of useful reactions are well known in the art, and those skilled in the art can employ specific active ingredients without any further work. Examples of such reactions are described in Behrens CR, Liu B. Methods for site-specific drug conjugation to antibodies. MAbs. 2014 Jan-Feb;6(1):46-53.

[0142] In diagnostic applications, the complex comprises a tag and a drug, preferably the tag being covalently bound to an iron-binding protein and the drug being non-covalently bound to the iron-binding protein and / or embedded in a lumen formed after the assembly of a TRBD variant ferritin polypeptide or a polypeptide multimer according to the second or third aspect of the invention.

[0143] In a sixth aspect, the present invention relates to a targeted delivery system comprising the separation of cells, wherein the cells comprise a polypeptide, a conjugate of the fifth aspect, or a complex of the sixth aspect of the invention.

[0144] The term "targeted delivery system" refers to a system capable of delivering an active ingredient to a target site, i.e., capable of targeted delivery, preferably capable of targeted delivery within a patient.

[0145] The term “targeted delivery” refers to the delivery of a therapeutic or diagnostic agent (collectively referred to herein as the “active ingredient”) to a subject such as a patient, particularly to cells within the patient’s body. Targeted delivery also includes “targeted therapeutic delivery,” which means the simultaneous delivery of a therapeutic and diagnostic agent, preferably to the site of disease, thereby allowing for simultaneous treatment and diagnosis and / or treatment monitoring.

[0146] Targeted delivery results in an increased concentration of the active ingredient in a specific part of the body compared to other parts of the patient's body. Preferably, the relative concentration is compared between one or more diseased sites of the body and other parts of the body that have a similar manner of entering the bloodstream. In a preferred embodiment, the concentration of the active ingredient in a given number of cells or a given biopsy volume from a diseased site is at least 10% higher than that from a non-diseased site, preferably 2 to 24 hours after application of the targeted delivery system of the invention. More preferably, after application of the targeted delivery system of the invention, preferably 2 to 24 hours later, the concentration of the active ingredient in the diseased site of the patient's body is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, more preferably at least 1000%, higher than that in a non-diseased site of the body. When assessed based on systemic distribution, preferably at least 5%, more preferably at least 10%, and more preferably at least 15% of the active ingredient administered to the patient is delivered to the affected site of the body. Targeted delivery of the active ingredient limits its potential adverse effects on the affected site of the body.

[0147] The targeted delivery system according to the invention enables the delivery of pharmaceutically active substances, labels, or pharmaceutically active substances and labels to tumors that would normally be unable to reach them (e.g., due to solubility issues). This allows for the precise application of active ingredients to tumor sites (especially hypoxic sites) and tumor masses, preventing their accumulation in other organs.

[0148] Targeted delivery includes direct targeting and indirect targeting. Direct targeting refers to the direct uptake of the active ingredient (as a conjugate according to the second aspect of the invention or a complex according to the third aspect of the invention) by diseased cells, such as cancer cells. Indirect targeting refers to the delivery of the active ingredient (as a conjugate according to the second aspect of the invention or a complex according to the third aspect of the invention) to diseased cells, such as cancer cells, via another cell, such as leukocytes.

[0149] The mutant Q11E exhibits at least a four-fold higher affinity for the CD71 receptor and a correspondingly slower release rate from its complex. Therefore, this mutant is readily absorbed by cancer cells overexpressing the CD71 receptor (direct targeting). Furthermore, it is readily taken up by CD45+ leukocytes capable of translocating ferritin mutants to target cells (indirect targeting).

[0150] In particular, if the cells in the targeted delivery system of the present invention are leukocytes, the targeted delivery system targets lymph nodes, making it particularly suitable for delivering antigens to dendritic cells located in lymph nodes. Lymph node targeting is especially significant if the cells carrying the complex are macrophages, particularly activated macrophages, even more preferably CCL-2-activated bone marrow-derived activated macrophages, or lymphocytes, particularly B cells or T cells. Therefore, in a preferred embodiment, the targeted delivery system is used to deliver one or more antigens to elicit a prophylactic and / or therapeutic immune response against one or more antigens. Preferred antigens are derived from pathogens, i.e., bacteria or viruses, or tumor-specific antigens. The term "tumor-specific antigen" refers to a protein or epitope (including peptides with altered glycosylation patterns) that is expressed at higher levels on tumor cells compared to non-tumor cells, preferably an antigen or epitope expressed only on tumor cells. Preferred antigens are selected from epidermal growth factor receptors (EGFR, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding protein (IGFBP), IGFR ligand family; platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; hepatocyte glycoprotein (EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1,2; Orphan receptor (ROR) family of tyrosine kinase-like receptors; Disk-domain receptor (DDR) family; RET family; KLG family; RYK family; Musk family; Transforming growth factor α (TGF-α) receptor, TGF-β; Cytokine receptors, class I (erythropoietin family) and class II (interferon / IL-10 family) receptors, Tumor necrosis factor (TNF) receptor superfamily (TNFRSF), death receptor family; Testicular cancer (CT) antigen, lineage-specific antigen, differentiation antigen, α-actin-4, ARTC1, split-cluster region-Abelson (Bcr-abl) fusion product, β-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-linkin (CTNNB1), cell cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, ETUD-2, elongation factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low-density lipid receptor / GDP-L fucose: β-Dgalactose 2-α-Lfucosyltransferase (LDLR / FUT) fusion protein, HLA-A2. The following are listed as potential mutations in HLA-A2 gene (HLA-A*201-R170I): arginine and isoleucine exchange at residue 170 of the α-helix of the α2 domain; HLA-A11; heat shock protein 70-2 mutation (HSP70-2M); KIAA0205; MART2; common melanoma mutations 1, 2, 3 (MUM-1, 2, 3); prostatic acid phosphatase (PAP); neo-PAP; myosin class I; NFYC; OGT; OS-9; pml-RARα fusion protein; PRDX5; PTPRK; K-ras (KRAS2); N-ras (NRAS); HRAS; RBAF600; SIRT2; SNRPD1; SYT-SSX1 or -SSX2 fusion protein; triose phosphate isomerase; BAGE; BAGE-1; BAGE-2, 3, 4, 5; GAGE-1, 2, 3, 4, 5, 6, 7.8. GnT-V (abnormal N-acetylglucosamine transferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized melanoma antigen (CAMEL), MAGE-A1 (MAGE-1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12 MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, Mucin 1 (MUC1), MART-1 / Melan-A (MLANA), gp100, gp100 / Pmel17 (SILV), Tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1, 2, 34. TRP2-INT2, Carcinoembryonic Antigen (CEA), Kallikrein 4, Pulmonary Globulin-A, OA1, Prostate-Specific Antigen (PSA), TRP-1 / gp75, TRP-2, Adipocyte Differentiation-associated Protein (AIM-2), Interferon-Inducible Protein 2 (AIM-2) is absent in melanoma, BING-4, CPSF, Cyclin D1, Epithelial Cell Adhesion Molecule (Ep-CAM), EphA3, Fibroblast Growth Factor-5 (FGF-5), Glycoprotein 250 (gp250), EGFR (ERBB1), HER-2 / neu (ERBB2), Interleukin-13 Receptor α2 Chain (IL13Rα2), IL-6 Receptor, Intestinal Carboxyesterase (iCE), Alpha-Fetoprotein (AFP), M-CSF, mdm-2, MUC1, p53 (TP53), PBF, PRAME, PSMA, RAGE-1, RNF4 3. RU2AS, SOX10, STEAP1, apoptosis inhibitor protein (BIRC5), human telomerase reverse transcriptase (hTERT), telomerase, Wilms tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC, MORC, SGY-1, SpO11, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE, TPTE, immunoglobulin idiotype, Bence-Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, cancer antigen 72-4 (CA 72-4), Cancer Antigen 15-3 (CA 15-3), Cancer Antigen 27-29 (CA 27-29), Cancer Antigen 125 (CA 125), Cancer Antigen 19-9 (CA 125) 19-9), β-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, saxaglastin, CTLA-4, 707 alanine-proline (707-AP), T-cell recognized adenocarcinoma antigen 4 (ART-4), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilic protein B (Cyp-B), human signet ring tumor 2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsid antigens, others), Epstein-Barr (EBV) proteins (EBV latent membrane proteins-LMP1, LMP2; others), hepatitis B or C virus proteins, and HIV proteins.

