FERRITIN VARIANTS WITH INCREASED STABILITY, COMPLEXATION 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 glutamine residues mutated into glutamic acid residues and/or one or more asparagine residues mutated into aspartic acid residues. 5 The invention further relates to a complex of this polypeptide and a label or drug, and an isolated cellular delivery system comprising the polypeptide or the complex of the invention as well as uses of such system for prophylaxis, therapy, diagnosis or theragnosis, in particular for therapy of cancer or inflammatory diseases.

Claims

1. A polypeptide comprising a transferrin receptor binding domain (TRBD) of a ferritin variant wherein within the TRBD the ferritin variant in comparison to the wild-type ferritin on which it is based comprises one or more glutamine residues mutated into glutamic acid residues and/or one or more asparagine residues mutated into aspartic acid residues, wherein the TRBD of the ferritin variant comprises at least the following amino acid sequence: TABLE-US-00004 (SEQ ID NO. 81) MTTASX.sub.1SZ.sub.1VRZ.sub.2BYHZ.sub.3DX.sub.2EAA X.sub.1=S or T, preferably T; X.sub.2=S or A, preferably S; Z.sub.1, Z.sub.2, and Z.sub.3=Q or E; and B=N or D; wherein at least one of Z.sub.2 and Z.sub.3 is E and/or B is D, which may further comprise one, two or three amino acid substitutions outside Z and/or B, and wherein the M at position 1 may be present or absent.

2. The polypeptide according to claim 1, wherein the TRBD of the ferritin variant comprises at least an amino acid sequence selected from the group comprising SEQ ID NO: 05 to 18, 20 to 33, 35 to 48 and 50 to 63, which may further comprise one, two or three amino acid substitutions outside amino acid positions 8, 11, 12 and/or 15, and wherein the M at position 1 may be present or absent.

3. The polypeptide according to claim 1, wherein the polypeptide is a ferritin variant polypeptide further comprising, an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% or 100% identity to a sequence selected from the group comprising SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80 and SEQ ID NO. 87.

4. The polypeptide according to a claim 3, wherein one, two, three or four, preferably four, lysine residues within the TRBD.

5. The polypeptide according to claim 3, wherein one or more cysteine residues are deleted or substituted.

6. The polypeptide according to claim 1, comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. 71-77 and SEQ ID NO. 85, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity to one of SEQ ID NO. 71-77 or SEQ ID NO. 85.

7. A nucleic acid encoding the polypeptide of claim 1 or a vector comprising said nucleic acid.

8. A conjugate comprising the polypeptide of claim 1 and at least one label and/or at least one drug.

9. A complex comprising at least one polypeptide of claim 1.

10. The complex of claim 9 further comprising at least one label and/or at least one drug.

11. The conjugate of claim 8, wherein the label is selected from the group consisting of a fluorescent dye, in particular a fluorescent dye selected from the group consisting of the following classes of fluorescent dyes: Xanthens, Acridines, Oxazines, Cynines, Styryl dyes, Coumarines, Porphines, Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals, Perylenes and Phtalocyanines as well as conjugates and combinations of these classes of dyes; a radioisotope/fluorescence emitting isotope, in particular a radioisotope/fluorescence emitting isotope selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as 65Tb, fluorescence emitting isotopes, such as 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 as well as conjugates and combinations of above with proteins, peptides, small molecular inhibitors, antibodies or other compounds; a detectable polypeptide, in particular an autofluorescent protein, preferably green fluorescent protein or any structural variant thereof with an altered adsorption and/or emission spectrum or nucleic acid encoding a detectable polypeptide; and a contrast agent, in particular a contrast agent comprising a paramagnetic agent, preferably selected from Gd, Eu, W and Mn, or ferrihydride; and/or the drug is selected from the group consisting of an anticancer drug, in particular a cytostatic drug, cytotoxic drug or prodrug thereof, an anti-arteriosclerotic drug, and an anti-inflammatory or immunomodulatory drug.

12. The conjugate of claim 8 comprising a drug, wherein the drug is auristatin, in particular monomethyl auristatin (MMAE), conjugated to the polypeptide via a maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl linker.

13. An isolated targeted delivery system comprising a cell, wherein the cell comprises the polypeptide of claim 1.

14. A pharmaceutical or diagnostic composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier and/or suitable excipient(s).