[0151] The targeted delivery system of this invention is particularly suitable for delivering active ingredients to hypoxic sites. Hypoxia is characteristic of various diseases, including cancer and inflammatory diseases, and therefore can be targeted at these diseases.

[0152] In addition to targeting, the use of an active ingredient activated under hypoxic conditions increases the specificity of the target and / or further reduces the side effects of the active ingredient. Therefore, in a particularly preferred embodiment, the active ingredient is a hypoxia-activated prodrug. The backbone of all hypoxia-activated prodrugs consists of one of five different chemical moieties (nitro, quinine, aromatic N-oxides, aliphatic N-oxides, and transition metals), which are enzymatically reduced under hypoxic conditions in tissues. A hypoxia-activated prodrug is any prodrug with low or no activity relative to the corresponding drug and contains the drug and one or more bioreducible groups. Such hypoxia-activated prodrugs include all prodrugs activated by a variety of reducing agents and reductases, including but not limited to single-electron transferases (such as cytochrome P450 reductase) and two-electron transferases (or hydride transferases). According to a preferred embodiment of the invention, the hypoxia-activated prodrug is TH-302. PCT applications WO 07 / 002931 and WO 08 / 083101 describe methods for synthesizing TH-302. Preferred examples of the prodrug are selected from Class I: benzotriazine N-oxide, apaquinone (EO9), telazamine (TPN) and SN30000; or Class II: nitro compounds PR-104A, TH-302, TH-4000 and AQ4N.

[0153] A surprising observation is the disease-specific homing of the targeted delivery system of this invention. Specifically, CD45... + Leukocytes appear to exhibit a preference for sites of hypoxia and oxidative stress. Hypoxia is a hallmark of various diseases, as is oxidative stress. Therefore, the present invention also relates to a separate targeted delivery system of the fourth aspect of the invention for the prevention, treatment, or diagnosis of diseases characterized by sites of hypoxia and / or oxidative stress within diseased tissues, particularly hypoxic tumors or hypoxic sites within tumors, or any site in an organism subjected to hypoxic conditions, such as during ischemic events, or undergoing inflammatory processes. Similarly, the present invention relates to a method for treating, preventing, or diagnosing diseases characterized by sites of hypoxia and / or oxidative stress within diseased tissues, particularly hypoxic tumors or hypoxic sites within tumors, or any site in an organism subjected to hypoxic conditions, such as during ischemic events, or undergoing inflammatory processes, by administering an effective amount of the separate targeted delivery system of the fourth aspect of the invention to a subject in need.

[0154] The ability of a given cell or population to internalize ferritin depends on the expression of the receptors involved in this internalization process. Receptors that lead to ferritin internalization include, for example, TfR, CXCR4, scavenger receptors, CD163, and TIM-2. Those skilled in the art are well aware of how to measure ferritin uptake, and preferred methods for measuring uptake are described in the Examples section below.

[0155] In a preferred embodiment of the isolated targeted delivery system according to the invention, the cells are CD45. + White blood cells, especially CD45 cells selected from monocytes, differentiated monocytes, monocytes / macrophages, lymphocytes, and granulocytes. + leukocyte.

[0156] In the context of this invention, the term "leukocyte" refers to cells of the immune system that participate in protecting the body from infectious diseases and foreign invaders. All leukocytes are produced and derived from pluripotent cells called hematopoietic stem cells in the bone marrow. Leukocytes are distributed throughout the body, including the blood and lymphatic systems. All leukocytes have a nucleus, which distinguishes them from other blood cells, anucleated red blood cells (RBCs), and platelets. Leukocytes can be classified in a standard manner. Two pairs of the broadest categories classify them by structure (granulocytes or agranulocytes) or cell division lineage (bone marrow cells or lymphocytes). These broadest categories can be further divided into five main types: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. These types are distinguished by their physical and functional characteristics. Monocytes and neutrophils are phagocytes. They can be further classified into subtypes; for example, lymphocytes are classified into B cells, T cells, and NK cells. The difference between granulocytes and agranulocytes lies in the shape of their nuclei (lobed vs. round, i.e., polymorphonuclear vs. mononuclear) and their cytoplasmic granules (present or absent, or more precisely, visible or therefore invisible under a light microscope). Another dichotomy is by lineage: bone marrow cells (neutrophils, monocytes, eosinophils, and basophils) are distinguished from lymphocytes (lymphocytes) by hematopoietic lineage (cell differentiation lineage).

[0157] CD45 + Expression is characteristic of leukocyte subsets, namely monocytes, monocytes / macrophages, lymphocytes, granulocytes, and NK cells, which are suitable for the targeted delivery system of this invention, especially because of CD45. + Leukocytes are attracted to specific tissues and cells in the body and are capable of delivering or introducing into cells one or more iron-binding proteins and one or more drug-active substances, tags, or drug-active substances and labeled complexes. This subset of leukocytes is referred to below as "CD45". +"White blood cells". Preferably, the monocytes are not dendritic cells whose differentiation is controlled by one or more of the following transcription factors: IFN-regulatory factor 8 (IRF8), nuclear factor interleukin (IL)-3-regulatory protein (NFIL3), basic leucine zipper transcription factor ATF-like 3 (BATF3) or transcription factor RelB (NF-KB subunit)-RELB, Spi-1 proto-oncogene (PU / 1), recombinant hairless binding protein repressor (RBPJ), interferon regulatory factor 4 (IRF4) or transcription factor E2-2 (also known as TCF4).

[0158] Those skilled in the art will understand that, unless it is of clone origin, CD45 as defined above... + White blood cells are cells that express CD45. + A mixed population of different leukocytes sharing common properties of surface antigens. Therefore, CD45 as defined above... + Cell subsets within different groups of leukocytes are determined throughout the specification by further functional and / or structural characteristics. The term "CD45" + This indicates that most or virtually all cells in the cell population express CD45. + Surface antigen.

[0159] In this context, "expression" means that most or virtually all cells within a cell population express the marker (also referred to herein as a surface antigen). In this context, and also with reference to other cell surface antigens, the term "expression" means that the surface antigen is produced within the cell and is detectably exposed on the cell surface. Expression levels, and therefore the amount of surface antigen detectably exposed on the cell surface, can vary considerably between different cells. Generally, a cell is considered positive if at least 5, preferably at least 10, copies of a cell surface antigen are detectably exposed on the cell surface, i.e., expressed as "expression" for the cell surface antigen. + Those skilled in the art are well aware of how to detect, quantify, and select cells that are positive (or negative) for a given cell surface antigen. Preferred methods include fluorescence-activated cell sorting (FACS). In this technique, fluorescently labeled antibodies are used to bind to cell surface antigens in a cell population, which are then separated into individual cells, and characterized as positive or negative for a given cell surface antigen based on the fluorescence intensity measured on the individual cells. In some embodiments of the invention, it is indicated that the expression of a given protein is either high or low. This means that the protein is detectably expressed in both cases, i.e., " +However, the expression levels differ. High expression and low expression respectively mean that the absolute number of different proteins per cell is different. Therefore, if there are more than 500 detectable copies of a protein per cell, a given protein can be considered to be expressed at a high level, and if there are 1 to 50 detectable copies per cell, the protein can be considered to be expressed at a low level. However, if there are more than 5000 detectable copies per cell, another protein can be considered to be expressed at a high level, and if there are 1 to 500 detectable copies per cell, another protein can be considered to be expressed at a low level. The use of flow cytometry and Becton Dickinson Quantibrite is well known in the art. TM Microbead method (see, for example, Pannu, KK, 2001, Cytometry.2001 Dec 1;45(4):250-8) or mass spectrometry (see, for example, Milo, R., 2013, Bioessays, 35(12):1050-1055) is used to quantify the amount of proteins expressed or produced in cells.

[0160] For the purposes of this invention, the term "high expression" for a given protein refers to the high expression of that protein in a healthy cell population, particularly CD45. + Detectable expression in a leukocyte population is defined as the highest expression level found, i.e., at least 70% of the number of copies per cell. The term "low expression" for a given protein refers to the protein being present in a healthy cell population, particularly CD45. + Detectable expression in a leukocyte population is defined as the highest expression level found, i.e., 30% or less of the protein copies per cell. Preferably, the "highest expression level" is defined as CD45 in healthy cells, particularly in different subjects. +The average of the highest expression levels found in leukocytes. In some embodiments, a preferred cell subpopulation is characterized by “producing” a given protein. This should be understood to mean that the protein is not necessarily detectable on the cell surface, but may only be present inside the cell. Those skilled in the art are well aware of how to detect and / or quantify the production of intracellular proteins and / or select cells that produce said proteins. Alternatively, a cell population can be defined by the expression of a specific transcription factor. The expression of a given protein or its encoding mRNA in a cell population or even a single cell is well known in the art, for example, by using an in vivo tag with an antibody alone, FISH assay, in vivo single-molecule fluorescence microscopy (Crawford et al. Biophys J. (2013) 105(11):2439) or in combination with fluorescence-activated cell sorting, or by PrimeFlow technology, (e Bioscience) (Adam S. Venable, et al., (2015) Methods in Molecular Biology).

[0161] In the context of this invention, the term "differentiated monocytes" refers to monocytes that differentiate from stereotyped precursors called macrophage-DC precursors (MDPs), which primarily reside in the bone marrow (but may also be found in the spleen) and differentiate into dendritic cells or macrophages. In mice, they consist of two main subsets: (i) those with high expression of CX3CR1, CCR2, and Ly6C... - Low expression of CD11b + Cells and (ii) cells with low expression of CX3CR1, CCR2 and Ly6C + High expression of CD11b + Cells. After leaving the bone marrow, mouse Ly6C + Monocytes differentiate into circulating Ly6C - Monocytes. Similarly, in human monocyte differentiation, CD14 is present in the peripheral blood circulation. ++ Classical monocytes leave the bone marrow and differentiate into CD14 cells. ++ CD16 + Intermediate monocytes and CD14 + CD16 ++ Non-classical monocytes (Yang et al. 2014; Biomark Res 2(1) doi.10.1186 / 2050-7771-2-1). Preferably, the differentiated monocytes are not dendritic cells whose differentiation is controlled by one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB, PU / 1, RBPJ, IIRF4 and / or TCF4, and more preferably not dendritic cells.

[0162] Macrophages are specialized phagocytic cells and antigen-presenting cells (APCs) that reside in tissues, distinct from circulating peripheral blood mononuclear cells (PBMs). In the context of this invention, the term "activated macrophage" refers to macrophages of arbitrary polarization. Macrophage activation is typically achieved through incubation with interleukins, cytokines, and / or growth factors. In particular, IL-4 and M-CSF can be used as activators. Activated macrophages of different phenotypes are classified as M1-macrophages, classical activated macrophages (CAMs), and M2-macrophages, or activated macrophages (AAMs). Classically activated M1 macrophages comprise immune effector cells with an acute inflammatory phenotype. These are highly aggressive against bacteria and produce large amounts of lymphokines (Murray and Wynn, 2011, J LeukocBiol, 89(4): 557-63). Alternative activated anti-inflammatory M2-macrophages can be divided into at least three subgroups. These subtypes have a variety of different functions, including immune regulation, maintenance of tolerance, and tissue repair / wound healing. In the context of this invention, the term "M1 inducer" refers to a compound that directs PBM differentiation into M1 macrophages. In the context of this invention, the term "M2 inducer" refers to a compound that directs PBM differentiation into M2 macrophages. Those skilled in the art understand numerous methods for promoting differentiation into M1 or M2 macrophages.

[0163] The term "macrophage phagocytosis" refers to the process by which macrophages engulf solid particles to form internal vesicles called phagosomes.

[0164] Preferably, CD45 + Monocytes are not dendritic cells whose differentiation is controlled by one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB, PU / 1, RBPJ, IIRF4 and / or TCF4, and more preferably not dendritic cells.

[0165] In a preferred embodiment of the separate targeted delivery system, CD45 + White blood cells can be converted from CD34 + Hematopoietic precursor cells are produced.

[0166] In a preferred embodiment of the separate targeted delivery system

[0167] (i) Monocytes are CD11b + Mononuclear cells, preferably selected from CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CD14 +CD16 + Monocytes, CD11b + CD14 + MHCII + Monocytes, CD11b + CD14 + CD115 + Monocytes, CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CCR1 + Monocytes, CD11b + CCR2 + Monocytes, CD11b + CX3CR + Monocytes, CD11b + CXR4 + Monocytes, CD11b + CXR6 + Monocytes and CD11b + CD14 + CD33 + Monocytes, preferably not dendritic cells whose differentiation is controlled by one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB, PU / 1, RBPJ, IRF4 and / or TCF4, more preferably not dendritic cells;

[0168] (ii) Differentiated monocytes or monocyte-macrophages differentiated from M-CSF and selected from macrophages, activated macrophages, preferably CD11b + Macrophages, preferably CD11b + CD16 + Macrophages, CD11b + CD32 + Macrophages, CD11b + CD64 + Macrophages, CD11b + CD68+ macrophages, preferably CD11b + CD86 + M1 macrophages, preferably producing inducible nitric oxide synthase (iNOS) and / or secreting interleukin-12 (IL-12) or preferably CD11b. + CCR2 + M2 macrophages, CD11b + CD204+M2 macrophages, CD11b + CD206+ M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + Major Histocompatibility Complex II + (MHCII + (Low or high expression) M2 macrophages, CD11b + CD200R + M2 macrophages, CD11b + CD163 + M2 macrophages or activated macrophages that produce and / or secrete arginase-1 and / or interleukin-10 (IL-10); preferably, the differentiated monocytes are not expressing lectin-like oxidized low-density lipoprotein receptor 1 (Lox1). + CXC chemokine receptor type 7 (CXCR7) + ) and nuclear factor-E2 related factor 2 (NRF2) + Foam cells are macrophages located on fatty deposits in the walls of blood vessels, where they take up low-density lipoprotein and load lipids, giving them a foamy appearance. These cells secrete various substances involved in plaque growth, and their death promotes inflammation, leading to cardiovascular disease.

[0169] (iii) Monocytes or activated monocytes or macrophages differentiated from M-CSF and preferably expressing at least one chemokine receptor, preferably selected from CCR1, CCR2, CXCR4 and CXCR6, or at least one growth factor receptor, preferably selected from macrophage colony-stimulating factor receptor (CD115), granulocyte colony-stimulating factor receptor (CD114) and granulocyte-macrophage colony-stimulating factor receptor (composed of CD116 and CD131); monocytes with these characteristics are particularly suitable for the treatment of inflammation and cancer;

[0170] (iv) Lymphocytes are selected from CD3 + and CD4 + or CD8 + T lymphocytes, or CD19 + CD20 + CD21 + CD19 + CD20 + CD19 + CD21 + CD20 + CD21 + or CD19 + CD20 + CD21+ B lymphocytes; or natural killer (NK) cells, preferably NK cells selected from CD56. + And does not express CD3 or CD16. + CD56 + CD56 + CD94 + CD56 + CD158a + CD56 + CD158f + CD56 + CD314 + CD56 + CD335 + Cells; or

[0171] (v) Granulocytes are selected from neutrophils, preferably CD66b. + Neutrophils, eosinophils, and basophils, preferably CD193. + Eosinophils.