15. A method of treatment comprising administration of the polypeptide of claim 1 to a patient in need thereof.

16. The polypeptide according to claim 4, wherein one, two, three or four of the lysine residues at position 54, 72, 87 and/or 144 indicated with respect to SEQ ID NO. 1 (human wild-type heavy chain ferritin) are deleted or substituted with a non-basic amino acid.

17. The polypeptide according to claim 4, wherein three or four of the lysine residues at position 54, 72, 87 and/or 144 indicated with respect to SEQ ID NO. 1 (human wild-type heavy chain ferritin) are deleted or substituted with a non-basic amino acid.

18. The polypeptide according to claim 17, wherein the lysine residues are substituted with E or Q.

19. The polypeptide according to claim 18, wherein K54 is substituted with E, K72 is substituted with E, K87 is substituted with Q and/or K144 is substituted with E.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0217] FIG. 1: In silico analysis of mutants: Hot spot prediction results by using PredHS2 for the human H-Ferritin-TfR1 complex and FoldX for the human H-Ferritin-DNA virtual complex. True positives, common for both DNA binding and TfR1 binding are represented as CPK.

[0218] FIG. 2: Sensorgrams corresponding to the interaction between the immobilized his-tagged TFRC receptor and human ferritin. Panel A: mutant Q11E, panel B: wild type. X-axis: time (s). The same amount of receptor was trapped onto the chip surface (see methods section for details). Five different analyte concentrations (0.0625, 0.125, 0.250, 0.5 and 1 mg/ml) were used. For all analyte concentrations measured, the amount of ferritin bound is higher for the Q11E mutant than for wild-type ferritin. The global fit using a simple 1:1 binding mode indicates a higher affinity and correspondingly a lower K.sub.D value.

[0219] FIG. 3: Native gel analysis of wild type ferritin and mutants showing that wild type ferritin mainly forms of double glued frames, aggregates of cages and larger forms of aggregates, however, mutations decrease the ability of ferritin to form aggregates, therefore the mutant ferritin variants are present as 24-mers (homogeneous cages). [0220] Lane 1: Ferritin wild type-dox concentrated on 10 kDa Amicon. [0221] Lane 2: Ferritin wild type-dox concentrated on 100 kDa Amicon [0222] Lane 3: Q11E mutant-dox concentrated on 10 kDa Amicon. [0223] Lane 4: Q11E mutant-dox concentrated on 100 kDa Amicon. [0224] Lane 5: Q11E-Q15E mutant-dox concentrated on 10 kDa Amicon. [0225] Lane 6: Q11E-Q15E mutant-dox concentrated on 100 kDa Amicon.

[0226] FIG. 4: Native gel analysis of wild type ferritin and mutants showing that storage conditions did not adversely affect the stability of the cages in the mutants, and the mutant ferritin variants are still present as 24-mers (homogeneous cages) after storage. [0227] Lane 1: Q11E mutant-dox concentrated on 10 kDa Amicon. [0228] Lane 2: Q11E mutant-dox concentrated on 100 kDa Amicon. [0229] Lane 3: Q11E-Q15E mutant-dox concentrated on 10 kDa Amicon. [0230] Lane 4: Q11E-Q15E mutant-dox concentrated on 100 kDa Amicon. [0231] Lane 5: Ferritin wild type-dox concentrated on 10 kDa Amicon. [0232] Lane 6: Ferritin wild type-dox concentrated on 100 kDa Amicon.

[0233] FIG. 5: Graphical representation of the calculation of doxorubicin loading efficiency for Ft wild type and Ft mutants. The average particle numbers per cage along with the median are marked on the graph.

[0234] FIG. 6: UV-Vis spectrum of Ft wild type and Q11E mutant after doxorubicin encapsulation. The initial concentration for both proteins was the same and equal 28.5 mg/ml. The final concentrations for Ft wild type and Q11E mutant were 10.2 mg/ml and 28.5 mg/ml. Each concentration and the recorded spectra are for a volume of 1 ml protein solution. The extinction coefficient for Ft wild type and Q11E mutant are the same and equal 18600 M.sup.−1cm.sup.−1 (λ=278 nm).

[0235] FIG. 7: Graphs of tumour cell viability after 72 h co-culture with macrophages. The concentration of ferritin cages filled with doxorubicin was the same for each variant of ferritin and was equal to 1 mg/ml.

[0236] FIG. 8: Picture of gel showing RNA association with Q11E ferritin mutant that is not observed in case of wild-type protein (wt).