[0172] In a preferred embodiment of the isolated targeted delivery system, activated macrophages:

[0173] (i) It can be prepared by incubating monocytes or macrophages or their precursors with factors that can alter the expression markers on macrophages in vitro, preferably by incubating with the following substances in vitro:

[0174] (a) at least one M1 inducer,

[0175] (b) at least one M2 inducer,

[0176] (c) or factors capable of altering the ability of macrophages to secrete cytokines, preferably IL-10 and IL-12, chemokines and / or the production of iNOS, arginase, or other immunomodulatory enzymes; examples of such factors include: activated platelets, IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated gamma globulin, glucocorticoids, tumor growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2), IL-6, macrophage colony-stimulating factor (M-CSF), peroxisome proliferator-activated receptor (PPR). Arginase-1 (AR) agonists, leukocyte inhibitory factor (LIF), adenosine, worm and fungal infections, lipopolysaccharide (LPS), interferon-γ (INF-γ), viral and bacterial infections; in this regard, it was observed that activation of monocytes with M1 inducers, especially LPS, led to the expression of iNOS, while activation of monocytes with M1 inducers, especially LPS, led to the non-expression of Arginase-1. Activation of monocytes with M2 inducers, especially IL-4, led to the expression of Arginase-1, and activation of monocytes with M2 inducers, especially IL-4, led to the non-expression of iNOS.

[0177] (ii) Characterized by the expression of at least one of the following antigens: high expression of CD64, CD86, CD16, CD32, MHCII and / or production of iNOS and / or IL-12;

[0178] (iii) It can be produced by incubating monocytes or macrophages with factors that can induce macrophage phagocytosis, such as IL-18, opsonins (e.g., complement-derived proteins, such as iC3b, immunoglobulin G), calcitonin gene-related peptide (CGRP), lipopolysaccharide (LPS), interferon-γ (INF-γ), viral infection and / or bacterial infection in vitro.

[0179] (iv) Characterized by expression of at least one of the following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC kinetic chemokine receptor 4 (CXCR4), CD163 and / or T cell immunoglobulin domain and mucin domain 2 (TIM-2), and / or exhibiting low expression of MHCII; activated macrophages with these properties are particularly suited to complexes containing ferritin as an iron-binding protein;

[0180] (v) Possesses the ability to devour; and / or

[0181] (vi) It is able to secrete cytokines, preferably IL-12 or IL-10, or produce inducible nitric oxide synthase (iNOS) (or other pro-inflammatory compounds), arginase or other immunosuppressive / anti-inflammatory compounds.

[0182] In a preferred embodiment of the isolated targeted delivery system, the M1 inducer for differentiating macrophages into M1 macrophages is selected from lipopolysaccharide (LPS), interferon-γ (INF-γ), viral and bacterial infections, while the M2 inducer for differentiating macrophages into M2 macrophages is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated gamma globulin, glucocorticoids, tumor growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2), IL-6, macrophage colony-stimulating factor (M-CSF), peroxisome proliferator-activated receptor (PPAR) agonists, leukocyte inhibitory factor (LIF), adenosine, worm and fungal infections.

[0183] In a preferred embodiment of the targeted delivery system of the present invention, mononuclear macrophages:

[0184] (i) Available from CD34 + Production of hematopoietic precursor cells;

[0185] (ii) It can be produced by in vitro incubation of mononuclear cells with at least one inducer, preferably M1 or M2 inducer, more preferably at least one M2 inducer;

[0186] (iii) Characterized by expressing at least one of the following antigens: TfR, CD163, TIM-2, CD14, CD16, CD33 and / or CD115;

[0187] (iv) Characterized by expression of at least one of the following antigens: TfR, CD163, TIM-2, CXCR4, CD14 and / or CD16; and / or

[0188] (v) Possesses the ability to devour; and / or

[0189] (vi) Dendritic cells that do not differentiate under the control of one or more of the following transcription factors: IRF8, NFIL3, BATF3 or RELB, PU / 1, RBPJ, IRF4 or TCF4.

[0190] In this embodiment of the targeted delivery system of the present invention, the M1 inducer for differentiating monocytes and macrophages is selected from LPS, INF-γ, or viral or bacterial infection, or the M2 inducer for differentiating monocytes is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated gamma globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPAR agonists, leukocyte inhibitory factor (LIF), cancer conditioned medium, cancer cells, adenosine, and worm or fungal infection.

[0191] In a preferred embodiment of the targeted delivery system of the present invention, lymphocytes:

[0192] (i) available from blood, spleen or bone marrow or, as known to those skilled in the art, from CD34 + Precursor cell generation has also been described in, for example, Lewisor and Kim, 2010, J Vis Exp 40:2017; Tassone and Fidler, 2012, Methods in Molecular Biology 882:351-357; Kouro et al., 2005, Current Protocols in Immunology, 66:F22F.1:22F.1.1-22F.1.9.

[0193] (ii) are lymphocytes with immune capabilities;

[0194] (iii) Expressing antigen-specific T cell receptors; and / or

[0195] (iv) Characterized by expressing at least one of the following antigens: (a) CD3 and CD4 or CD8 or (b) CD19, CD20, CD21, CD19 CD20, CD19 CD21, CD20 CD21, or CD19 CD20 CD21 antigen, and preferably capable of producing immunoglobulins.

[0196] In a particularly preferred embodiment, CD45+ lymphocytes are NK cells.

[0197] (i) Available from blood, spleen or bone marrow or from CD34 + Precursor cell production; and / or

[0198] (ii) Characterized by the absence of CD3 expression and the expression of the following CD56 + and / or CD94 + CD158a + CD158f + CD314 + CD335 + At least one of them.

[0199] In a preferred embodiment of the targeted delivery system of the present invention, granulocytes:

[0200] (i) As described, for example, Kuhs et al. 2015, CurrProtocImmunol 111:7.23-1-7.23.16; Coquery et al. 2012, Cytometry A 81(9):806-814; Swemydas and Lionakis 2013, J Vis Exp 77:50586., it can be obtained from blood, spleen or bone marrow or from CD34. + Precursor cells are produced;

[0201] (ii) Characterized by expressing at least one of CD66b and / or CD193:

[0202] (iii) is a polymorphonuclear leukocyte characterized by the presence of granules in its cytoplasm; and / or

[0203] (iv) Characterized by expressing at least one of the following: TfR, CD163, TIM-2 and / or CXCR4.

[0204] Cells included in the isolated targeted delivery system can also be mesenchymal stem cells. In the context of this invention, the term "mesenchymal stem cell" or "MSC" refers to adult stem cells, which are non-hematopoietic pluripotent stem cells capable of differentiating into mesodermal lineages such as osteocytes, adipocytes, and chondrocytes, as well as ectodermal lineages (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers such as differentiation clusters (CD)73, CD90, and CD105, and lack expression of CD45, CD34, CD14 / CD11b, CD19 / CD20 / CD79α, and HLA (human leukocyte antigen)-DR. Human MSCs were first reported in bone marrow, and to date, they have been isolated from various tissues, including adipose tissue, placenta, amniotic fluid, endometrium, dental pulp tissue, umbilical cord blood, and umbilical cord tissue (Wharton's jelly). They are also derived from (i.e., differentiated into) induced pluripotent stem cells (iPSCs). Therefore, in a preferred embodiment of the invention, the MSC is selected from umbilical cord MSCs, bone marrow MSCs, adipose MSCs, placental MSCs, dental pulp MSCs, amniotic fluid MSCs, endometrial MSCs, and iPSC-derived MSCs. Preferably, it is umbilical cord blood MSCs or umbilical cord tissue (or Wharton's jelly) MSCs. Furthermore, the MSCs are preferably human MSCs. The inventors have observed that the MSC stem cells can be loaded with the complex according to the invention and delivered to cancer cells.

[0205] In a seventh aspect, the present invention relates to pharmaceutical or diagnostic compositions comprising a polypeptide of the first aspect, a conjugate of the second aspect, a complex of the third aspect, or a separate targeted delivery system of the fourth aspect, as well as a pharmaceutically acceptable carrier and / or a suitable excipient.

[0206] In cases where the pharmaceutical or diagnostic composition contains live cells, the carrier and excipient are preferably selected to maintain cell viability.

[0207] "Pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more specifically for use in humans.

[0208] As used herein, the term "carrier" refers to a pharmaceutically active substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer solution, or carrier administered with the pharmaceutically active substance. The pharmaceutical carrier may be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as water and saline solutions in oil, wherein the oil includes, but is not limited to, those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous solutions of dextran and glycerol may also be used as liquid carriers, particularly for injectable solutions. Saline solutions are a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in EW Martin's "Remington's Pharmaceutical Sciences".

[0209] Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc.

[0210] "Surfactants" include anionic, cationic and nonionic surfactants, such as, but not limited to, sodium deoxycholate, sodium lauryl sulfate, Triton X-100 and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0211] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and / or nuclease antagonists.

[0212] "Physiological buffer solutions" include, but are not limited to, sodium chloride solutions, softened water, and suitable organic or inorganic buffer solutions such as, but not limited to, phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4(2-hydroxyethyl)piperazinyl]ethanesulfonic acid), or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of the appropriate buffer usually depends on the required molar concentration. For example, phosphate buffer is suitable for both injection and infusion solutions.

[0213] The term "adjuvant" refers to an agent that enhances, stimulates, activates, strengthens, or modulates the immune response to a pharmaceutically active substance contained in a composition at the cellular or humoral level. For example, an immune adjuvant stimulates the immune system's response to an actual antigen but has no immune effect itself. Examples of adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virions, bacterial adjuvants (e.g., monophospholipid A or muramyl peptide), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine).

[0214] As stated above, the term "CD45" is used throughout this specification. + "White blood cells" refers to CD45 + Monocytes, CD45 + Monocytes and macrophages, CD45 + Lymphocytes and / or CD45 + Preferably, the monocytes are not dendritic cells whose differentiation is controlled by one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB, PU / 1, RBPJ, IIRF4, and / or TCF4, and more preferably not dendritic cells. Preferred subgroups of these general leukocyte classes are defined below by structural parameters, such as the presence or absence of a given protein, its functional properties, and / or its preparation / differentiation methods. As mentioned above, the targeted delivery system of the present invention still provides the aforementioned advantages if not every cell in the cell population possesses a specific property, but as long as the majority of cells in the population possess that property. Therefore, the properties of a preferred cell for the targeted delivery system of the present invention are described below. Those skilled in the art will understand that the pharmaceutical compositions of the present invention will comprise millions of cells, and not every cell in the population will possess the functional and / or structural properties described herein, but the pharmaceutical composition can still be used to treat diseases if the majority of cells possess their respective functional and / or structural properties.

[0215] Cells contained in targeted delivery systems, especially CD45 +Leukocytes or MSCs, derived from the patient to be treated. In this case, the cells loaded with the complex are autologous to the patient. It is also envisioned that the patient be MHC-generated prior to treatment with the targeted delivery system of the present invention, and that the cell type used for a given patient be a patient-matched MHC. In both preferred embodiments, the cells are nascent cells or derived from nascent cells through a small number of differentiation steps. Alternatively, the cells may be derived from immortalized but preferably untransformed cell lines.

[0216] Used to separate CD45 + White blood cells, specifically CD45 + Monocytes, CD45 + Monocytes and macrophages, CD45 + Granulocytes or CD45 + Lymphocytes, especially CD45 + The blood used to generate NK cells is preferably obtained from the patient to be treated or from a healthy donor. Alternatively, blood can be obtained from a blood bank. The use of umbilical cord blood is also considered in this study.

[0217] In an eighth aspect, the present invention relates to a polypeptide of the first aspect, a conjugate of the second aspect, a complex of the third aspect, or a separate targeted delivery system of the fifth aspect, which is used as a drug.

[0218] In a ninth aspect, the present invention relates to the polypeptide of the first aspect, the conjugate of the second aspect, the complex of the third aspect, or the isolated targeted delivery system of the fifth aspect, for the treatment, prevention, and diagnosis of tumors, preferably solid tumors and / or metastatic tumors, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, or tumors with hypoxic regions; inflammatory diseases or ischemic sites, particularly at the site of skin wounds or organ infarction (heart) or ischemic retinal inflammatory diseases or ischemic sites; or for prophylactic or therapeutic vaccination, particularly for the prevention or treatment of infectious diseases or cancer. This aspect also includes the targeted delivery of antigens to physiological or non-physiological lymph nodes for the purpose of vaccinating an individual or inducing immune memory.

[0219] In a tenth aspect, the invention relates to a method for treating, preventing, or diagnosing tumors, preferably solid tumors and / or metastatic tumors, particularly breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, liver cancer, glioma / glioblastoma, or tumors with hypoxic areas; inflammatory diseases or ischemic areas, particularly skin wounds or organ infarctions (cardiac) or ischemic retinal inflammatory diseases or ischemic areas; or methods for prophylactic or therapeutic vaccination, particularly by administering an effective amount of the polypeptide of the first aspect, the conjugate of the second aspect, the complex of the third aspect, or the isolated targeted delivery system of the fifth aspect to a subject in need to prevent or treat infectious diseases or cancers. This aspect also includes targeted delivery of antigens to physiological or non-physiological lymph nodes to vaccinate an individual or induce immune memory.

[0220] The term “treatment” as used in this article includes all types of preventive and / or therapeutic interventions permitted by medicine, with the aim of curing, temporarily relieving, preventing, etc., for various purposes, including delaying or stopping the progression of disease, causing lesions to subside or disappear, preventing attacks or suppressing recurrence. Attached Figure Description

[0221] Figure 1 Computer analysis of mutants: Hotspot prediction results for the human H-ferritin-TfR1 complex using PredHS2 and the human H-ferritin-DNA virtual complex using FoldX. Common true positives for DNA binding and TfR1 binding are represented by CPK.

[0222] Figure 2 Figure 1: Sensing plots corresponding to the interaction between immobilized his-tagged TFRC receptors and human ferritin. Figure A: Q11E mutant, Figure B: wild type. X-axis: time (s). The same number of receptors were captured onto the chip surface (see Methods section for details). Five different analyte concentrations were used (0.0625 mg / ml, 0.125 mg / ml, 0.250 mg / ml, 0.5, and 1 mg / ml). For all measured analyte concentrations, the Q11E mutant showed higher ferritin binding than the wild-type ferritin. Global fitting using a simple 1:1 binding pattern indicated higher affinity and a correspondingly lower Kc. D value.

[0223] Figure 3 Non-denaturing gel analysis of wild-type ferritin and mutants showed that wild-type ferritin mainly exhibited a doublet framework, cage-like aggregates, and larger aggregates. However, the mutation reduced the ability of ferritin to form aggregates, so the mutant ferritin variant existed in the form of 24-mers (homogeneous cages).

[0224] Lane 1: Wild-type ferritin-dextrin concentrated in 10 kDa Amicon.

[0225] Lane 2: Wild-type ferritin-dextrin concentrated in 100kDa Amicon.

[0226] Lane 3: Q11E mutant-doxorubicin concentrated in 10 kDa Amicon.

[0227] Lane 4: Q11E mutant doxorubicin concentrated in 100 kDa Amicon.

[0228] Lane 5: Q11E-Q15E mutant doxorubicin concentrated in 10 kDa Amicon.

[0229] Lane 6: Q11E-Q15E mutant doxorubicin concentrated in 100 kDa Amicon.

[0230] Figure 4 Non-denaturing gel analysis of wild-type ferritin and mutants showed that storage conditions did not adversely affect the stability of cages in mutants, and that mutant ferritin variants remained in the form of 24-mers (homogeneous cages) after storage.

[0231] Lane 1: Q11E mutant-doxorubicin concentrated in 10 kDa Amicon.

[0232] Lane 2: Q11E mutant-doxorubicin concentrated in 100 kDa Amicon.

[0233] Lane 3: Q11E-Q15E mutant doxorubicin concentrated in 10 kDa Amicon.

[0234] Lane 4: Q11E-Q15E mutant doxorubicin concentrated in 100 kDa Amicon.

[0235] Lane 5: Wild-type ferritin-dextrin concentrated in 10kDa Amicon.

[0236] Lane 6: Wild-type ferritin-dextrin concentrated in 100kDa Amicon.

[0237] Figure 5 : A graphical representation of the calculation of doxorubicin loading efficiency for Ft wild-type and Ft mutant. The average number of particles per cage and the median are labeled in the figure.

[0238] Figure 6UV-Vis spectra of Ft wild-type and Q11E mutants encapsulated with doxorubicin. The initial concentrations of both proteins were the same, equal to 28.5 mg / ml. The final concentrations of Ft wild-type and Q11E mutants were 10.2 mg / ml and 28.5 mg / ml, respectively. Each concentration and the recorded spectrum correspond to a volume of 1 ml of protein solution. The extinction coefficients of Ft wild-type and Q11E mutants were the same, equal to 18600 M. -1 cm -1 (λ=278nm).