[0237] FIG. 9. The LC-MS spectrum of ferritin after conjugation with vcMMAE. The spectrum shows that it is mostly a ferritin fraction with two drug molecules attached. The molar drug to protein ratio of the conjugate is equal to 1.95.

EXAMPLE SECTION

Example 1—in Silico Analysis of Mutants

[0238] Identification of small portions of a protein-protein interface that contribute to the majority of the binding free energy can provide crucial information for understanding the nature of the interaction and recognition properties. These portions are referred to as “hot spots” in recent computational chemistry approaches (Hao Wang, et al., Sci. Rep. 8, 14285 2018). Here, the inventors describe the application of the PredHS2 software (http://predhs2.denglab.org) coupled to MD minimization in order to predict hot spots from the complex of human H chain ferritin and Transferrin receptor (PDB ID:6H5I). Based on PredHSmethod (Wei, L, et Al. Comb. chemistry & high throughput screening 19, 144-152 2016), the inventors built a dataset of 14 interface residues on H Ferritin interface that corresponds to the contacts obtained from the CD71/H-ferritin complex recently identified by Montemiglio et al., (Montemiglio et al., 2019 Nat Comm 10 1121-1121). Then the inventors generated a set of 476 sequences (single mutants of the 14 positions) obtained after removal of redundant and irrelevant sequences utilizing a two-step feature selection method, which consists of a minimum Redundancy Maximum Relevance (mRMR) procedure and a sequential forward selection process that eliminates all mutations that are considered not compatible with folding properties (e.g. Gly or Pro within alpha helices regions). Thereafter, energy minimization of the relevant structure, exposure to solvent and energy features, together with Euclidean and Voronoi neighbourhood properties was carried out. In order to assess the performance of the prediction model, the inventors adopted 10-fold cross-validation together with commonly used measures, such as specificity (SPE), precision (PRE), sensitivity (SEN/Recall), accuracy (ACC), F1-score (F1) and Matthews correlation coefficient (MCC). Data relative to the best energy matches (ZAPP) and measures are listed in Table 1. As apparent, the isosteric glutamine substitutions with a glutamate residue predicted a higher binding free energy contribution whereas non-isosteric mutations (even when bearing the same charge) invariably led to a decrease in binding free energy contribution (RMSE). Apparently, Voronoi contribution (polynomial squared distances minimization), played a key role in contributing to the binding free energy as it confers energy penalties to the voids generated by missing atoms in non-isosteric mutants, or even higher gaps in the case of bulkier residues. As shown in FIG. 1, four hot spots (8, 11, 12 and 15) have been experimentally determined at the binding interface. These residues were individually taken into account. Multiple mutants have not been considered in these calculations as the resulting binding free energies appeared to be unrealistically high.

TABLE-US-00002 TABLE I Prediction of “hot spots” for the complex of human H chain ferritin and Transferrin receptor. Composition of ZAPP and Performance of Energy Functions with Terms RMSE Mutant (Kcal mol.sup.−1) ACC SPE PRE SEN F1 MCC Q8E 7.2 0.89 0.91 0.85 0.78 0.79 0.70 Q11E 8.1 0.87 0.95 0.84 0.83 0.80 0.70 N12D 4.9 0.91 0.93 0.85 0.81 0.79 0.70 Q15E 5.3 0.94 0.94 0.86 0.87 0.82 0.70 WT 4.3 0.88 0.94 0.86 0.79 0.80 0.70 The four residues in positions 8, 11, 12 and 15 (PDB ID: 6H5I) were found to be the most important contributors to the H-ferritin/CD71 receptor complex formation. Here the inventors show that individual, isosteric mutations of these “hot spots” provide a further binding free energy gain to the complex.

[0239] A second algorithm has been applied based on Protein-assisted DNA assembly (PADA1) algorithm to predict and model the binding of double-stranded DNA (dsDNA) to proteins. PADA1 includes an empirical interaction model generator in combination with an ultra-fast statistical knowledge-based force field, which act in synergy in order to perform dsDP docking (Blanco J D, et al., Nucleic Acids Res. 2018 May 4; 46(8):3852-3863). This algorithm uses fragment pairs (peptide paired to short dsDNA) that represent empirical, compatible backbone conformations found in nature. DNA-protein structures modeled by PADA1 have been used in combination with FoldX (protein design software) to predict DNA recognition sequences. The cooperative action between PADA1 and FoldX, for side chain refinement and interface optimization, turns ModelX in a powerful modelling tool for predicting key residues at the core of ferritin-DNA interaction. In the case of Human H ferritin, we measured the atomic “all-to-all” distances between the protein fragment and the corresponding dnaX fragment. Then, using the atomic distance distributions, we obtained the statistical parameters (mean and standard deviation of the distances) for all possible contacts between the protein and dsDNA fragments included in the interaction database. All-to-all distances between contacting nucleotide-amino acid pairs were measured according to the limits suggested by Blanco J D et al., (a contact is considered when at least one atom of the amino acid, including side chains, is less than 4 Å from any atom in the nucleotide). Most interestingly, 7 residues were found to be responsible for nucleic acid binding properties, which comprise the 4 glutamines already demonstrated to contribute to the receptor binding interface plus glutamine 83 together with lysines 86 and 87 (see FIG. 1).