[0239] Figure 7 : Tumor cell viability after co-culturing with macrophages for 72 hours. For each ferritin variant, the concentration of ferritin cages filled with doxorubicin was the same, equal to 1 mg / ml.

[0240] Figure 8 The gel image shows the binding of RNA to the Q11E ferritin mutant, which was not observed in the wild-type protein (wt).

[0241] Figure 9 LC-MS spectrum of ferritin conjugated with VCMMAE. The spectrum shows that it mainly consists of the ferritin moiety with two drug molecules attached. The molar ratio of drug to protein in the conjugate is 1.95.

[0242] Example Section

[0243] Example 1 - Computer Analysis of Mutants

[0244] Identifying a small subset of the protein-protein interface that contributes to the majority of binding free energy can provide important information for understanding the nature of interactions and recognition properties. In recent computational chemistry approaches, these subsets are referred to as "hot spots" (Hao Wang et al., Sci. Rep. 8, 14285 2018). Here, the inventors describe the PredHS2 software with MD minimization (…). http: / / predhs2.denglab.orgThe invention employs a method to predict hotspots from the complex of human H-chain ferritin and transferrin receptor (PDB ID: 6H5I). Based on the PredHS method (Wei, L et al., Comb. chemistry & high throughput screenshot 19, 144–152 2016), the inventors constructed a dataset of 14 interface residues on the H-ferritin interface, corresponding to touchpoints recently identified by Montemiglio et al. for the CD71 / H-ferritin complex (Montemiglio et al., 2019 Nat Comm 10 1121-1121). The inventors then used a two-step feature selection method to generate a set of 476 sequences (single mutants at 14 positions) after removing redundant and irrelevant sequences. This method consists of a minimum redundancy maximum relevance (mRMR) process and a sequential forward selection process that eliminates all mutations considered incompatible with folding properties (e.g., Gly or Pro within the α-helical region). Subsequently, energy minimization was performed on related structures exhibiting Euclidean and Voronoi neighborhood properties under exposure to solvent and energy characteristics. To evaluate the performance of the prediction model, the inventors employed 10-fold cross-validation and commonly used measurement methods such as specificity (SPE), precision (PRE), sensitivity (SEN / Recall), accuracy (ACC), F1-score (F1), and Matthews correlation coefficient (MCC). Table 1 lists the data associated with the optimal energy match (ZAPP) and measurements. Clearly, replacing isoparaffinic glutamine with glutamate residues predicted a higher binding free energy contribution, while non-allelic mutations (even with the same charge) always resulted in a decrease in the binding free energy contribution (RMSE). The Voronoi contribution (polynomial squared distance minimization) clearly plays a crucial role in the binding free energy contribution because it imparts an energy penalty to the higher band gaps created by missing atoms in non-allelic mutants or in the case of larger residues. Figure 1 As shown, four hotspots (8, 11, 12, and 15) have been experimentally determined at the binding interface. These residues are considered individually. Multiple mutants are not considered in these calculations because the resulting binding free energies appear unrealistically high.

[0245] Table I - Prediction of "hotspots" in the complex of human H-chain ferritin and transferrin receptor.

[0246] Four residues at positions 8, 11, 12, and 15 (PDB ID: 6H5I) were found to be the most important contributors to the formation of the H-ferritin / CD71 receptor complex. Here, the inventors demonstrate that single allelic mutations at these "hotspots" provide a further gain in binding free energy for the complex.

[0247]

[0248] The second algorithm has been used to predict and model the binding of double-stranded DNA (dsDNA) to proteins based on the Protein-Assisted DNA Assembly (PADA1) algorithm. PADA1 combines an empirical interaction model generator with a knowledge-based ultrafast statistical force field, which work together to perform dsDP docking (Blanco JD et al., Nucleic Acids Res. 2018 May4;46(8):3852-3863). The algorithm uses fragment pairs (peptides paired with short dsDNA) representing empirically compatible skeletal structures found in nature. DNA-protein structures modeled by PADA1 in conjunction with FoldX (a protein design software) have been used to predict DNA recognition sequences. The collaboration between PADA1 and FoldX for side-chain refinement and interface optimization makes ModelX a powerful modeling tool for predicting key residues at the core of ferritin-DNA interactions. In the case of human H ferritin, the all-to-all atomic distances between protein fragments and corresponding dnaX fragments were measured. Then, using atomic distance distributions, statistical parameters (mean and standard deviation of distances) for all possible touchpoints between proteins and dsDNA fragments included in the interaction database were obtained. The limit for measuring touchpoints was determined according to the all-pair, all-distance measurements between nucleotide-amino acid pairs (touchpoints are considered when at least one amino acid atom, including the side chain, is less than 4 Å from any atom in the nucleotide). Most interestingly, seven residues were found to be associated with nucleic acid binding properties, including four glutamine residues already shown to contribute to the receptor-binding interface, plus glutamine 83, and lysines 86 and 87 (see [link to relevant documentation]). Figure 1 ).

[0249] Example 2—In vitro binding with ferritin receptors

[0250] The binding of wild-type and Q11E mutant ferritin to TfR1 was analyzed by surface plasmon resonance.

[0251] Examples 3 to 8

[0252] The inventors also generated ferritin variants based on the Q11E and Q11E-Q15E mutants by adding the mutants K54E, K72E, K87Q, K144E, C91S, and C103S. The mutant C131S was also added to the Q11E mutant. The properties of these ferritin variants were analyzed in Examples 3 through 8.

[0253] Example 3

[0254] To examine the size of the protein after encapsulation and purification, non-denaturing PAGE gels were performed. It clearly showed that the wild-type protein exhibited heterogeneity in form, containing not only cages but also aggregates of cages and larger aggregates, in contrast to the mutant protein, which existed as 24-mers (homogeneous cages). This greater degree of aggregation adversely affected the loading efficiency and protein recovery after doxorubicin loading. Figure 3 ).

[0255] Example 4

[0256] Wild-type ferritin has limited storage conditions. Storage in a refrigerator (-80°C) and thawing lead to increased cage aggregation, preventing their separation in native electrophoresis. In contrast, the Q11E mutant reduces the presence of aggregates and allows ferritin cages containing doxorubicin to be stored in a refrigerator. Figure 4 ).

[0257] Example 5

[0258] Encapsulation efficiency calculations showed that, compared to the Ft wild-type, the Ft mutants Q11E and Q11E-Q15E were able to load an average of more doxorubicin molecules into their cages, with the Ft mutant Q11E, Ft mutant Q11E-Q15E, and Ft wild-type containing 55, 63, and 23 molecules in their respective cages, respectively. Figure 5 ).

[0259] Example 6

[0260] UV-Vis spectral analysis showed that the protein recovery rate after loading the mutant Q11E was 100%, while the protein recovery rate of wild-type ferritin was only 38%. Figure 6 ).

[0261] Example 7

[0262] In vitro experiments showed that the ferritin mutant exhibited better cytotoxicity against tumor cells. Doxorubicin-packaged proteins were inserted into macrophages and then co-cultured with breast cancer and ovarian cancer cell lines: MDA-MB 231, Skov3, and 4T1. The Q11E-Q15E mutant showed the lowest number of viable cells after co-culture. Figure 7 ).

[0263] Example 8

[0264] 1 ml of reaction solution containing 2 mg Ft and 4 μg siRNA in DPBS was prepared, and the pH was lowered to the values ​​shown (2.5, 3.2, 4.4, 5.6, and 6.8, respectively). The samples were incubated at room temperature for 15 minutes, and then NaOH was added to adjust the pH to neutral (pH 7). Unbound free siRNA was removed by four centrifugation steps on an Amicon centrifuge with a 100 kDa cutoff. Figure 8 As shown, ferritin Q11E mutants associated with siRNA were observed when incubated within a pH range of 4.4 to 6.8 (arrows), but not at pH conditions of 2.4 and 3.2, where the ferritin cages might be disrupted. This observation suggests that the integrity of the Ft nanocages may be crucial for the observed association. Conversely, no association between wild-type ferritin variants and siRNA was observed, regardless of the pH conditions during Ft incubation with siRNA.

[0265] Example 9

[0266] Human heavy chain ferritin can covalently bind to host hydrophobic drug molecules within cysteine ​​residues. Maleimide-functionalized drugs, such as the microtubule inhibitor monomethylaurestatin (MMAE), are among the most prominent examples of potent cytotoxic drugs that can attach readily and specifically. The inventors have conjugated this drug to ferritin according to the following procedure: obtaining an aurestatin E analogue, maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethylaurestatin E (vcMMAE), from MedChem Express (Princeton, NJ). The ferritin vcMMAE adduct was prepared using human heavy chain ferritin according to SEQ ID NO: 77. The ferritin solution was adjusted to 125 µM using reaction buffer (20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)), 0.04% polysorbate 80, pH 7.0) and conjugated with VCMMAE by a 5-molar excess at room temperature (4°C) for 4 hours. The maleimide group reacts efficiently and specifically with free (reduced) thiol groups at pH 6.5 to pH 7.5 to form stable thioether bonds. The final conjugate was dialyzed in wash buffer (20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)), 0.02% polysorbate 80, 2% glycerol, pH 7.0) to remove unbound VCMMAE and concentrated using an Amicon® centrifugal filter. The drug-to-protein molar ratio of the resulting conjugate, as determined by LC-MS, was 1.95 (see [link to LC-MS]). Figure 9 The concentration of the Ft-vcMMAE conjugate was determined by BCA colorimetric assay based on absorbance at 562 nm.

[0267] project

[0268] 1. A ferritin variant polypeptide wherein at least one, at least two, at least three, or at least four, preferably four lysine residues, preferably at positions 54, 72, 87, and / or 144, of the lysine residues shown in SEQ ID NO.1 (human wild-type heavy chain ferritin) are missing or replaced by a non-basic amino acid, preferably E or Q.

[0269] 2. The ferritin variant polypeptide according to Project 1, wherein K54 is replaced by E, K72 is replaced by E, K87 is replaced by Q and K144 is replaced by E.

[0270] 3. The ferritin variant polypeptide according to item 1 or 2, wherein the ferritin variant polypeptide has a sequence according to SEQ ID NO. 82, SEQ ID NO. 1 or SEQ ID NO. 2, wherein at least one of the 54th, 72nd, 87th and / or 144th positions, preferably all lysine residues are deleted or replaced by non-basic amino acids, preferably E or Q, and wherein the sequence according to SEQ ID NO. 82, SEQ ID NO. 1 and SEQ ID NO. 2 further comprises one to five, one to ten, one to fifteen, one to twenty or one to twenty-five amino acid mutations other than the 54th, 72nd, 87th and / or 144th positions.

[0271] 4. The ferritin variant polypeptide according to any one of items 1 to 3, wherein one or more cysteine ​​residues shown in SEQ ID NO.1, particularly the cysteine ​​residues at positions 91, 103 and / or 131, are missing or substituted, preferably substituted with serine residues.

[0272] 5. The ferritin variant polypeptide according to any one of items 1 to 4, wherein the ferritin variant polypeptide has a sequence according to SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 75, SEQ ID NO. 76 or SEQ ID NO. 77, or contains one to five, one to ten, one to fifteen, one to twenty or one to twenty amino acid mutations other than position 54, position 72, position 87 and / or position 144, according to the sequence according to SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 75, SEQ ID NO. 76 or SEQ ID NO. 77.

[0273] 6. A ferritin variant polypeptide wherein one or more cysteine ​​residues shown in SEQ ID NO.1, particularly the cysteine ​​residues at positions 91, 103 and / or 131, are deleted or substituted, preferably substituted with serine residues.

[0274] 7. The ferritin variant polypeptide according to item 6, wherein the ferritin variant polypeptide has a sequence according to SEQ ID NO. 82, SEQ ID NO. 1 or SEQ ID NO. 2, wherein at least one of the 91st, 103rd and / or 131st positions, preferably all cysteine ​​residues are deleted or substituted, preferably substituted with serine residues, and wherein the sequence according to SEQ ID NO. 82, SEQ ID NO. 1 and SEQ ID NO. 2 further comprises one to five, one to ten, one to fifteen, one to twenty or one to twenty-five amino acid mutations other than the 91st, 103rd and / or 131st positions.

[0275] 8. The ferritin variant polypeptide according to item 6 or 7, wherein one, two, three or four, preferably four lysine residues, are shown with respect to SEQ ID NO.1 (human wild-type heavy chain ferritin), preferably four lysine residues, preferably lysine residues at positions 54, 72, 87 and / or 144 are missing or replaced by non-basic amino acids, preferably E or Q, most preferably, wherein K54 is replaced by E, K72 by E, K87 by Q and K144 by E.

[0276] 9. The ferritin variant polypeptide according to any one of items 6 to 8, wherein the ferritin variant polypeptide has a sequence according to SEQ ID NO. 75 or SEQ ID NO. 76, or contains 1 to 5, for example 1, 2, 3, 4 or 5, or 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations according to SEQ ID NO. 75 or SEQ ID NO. 76, other than positions 91, 103 and / or 131.

[0277] 10. The ferritin variant polypeptide according to any one of items 1 to 9, further comprising a transferrin receptor-binding domain (TRBD) of the ferritin variant, wherein, compared with the wild-type ferritin on which it is based, the TRBD comprises one or more glutamine residues mutated to glutamate residues and / or one or more asparagine residues mutated to aspartic acid residues, wherein particularly at least one, preferably all mutations are contained in the 20 N-terminal amino acids of wild-type ferritin.

[0278] 11. The ferritin variant according to Item 10, wherein TRBD contains at least the following amino acid sequence:

[0279] MTTASX1SZVRZBYHZDX2EAA(SEQ ID NO.3)

[0280] X1 = S or T, with T being preferred;

[0281] X2 = S or A, with S being the preferred choice;

[0282] Z = Q or E; and

[0283] B = N or D;

[0284] At least one of Z or B is E or D.

[0285] It may also contain one, two or three amino acid substitutions other than Z and / or B, and the M at position 1 may or may not be present.

[0286] 12. A ferritin variant according to item 10 or 11, wherein the TRBD comprises at least a subset selected from SEQ ID NO. 04 to SEQ ID NO. 63, particularly from SEQ ID NO. 05, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 30, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 45, SEQ ID NO. 50, SEQ ID NO. 56, SEQ ID NO. 57 and SEQ ID NO. 60, and more particularly from SEQ ID NO. 05, SEQ ID NO. 12, SEQ ID NO. 20, SEQ ID NO. 27, SEQ ID NO. 35, SEQ ID NO. 42, SEQ ID NO. 50 and SEQ ID NO. 63. The amino acid sequence of NO.57 may also contain one, two or three amino acid substitutions other than the amino acids at positions 8, 11, 12 and / or 15, and the M at position 1 may or may not be present.

[0287] 13. A ferritin variant according to any one of items 10 to 12, wherein, compared with wild-type ferritin, the TRBD exhibits an increased affinity for TfR-1 of at least (≥) 1.5 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥30 times, ≥40 times, or ≥50 times, but less than (≤) 100 times, ≤75 times, ≤50 times, ≤40 times, or ≤30 times. 20 times, ≤10 times, or ≤5 times, in particular, compared with wild-type ferritin, TRBD has an increased affinity for TfR-1 of 1.5 times to 50 times, 2 times to 50 times, 3 times to 50 times, 4 times to 50 times, 5 times to 50 times, 10 times to 50 times, 20 times to 50 times, 30 times to 50 times, 40 times to 50 times, 1.5 times to 10 times, 2 times to 20 times, or 5 times to 30 times.

[0288] 14. A nucleic acid encoding any one of items 1 to 13.

[0289] 15. A vector containing the nucleic acid of item 14.

[0290] 16. A conjugate comprising the polypeptide described in items 1 to 13 and at least one tag and / or at least one drug.