Example 2—In Vitro Binding to Ferritin Receptor

[0240] Binding of wild type and Q11E mutant ferritin to TfR1 was analysed by surface plasmon resonance.

Examples 3-8

[0241] The inventors generated further ferritin variants based on the Q11E and Q11E-Q15E mutants by adding the mutations K54E, K72E, K87Q, K144E, C91S and C103 S. Mutation C131S was further added to mutant Q11E. The properties of these ferritin variants were further analysed in examples 3-8.

Example 3

[0242] Native PAGE gel has been performed in order to check the size of the protein after encapsulation and purification. It clearly shows that wild type protein shows a heterogeneity of forms, in addition to cages it contains aggregates of cages and larger forms of aggregates, in contrary to mutated protein, which is are present as 24-mers (homogeneous cages). A greater degree of aggregation adversely affects the loading efficiency and protein recovery after loading with doxorubicin (FIG. 3).

Example 4

[0243] Storage conditions for wild type ferritin are limited. Storage in a freezer (−80 degrees Celsius) and thawing causes an increase of cages aggregation, which prevents their separation on native electrophoresis. In contrast, the Q11E mutant reduces the presence of aggregates and allows the storage of ferritin cages with doxorubicin in the freezer (FIG. 4).

Example 5

[0244] The calculation of the encapsulation efficiency indicates that Ft mutants Q11E and Q11E-Q15E are able to load on average more doxorubicin molecules into their cages compared to Ft wild type, 55, 63, 23 molecules, respectively (FIG. 5).

Example 6

[0245] UV-Vis spectrum analysis has shown that protein recovery after loading was 100% for mutant Q11E and only 38% for wild ferritin (FIG. 6).

Example 7

[0246] In vitro experiments have shown that ferritin mutants reveal better cytotoxicity against tumor cells. The doxorubicin packed proteins were inserted into macrophages and then co-cultured with breast and ovarian cancer cell lines: MDA-MB 231, Skov3 and 4T1. The number of viable cells after co-culture was the lowest for the Q11E-Q15E mutant (FIG. 7).

Example 8

[0247] 1 ml reaction containing 2 mg Ft and 4 ug siRNA in DPBS, pH lowered to values as indicated (2.5, 3.2, 4.4, 5.6 and 6.8, respectively). Samples were incubated 15 min in RT, then NaOH was added to adjust pH to neutral (pH 7). Non-associated free siRNA was removed by 4 centrifugation steps on Amicons with 100 kDa cut-off. As shown in FIG. 8, Ferritin Q11E mutant associated with siRNA when incubated in pH range 4.4 to 6.8 (arrow), but not in 2.4 and 3.2, conditions in which Ferritin cage is likely to be disrupted. This observation suggests that Ft nanocage integrity might be crucial for observed association. In contrast, no association with siRNA was observed for wild type Ferritin variant, independently from pH conditions during Ft incubation with siRNA.

Example 9

[0248] Human heavy chain ferritin can be covalently linked to host hydrophobic drug molecules within the cysteine residues. Maleimide functionalized drugs, such as a tubulin inhibitor Monomethyl Auristatin (MMAE) is one of the most notable examples of potent cytotoxic that can be readily and specifically attached. The inventors have conjugated this drug to ferritin according to the following procedure: The auristatin E analogue, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl auristatin E (vcMMAE) was obtained from MedChem Express (Princeton, N.J.). The ferritin vcMMAE adduct was prepared as follows: Human heavy chain ferritin according to SEQ ID NO: 77 was used. Ferritin solution was adjusted to a concentration of 125 μM with reaction buffer (20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)), 0.04% Polysorbate 80, pH 7.0) and conjugated with 5-fold molar excess of vcMMAE at room temperature at 4° C. for 4 hours. Maleimide groups react efficiently and specifically with free (reduced) sulfhydryls at pH 6.5-7.5 to form stable thioether bonds. The final conjugate was dialyzed in washing buffer (20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)), 0.02% Polysorbate 80, 2% Glycerin, pH 7.0), to remove unbound vcMMAE, and concentrated at Amicon® centrifugal filter device. The molar drug to protein ratio of the obtained conjugate was determined by LC-MS analysis and it was equal to 1.95 (see FIG. 9). The concentration of Ft-vcMMAE conjugate was determined by BCA colorimetric assay based on the absorbance at 562 nm.