[0291] 17. A complex comprising at least one polypeptide of items 1 to 13 and / or at least one conjugate of item 16.

[0292] 18. The complex according to item 17 further comprises at least one label and / or at least one drug.

[0293] 19. The conjugate according to item 16 or the complex according to item 17 or 18, wherein the label is selected from

[0294] a. Fluorescent dyes, especially fluorescent dyes selected from the following categories of fluorescent dyes: xanthanides, acridines, oxazines, anthocyanins, styryl dyes, coumarins, porphyrins, metal-ligand-complexes, fluorescent proteins, nanocrystals, perylenes and phthalocyanines, as well as conjugates and combinations of these categories of dyes;

[0295] b. Radioactive isotopes / fluorescent isotopes, especially those selected from the following substances: alpha radiation emission isotopes, gamma radiation emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, fluorescent isotopes such as 65Tb, and fluorescent isotopes such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47S c, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag, as well as conjugates and combinations of the above substances with proteins, peptides, small molecule inhibitors, antibodies or other compounds;

[0296] c. Detectable peptides, particularly autofluorescent proteins, preferably green fluorescent proteins or any structural variants thereof having altered adsorption and / or emission spectra, or nucleic acids encoding the detectable peptide; and

[0297] d. Contrast agents, particularly contrast agents containing paramagnetic agents, preferably selected from Gd, Eu, W and Mn, or iron hydride.

[0298] 20. The conjugate of item 16 or 19 or the complex of items 17 to 18, wherein the drug is selected from anticancer drugs, particularly cell-inhibiting drugs, cytotoxic drugs or their prodrugs, anti-atherosclerotic drugs, and anti-inflammatory or immunomodulatory drugs.

[0299] 21. The conjugate according to items 16, 19 or 20, comprising a drug, wherein the drug is an auristatin, particularly monomethyl auristatin (MMAE), conjugated to a polypeptide via a maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarbonyl linker.

[0300] 22. A targeted delivery system comprising the separation of cells, wherein the cells comprise the polypeptides described in items 1 to 13, the conjugates described in items 16 or 19 to 21, or the complexes described in items 17 to 21, wherein the cells are particularly CD45+ leukocytes, more particularly CD45+ leukocytes selected from monocytes, differentiated monocytes, lymphocytes and granulocytes.

[0301] 23. A pharmaceutical composition or diagnostic composition comprising the polypeptides described in items 1 to 13, the conjugates described in items 16 or 19 to 21, the complexes described in items 17 to 21, or the isolated targeted delivery system described in item 22, and a pharmaceutically acceptable carrier and / or a suitable excipient.

[0302] 24. The polypeptides described in items 1 to 13, the conjugates described in items 16 or 19 to 21, the complexes described in items 17 to 21, or the isolated targeted delivery system described in item 22, used as a drug.

Claims

1. A ferritin variant polypeptide, comprising: - A transferrin receptor-binding domain (TRBD), wherein the transferrin receptor-binding domain comprises the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 12, wherein M may or may not be present at position 1; and - Other amino acid sequences located at the C-terminus of the TRBD, wherein the other amino acid sequences are selected from SEQ ID NO.67 to SEQ ID NO.70, SEQ ID NO.78 to SEQ ID NO.80 and SEQ ID NO.

87.

2. The ferritin variant polypeptide according to claim 1, wherein the transferrin receptor binding domain is composed of the amino acid sequence of SEQ ID NO.

12.

3. The ferritin variant polypeptide according to claim 1, wherein the other amino acid sequence at the C-terminus of TRBD is SEQ ID NO.

80.

4. The ferritin variant polypeptide according to claim 1, wherein the composition is as follows: A TRBD consisting of the amino acid sequence of SEQ ID NO. 5 and other amino acid sequences selected from SEQ ID NO. 68 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80 and SEQ ID NO. 87; or The TRBD consists of the amino acid sequence of SEQ ID NO.12 and other amino acid sequences selected from SEQ ID NO.67 to SEQ ID NO.70, SEQ ID NO.78 to SEQ ID NO.80 and SEQ ID NO.

87.

5. The ferritin variant polypeptide according to claim 1, which consists of the amino acid sequence of SEQ ID NO.77, wherein M at position 1 may or may not be present.

6. A nucleic acid encoding a ferritin variant polypeptide according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid according to claim 6.

8. A conjugate comprising a ferritin variant polypeptide according to any one of claims 1 to 5 and at least one small molecule drug.

9. The conjugate of claim 8, wherein the small molecule drug is linked to the ferritin variant polypeptide via a linker portion that is reactive to thiols or amino groups.

10. The conjugate according to claim 9, wherein the small molecule drug is auratestatin conjugated to a ferritin variant polypeptide via a maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarbonyl linker.

11. A complex comprising a ferritin variant polypeptide according to any one of claims 1 to 5 and at least one small molecule drug.

12. The complex according to claim 11, wherein the small molecule drug is selected from anticancer drugs, anti-atherosclerotic drugs, and anti-inflammatory or immunomodulatory drugs.

13. The complex according to claim 11, wherein the small molecule drug is doxorubicin.

14. A targeted delivery system comprising the separation of cells, wherein the cells comprise a conjugate according to any one of claims 8 to 10 or a complex according to any one of claims 11 to 13.

15. The isolated targeted delivery system of claim 14, wherein the cells are CD45+ leukocytes.

16. The isolated targeted delivery system of claim 15, wherein the CD45+ leukocytes are monocytes or macrophages.

17. A pharmaceutical composition comprising a conjugate according to any one of claims 8 to 10, or a complex according to any one of claims 11 to 13, or a separate targeted delivery system according to any one of claims 14 to 16, and a pharmaceutically acceptable carrier and / or a suitable excipient.

18. Use of the conjugate according to any one of claims 8 to 10, or the complex according to any one of claims 11 to 13, or the isolated targeted delivery system according to any one of claims 14 to 16 in the preparation of a medicament.

19. A conjugate comprising a ferritin variant polypeptide according to any one of claims 1 to 4 and at least one tag, said tag being selected from... Small molecule fluorescent dyes, Radioactive isotopes / fluorescent emission isotopes, alpha radiation emission isotopes, gamma radiation emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, fluorescent isotopes, and Small molecule contrast agents.

20. The conjugate according to claim 19, wherein The small molecule fluorescent dyes are selected from xanthines, acridines, oxazines, anthocyanins, styryl dyes, coumarins, porphyrins, metal-ligand-complexes, nanocrystals, perylenes, and phthalocyanines; The radioactive isotopes / fluorescent emission isotopes are selected from 65Tb, 18F, 51Cr, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au, and 199Ag; and The small molecule contrast agent is a paramagnetic agent or iron hydride.

21. The conjugate according to claim 19 or 20, wherein the tag is linked to the ferritin variant polypeptide via a linker portion that is reactive to thiols or amino groups.

22. A complex comprising a ferritin variant polypeptide assembled into a cage-like structure according to any one of claims 1 to 5, and at least one tag encapsulated within said cage-like structure.

23. The composite of claim 22, wherein the label is selected from... Fluorescent dyes Radioactive isotopes / fluorescent emission isotopes, alpha radiation emission isotopes, gamma radiation emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, fluorescent isotopes. Detectable peptides, and Contrast agent.

24. The composite according to claim 23, wherein The fluorescent dyes are selected from oxazine, acridine, oxazine, anthocyanin, styryl dyes, coumarin, porphyrin, metal-ligand-complex, fluorescent protein, nanocrystal, perylene, and phthalocyanine. The radioactive isotopes / fluorescent emission isotopes are selected from 65Tb, 18F, 51Cr, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au, and 199Ag; The detectable peptide is an autofluorescent protein; and The contrast agent is a paramagnetic agent or ferric hydride.

25. A targeted delivery system comprising the separation of cells, wherein the cells comprise a ferritin variant polypeptide according to any one of claims 1 to 5, a conjugate according to any one of claims 19 to 21, or a complex according to any one of claims 22 to 24.

26. The isolated targeted delivery system of claim 25, wherein the cells are CD45+ leukocytes.

27. The isolated targeted delivery system of claim 26, wherein the CD45+ leukocytes are monocytes or macrophages.

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