Items

[0249] 1. A ferritin variant polypeptide, wherein at least one, at least two, at least three or at least four, preferably four, lysine residues, preferably lysine residues at position 54, 72, 87 and/or 144 indicated with respect to SEQ ID NO. 1 (human wild-type heavy chain ferritin), are deleted or substituted with a non-basic amino acid, preferably E or Q. [0250] 2. The ferritin variant polypeptide of item 1, wherein K54 is substituted with E, K72 is substituted with E, K87 is substituted with Q and K144 is substituted with E. [0251] 3. The ferritin variant polypeptide of 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, preferably all, lysine residues at position 54, 72, 87 and/or 144 are deleted or substituted with a non-basic amino acid, preferably E or Q, and wherein the sequences according to SEQ ID NO. 82, SEQ ID NO. 1 and SEQ ID NO. 2 may further comprise 1-5, 1-10, 1-15, 1-20 or 1-25 amino acid mutations outside position 54, 72, 87 and/or 144. [0252] 4. The ferritin variant polypeptide of any one of items 1 to 3, wherein one or more cysteine residues, in particular cysteine residues at position 91, 103 and/or 131 indicated with respect to SEQ ID NO. 1, are deleted or substituted, preferably substituted with serine residues. [0253] 5. The ferritin variant polypeptide of 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 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 comprising 1-5, 1-10, 1-15, 1-20 or 1-25 amino acid mutations outside position 54, 72, 87 and/or 144. [0254] 6. A ferritin variant polypeptide, wherein one or more cysteine residues, in particular cysteine residues at position 91, 103 and/or 131 indicated with respect to SEQ ID NO. 1, are deleted or substituted, preferably substituted with serine residues. [0255] 7. The ferritin variant polypeptide of 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, preferably all, cysteine residues at position 91, 103 and/or 131 are deleted or substituted, preferably substituted with serine residues, and wherein the sequences according to SEQ ID NO. 82, SEQ ID NO. 1 and SEQ ID NO. 2 may further comprise 1-5, 1-10, 1-15, 1-20 or 1-25 amino acid mutations outside position 91, 103 and/or 131. [0256] 8. The ferritin variant polypeptide of item 6 or 7, wherein one, two, three or four, preferably four, lysine residues, preferably lysine residues at position 54, 72, 87 and/or 144 indicated with respect to SEQ ID NO. 1 (human wild-type heavy chain ferritin), are deleted or substituted with a non-basic amino acid, preferably E or Q, most preferably wherein K54 is substituted with E, K72 is substituted with E, K87 is substituted with Q and K144 is substituted with E. [0257] 9. The ferritin variant polypeptide of 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 a sequence according to SEQ ID NO. 75 or SEQ ID NO. 76 comprising 1-5, e.g. 1, 2, 3, 4 or 5, or 1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations outside position 91, 103 and/or 131. [0258] 10. The ferritin variant polypeptide of any one of items 1 to 9, further comprising a transferrin receptor binding domain (TRBD) of a ferritin variant wherein the TRBD in comparison to the wild-type ferritin on which it is based comprises one or more glutamine residues mutated into glutamic acid residues and/or one or more asparagine residues mutated into aspartic acid residues, wherein in particular at least one, preferably all mutations are comprised in the 20 N-terminal amino acids of the wild-type ferritin. [0259] 11. The ferritin variant polypeptide of item 10, wherein the TRBD comprises at least the following amino acid sequence:

TABLE-US-00003 (SEQ ID NO. 3) MTTASX.sub.1SZVRZBYHZDX.sub.2EAA [0260] X.sub.1=S or T, preferably T; [0261] X.sub.2=S or A, preferably S; [0262] Z=Q or E; and [0263] B=N or D; [0264] wherein at least one Z or B is E or D, [0265] which may further comprise one, two or three amino acid substitutions outside Z and/or B, and wherein the M at position 1 may be present or absent. [0266] 12. The ferritin variant polypeptide according to item 10 or 11, wherein the TRBD comprises at least an amino acid sequence selected from the group comprising SEQ ID NO. 04 to SEQ ID NO. 63, particularly from the group consisting of SEQ ID NO. 05, 11, 12, 15, 20, 26, 27, 30, 35, 41, 42, 45, 50, 56, 57 and 60, more particularly from the group consisting of SEQ ID NO. 05, 12, 20, 27, 35, 42, 50 and 57, which may further comprise one, two or three amino acid substitutions outside amino acid positions 8, 11, 12 and/or 15, and wherein the M at position 1 may be present or absent. [0267] 13. The ferritin variant polypeptide according to any of items 10 to 12, wherein the affinity of the TRBD to TfR-1 is increased in comparison to the TRBD of the wild-type ferritin at least (≥) 1.5×, ≥2×, ≥3×, ≥4×, ≥5×, ≥10×, ≥20×, ≥30×, ≥40×, ≥50×, but less than (≤) 100×, ≤75×≤50×, ≤40×, ≤30×, ≤20×, ≤10×, or ≤5×, in particular the affinity of the TRBD to TfR-1 is increased between 1.5×-50×, 2×-50×, 3×-50×, 4×-50×, 5×-50×, 10×-50×, 20×-50×, 30×-50×, 40×-50×, 1.5×-10×, 2×-20× or 5×-30× in comparison to the TRBD of the wild-type ferritin. [0268] 14. A nucleic acid encoding the polypeptide of any of items 1 to 13. [0269] 15. A vector comprising the nucleic acid of item 14. [0270] 16. A conjugate comprising the polypeptide of items 1 to 13 and at least one label and/or at least one drug. [0271] 17. A complex comprising at least one polypeptide of items 1 to 13 and/or at least one conjugate of item 16. [0272] 18. The complex of item 17 further comprising at least one label and/or at least one drug. [0273] 19. The conjugate of item 16 or the complex of items 17 or 18, wherein the label is selected from the group consisting of [0274] a. a fluorescent dye, in particular a fluorescent dye selected from the group consisting of the following classes of fluorescent dyes: Xanthens, Acridines, Oxazines, Cynines, Styryl dyes, Coumarines, Porphines, Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals, Perylenes and Phtalocyanines as well as conjugates and combinations of these classes of dyes; [0275] b. a radioisotope/fluorescence emitting isotope, in particular a radioisotope/fluorescence emitting isotope selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as 65Tb, fluorescence emitting isotopes, such as 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 as well as conjugates and combinations of above with proteins, peptides, small molecular inhibitors, antibodies or other compounds; [0276] c. a detectable polypeptide, in particular an autofluorescent protein, preferably green fluorescent protein or any structural variant thereof with an altered adsorption and/or emission spectrum or nucleic acid encoding a detectable polypeptide; and [0277] d. a contrast agent, in particular a contrast agent comprising a paramagnetic agent, preferably selected from Gd, Eu, W and Mn, or ferrihydride. [0278] 20. The conjugate of item 16 or 19 or the complex of items 17 to 18, wherein the drug is selected from the group consisting of an anticancer drug, in particular a cytostatic drug, cytotoxic drug or prodrug thereof, an anti-arteriosclerotic drug, and an anti-inflammatory or immunomodulatory drug. [0279] 21. The conjugate of item 16, 19 or 20 comprising a drug, wherein the drug is auristatin, in particular monomethyl auristatin (MMAE), conjugated to the polypeptide via a maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl linker. [0280] 22. An isolated targeted delivery system comprising a cell, wherein the cell comprises the polypeptide of items 1 to 13, the conjugate of item 16 or 19 to 21, or the complex of item 17 to 21, wherein particularly the cell is a CD45+ leukocyte, more particularly a CD45+ leukocyte selected from the group consisting of a monocyte, a differentiated monocyte, lymphocyte and a granulocyte. [0281] 23. A pharmaceutical or diagnostic composition comprising the polypeptide of items 1 to 13, the conjugate of item 16 or 19 to 21 or the complex of item 17 to 21 or the isolated targeted delivery system of item 22 and a pharmaceutically acceptable carrier and/or suitable excipient(s). [0282] 24. The polypeptide of items 1 to 13, the conjugate of item 16 or 19 to 21 or the complex of item 17 to 21 or the isolated targeted delivery system of item 22 for use in medicine.